Giornate di chimica analitica

in memoria del Prof. Francesco Dondi

Recenti sviluppi in Scienze delle Separazioni e Bioanalitica

FERRARA, 10-11 Luglio 2017

Polo Chimico-Bio-Medico, via Luigi Borsari 46

scf.unife.it/it/chimica2017

Lettera di Benvenuto Caro Collega, caro Amico, queste due giornate di Chimica Analitica organizzate in collaborazione tra il Gruppo Interdivisionale di Scienza delle Separazione e quello Divisionale di Bioanalitica della Società Chimica Italiana (SCI) vogliono ricordare la figura di Francesco Dondi, professore di Chimica Analitica presso l’Università degli Studi di Ferrara dal 1986 al 2013, prematuramente scomparso il 30 Ottobre 2015. Francesco è stato uno dei fondatori della moderna Scienza delle Separazioni in Italia. Formatosi alla scuola di due tra i più grandi scienziati a livello mondiale in questo campo J. Calvin Giddings e Georges Guiochon i suoi maggiori contributi scientifici sono stati nell’ambito della modellizzazione della separazione cromatografica da un punto di vista microscopico-molecolare (la teoria stocastica della cromatografia) e nell’interpretazione di miscele complesse multicomponente. Nell’ultimo periodo della sua carriera, Francesco si è dedicato assiduamente allo studio di tematiche di etica e scienza per uno sviluppo sostenibile della società, ruolo in cui è stato anche Delegato del Rettore. Questa attività è solo una delle dimostrazioni del costante servizio che Francesco ha reso al suo Ateneo durante tutta la sua carriera. Ferruccio Trifirò e Francesco Gasparrini, scienziati di fama internazionale nonché cari amici di Francesco, ricorderanno la figura di Francesco all’apertura delle giornate di Chimica Analitica. Siamo lieti di annunciare che alle giornate di Chimica Analitica si sono registrati più di 120 ricercatori a testimonianza del grande interesse e delle grandi motivazioni che, malgrado le numerose difficoltà in cui versa il sistema della ricerca pubblica in Italia, continuano a caratterizzare la nostra comunità. Durante le giornate, verranno consegnate tre medaglie di nuova istituzione. Due medaglie “Giovanni Dugo” saranno consegnate rispettivamente alle professoresse Rosangela Marchelli e Paola Dugo per i loro contributi nelle Scienza delle Separazioni con applicazioni nel campo della Chimica degli Alimenti. La medaglia “Alessandro Mangia” verrà assegnata al professor Aldo Roda per la sua ricerca nel campo della Bioanalitica. Verranno anche premiati due giovani ricercatori che si sono distinti sia nel campo delle Scienze delle Separazioni che della Bionalitica. Un ringraziamento speciale va agli sponsors di queste giornate che, tramite il loro generoso contributo, hanno consentito di offrire a tutti i soci SCI appartenenti al Gruppo Interdivisionale di Scienza delle Separazione e/o al Gruppo Divisionale di Bioanalitica l’iscrizione gratuita alle giornate di Ferrara. Siamo coscienti che senza di loro questo evento non sarebbe stato lo stesso. Vi auguro un proficuo e sereno soggiorno a Ferrara. Con viva cordialità, Alberto Cavazzini Chairman

SPONSORS

Platinum

Gold

Silver

Comitato Scientifico Scienze delle Separazioni

Aldo Laganà, Università di Roma La Sapienza

Luigi Mondello, Università di Messina

Anna Laura Capriotti, Università di Roma La Sapienza

Achille Cappiello, Università di Urbino Carlo Bo

Alberto Cavazzini, Università di Ferrara

Danilo Corradini, CNR, Ist. di Metodologie Chimiche, Roma

Massimo Del Bubba, Università di Firenze

Comitato Scientifico BioAnalitica

Mara Mirasoli, Università di Bologna

Maria Careri, Università di Parma

Chiara Cavaliere, Università di Roma La Sapienza

Claudio Baggiani, Università di Torino

Ilaria Palchetti, Università di Firenze

Comitato Organizzatore

Alberto Cavazzini Maurizio Remelli Maria Chiara Pietrogrande Nicola Marchetti Luisa Pasti Catia Contado Antonella Pagnoni Valentina Costa Annalisa Maietti Paola Tedeschi Caterina Bergantin Martina Catani Simona Felletti Tatiana Chenet Francesco Manarini Paolo Oliveri

PROGRAMMA

9:30 11:00

11:00 11:20

11:20 11:40 Trifirò Ferruccio Etica e Chimica: la passione e l'impegno di Francesco Dondi Plenary lecture

11:40 12:00 Gasparrini Francesco

On the use of the sub‐2µm Chiral Stationary Phases in 

enantioselective Ultra‐High Performance Chromatography 

(UHPLC/UHPSFC)

Plenary lecture

12:00 12:20 Dugo  GiovanniUn viaggio di mezzo secolo attraverso la Chimica degli alimenti e la 

Scienza delle SeparazioniPlenary lecture

12:20 12:40 Mangia  Alessandro Un percorso nella Chimica Analitica Plenary lecture

12:40 12:45

12:45 12:50

12:50 12:55

13:00 14:30

14:35 14:55 Capriotti Anna Laura Recent trends in bioactive peptides analysis Vincitore giovane ‐ Sezione Scienza Separazioni 

14:55 15:15 Piovesana SusyShotgun Phosphoproteomics of Complex Real Samples by New 

Magnetic MaterialsVincitore giovane ‐ Sezione Bioanalitica

Chairpersons: Achille Cappiello, Giampiero Adami Chairpersons: Maria Careri, Giuseppe Palleschi

15:15 15:35Sciarrone Danilo           

keynote

Three‐Dimensional GC‐Prep Coupled To Spectroscopic Analysis As 

A Tool For Structural Identification Of Unknown Molecules15:15 15:35

Cinti Stefano          

keynote

Paper as Substrate in Bioelectroanalysis for Healthcare 

Applications

15:35 15:55 Vecchietti Davide      

Shimadzu

Enhancing MS/MS spectral libraries: improved approaches using 

triple quadrupole mass spectrometer15:35 15:50 Baggiani Claudio

How Can We Evaluate the Binding Properties of Molecularly 

Imprinted Polymers without the Misleading Current Approach?

15:55 16:15 Sciarrone  Danilo           

DANI

Reliable Compound Identification by Means of GC‐TOF‐ MS 

Analysis and Spectral Search with the Simultaneous Use of Linear 

Retention Index

15:50 16:05 Giannetto Marco

Competitive amperometric immunosensor for determination of 

p53 protein in urine with carbon nanotubes/gold nanoparticles 

screen printed electrodes: a rapid and noninvasive screening tool 

for early diagnosis of bladder carcinoma

16:05 16:20 Risoluti RobertaTGA/Chemometrics Approach In Bioanalytical Investigations: The 

Screening of Sickle Cell Anemia

16:25 16:50

Chairpersons: Massimo del Bubba, Alberto Cavazzini Chairpersons: Sara Bogialli, Chiara Cavaliere

16:50 17:05 Frapiccini EmanuelaPAHs in Mullus barbatus of North Adriatic Sea: Determination by 

HPLC, After QuEChERS Method Extraction16:50 17:05 Arigò Adriana

Supercritical Fluid Chromatography coupled to Tandem Mass 

Spectrometry for Limonoid Aglycones Detection in Citrus Essential 

Oils

17:05 17:20 Ancillotti Claudia LC‐MS/MS Methods for Metabolomics: from Fruits to Biofluids 17:05 17:20 Orsini Francesca

Identification of Plant Secondary Metabolites by HPTLC‐ESI‐MS 

and Assessment of their Antioxidant Activity by a HPTLC‐DPPH 

Method

17:20 17:35 Catani Martina

Kinetic Performance Of New Core‐Shell And Sub‐2μm Fully Porous 

Pirkle‐type Chiral Stationary Phases For Ultrafast 

Enantioseparations

17:20 17:35Calvano Cosima 

Damiana

Structural Characterization of Croconaine Dyes by MALDI‐Tof/Tof 

Mass Spectrometry Analysis

17:35 17:50 Micalizzi Giuseppe

Method Optimization for Elucidation of Human Blood Fatty Acid 

Methyl Esters by Using Conventional and Fast Gas 

Chromatography

17:35 17:50Zenezini Chiozzi 

Riccardo

Protein Corona Sensor Array Nanosystem for Multivariate Cancer 

Detection

17:50 18:05 Oteri Marianna

Carotenoid Fingerprinting in a Paprika Sample by Supercritical 

Fluid Chromatography×Ultra High Pressure Liquid 

Chromatography and Mass Spectrometry Detection

17:50 18:05 Robotti ElisaMitochondrial Proteome Modifications due to eIF6 Depletion by

UHPLC‐QTOF MS/MS with SWATH‐MS Acquisition

18:05 18:20 La Barbera GiorgiaUntargeted metabolic profiling by means of UHPLC‐QTOF/MS for 

the identification of meat and dairy products biomarkers.18:05 18:20 Ferrone Vincenzo

Three‐Dimensional Graphene/Fe3O4 Nanocomposite Based 

Dispersive Magnetic Solid Phase Extraction Coupled With 

UHPLCPDA For Simultaneous Determination Of NSAIDs In Human 

Plasma

18:20 18:35 Felletti SimonaEvaluation Of Excess And Adsorption Isotherms In Chiral 

Stationary Phases

20:00

Consegna medaglia "G. Dugo" a prof.ssa Dugo Paola

pranzo e sessione poster

coffee break e sessione poster

Consegna medaglia "A. Mangia" a prof. Roda Aldo

Lunedì 10 Luglio

SCIENZA SEPARAZIONI   ‐  AULA E2 BIOANALITICA   ‐   AULA E1

cena sociale

Registrazione

 AULA E2

Benvenuto e Saluti delle Autorità

Chairpersons: Aldo Laganà, Danilo Corradini, Mara Mirasoli

Chairpersons: Luigi Mondello, Mara Mirasoli

 AULA E2

Consegna medaglia "G. Dugo" a prof.ssa Marchelli Rosangela

 Prof. Luigi Dei ‐ Magnifico Rettore dell'Università degli Studi di Firenze 

Prof. Giorgio Zauli ‐ Magnifico Rettore dell'Università degli Studi di Ferrara

Chairpersons: Aldo Laganà, Danilo Corradini Chairpersons: Claudio Baggiani, Danila Moscone

9:00 9:20Bianchi Federica         

keynote

The role of selective materials in sample treatment and analyte 

detection9:00 9:20

Zangheri Martina       

keynote

Latest advances in ultrasensitive chemiluminescent lateral flow 

immunoassay biosensors for the multiplex detection of 

mycotoxins in food and beverage

9:20 9:35 Montone Carmela Maria

Determination of Mycotoxins in Cereals by a Rapid Magnetic Solid 

Phase Extraction Method followed by Liquid Chromatography‐

Tandem Mass Spectrometry Analysis

9:20 9:35 Anfossi LauraDual Signal Readout for Sensitive Detection Of Fluorescence 

Immunochromatographic Test Strip

9:35 9:50 Bergantin Caterina

Qualitative And Quantitative Differences In Carotenoid Profile 

Among Two Varieties Of Pumpkins By HPLC‐DAD Analysis with a 

C30 column

9:35 9:50 Di Nardo FabioRecent Strategies for Multiplex Immunochromatographic Strip 

Test Based on Noble Metal Nanoparticles

9:50 10:10 Barbieri Marica            

Agilent

Il potere della Cromatografia Bidimensionale 2D‐LC per l’analisi 

quantitativa e qualitativa in matrici complesse.09:50 10:05 Arduini Fabiana

Paper‐based (bio)sensors for the detection of chemical warfare 

agents

10:10 10:25 Schepis Antonino

Novel Simultaneous Detection by Isotope Ratio Mass 

Spectrometry and Quadrupole Mass Spectrometry Coupled to 

Multidimensional Gas Chromatography for the Analysis of 

Valuable Food Products

10:05 10:20 Calabria Donato

Smartphone–based reflectance biosensor for rapid detection of 

oxidase substrates using confined “wafer‐like” multilayer 

paperbased enzymatic assays.

10:25 10:45Pantò Sebastiano           

LECO

Development of a GCXGC‐TOFMS methods for the quantification 

of the extended list of suspected allergens in fragrances materials10:20 10:35 Costantini Francesca A Versatile Aptasensor Material for Lab‐on‐Chip Applications

10:45 11:00 Vincenti FlaminiaDifferent Strategies For Extraction Of Illicit Drugs In Hair By Means 

UHPLC‐HRMS/MS10:35 10:50 Badocco Denis

Prototype of an optical sensor for oxygen measurements in 

oenological matrices

11:00 11:15 Pasquini Benedetta

Enantioseparation and Impurity Determination of Cinacalcet using 

Solvent Modified Capillary Zone Electrophoresis and Quality by 

Design: Study of the Complexation with Cyclodextrins by 

Molecular Modeling and NMR

10:50 11:05 Marassi ValentinaSeparation And Characterisation Of Exosomal Subpopulations 

Through Flow Field‐Flow Fractionation

11:15 11:50

Chairpersons: Paola Dugo, Paolo Pastore Chairpersons: Mara Mirasoli, Renato Seeber

11:50 12:05 Tripodo GiusyAnalysis of bioactive phenolic compounds in different hazelnut 

kernels by RP‐HPLC/PDA/ESI‐MS.11:50 12:05 Giovannoli Cristina

Effect of Surfactants on the Binding Properties and Selectivity of 

Molecularly Imprinted Polymers

12:05 12:20 Termopoli Veronica Novel aspects and performance evaluation of a liquid‐EI LC‐MS 

interface12:05 12:20 Zanardi Chiara Nanosized materials in amperometric sensing

12:20 12:40Volders Filip               

Elementar

An LC‐IRMS Interface for Flexible Compound‐specific Stable 

Isotope Analysis12:20 12:35 Bettazzi Francesca

Evaluation of sample preparation methods for the determination 

of PBDE in foodstuff by immuno‐assay‐based screening methods

12:40 12:55 Chenet TatianaBivalve Mollusk Shells as a Low‐Cost Biosorbent for Water 

Remediation12:35 12:50 Della Pelle Flavio

Microextraction techniques coupled to different carbon black 

based electrochemical detection strategy: application to 

carbamates analysis

12:55 13:10 Piparo Marco

Fast Cryogenic Comprehensive Two‐dimensional Gas 

Chromatography‐mass Spectrometry: Concept, Method 

Optimization and Applications in the Lipid Field

12:50 13:05Battaglia Ivano  

LabService

Environmental odor pollution. A GC‐MS/O study with OdorPrep 

sampling approach

13:10 14:05

Chairpersons: Tommaso Cataldi, Chiara Fanali

14:05 14:20 Agostini Alessandro        

FKV

Nuovi sviluppi nella Spettrometria NMR da 

banco: affidabilità analitica, precisione di misura e sicurezza di 

utilizzo

14:20 14:35 Piergiovanni Maurizio Determination Of Benzodiazepines In Beverages Using Green 

Extraction Methods And HPLC‐UV Detection

14:35 14:50 Mangraviti Domenica

Comprehensive Two‐dimensional Liquid Chromatography Coupled 

to Mass Spectrometry for Elucidation of the Polyphenolic Fraction 

of Pistacia vera from Different Geographical Origin

14:50 15:05 Rivoira LucaChromatographic determination of biogenic amines in wines by 

novel detection approaches

15:05 15:20 Truzzi Cristina

Global Warming: Influence of Raising Temperature on Fatty Acid 

Composition of Muscle of Antarctic Teleost Trematomus 

Bernacchii, Analysed by Gas‐Chromatography Mass‐Spectrometry

15:20 15:35 Ventura Giovanni Characterization of Vitamin B12–Pt(II) Conjugates by RPLC‐ESI‐MS

15:35 15:50 Di Gangi Iole MariaLiquid Chromatography‐High Resolution Mass Spectrometry For 

The Correct Identification Of The Cyanotoxin BMAA

15:50 16:05 Manarini FrancescoOptimization of an ultrasound‐assisted derivatization for GC/MS 

analysis of oxygenated organic species in atmospheric aerosol

16:05 16:20 Ismail Omar

Teicoplanin Chiral Stationary Phases on 2.0 µm and 2.7 µm 

Superficially Porous Particles: Chromatographic Evaluation And 

Comparison With Teicoplanin On 1.9 µm Fully Porous Particles

16:20 16:30 chiusura lavori

coffee break e sessione poster

pranzo e sessione poster

SCIENZA SEPARAZIONI  ‐  AULA E2 BIOANALITICA   ‐   AULA E1

Martedì 11 Luglio

VINCITORI MEDAGLIE “GIOVANNI DUGO”

E “ALESSANDRO MANGIA”

Medaglia “Giovanni Dugo” Prof.ssa Paola Dugo Prof.ssa Rosangela Marchelli

Medaglia “Alessandro Mangia” Prof. Aldo Roda

VINCITORI PREMIO GIOVANE RICERCATORE

Abstract

Recent trends in analysis of bioactive peptides in food matrices

Capriotti Anna Laura

aDipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy

Keywords: bioactive peptides, multidimensional chromatography, nutraceutical, food and by-products, mass spectrometry, bioinformatics tools

Food-derived constituents represent important sources of several classes of bioactive compounds. Among them

peptides have gained great attention in the last two decades thanks to the scientific evidence of their beneficial effects on health in addition to their established nutritional value. Several functionalities for bioactive peptides have been described, including antioxidative, antihypertensive, anti-

inflammatory, immunomodulatory, and antimicrobial activity. They are now considered as novel and potential dietary ingredients to promote human health, though in some cases they may also have detrimental effects on health. Bioactive peptides can be naturally occurring, produced in vitro by enzymatic hydrolysis, and formed in vivo during gastrointestinal digestion of proteins.

This keynote provides an overview of my research activity focusing mainly on the major developments in the field of peptidomic sciences, telling some success stories as well as challenges that are currently being faced.

The keynote highlights the prospects of bioinformatics and a proposed integrated approach for enhancing the production of existing and new bioactive peptides from sustainable food protein sources, followed by a critical evaluation of the major challenges that may impact prospective commercialization of food bioactive peptides for use in human health promotion. Examples of promising applications of these peptides in food, nutraceuticals and cosmeceuticals will be also discussed with an insight to the future research needs.

Moreover, a new on-line multidimensional system for sequential trapping and individual elution and separation of peptides based on their molecular weight is described. By sequentially using two chemically different trapping columns, a polymethacrylate monolith and a packed C18 one, peptides from complex samples can be on-line trapped and divided into two fractions, containing respectively mainly medium-large peptides and smaller peptides. This simple approach can contribute to further extending the strategies of peptide identification in food peptidomics.

Abstract

Shotgun Phosphoproteomics of Complex Real Samples by New Magnetic Materials

Susy Piovesana

Dipartimento di Chimica, Sapienza Universita di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy

Keywords: Phosphopeptide enrichment; affinity chromatography; magnetic nanoparticles; nanoHPLC-M/MS; phosphoproteomics.

Nanomaterials are attracting a considerable attention in several fields, including analytical chemistry, where they can provide new sorbents for extraction and concentration of target compounds (1). Protein phosphorylation is fundamental in living organisms, because it is an on and off switch for a myriad of processes, strictly related to biological pathways within cells themselves; thus, the knowledge of the phosphorylation state provides valuable information, useful to elucidate disease mechanisms (2) and plant metabolism (3). Nevertheless, the characterization of phosphoproteins and endogenous phosphopeptides, together with the quantification of their changes, still represents an analytical challenge in shotgun phosphoproteomics analysis, especially for complex real samples (4). Among the different approaches, immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) represent the most exploited ones, but still a comprehensive enrichment strategy is lacking, thus new materials continue to be developed and tested (5,6). In this context, magnetic solid-phase extraction can be advantageously coupled with nanomaterials. The main advantage of such an approach is the possibility of enriching the target compound directly in the sample solution; then, the magnetic propertied of the material allow to easily retrieve it by application of an external magnetic field. This workflow is a very versatile one and suitable for large-volumes as well, thus making the isolation convenient, rapid and effective, while increasing the interfacial area between the solid adsorbent and the sample solution, thus shortening the extraction time (7). In this regard, the keynote will present some new materials which exploit magnetic solid phase extraction and affinity chromatography to set up methods for phosphopeptide enrichment. A special emphasis is given to the application to real matrices, such as yeast extracts and serum, and the need to embed the enrichment protocols within a typical shotgun proteomics workflow, to move from the proof of principle level to the real world application one.

REFERENCES

1. M. Ahmadi, H. Elmongy, T. Madrakian, M. Abdel-Rehim, Nanomaterials as Sorbents for Sample Preparation in Bioanalysis: A Review, Anal. Chim. Acta 2016, 958: 1–21.

2. P. R. Cutillas, Role of Phosphoproteomics in the Development of Personalized Cancer Therapies, Proteomics - Clin. Appl. 2015, 9:383–395.

3. C. Silva-Sanchez, H. Li, s. Chen, Recent Advances and Challenges in Plant Phosphoproteomics, Proteomics 2015, 15:1127–1141. 4. X.-S. Li, B.-F. Yuan, Y.-Q. Feng, Recent Advances in Phosphopeptide Enrichment: Strategies and Techniques, TrAC Trends Anal.

Chem. 2016, 78:70–83. 5. S. Piovesana, A. L. Capriotti, C. Cavaliere, F. Ferraris, D. Iglesias, S. Marchesan, A. Laganà, A New Magnetic Graphitized Carbon

Black TiO 2 Composite for Phosphopeptide Selective Enrichment in Shotgun Phosphoproteomics. Anal. Chem. 2016, 88:12043–12050.

6. S. Piovesana, A. L. Capriotti, C. Cavaliere, F. Ferraris, R. Samperi, S. Ventura, A. Laganà, Phosphopeptide Enrichment: Development of Magnetic Solid Phase Extraction Method Based on Polydopamine Coating and Ti4+-IMAC. Anal. Chim. Acta 2016, 909:67–74.

7. X.-S. Li, G.-T. Zhu, Y.-B. Luo, B.-F. Yuan, Y. Q. Feng, Synthesis and Applications of Functionalized Magnetic Materials in Sample Preparation. TrAC - Trends Anal. Chem. 2013, 45:233–247.

PLENARY

Abstract

Etica e Chimica ; la passione e l’impegno di Francesco Dondi

Ferruccio Trifirò

Presidente Accademia delle Scienze dell’Istituto di Bologna

Nel campo dei rapporti fra etica e scienza, Francesco Dondi, professore di analitica all' Università di Ferrara, ha dato un contributo significativo, riconosciuto anche a livello europeo.. Per ricordarlo accenneremo in questa nota a due aspetti dei rapporti fra etica c scienza che lui ha approfondito: come e perché insegnare l'etica al chimici' e come comportarsi nei riguardi dei grandi rischi per l'umanità, in particolare il nucleare Per Dondi un corso di etica è l'occasione per insegnare agli studenti una rinnovata visione dei grandi problemi attuali del mondo : l’ambiente il cibo le risorse di acqua i cambiamenti climatici i rifiuti il bioaccumulo di sostanze tossiche ecc .I corsi di etica devono trattare gli aspetti dell'impatto della chimica sulla natura creando una cultura della responsabilità, la sola capace di risolvere gli enormi e complessi problemi del mondo e per questo occorre una interazione fra chimica, politica, scienza e ambiente mettendo insieme la cultura umanistica con quella scientifica. Per Dondi i diversi disastri ambientali e tecnologici e i loro tragici effetti, impongono un approccio serio all'identificazione delle responsabilità attraverso una sistematica applicazione delle metodologie etiche. I sistemi a rischio, come gli impianti nucleari, non possono mai considerati a basso rischio, devono essere gestiti e ispirati al "principio di precauzione" perché coinvolgono le generazioni future, le quali non avendo voce in capitolo ora, hanno pur tuttavia il diritto di essere tutelate secondo Ie nostre norme etiche. Esaminare gli errori etici fatti nella gestione del nucleare consente di meglio comprendere questa società che, accanto all'abbondanza di uno sfrenato consumismo, sembra altresì produrre una serie spettrale di rischi, imprevisti e inimmaginabili. Gli aspetti etici relativi ai grandi rischi sono assai rilevanti, e non solo in relazione alla scelta nucleare, ma anche per i molteplici aspetti della "società del rischio". La società dovrebbe investire quindi più risorse in questo importante approccio scientifico alla critica sociale e tecnologica per andare verso una civiltà della "sostenibilità", consapevole cioè dei rischi e delle criticità presenti e future. Ciò richiede una sintesi degli aspetti culturali, ambientali ed economici: in tutto ciò I’etica deve essere integrata con la cultura scientifica.

REFERENCES

1. F.Dondi “Why and how to teach ethics in chemical education” La Chimica & L’Industria 2009, 9, 100. 2) F Dondi “Etica, responsabilità e sostenibilità nella cultura chimica” La Chimica & L’Industria 2011, 9, 106. 3) F Dondi “Etica e Nucleare le scelte della Società del rischio” La Chimica & L’Industria, 2012 ,2 ,89. 4) F Moser e F Dondi “On the road to Rio+20 the evolution of environmental ethics for a safe world” Toxicological & Environmental

Chemistry 2012, 84(5), 807.

Abstract

On the use of the sub-2µm Chiral Stationary Phases in enantioselective Ultra-High Performance Chromatography

(UHPLC/UHPSFC)

F. Gasparrini

Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma (Italy)

This presentation cannot begin without a necessary remembrance of Francesco Dondi, a precious colleague and rigorous scientist, with whom for a decade I have shared a fruitful research experience in the study of mechanisms of molecular recognition, chiral discrimination and chemical-physical properties of Stationary phases. His example is certainly a precious inheritance for those who have followed his way. The extraordinary progress in developing highly efficient packing materials for liquid chromatography (LC) has only partly touched chiral separations. This is due, in part, to practical problems in synthesis and functionalization of sub-2μm particles with chiral selectors, and, in part, to the lack of clear understanding of mass-transfer mechanisms in enantioselective chromatography. However, there is an increasing demand for ultrafast high performance chiral separations, mainly from fine chemical and pharmaceutical companies, and from researchers active in life science. This talk revisits the most important achievements in enantioselective Ultra High Performance Chromatography (eUHPC) focused on brush-type Chiral Stationary Phases (CSPs). From the kinetic point of view, this kind of CSPs represent a very promising material for the transition from the traditional enantioselective High Performance LC (eHPLC) to the Ultra-High Speed and Ultra-High Pressure regime, i.e. enantioselective “e” Ultra High Performance Chromatography (eUHPC). In particular we will show the results obtained by using the well-known broad-spectrum Whelk-O1 selector. This selector, introduced by Pirkle and Welch on 1992 for enantioselective HPLC, was recently chemically-bonded on the surface of sub-2μm totally porous, high surface area, silica particles. The potential of ultra-fast chiral separations by employing the sub-2µm Whelk-O1 CSP has been then investigated in both UHPLC and Ultra High Performance Supercritical Fluid Chromatography, UHPSFC[1]. Evaluation of the column kinetic performance demonstrated that higher linear velocities can be used in UHPSFC compared to UHPLC. The benefit of UHPSFC over normal-phase UHPLC with sub-2μm Whelk-O1 chiral particles for ultra-fast separations is evident as, in the regime of very fast separations, it is possible to achieve larger efficiency and, consequently, even larger resolution. In addition, in order to obtain at the same time ultra-fast and high efficiency separations, we packed ultra-short columns (1-cm long) that allowed analysis times, in same case, shorter than 1 seconds . Finally, focusing on the development of high-throughput screening approaches, the sub-2µm Whelk-O1 has been successfully employed in the enantioresolution of a large group of racemates with noticeable results.

REFERENCES

1. D. Kotoni, A. Ciogli, C. Molinaro, I. D'Acquarica, J. Kocergin, T. Szczerba; H. Ritchie; C. Villani; F. Gasparrini. Anal. Chem. 2012, 84, 6805; L. Sciascera, A. Ciogli, O. Ismail, D. Kotoni, A. Cavazzini, L. Botta, T. Szczerba, C. Villani, F. Gasparrini. J Chromatogr A 2015, 1383, 160-8; O. Ismail, L. Pasti, A. Ciogli, C. Villani, J. Kogercin, S. Anderson, F. Gasparrini, A. Cavazzini, M. Catani, J Chromatogr A 2016, 1466, 96-104.

KEYNOTE

SCIENZA DELLE SEPARAZIONI

Abstract

Three-Dimensional GC-Prep Coupled To Spectroscopic Analysis As A Tool For Structural Identification Of Unknown Molecules

Danilo Sciarronea, Antonino Schepisa and Luigi Mondelloa,b,c

a Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università degli Studi di Messina, Polo Annunziata, Viale Annunziata - 98168 Messina

b Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali”, University of Messina, Polo Annunziata, viale Annunziata, 98168 Messina, Italy

c Unit of Food Science and Nutrition, Department of Medicine - University Campus Bio-Medico of Rome, via Álvaro del Portillo 21, 00128 - Rome, Italy

Keywords: MDGC, prep-GC, NMR

Conventional GC analysis for preparative purpose presents different limitations when highly pure compounds have to be collected at milligrams level in reasonable time. The continuous research for novel molecules represents nowadays a “hot topic” for many academic and industrial fields as pharmaceutical, nutraceutical and flavour & fragrance just to consider some of them. The never-ending availability of new plant species from all over the world for example can be considered one of the most important of source for new molecules. The collection of high amount of compounds from natural sources, often characterized by an high complexity level, presents some issues: A) a limited amount of neat or diluted oil can be analyzed in each run due to the GC column sample capacity and efficiency, even if wide bore columns are used, and as a consequence, B) the total analysis time to collect a certain amount is greatly affected by the sample injection volume and finally, C) the purity degree of the collected fraction is often unsatisfactory due to the presence of coeluted compounds. As expected, the higher is the injection volume, the lower is the total time required to collect a specific compound thus the highest injection volume should be always used. With the intention to improve the productivity of the system a multidimensional prep-GC instrument is presented with the goal to reduce the total collection time and to improve the purity of the components collected. The prep-MDGC system consisted of three chromatographs1, equipped with three Deans switch transfer devices. a highly productive multidimensional GC-prep system demonstrating the capability to collect highly pure sample amounts (mgs) in a short time. The system can be operated in different configurations, based on the complexity of the sample, exploiting a front-end LC pre-separation (whenever required by the complexity of the sample) and two or three GC dimensions with the aim to purify the fraction of interest prior to the collection step. Different case-of-study are reported describing the potentiality of such an approach to provide a useful starting point for the identification of possible highly valuable molecules for industrial and biological evaluations.

REFERENCES

Danilo Sciarrone et al., Trends in Analytical Chemistry 71 (2015) 65-73

Abstract

The role of selective materials in sample treatment and analyte

detection

Federica Bianchi, Nicolò Riboni and Maria Careri

Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma Parco Area delle Scienze 17/A, 43124 Parma

Keywords: sol-gel process; supramolecular chemistry; solid-phase microextraction; microelectromechanical sensors; ambient detection techniques

The development of new materials for analytical purposes is of paramount importance both to improve performance and to shorten analysis time. The main goal is to increase the enrichment capabilities of the pre-concentration units, thus reducing the presence of potential interfering compounds.

Our research group has a longstanding experience in the rational design and use of supramolecular receptors as sensing materials to address the selectivity issue via specific π−π and CH−π interactions and controlled cavity dimension. Conformationally mobile1, blocked2 and properly functionalized3 tetraquinoxaline cavitands have been proposed as solid-phase microextraction (SPME) coatings for environmental detection of BTEX, drugs and nitroaromatic explosives showing superior performances compared to available commercial materials. A new device for ppbv level detection of benzene in air by coupling a microelectromechanical sensor-integrated supramolecular concentration unit to a miniaturized PID detector is currently being studied. This

configuration allowed to develop a simple, stand-alone, and unsupervised sensing device, in which the cavitand receptor acts at the same time as selective pre-concentrator and GC-like separation device. Sol-gel technology can be proposed as a valid alternative to the commonly used coating procedures to produce high thermallly and chemically stable materials4. Interesting instrumental innovations based on the development of sol-gel coated ion sources have been recently tested by interfacing liquid chromatography and electron ionization mass spectrometry for the determination of environmental pollutants5. The capabilities of sol-gel materials were exploited also for the development of planar SPME devices for the rapid detection of explosives by ion mobility spectrometry6. Finally, miniaturized-solid-phase extraction cartridges proved to be suitable for the desorption electrospray ionization high resolution mass spectrometry determination of explosives in soil with minimal sample preparation and fast analysis time, thus allowing high-throughput analyses for screening purposes7.

REFERENCES

1. F. Bianchi, M. Mattarozzi, P. Betti, F. Bisceglie, M. Careri, A. Mangia, L. Sidisky, S. Ongarato, E. Dalcanale, Anal. Chem., 2008, 80, 6423.

2. N. Riboni, J. Trzcinski, F. Bianchi, C. Massera, R. Pinalli, L. Sidisky, E. Dalcanale, M. Careri, Anal. Chim. Acta, 2016, 905, 79. 3. F. Bianchi, A. Bedini, N. Riboni, R. Pinalli, A. Gregori, L. Sidisky, E. Dalcanale, M. Careri, Anal. Chem., 2014,86, 10646. 4. C. Shende, A. Kabir, E. Townsend, A. Malik, Anal. Chem. 2003, 75, 3518. 5. N. Riboni, L. Magrini, F. Bianchi, M. Careri, A. Cappiello, Anal. Chim. Acta, 2017, 978, 35. 6. M. Mattarozzi, F. Bianchi, F. Bisceglie, M. Careri, A. Mangia, G. Mori, A. Gregori, Anal. Bioanal. Chem., 2011, 399, 2741. 7. F. Bianchi, A. Gregori, G. Braun, C. Crescenzi, M. Careri, Anal. Bioanal. Chem., 2015, 407, 93

KEYNOTE

BIOANALITICA

Abstract

Paper as Substrate in Bioelectroanalysis for Healthcare Applications

Stefano Cinti, Fabiana Arduini, Danila Moscone, Giuseppe Palleschi

Dipartimento di Scienze e Tecnologie Chimiche, Università degli Studi di Roma “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy

Keywords: Sustainability, Electroanalysis, Screen-printing, Paper, Healthcare.

This is In the era of sustainability, the reduction of both the environmental and the economic impact related to mass-scale processes represents the leitmotiv. Regarding the analytical methodologies, the development of real-time, in-process monitoring, and environmental friendly analysis have been placed in top of the list of required features. A sustainable analytical method should minimize the production of hazardous waste during the analysis to reduce environmental impact and it should provide a more sustainable use of recyclable materials. Furthermore, the measurement should be cost-effective allowing for cost-effective analysis. The electroanalytical techniques, compared with the other analytical methods, require non-sophisticated equipment, small amount of sample, and are suitable for measurements on field. In addition, screen-printed electrodes own high adaptability: customizing shape, dimension, conductive-ink material, and substrate, allow for selective and finely calibrated electrodes to be fabricated for specific target analytes. However, even if glucometers represent a keystone as self-monitoring device, drawbacks related to their production cost and waste removal need to be carefully evaluated. In this keynote, paper-based substrates are proposed as novel materials for the sustainable production of printed electroanalytical platforms. An overview regarding the various manufacturing processes will be provided, and the properties of both chromatographic and office papers will be showed, highlighting the diverse experimental setup that are adopted depending on the type of involved paper. Herein, paper patterning will be focused on the well-known wax printing technology which allows to create hydrophobic/hydrophilic areas, making paper an all-in-one platform. The analytical relevance of the proposed approach will be proposed in terms of healthcare applications. More specifically, paper-based (bio)sensors to detect chloride, zinc, and DNA in biological fluids will be taken into account. By merging type of printing substrate, conductive inks, (nano)modifiers, and biological components, paper is a candidate towards the development of a low-cost and reagentless substrates, being capable to load, react, and filter the samples.

Abstract

A Smartphone-Based Biosensor for Ultrasensitive Chemiluminescent-Lateral Flow Immunoassay for the

Quantification of Ochratoxin-A in Wine and Istant Coffee

Martina Zangheria, Mara Mirasolia, Laura Anfossib, Fabio Di Nardob, Cristina Giovannolib, Massimo Guardiglia, Claudio Baggianib, Aldo Rodaa

aDepartment of Chemistry “Giacomo Ciamician”, Alma Mater Studiorum- University of Bologna, Via Selmi, 2 - 40126 Bologna, Italy

bDepartment of Chemistry, University of Turin, Via Giuria 5-10125, Turin, Italy

Keywords: Biosensor; Chemiluminescence; Mycotoxins; Lateral Flow Immunoassay, Ochratoxin-A Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi that is detected worldwide in various food and feed sources. Since OTA represents a potential hazard for human health, the European Community (EC) has established a maximum level for OTA in various feed and foods, in particular 2 g L-1 in grape juices, wine and must and 10 g L-1 in instant coffee. Several instrumental analytical methods are currently available for detecting these toxins in foodstuff, but they require complex sample preparation and dedicated laboratory equipment. Biosensors are very promising analytical tools for rapid on-site detection of analytes in complex matrices. We recently described a biosensor for multiplex detection of type-B fumonisins and B1 aflatoxin in maize samples based on a chemiluminescence Lateral Flow ImmunoAssay (CL-LFIA) coupled with a portable ultrasensitive CCD-based “contact” imaging device (1). The use of CL detection allowed accurate and objective analytes quantification, down to picomoles, rather than qualitative or semi-quantitative information usually obtained employing conventional LFIAs based on colloidal gold labelling. Recently, thanks to the technological advance in complementary metal oxide semiconductor (CMOS) camera technology, smartphones are emerging as detectors suitable for CL-based bioassays (2). Here, we report on the development of a smartphone-based simple, rapid and accurate biosensor based on CL-LFIA method for quantitative detection of OTA in wine and instant coffee. The biosensor is based on a direct competitive immunoassay employing horseradish peroxidase (HRP)-OTA conjugate as a tracer, which is detected by adding the luminol/enhancer/hydrogen peroxide CL cocktail and by using a smartphone’s camera for image acquisition and data handling. A self-standing microfluidic cartridge was developed, which houses the LFIA membrane and all the reagents necessary for the analysis. For CL signal detection, a smartphone cover-like adaptor, containing a plano-convex lens aligned with the camera, was developed and produced by 3D printing. Once the operator has carried out the assay using the LFIA cartdridge, both the smartphone and the cartridge are assembled with the smartphone adapter creating a mini-dark box to perform measurement of the CL signal. Calibration curves were generated by adding known amounts of OTA standard solutions to wine and instant coffee OTA free founding a limit of detection of 0.3 g L-1 in wine and 1.0 g L-1 in instant coffee and dynamic ranges were respectively 0.3-50 µg L−1 and 1.0-56 µg L−1. The developed system is suitable for screening procedures aimed at the quantitative detection of OTA in wine and instant coffee samples complying with EC legislation requirements.

REFERENCES

1. M. Zangheri, F. Di Nardo, L. Anfossi, C. Giovannoli, C. Baggiani, A. Roda, M. Mirasoli, Analyst 140 (2015) 358-365. 2. M. Zangheri, L. Cevenini, L. Anfossi, C. Baggiani, P. Simoni, F. Di Nardo, A. Roda, Biosensors and Bioelectronics 64 (2015) 63-68.

COMUNICAZIONI ORALI SCIENZA DELLE SEPARAZIONI

Abstract

Enhancing MS/MS Spectral Libraries: Improved Approaches Using Triple Quadrupole Mass Spectrometer.

Davide Vecchiettia

aShimadzu Italia, via G.B Cassinis , Milano, Italy

Keywords: MRM, Spectral Library, Spectrum Mode

Liquid Chromatography coupled with tandem mass spectrometry is a powerful technique for the quantifications and identification of small molecules at trace level in different matrix. Nevertheless the problem of false positive and false negative reporting could affect the reliability of this approach. Nowadays many scientists highlight the need to use stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, in order to prevent the occurrence of false positive and false negative results (1). Moreover the use of ESI ionization mode lack of standardization in the creation/use of spectral libraries, due to spectral differences obtained from different instruments. In order to overcome this limitation we reports a novel acquisition mode, based on Multiple Reaction Monitoring, able to produce high quality and reliable spectra that can be used both for spectral confirmation and, in future, for spectral libraries standardization. This approach resulted suitable for toxicological analysis and could be easily extended to many other applications.

REFERENCES

1. Francois Ludovique Sauvage, J M Gauller, G Lachatre Pitfall and prevention strategies for Liquid Chromatography Tandem Mass spectrometry in Selected Reaction – Monitoring mode for Drug analysis, Drug and monitoring Toxicology. 2008.

Abstract

Reliable Compound Identification by Means of GC-TOF-MS Analysis and Spectral Search with the Simultaneous Use of Linear

Retention Index

Danilo Sciarronea, Luigi Mondelloa,b,c

a Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università degli Studi di Messina, Polo

Annunziata, Viale Annunziata - 98168 Messina

b Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali”, University of Messina,

Polo Annunziata, viale Annunziata, 98168 Messina, Italy

c Unit of Food Science and Nutrition, Department of Medicine - University Campus Bio-Medico of Rome, via Álvaro del

Portillo 21, 00128 - Rome, Italy

All mass spectrometers combine ion formation, mass analysis, and ion detection. Each mass analyzer has its own special characteristics, potential applications as well as its own benefits and limitations. Consequently, the choice of mass analyzer should be based upon the application, cost, and performance desired. Among the different MS detectors, quadrupole- and TOF-based ones are preferred in the case of gas chromatography (GC) front-end separation, especially in fast or comprehensive approaches where fast scanning rates (>50Hz)are needed to properly detect sharp chromatographic bands eluted. Unlike quadrupole analyzer, where the mass range allowed needs to be narrowed in order to allow for faster acquisition rates, TOF analyzer requires no compromise in terms of speed and mass range amenable. On the other hand, TOF-MS spectra often suffer from a low spectral similarity, when searched against currently marketed spectral libraries that are based mostly on qMS. A quadruole-suitable MS spectra can be obtained using a new generation GC-TOF-MS system demonstrating the possibility to exploit the mass spectra databases with high-similarity hits. Furthermore, the simultaneous use of linear retention indices as an additional filter for identification

purposes is discussed.

Abstract

PAHs in Mullus barbatus of North Adriatic Sea: Determination by HPLC, After QuEChERS Method Extraction.

Emanuela Frapiccinia, Anna Annibaldib, Mattia Bettia, Mattia Bernardia, Monica Panfilia, Stefano Guicciardia, Silvia Illuminatib, Cristina Truzzib, Giuseppe

Scarponib, Mauro Marinia

a National Research Council (CNR), Institute of Marine Science (ISMAR), Largo Fiera della Pesca, 2, 60125 Ancona, Italy bDepartment of Life and Environmental Sciences, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy

Keywords: QuEChERS, polycyclic aromatic hydrocarbons, Mullus barbatus, Adriatic Sea

PAHs (polycyclic aromatic hydrocarbons) represent a wide class of organic compounds that derive mainly from incomplete combustion and pyrolysis of organic materials and are ubiquitous environmental pollutants. In the marine environment, PAHs are present in water column where, due to their high hydrophobicity and molecular mass, they tend to accumulate in sediments and biota becoming bioavailable to marine species via the food chain (1). Here, for the first time, a simple and fast method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction followed by HPLC-FLD (High Performance Liquid Chromatography-Fluorescence Detection) analysis was adopted for sixteen US EPA priority PAHs detection in fish tissue of red mullet, Mullus barbatus. The methodology used in this work (2) represents a valid alternative to traditional or novel methods, since it employs a multiresidue sample preparation procedure adapted for extraction and clean-up. The purpose of this work was both to test QuEChERS method for the extraction of these class of priority pollutants and to estimate the level of PAHs contents of a demersal species of Adriatic Sea: this species is of high commercial interest, consumed in large quantities by the local population and used as sentinel species for assessing the pollution along the western Mediterranean coast (3). Biological variables (age, weight, sex, sexual maturity) that would affect the PAHs accumulation were also investigated. The specimens (n=215) were captured during the period from August 2015 to March 2016 in the Central and Northern Adriatic Sea (geographical sub-area [GSA] 17), a wide continental shelf and eutrophic shallow waters, characterized by the presence of several rivers and by anthropogenic activities. Muscle samples were dissected out using acid-cleaned bistouries and scissors in the laboratory present on board, weighed (wet weight) and then stored at -18 °C for PAH determinations. QuEChERS method was successfully applied for PAHs determination from fish fillet of Mullus barbatus, the percentage of recovery was 85 ± 13.3. About PAHs content three of them (naphthalene, phenanthrene and fluoranthene) were found in all analyzed samples in a remarkable level; the mean concentrations were 6.5 ± 3.9 ng g-1, 0.8 ± 0.3 ng g-1 e 9.1 ± 3.1 ng g-1, for naphthalene, phenanthrene and fluoranthene, respectively. Among the biological variables, age, weight and sexual maturity of fish show an influence on the PAHs accumulation. In conclusion, QuEChERS method as an efficient alternative for extraction of PAHs in fish fillet; polycyclic aromatic hydrocarbons show safety levels for human consumption of red mullet Mullus barbatus of Adriatic Sea.

REFERENCES

1. M. Marini, E. Frapiccini, Do lagoon area sediments act as traps for polycyclic aromatic hydrocarbons? Chemosphere, 2014, pp. 80-88. 2. M. J. Ramalhosa, P. Paìga, S. Morais, C. Delerue-Matos, M. B. P. P. Oliveira, Analysis of polycyclic aromatic hydrocarbons in fish:

evaluation of a quick, easy, cheap, effective, rugged, and safe extraction method: Journal of Separation Science, 2009, pp. 3529-3538. 3. C. Porte, E. Escartìn, L. M. Garcìa de la Parra, X. Biosca, J. Albaigés, Assessment of coastal pollution by combined determination of

chemical and biochemical markers in Mullus barbatus: Marine Ecology Progress Series, 2002, pp. 205-216.

Abstract

LC-MS/MS Methods for Metabolomics: from Fruits to Biofluids

Claudia Ancillottia, Lorenzo Ciofia, Daniele Rossinia, Lapo Renaia, Leonardo Checchinia, Massimo Del Bubbaa

a Department of Chemistry, University of Florence, Via della Lastruccia, 3 - 50019 Sesto Fiorentino, Florence, Italy

Keywords: Polyphenolic profile, Berries, Mass Spectrometry, Metabolomics

Metabolomics is frequently applied to the investigation of bioactive compounds in functional foods, in order to assess their quality and possibly explain nutraceutical properties1. Among functional foods, small berries (e.g. bilberry, blueberry and cranberry) are well-known for their healthy properties, mostly related to the high concentration of antioxidant compounds, such as polyphenols. Besides the assessment of the native composition of antioxidants in functional foods, it is probably still more important to obtain information regarding metabolites originated in human body from native bioactive compounds, after fruit ingestion, since they are those actually responsible of the health effects2. In our study LC-MS/MS methods were developed and applied for the untargeted metabolomics analysis of small berries from three different Vaccinium species in order to discriminate them according to their polyphenolic profile. The investigated species were (i) the wild bilberry (V. myrtillus) well-known for its high content of antioxidant compounds (ii) the “false bilberry” (V. uliginosum L. subsp. gaultherioides) which is supposed to have a lower nutritional value compared to V. myrtillus and recently found in the same growing areas of bilberry (iii) the cultivated blueberry (V. corymbosum) largely commercialized in markets. The analysis of Vaccinium berries led to the identification of more than two hundred polyphenols, among which anthocyanins, flavonols, flavanols and phenolic acids, some of them found for the first time in these berry species3. Then, the quantification of the most abundant identified polyphenols confirmed a different composition and a higher concentration of antioxidant compounds in bilberry compared to false bilberry4. After fruit characterization, in order to assess the bioavailability of polyphenols present in the consumed berries, a clinical study involving volunteers was performed. A half of the volunteers assumed a V. myrtillus-based supplement, while a V. corymbosum-based supplement was administered to the others. Afterwards, untargeted LC-MS/MS metabolomics analysis of plasma and urine, collected at different time points from the assumption, was performed with the aim of identifying polyphenol metabolites and clarifying the pharmacokinetic of these bioactive compounds. The preliminary results highlighted different markers of fruit species probably due to the different native quali-quantitative polyphenolic composition of berries and confirmed the key-role of untargeted LC/MS-MS metabolomics approach for discovering new metabolites.

REFERENCES

1. D. S. Wishart, Metabolomics: applications to food science and nutrition research, Trends Food Sci Technol 2008, 19:482-493. 2. M.A. Lila, B. Burton-Freeman, M. Grace, W. Kalt, Unraveling Anthocyanin Bioavailability for Human Health, Annu. Rev. Food Sci.

Technol. 2016. 7:17.1–17.19. 3. C. Ancillotti, L. Ciofi, D. Rossini, U. Chiuminatto, J. Stahl-Zeng, S. Orlandini, S. Furlanetto, M. Del Bubba, Liquid

chromatographic/electrospray ionization quadrupole/time of flight tandem mass spectrometric study of polyphenolic composition of different Vaccinium berry species and their comparative evaluation, Anal Bioanal Chem 2017, 409:1347-1368.

4. C. Ancillotti, L. Ciofi, D. Pucci, E. Sagona, E. Giordani, S. Biricolti, M. Gori, W.A. Petrucci, F. Giardi, R. Bartoletti, U. Chiuminatto, S. Orlandini, S. Mosti, M. Del Bubba, Polyphenolic profiles and antioxidant and antiradical activity of Italian berries from Vaccinium myrtillus L. and Vaccinium uliginosum L. subsp. gaultherioides (Bigelow) S.B. Young, Food Chem 2016, 204:176–184.

Abstract

Kinetic Performance Of New Core-Shell And Sub-2μm Fully Porous Pirkle-type Chiral Stationary Phases For Ultrafast

Enantioseparations

Martina Catania, Omar H. Ismailb, Simona Fellettia, Francesco Gasparrinib and Alberto Cavazzinia

aDipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Ferrara, via L. Borsari 46, 44121, Ferrara bDipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, P.le A. Moro 5, 00185, Roma

Keywords: Mass Transfer Phenomena, Chiral Stationary Phases, Enantioseparations, Band Broadening, Ultrafast

During the last decade, pressing requirements by pharmaceutical and medicinal industries – e.g., for the screening of large libraries of chiral molecules or, in general, for high-throughput analysis – have pushed towards the development of increasingly faster and efficient chiral separations, which could not be achieved on chiral stationary phases (CSPs) developed on typical 3-5 micron FPPs. To this purpose, kinetically high efficient particle formats, such as sub-2µm fully porous particles (FPPs) and superficially porous ones (SPPs), have been introduced in chiral chromatography and functionalized with already known chiral selectors. This transition has been unfortunately slowed down by the lack of complete understanding of the complex mass transfer phenomena in chiral chromatography and by the practical difficulties during the functionalization process of small particles with chiral selectors. (1) In this study, a Pirkle-type chiral selector (Whelk-O1) was used to functionalize both SPPs and FPPs of different diameter (including also sub-2μm fully porous ones). The CSPs were slurry packed at high pressure into columns of different geometries. The kinetic performance of these columns was evaluated in normal phase conditions for the separation of the two trans-stilbene oxide (TSO) enantiomers. (2) Mass transfer phenomena were investigated by interpreting the results of peak parking experiments in the light of a proper model of diffusion in porous media (the so called time-averaged or parallel model was used) (3) and each contribution to band broadening was individually determined. The results shown that eddy dispersion, adsorption-desorption kinetics and frictional heating effects are the most important contributions that significantly affect column performance. Additionally, to further assess the potential of these new CSPs towards ultrafast enantioseparations, short columns (10 mm long) were packed with both sub-2µm and core-shell Whelk-O1 CSPs and operated at a very high flow rate (8 mL/min) to perform the separation of TSO enantiomers in less than one second. (2)

REFERENCES

1. M. Catani, O. H. Ismail, F. Gasparrini, M. Antonelli, L. Pasti, N. Marchetti, S. Felletti and A. Cavazzini, Recent advancements and future directions of superficially porous chiral stationary phases for ultrafast high-performance enantioseparations, Analyst 2017, 142:555-566.

2. O. H. Ismail, L. Pasti, A. Ciogli, C. Villani, J. Kocergin, S. Anderson, F. Gasparrini, A. Cavazzini and M. Catani, Pirkle-type chiral stationary phase on core–shell and fully porous particles: Are superficially porous particles always the better choice toward ultrafast high-performance enantioseparations?, J. Chromatogr. A 2016, 1466:96-104.

3. J. H. Knox and H. P. Scott, B and C terms in the Van Deemter equation for liquid chromatography, J. Chromatogr. 1983, 282: 297–313.

Abstract

Method Optimization for Elucidation of Human Blood Fatty Acid Methyl Esters by Using Conventional and Fast Gas

Chromatography.

Giuseppe Micalizzia, Paola Dugoa,b,c, Luigi Mondelloa,b,c aDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina – Polo Annunziata, Viale Annunziata, Messina, Italy

bChromaleont S.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina – Polo Annunziata, Viale Annunziata, Messina, Italy

cUniversity Campus Bio-Medico of Rome, Via Alvaro del Portillo, 28, Rome, Italy

Keywords: ionic liquid column, fatty acid methyl ester, Fast GC analysis, human blood.

The complete elucidation of fatty acids methyl esters (FAMEs) in human blood is a useful and routine tool to understand the health state. The aim of this work was to develop a simple and versatile analytical strategy for the comprehensive FAMEs profile characterization of whole blood. The research was focused on analytical method optimization evaluating different sample preparation approaches (Folch extraction and MeONa/BF3 derivatization, TMSH direct derivatization , MeONa/BF3 direct derivatization. The choice of the most appropriate approach was carried out by testing a certified human plasma sample (Standard Reference Material 1950). Better results were obtained with a solvent free, direct derivatization procedure using MeONa in methanol solution and BF3 reagents. Conventional GC technique was used for FAMEs analysis, with a flame ionization detector and with an electronic impact mass spectrometer. A ionic liquid polar column was used. The peaks assignment was carried out based on a double filter, namely the MS similarity spectra (similarity between experimental and Shimadzu LIPIDS library spectra) and a Linear Retention Index (LRI) ±10 range compared to the value obtained by injection of standard mixtures of FAMEs. A fast GC approach that allowed analysis of FAMEs in about 3 minutes, maintaining equal resolving power of the conventional approach, was also developed. The analytical strategy enables the complete characterization with a considerable reduction of the time and costs of analysis.

Abstract

Carotenoid Fingerprinting in a Paprika Sample by Supercritical Fluid Chromatography×Ultra High Pressure Liquid

Chromatography and Mass Spectrometry Detection

Marianna Oteria, Paola Donatob, Daniele Giuffridab, Paola Dugoa,c,d and Luigi Mondelloa,c,d

a Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina - Polo Annunziata - viale Annunziata, 98168 Messina, Italy

b Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali, University of Messina, Via Consolare Valeria, 98125 Messina, Italy

c Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina - Polo Annunziata - viale Annunziata, 98168 Messina, Italy

d Unit of Food Science and Nutrition, Department of Medicine, University Campus Bio-Medico of Rome, via Álvaro del Portillo 21, 00128 Rome, Italy

Keywords: Supercritical fluid chromatography; Ultra high pressure liquid chromatography; Mass spectrometry; Carotenoids.

The aim of this work was the development of a two-dimensional comprehensive separation platform, consisting of the on-line coupling of supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RP-LC), the latter operated under ultra high pressure. The system was employed for the analysis of the intact (unsaponified) carotenoid fraction of a paprika sample, by the complementary information gathered from the use of photodiode array (PDA), and quadrupole time-of-flight (QToF) mass spectrometry (MS) detection. A further separation/identification dimension may be provided by ion mobility (IMS), discriminating analytes based on analyte mass, shape and size. The two separation dimensions were interfaced by means of two symmetrical 2-position, six-port high pressure switching valves, equipped with two packed octadecyl silica cartridges for effective trapping and focusing of the analytes after elution from the first dimension(1D), in which elution of the carotenoids was achieved by a gradient of organic modifier (methanol) into carbon dioxide (CO2).After depressurization of the effluent by the backpressure regulator (BPR), a water make-up flow was added prior to entering the loops, to efficiently focus the solutes on the sorbent material and reduce interferences of expanded CO2 gas on the second dimension separation. The latter consisted of rapid (2 minutes) repetitive gradients of isopropanol into water/methanol. The use of SFC offered a number of advantages over normal-phase (NP-LC) typically employed as 1D for carotenoid separation, since immiscibility of the mobile phases is avoided, and RP-LC column lifetime is extended, since compatible solvents are transferred. Most important, SFC is capable of delivering complementary selectivity to RP-LC, with remarkably reduced analysis time (with respect to NP-LC×RP-LC), and significant reduction in organic solvent consumption and, thus, toxicity, costs, and environmental impact.

Abstract

Untargeted metabolic profiling by means of UHPLC-QTOF/MS for the identification of meat and dairy products biomarkers.

Giorgia La Barberaa, Giulia Praticòbc, Göezde Gurdenizb, Maj Britt Schmidt Andersen b, Lars Ove Dragstedb.

aDipartimento di Chimica, Università degli Studi di Roma “La Sapienza”, P.le Aldo Moro, 5 00185-Roma; bDepartment of Nutrition, Exercise and Sports, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C;

cDepartment of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C;

Keywords: Metabolomics; Meat; Untargeted; Biomarkers; High resolution mass spectrometry.

Meat intake has been associated with positive as well as negative health outcomes. Meat is essential from a nutritional point of view but, on the other hand, has been associated to increased risk of several diseases. However, a causal relation between meat consumption and negative health outcomes is still controversial (1). In order to shed light on this issue a reliable assessment of meat intake is needed. Meat intake has often been assessed using very imprecise methods (2). Dietary biomarkers measured in biological fluids by means of untargeted metabolomics have been proposed as possible objective measurements of the actual intake of specific foods (3). A meat intake marker should be able to differentiate meat consumption from that of other protein-rich food such as dairy products. For this purpose, a randomized, controlled, cross-over single meal study was conducted in 17 subjects to compare urine samples after the ingestion of red meat or dairy product based meals. An ultra-high-performance-liquid chromatography coupled via electrospray source to a Q-TOF mass spectrometer was used for the separation and the metabolic profiling of the samples. Urine samples were analysed by both univariate and multivariate data analysis. The 78 compounds resulting significant for discriminating the two diets in both analysis have been selected as meat or dairy products intake biomarkers. The selected markers were fragmented and tentatively identified. Some of them were synthesized to allow the identity confirmation. Combinations of these markers are proposed as specific markers for meat or dairy product intake.

REFERENCES

1. D. M. Klurfeld, Research gaps in evaluating the relationship of meat and health. Meat Sci. 2017, 109: 86–95. 2. V. Kipnis, D. Midthune, L. Freedman, S. Bingham, N.E. Day, E. Riboli, P. Ferrari, R.J. Carroll, Long-term reproducibility of a

food-frequency questionnaire and dietary changes in European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg cohort, Public Health Nutr. 2002, 109: 915–923.

3. A. O’Sullivan, M. J. Gibney, L. Brennan, Dietary intake patterns are reflected in metabolomic profiles: potential role in dietary assessment studies, Am. J. Clin. Nutr. 2011, 93: 314–321.

Abstract

Evaluation Of Excess And Adsorption Isotherms In Chiral Stationary Phases

Simona Fellettia, Martina Catania, Omar H. Ismailb, Francesco Gasparrinib and Alberto Cavazzinia

a Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Ferrara, via L. Borsari 46, 44121, Ferrara;

b Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, P.le A. Moro 5, 00185, Roma;

Keywords: Excess isotherms, Adsorption isotherms, Chiral stationary phases

The adsorption equilibria of trans-stilbene oxide (TSO) enantiomers have been studied on Whelk-O1 chiral stationary phases (CSPs) prepared on both core-shell and fully porous particles (FPPs) (1). Three columns were used in this study: a 100x4.6 mm column packed with 1.8 μm FPPs, a 150x4.6 packed with 2.5 μm FPPs and, finally, a 150x4.6 packed with 2.6 μm superficially porous particles (SPPs). Firstly, the preferential adsorption of the binary eluent components on the stationary phase (or, more precisely, their excess adsorption isotherms) was evaluated through the so-called minor disturbance method. This method is based on the perturbation of the adsorption equilibria of mobile phase components (in this case hexane and ethanol) through small injections of an excess of one of the two components, when a steady-state equilibrium between mobile and stationary phase is reached. The excess isotherm is then obtained by studying the relationship between the retention times of the so-called disturbance peaks and the composition of the mobile phase (2). Afterwards, the adsorption isotherms of TSO enantiomers have been studied by modeling overloaded peaks measured under different experimental conditions with mobile phases made of pure hexane and 90/10 hexane/ethanol % (v/v). The isotherm parameters were obtained by iteratively solving the chromatographic mass balance equation adjusting the adsorption parameters until optimal matching between calculated and experimental profiles is reached (3). Different isotherm models have been used to determine the best adsorption isotherm for TSO on the three columns studied. At 90/10 hexane/ethanol % (v/v) the best adsorption model was found to be the bi-Langmuir one (accounting for two different adsorption sites on the adsorption surface) for all the columns. On the contrary, at 100% hexane the heterogeneous Toth model (predicting a continuous adsorption energy probability density function) best describes the adsorption.

REFERENCES

1. O. H. Ismail, L. Pasti, A. Ciogli, C. Villani, J. Kocergin, S. Anderson, F. Gasparrini, A. Cavazzini and M. Catani, J. Chromatogr. A, 1466, 2016, pp. 96-104.

2. Y. Kazakevich, H.M. McNair, J. Chromatogr. Sci. 33, 1995, pp.321. 3. N. Marchetti, A. Cavazzini, L. Pasti, F. Dondi, J. Sep. Sci. 2009, 32, pp. 727-741.

Abstract

Determination of Mycotoxins in Cereals by a Rapid Magnetic Solid Phase Extraction Method followed by Liquid

Chromatography-Tandem Mass Spectrometry Analysis

Carmela Maria Montone, Francesca Ferraris, Susy Piovesana, Aldo Laganà

Keywords: mycotoxins; magnetic solid phase extraction; graphitized carbon black; liquid chromatography-tandem mass spectrometry; Gramineae

Mycotoxins can contaminate various food commodities, including cereals. Moreover, mycotoxins of different classes can co-contaminate food, increasing human health risk. Several analytical methods have been published in the literature dealing with mycotoxins determination in cereals (1). Nevertheless, in the present work, the aim was to propose an easy and effective system for the extraction of six of the main mycotoxins from corn meal and durum wheat flour, i.e., the main four aflatoxins, ochratoxin A, and the mycoestrogen zearalenone. The developed method exploited magnetic solid phase extraction (SPE), a technique that is attracting an increasing interest as an alternative to classical SPE. Therefore, the use of magnetic graphitized carbon black as a suitable extracting material was tested. The same magnetic material proved to be effective in the extraction of mycoestrogens from milk, but has never been applied to complex matrices as cereals. Ultra high–performance liquid chromatography tandem mass spectrometry was used for detection. Recoveries were >60% in both cereals, even if the matrix effects were not negligible. The limits of quantification of the method results were comparable to those obtained by other two magnetic SPE-based methods applied to cereals, which were limited to one or two mycotoxins, whereas in this work the investigated mycotoxins belonged to three different chemical classes.(2) The suitability of mGCB for the extraction of AFB1, AFB2, AFG1, AFG2, OTA, and ZEN from corn meal and durum wheat flour samples was demonstrated. The overall process efficiency of the developed method was a compromise between performance and easiness and rapidity of application. Compared with a classic on-column SPE, the time required for a single extraction was about the same. Nevertheless, mSPE was less labor intensive, and more than ten extractions can be managed simultaneously. Even if in the present work, the limits of quantification were comparable to or higher than those of other mSPE methods. However, this method allows for the simultaneous investigation of a larger number of mycotoxins. Moreover, due to the different detection technique (fluorescence in the other works and tandem mass spectrometry in the present one), MLOQ calculation modes are very different. This is the first application of mGCB in an mSPE procedure for extraction of mycotoxins from cereals. The potentiality of this material has been exploited before for the extraction of mycotoxins belonging to the same chemical class, but from milk (2).

REFERENCES

1. N.W Turner,H. Bramhmbhatt, M. Szabo-Vezse, A. Poma, R. Coker, S. A. Piletsky, Analytical methods for determination of mycotoxins: An update (2009–2014). Anal. Chim. Acta 2015, 901, 12–33;

2. G. La Barbera, A. L. Capriotti, C. Cavaliere, P. Foglia, C. M. Montone, R. Zenezini Chiozzi and Aldo Laganà, A Rapid Magnetic Solid Phase Extraction Method Followed by Liquid Chromatography-Tandem Mass Spectrometry Analysis for the Determination of Mycotoxins in Cereals, Toxins 2017, 9(4), 147;

Abstract

Qualitative And Quantitative Differences In Carotenoid Profile Among Two Varieties Of Pumpkins By HPLC-DAD Analysis with a

C30 column

Caterina Bergantin, Paola Tedeschi, Edison Vasquez Corales, Alberto Cavazzini, Nicola Marchetti and Annalisa Maietti

Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17, 44121 Ferrara, Italy

Keywords: carotenoids, pumpkins, HPLC, C30 column.

Carotenoids are a widespread group of naturally fat-soluble pigments. The nutritional importance of these compounds comes from the pro-vitamin A activity. Carotenoids are antioxidants and may differently contribute to the nutraceutical role of foods, since they have been involved in the prevention against human disorders. They are found in a large number of fruits and vegetables, spices, some animal products and seafood. Carotenoids are usually C40 tetraterpenoids built from eight C5 isoprenoid units joined head-to-tail, except at the center where there is a tail-to-tail connection that reverse the order and bestow a symmetrical configuration to the molecule. The elementary skeleton can be cyclized at one or both ends or modified in many ways, including isomerization, hydrogenation, dehydrogenation, introduction of oxygen groups, double-bond migration, chain shortening or extension, rearrangement or a combination of these possibility. This involves a large number of possible structures and in fact more than 600 carotenoids, not including trans-cis isomer, have been isolated and characterized from natural sources. An important property is an extended conjugated double-bond system which constitutes the light-absorbing chromophore. This feature is responsible for the typical yellow, orange or red color of these molecules: carotenoids exhibit intense absorption bands in the visible, or in some cases, UV region. The UV-VIS spectrum is the first diagnostic tool for the identification because both the wavelengths of maximum absorption (λmax) and the shape of the spectrum (spectral fine structure) are characteristic of the chromophore [1]. The present work had a twofold objective: (a) the characterization of major carotenoids in two pumpkin varieties from southern Po Delta area (Massenzatica, Ferrara), compared with the same varieties cultivated in another territory (Sermide, Mantova); (b) the different concentration of same target compounds depending on the cooking methods used (baking and boiled pumkin). High-performance liquid chromatography (HPLC) has become the method of choice for carotenoid analysis and in agreement with the last studies about the selectivity towards carotenoids isomers, the separation was investigated on a C30 column (Develosil, 3µm, 150 x 3.0mm, 140Å) [2]. Before analysis carotenoids were extracted according to literature recommendations and the HPLC conditions were optimized to obtain a better separation. The identification of the carotenoids was carried out considering: (a) the combined informations from chromatographic parameters (retention time and elution order); (b) the comparison of UV-visible spectra between standards and data available in literature (λmax and the ratio of the height of the longest wavelength absorption peak to that of the middle one, % III/II). The quantification of some target compounds was performed with analytical standards.

REFERENCES

1. D. B. Rodriguez-Amaya and M. Kimura, HarvestPlus Handbook for Carotenoids Analysis, HarvestPlus Technical Monograph 2, 2004.

2. S. Bijttebier, E. D’Hondt, B. Noten, N. Hermans,S . Apers, S. Voorspoels, Ultra high performance liquid chromatography versus high-performance liquid chromatography: Stationary phase selectivity for generic carotenoid screening, Journal of Chromatography A, 2014, 1332: 46-56

Abstract

The power of the bidimensional chromatography (2D-LC) in the qualitative and quantitative analysis of complex matrices

Marica Barbieria, Gerd Vanhoenacker b, Frank David b, Koen Sandra b and Pat Sandra b

a Agilent Technologies, Via P. Gobetti, 2/C, 20063 Cernusco sul Naviglio bResearch Institute for Chromatography President Kennedypark 26, B-8500 Kortrijk, Belgium

Keywords: Liquid bidimensional chromatography, peak capacity, complex mixtures

Extracts of the natural products are complex mixtures containing a diversity of organic molecules at different concentration levels. The analysis of such complex extracts requires chromatographic methods with high resolution and extended peak capacity. Coelution of compounds needs to be avoided to enable accurate quantification and evaluate the purity of extracts and fractions. The liquid bidimensional chromatography (2D-LC) provides the chromatographic power to overcome not only the challenge of separating the analytes from the matrix, but also overlapping peaks from the first dimension. With the improvements introduced in the recent years, the 2D-LC is not anymore an highly complicated manual system as it was in the past and now every laboratory can take advantage of this powerful technique to solve the biggest analytical challenges. The analysis of xanthones present in the extracts of mangosteen pericarp (Garcinia mangostana L.) and the analysis of Taxus sp. extracts will be presented as examples of the high resultion sampling and the comprehensive two-dimensional liquid chromatography technique respectively. 1,2

REFERENCES

1. G.Vanhoenacker, F. David, K. Sandra and P. Sandra, Agilent Application Note , 5991-7552EN, Published in the USA, November 1, 2016. 2. G.Vanhoenacker, F. David, K. Sandra and P. Sandra, Agilent Application Note , 5991-3576EN, Published in the USA, September 15,

2014.

Abstract

Novel Simultaneous Detection by Isotope Ratio Mass Spectrometry and Quadrupole Mass Spectrometry Coupled to

Multidimensional Gas Chromatography for the Analysis of Valuable Food Products

Antonino Schepisa, Danilo Sciarronea and Luigi Mondelloa,b.c

a Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina - Polo Annunziata - viale Annunziata, 98168 Messina, Italy

b Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina - Polo Annunziata - viale Annunziata, 98168 Messina, Italy

c Unit of Food Science and Nutrition, Department of Medicine, University Campus Bio-Medico of Rome, via Álvaro del Portillo 21, 00128 Rome, Italy

Keywords: MDGC, IRMS, qMS

Isotope Ratio Mass Spectrometry (IRMS) is commonly recognized to be able to provide information about the geographical, chemical, and biological origins of substances. The ability to determine the source of substances stems from the relative isotopic abundances of the elements which comprise the material. Hyphenated techniques such as GC-C-IRMS, can provide isotopic analysis of a complex mixture, thereby providing additional information and higher discriminatory power. Since its introduction, the use of this analytical approach was not widespread due to a series of drawbacks related to chromatographic and isotopic issues. Dead volumes due to the typical instrumental setup lead to an increased band broadening and peak asymmetry producing peak coelutions, thus falsify the measurements. Moreover, the reduced chromatographic performance increases the gas chromatographic isotope effect that generates GC peak not isotopically consistent because composed of lighter isotopes (12C, 1H and 16O) that elute after the isotopomers containing heavier organic compounds because of their higher volatility. The present research deals with the investigation of the 13C ratio of several high-value white truffles collected in Italy, and commercial products flavoured with different truffle species exploiting an MDGC-MS/IRMS prototype characterized by the improved resolution capability of the heart-cut mode and the simultaneous qMS and IRMS detection of the 2D chromatographic bands. The IRMS system was optimized in terms of dead volumes enabling to overcome the extra-column band broadening effect that usually affects the commercial systems. Different commercial products as flavoured pasta, cheese, sauce and olive oil samples have been investigated in terms of bis methylthio methane showing the enhanced performances of the prototype described.

Abstract

Development of a GCxGC-TOFMS Method for the Quantification of the Extended List of Suspected Allergens in Fragrance

Materials

Sebastiano Pantò

LECO EATC, Max-Dohrn-Str. 8-10, Building B 5.2, Berlin, Germany

e-mail: sebastiano_panto@leco.com

In 2011, the Scientific Committee on Consumer Safety (SCCS) proposed to extend the list of 24 suspected allergens to 57 compounds, including all the isomeric forms [1]. As a consequence, a new legislation is expected in the next future regarding the information to be provided to consumers. The impact for the companies involved in this field will be high; they need to adapt/modify their analytical methods in order to accomplish the challenge of quantify the updated list of substances.

The method currently applied for the 24 allergens is based on two parallel GC-MS separations with two different stationary phases (apolar and mid-polar stationary phases) [2]. This approach usually overcomes coelutions of the current suspected allergens with matrix. However this becomes more difficult when considering the new list of allergens, especially if isomers, allergen precursors and additional regulated substances are considered. In order to overcome these issues, especially with most complex matrices, a comprehensive two-dimensional gas chromatography approach (GCxGC-TOFMS) has been developed. Different parameters have been tested in order to reach a satisfactory separation between the target compounds. Finally, an apolar x mid-polar column set has been exploited for the development of the GCxGC method.

The method has been successfully applied to different commercial fragrances and their respective commercial copy products.

REFERENCES

[1] SCCS Opinion on fragrance allergens in cosmetic products (SCCS/1459/11). [2] A. Chaintreau et al., J. Agric. Food Chem., 51, 2003, p. 6398.

Abstract

Different Strategies For Extraction Of Illicit Drugs In Hair By Means UHPLC-HRMS/MS

Flaminia Vincenti,a Federico Fanti a, Gabriele Vannutelli a, Adolfo Gregorib, Manuel Sergic, Dario Compagnonec, Roberta Curinia

aSapienza University of Rome, Department of Chemistry, 00185 Rome, Italy

bCarabinieri, Department of Scientific Investigation (RIS), 00191 Rome, Italy cUniversity of Teramo, Faculty of Bioscience and Technology for Food, Agriculture and Environment, 64100 Teramo, italy

Keywords: Illicit drugs, Hair, PLE, dLLME, UHPLC-HRMS

Hair testing has gained increasing attention and recognition as a complement to blood and urine analysis, since is a unique material for the retrospective detection of drugs, due to its large detection window (1). To avoide false-positive results, caused by passive exposure to the drug, for this reason starts with a wash step to remove external contamination is foundamental (2). For the analyte extraction there are two main strategies: dissolution of the matrix (3) or solvent extraction (4); the first one may cause analytes degradations due to the extreme conditions used in the method, on the other hand the second one takes long time in order to obtain the final samples. In this work, we developed a new method based on pressurized liquid extraction (PLE), which allows a rapid and effective extraction (5), followed by a quick clean-up performed by dispersive liquid-liquid microextraction (dLLME) and then we compared our method with different methods commonly used in hair testing. In order to evaluate the method performances, three different types of samples were prepared: the first one simply by spiking the standard solution on the hair; the second one was performed by soaking; real samples obtained from volunteers. By using a HPLC-ESI-MS/MS system, we were able to ascertain that soaking samples were better than spiked to simulate a real sample conditions. Through to PLE-dLLME method we were able to obtain a reliable extract in few minutes, highlighting the method as alternative technique compared to the common ones reported in the literature. The following step was the analysis of 60 psychoactive substances in the keratinic matrix by means UHPLC-HRMS/MS. Furthermore the technique revealed it-self as useful method to multiclass substances analysis, and in addiction to this the using of solvent was smaller than other methods. The application of a multiclass method, including different classes of both classical drugs and new psychoactive substances, is very important as it provides cost and time of analysis reduction, especially on keratinic matrix, which provides important information on history of abuse, being very challenging from the analytical point of view.

REFERENCES

1. A. Verstraete, Detection time of drugs of abuse in blood, urine and oral fluids. Therapeutic Drug Monitoring, 2005, pp. 200-205. 2. G.A.A. Cooper, R. Kronstrand, P. Kintz. Society of Hair Testing guidelines for drug testing in hair, Forensic Science International 2012,

pp. 20-24. 3. M. Cheze, M. Deveaux, C. Martin, M. Lhermitte, G. Pepin. Simultaneous analysis of six amphetamines and analogues in hair, blood and

urine by LC- ESI-MS/MS Application to the determination of MDMA after low Ecstasy intake. Forensic Science International, 2007, pp. 100-104.

4. M. Fisichella, L. Morini, C. Sempio, A. Groppi. Validation of a multi-analyte LC–MS/MS method for screening and quantification of 87 psychoactive drugs and their metabolites in hair, Anaytical andl Bioanalytical Chemestry, 2014, pp. 3497–3506.

5. C. Montesano, M.C. Simeoni, G. Vannutelli, A. Gregori, L. Ripani, M. Sergi, D. Compagnone, R. Curini, Pressurized liquid extraction for the determination of cannabinoids and metabolites in hair: Detection of cut-off values by high performance liquid chromatography–high resolution tandem mass spectrometry, Journal of Chromatography A, 2015, pp. 192–200.

Abstract

Enantioseparation and Impurity Determination of Cinacalcet using

Solvent Modified Capillary Zone Electrophoresis and Quality by Design: Study of the Complexation with Cyclodextrins by

Molecular Modeling and NMR

Benedetta Pasquinia, Serena Orlandinia, Fabrizio Melanic, Claudia Caprinia, Massimo Del Bubbab, Sandra Furlanettoa

Department of Chemistry “U. Schiff”, University of Florence, aVia U. Schiff 6, bVia della Lastruccia 3-13, 50019 Sesto Fiorentino, Florence, Italy

cDept of Neurofarba, University of Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

Keywords: Quality by Design, Capillary Electrophoresis, NMR, Molecular Modeling, Job Plot, Cyclodextrins, Inclusion Complex.

The combination of capillary electrophoresis (CE), NMR and Molecular Modeling is presented to evaluate in depth the

enantioselective complexation with cyclodextrins (CDs) of the new therapeutic agent Cinacalcet Hydrochloride (CINA), used for the treatment of chronic kidney disease. In our previous pharmaceutical quality control study, the enantioseparation and impurity determination of CINA was carried out using as operating mode solvent modified capillary zone electrophoresis with the addition of 2-hydroxypropyl-γ-cyclodextrin (HPγCD). The method was developed following Quality by Design (QbD) approach, leading to visualize the design space (1). The incorporation of QbD strategy into CE method development enabled dealing with optimization challenges in a rational and systematic way, providing the key for a better comprehension of the separation. In the scouting phase neutral and charged CDs were tested, showing different separation abilities. In electromigration techniques the separation depends on the affinity towards the chiral selector, but also on the different electrophoretic mobility of the formed enantiomer-chiral selector complexes. NMR spectroscopy provides a powerful tool to gain insight into the fine interactions between the analytes and the chiral selectors. Unlike other techniques, the main advantage of NMR spectroscopy is that it can gather information in conditions that mimic those of the CE runs, in particular pH, temperature and ionic strength. The molecular association of CINA with CDs, expressed by the binding constant of the inclusion complex, was calculated from the changes in the 1H NMR spectra of the drug in the presence of the cyclodextrin. The stoichiometry of the complex was calculated by use of the continuous variation method (Job Plot). Molecular Modeling studies complemented the obtained results in order to achieve a better understanding of the intermolecular affinities and recognition mechanism. The complexation behavior of CINA enantiomers and CDs was evaluated by dynamic simulations. Different geometries and conformations were designed and the stability of the inclusion complexes were calculated. The docking energies indicated that the most stable complexes were obtained with (2-carboxyethyl)-β-cyclodextrin. The obtained data showed a good agreement with the CE results.

REFERENCES

1. S. Orlandini, S. Pinzauti, S. Furlanetto, Application of quality by design to the development of analytical separation methods, Anal. Bioanal. Chem. 2013, 405:443-450.

Abstract

Analysis of bioactive phenolic compounds in different hazelnut kernels by RP-HPLC/PDA/ESI-MS.

Giusy Tripodoa, Marina Russoa, Valentina Pasqualetti a, Laura De Garaa, Chiara Fanalia

a Department of Medicine - University Campus Bio-Medico of Rome, Via Álvaro del Portillo 21, 00128 Rome, Italy

Keywords: Hazelnut, Corylus avellana, phenolic compounds, liquid chromatography, mass spectrometry.

Hazelnut (Corylus avellana L.) is one of the nuts most consumed in many countries including Turkey, Italy, Spain and United States. It is a rich source of dietary fibers and beneficial nutrients such as lipids, proteins, but also significant micronutrients like essential minerals, vitamin E, B complex vitamins and phenolic compounds, which contribute to its organoleptic properties such as astringent and bitter taste. Particularly, phenolic compounds play an important role on human health for their beneficial effects, such as antioxidant acitivity. The content of phenolic compounds may be a significant parameter in the assessment of hazelnuts quality. In fact, it depends on several factors like cultivar, geographical origin and processing condition such as roasting [1, 2]. The present study aims to characterize phenolic compounds in hazelnut kernels of different cultivars by high performance liquid chromatography (HPLC) coupled to photodiode array detector (PDA) and mass spectrometry detector (MS) using ESI as interface in positive and negative mode. Phenolic compounds were extracted from natural and roasted hazelnut kernels by solid-liquid extraction using a mixture of solvents. Samples were examined for the total phenolic content and antioxidant capacity by Folin-Ciocalteau and TEAC (trolox equivalent antioxidant capability) assays, respectively. Considering the nature of compounds, the analytes were separated on a C18 column using a binary mobile phase composed of aqueous solution with 0.1 % (v/v) formic acid and acetonitrile with 0.1 % (v/v) formic acid in a gradient elution mode. The analytical method was developed and then validated using a mixture of 15 different phenolic acids and flavonoids standards. RSD % for intra-day e inter-day, LOD and LOQ values for extraction validation were evaluated. Separation conditions were optimized obtaining a baseline separation of all standard compounds. Calibration curves were constructed with a good linearity and satisfactory determination coefficients R2. Developed and validated method was then applied to the analysis of phenolic compounds in extracts from natural and roasted hazelnut kernels of different cultivars. Samples identification of compounds was assessed by UV-Vis spectrum, mass spectra, comparison with available commercial standards when possible and data reported in the literature. The extracts showed the presence of phenolic acids, flavan-3-ols and procyanidins. A similar qualitative phenolic compounds profile among analyzed samples was observed. Differences in the quantitative analysis between the different cultivars were reported. Moreover roasted hazelnut kernel extracts showed a lower concentration of phenolic compounds respect to the natural ones. From the characterization of bioactive phenolic compounds by RP-HPLC/PDA/ESI-MS in different hazelnuts, it has been found that this fruits are a rich source of nutraceutical molecules.

REFERENCES

1. N. Tas, V. Gokmen, Bioactive compounds in different hazelnut varieties and their skins, J. Food Compost. Anal. 2015, 43:203-208.

2. E. Pelvan, C. Alasavar, S. Uzman, Effects of roasting on the antioxidant status and phenolic profiles of commercial Turkish hazelnut varieties (Corylus avellana L.), J. Agric. Food Chem. 2012, 60:1218-1223

Acknowledgments: the authors gratefully acknowledge Soremartec Italia s.r.l. - FERRERO Group for the financial support, and

Shimadzu Corporations for the continuous support.

Abstract

Novel aspects and performance evaluation of a liquid-EI LC-MS interface

Veronica Termopolia , Giorgio Famiglinia , Pierangela Palmaa , Maurizio Piergiovannia , Achille Cappielloa

a University of Urbino, Department of Pure and Applied Sciences, Urbino, Italy

The performance and the applicability of the recent liquid chromatography-mass spectrometry (LC-MS) interface called Liquid-EI (LEI) is thoroughly investigated and evaluated. LEI is based on electron ionization (EI), but the vaporization of solutes and mobile phase occurs at atmospheric pressure into a specially designed region called “vaporization micro-channel”, outside the high-vacuum ion source. The gas-phase molecules are driven into the ion source by an inert gas flow. The interface is a stand-alone device that can be connected to a GC-MS for its fast conversion into an LC-MS system. LEI stands alone in terms of robustness, ease-of-use, and sensitivity. Agilent 1290 Infinity UHPLC coupled to 7010 QQQ triple quadrupole mass spectrometer equipped with a High Efficiency Ion Source (HEIS) (Agilent Technologies Inc., Santa Clara, CA). 4,5-dichloroisophthalodinitrile and cholesterol analyzed with Agilent Eclipse C18 column (2.1 mm x 50 mm) 1.7 µm particle size. Injection volume was 2 µL for system evaluation (FIA 50% ACN) and 100 µL for performance evaluation. Flow rate: 100 µL/min; split ratios: 1:200 and 1:125. Gradient: 1 min at 0% ACN, from 0% to 50 % ACN in 30 s, then up to 90% ACN in 5 min. Ion source temperature: 280 °C. Vaporization micro-channel temperature: 350 °C. Data acquisition: MRM. Full scan analyses: m/z 80-400;1 cyc/s, threshold 10. Vaporization micro-channel is entirely lined with a 0.4 mm i.d., 0.8 mm o.d. removable fused silica capillary (internal volume 19 µL): 170 mm long, for the vaporization and transport of the LC eluate into the EI ion source, connected on one side to the GC-MS inlet port of an EI source and to the LC splitter on the other one. The micro-channel, built modifying a standard GC transfer line, can be heated up to 400 °C, was. A 150 µm o.d., 75 µm i.d. fused silica capillary (inlet) penetrates in the first portion of the liner to release the LC eluate. The liner separates the high-vacuum zone (ion source) from the atmospheric pressure at the end of the LC capillary. The eluate reaches the hot zone of the micro-channel and vaporizes immediately. A helium gas flow around the inlet speeds up the vapors into the ion source. A Peltier unit placed at the entrance of the vaporization micro-channel prevents the heat diffusion backwards from the hot region of the interface. The evaluation has already considered different inlet capillary dimensions (50 and 75 µm) and positions to test the nebulization process, demonstrating good performances of both capillaries; 20 consecutive injections of 4,5-dichloroisophthalodinitrile diluted in pure solvent and in soil matrix showed an excellent repeatability even in presence of a complex matrix; overlapped linear regression plots of 4,5-dichloroisophthalodinitrile in pure solvent and in soil matrix demonstrated the absence of matrix effects. Other tests will allow to define the best interface setting. Identification of LC amenable endocrine disruptors and medium-long chain fatty acids methyl esters (FAMEs) are planned as possible real-world applications. LEI proves to be a new and effective strategy in the exploitation of EI in many fields of LC-MS analysis.

Abstract

An LC-IRMS Interface for Flexible Compound-specific Stable Isotope Analysis

Filip Volders, Christian Schmidt, Sam Barker, Lutz Lange and Hans-Peter Sieper

Elementar Analysensysteme GmbH, Elementarstrasse 1, Langenselbold (Germania)

Keywords: Instrumentation, LC-IRMS, IRMS interface, stable isotope analysis.

Introduction: In aqueous samples compound-specific stable isotope analysis (CSIA) plays an important role. Environmental and forensic sciences are prominent examples of such applications, utilizing naturally occurring fractionation processes during transport and transformation processes to, e.g., allocate contaminants or drugs sources. The broad range of involved application areas includes e.g. the food industry (food fraud) and sport (doping). However, the currently available LC-IRMS solutions are limited to stable carbon isotope analysis only and therefore the use of pure aqueous solvent. This considerably limits the application possibilities and analyzable compound classes. No direct method (without sample preparation) for stable isotope analysis of nitrogen and sulfur of non-volatile compounds is known yet.

Methods: A novel high-temperature combustion interface was developed to hyphenate high-performance liquid chromatography with isotope ratio mass spectrometry in a more flexible way. The system is capable to analyze stable isotopes other than carbon, which also abolish the limitation of pure aqueous solvent usage. In continuous operation virtually for all peaks in a chromatogram the stable isotope ratio can be analyzed.

Results: Experimental data of different examples proof the performance and flexibility of such a system. Compounds were determined typically with a precision and trueness of ≤0.5‰ for different stable isotopes.

Conclusion: The development of a novel LC-IRMS interface resulted in the first system reported that is not limited to stable carbon isotopes anymore. Furthermore the use of organic solvents is possible which open up new possibilities in CSIA-based research fields.

Abstract

Bivalve Mollusk Shells as a Low-Cost Biosorbent for Water Remediation

Tatiana Cheneta, Claudia Stevanina, Michele Mistria, Alberto Cavazzinia, Luisa Pastia

aUniversity of Ferrara, Department of Chemistry and Pharmaceutical Sciences, via L. Borsari, 46, Ferrara tatiana.chenet@unife.it

Keywords: Biosorbent, Scallop shell, Heavy metals.

Shellfish cultivation represents a vast economic activity worldwide. Seafood industries produce every year a large amount of waste, mainly shells, which can constitute a serious environmental problem if not properly disposed. Treating these residues as special waste means additional costs for the industries, so finding an efficient use of these raw materials can potentially resolve disposal and environmental problems. This study is focused on the use of scallop shell powder as a biosorbent for the removal of heavy metals ions from natural waters, in particular the adsorption behavior of cadmium and nickel is investigated. Heavy metals represent common pollutants of natural waters, especially nearby mining sites and metalworking industries. Cadmium contamination also derives from the use of this element in batteries, electroplating and pigments, it is toxic even at low concentration for humans and biota since it can substitute essential elements such as calcium and zinc in biological processes leading to the alteration of cellular metabolism [1,2]. Even though nickel is considered to be an essential element for some animals, microorganism and plants, it is not for humans and its increasing use for the production of stainless steel, Ni-based alloys and Ni-based rechargeable batteries can cause severe environmental pollution and health problems. Exposure to highly Ni-polluted environments has the potential to produce a variety of pathological effects in humans from contact dermatitis to kidney diseases, and even cancer [3]. In the first part of this research project different techniques, such as inductively coupled plasma, X-ray powder diffraction, scanning electron microscopy and dynamic light scattering, were used for the characterization of the adsorbent material to determine its chemical composition and its structure and dimension. Batch experiments were carried out by placing in contact a given amount of shell powder with a solution containing a known concentration of metal cations. After equilibrium was reached the solution was filtered and analyzed with inductively coupled plasma atomic emission spectroscopy to quantify the amount of not adsorbed cation. Kinetic and thermodynamic properties of the biosorbent, regarding cadmium and nickel adsorption, were investigated and the kinetic equation and adsorption isotherms describing the process were obtained. In order to better understand the adsorption mechanism, the effects of some parameters as pH, temperature and salinity were also investigated.

REFERENCES

1. J. E. Fergusson, The heavy elements: chemistry, environmental impact and health effects, Pergamon Press (1990). 2. M. Remelli, V. M. Nurchi, J. I. Lachowicz, S. Medici, M. A. Zoroddu, M. Peana / Coordination Chemistry Reviews 327-328 (2016)

55-69. 3. V. Coman, B. Robotin, P. Ilea / Resources, Conservation and recycling 73 (2013) 229-238.

Abstract

Fast Cryogenic Comprehensive Two-dimensional Gas Chromatography-mass Spectrometry: Concept, Method

Optimization and Applications in the Lipid Field

Marco Piparoa, Barbara Giocastroa, Fabrizio Cincottaa, Peter Q. Tranchidaa, Luigi Mondelloa,b,c

aDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina – Polo Annunziata, Viale Annunziata, Messina, Italy

bChromaleont S.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina – Polo Annunziata, Viale Annunziata, Messina, Italy

cUniversity Campus Bio-Medico of Rome, Via Alvaro del Portillo, 28, Rome, Italy

Keywords: GC×GC-MS, comprehensive gas chromatography, fatty acids, oil.

The investigation is focused on the concept of fast comprehensive two dimensional gas chromatography mass spectrometry (GC×GC-MS) separations. More in detail, a 8.9 m × 0.1 mm ID low-polarity column was used as first dimension, while a 1.1 m × 0.1 mm ID medium-polarity one was used as second dimension. The main scope of the research was to develop a high-resolution GC×GC-MS method, with an analysis time of approx. 10 min. to be used for high throughput lipid analysis. Various aspects related to method optimization are discussed, as well as separation parameters such as peak capacity (in each dimension), efficiency, peak widths, modulation ratio, and sensitivity enhancement. A series of applications are shown involving the analysis of fatty acid methyl esters in a series of edible oils The GC×GC-MS approach proposed enables high-resolution separations in a short time, as well as a considerable reduction of the consumption of gases for modulation cooling and heating.

ACKNOWLEDGEMENTS

The research was performed within the context of the project AGER2-Rif 2016-0169, “Valorizzazione dei prodotti italiani derivanti dall’oliva attraverso Tecniche Analitiche Innovative” – “Violin”

Abstract

Nuovi sviluppi nella Spettrometria NMR da banco: affidabilità analitica, precisione di misura e sicurezza di utilizzod

Alessandro Agostini

FKV Srl, +39 346 7934509, a.agostini@fkv.it

Le origini degli spettrometri NMR da banco risalgono agli anni 1960-70 in cui sono stati sviluppati i primi sistemi, che lavoravano ad una frequenza di 60-90 MHz per il protone. Tale tecnologia è stata abbandonata fino ai primi anni duemila, quando sono tornati in auge gli spettrometri NMR da banco. I nuovi sistemi presentano numerosi vantaggi in termini di risoluzione, sensibilità, stabilità e affidabilità. In questo contesto Magritek propone Spinsolve come nuovo concetto di spettrometro NMR da banco. A differenza di tutti gli spettrometri NMR da banco esistenti, Spinsolve è basato sull’utilizzo di un Magnete di Halbach, che consente di ottenere una stabilità di campo magnetico unica nel suo genere da cui deriva, a parità di frequenza operativa, una risoluzione spettrale senza pari. Rispetto ai tradizionali spettrometri ad elevato campo magnetico, basati sull’utilizzo di elettromagneti, Spinsolve non prevede l’utilizzo di gas criogenici, è in grado di lavorare senza solventi deuterati e può essere direttamente accoppiato al batch di reazione, tramite una specifica cella a flusso, per monitorare in tempo reale il processo reattivo. Infine, le ridotte dimensioni danno la possibilità di posizionare lo spettrometro direttamente sotto una cappa o su di un bancone di laboratorio (a prescindere dai campi elettromagnetici presenti nell’edificio). Tutto questo è possibile grazie all’utilizzo di un magnete permanente estremamente stabile e alla possibilità di lavorare con un lock esterno. Da un punto di vista operativo Spinsolve lavora con i tradizionali tubi NMR da 5,0 mm e implementa praticamente tutte le sequenze di impulsi (mono e bi-dimensionali) disponibili per i tradizionali NMR high field. Normalmente, oltre a 1H e 19F, sono previste configurazioni che consentono di lavorare con gli eteronuclei di 13C e 31P (anche esperimenti HETCOR); in più è possibile customizzare la macchina per lavorare anche su diversi eteronuclei. Esistono particolari configurazioni di Spinsolve che consentono di lavorare in gradiente di campo magnetico e quindi di realizzare esperimenti di diffusione (DOSY).

Abstract

Determination Of Benzodiazepines In Beverages Using Green Extraction Methods And HPLC-UV Detection.

Maurizio Piergiovanni, Achille Cappiello, Giorgio Famiglini, Veronica Termopoli, Pierangela Palma

DiSPeA - University of Urbino, Piazza Rinascimento 6, 61029 Urbino, Italy

m.piergiovanni1@campus.uniurb.it

Keywords: Benzodiazepines, MEPS extraction, DLLME extraction, LC-UV analysis, Forensic analysis.

Microextraction by packed sorbent (MEPS) and Dispersive Liquid–Liquid Micro Extraction (DLLME) with and without ultrasound assistance (UA-DLLME) were used as “green” extraction methods for the determination of benzodiazepines (BDZ) in beverages followed by HPLC-UV detection. BDZ are pharmaceutical compounds usually employed for their tranquilizing and anti-depressive effect. However, their simple availability and reduced cost make them attractive for criminal intent (1). The very low amount of sample usually available for the analyses makes the determination suitable for micro–scale extraction techniques. MEPS and DLLME are emerging techniques based on different principles, and they can be considered “green” thanks to low solvent consumption, reduced execution time and good recovery values (2,3). MEPS, DLLME, UA-DLLME were used for the extraction of 8 BDZ (chlordiazepoxide, oxazepam, lorazepam, bromazepam, flurazepam, flumitrazepam, clobazam, and clonazepam) in three common beverages (tonic water, Spritz and red fruit juice). MEPS extraction was optimized testing various elution mixtures of solvents to yield the maximum recovery percentage. Several parameters influencing DLLME, such as type and dispersive solvent volumes, type and extraction solvent volumes, and ionic strengths were investigated and optimized to yield the highest recoveries. The analyses were performed with a Agilent series 1100 capillary pump system with a Thermo Scientific DionexUltiMate 3400 Variable Wavelenght UV detector, 45 nL flow cell, 100 nL injection volume and an Agilent Zorbax XDB C18 (3,5 µm x 300 µm x 150 mm) column. The chromatographic separation was performed with a 4 µL/min multi-step gradient with H2O (0,1% HCOOH) and Acetonitrile (0,1% HCOOH) and the detection was carried out at 254 nm; capillary HPLC separation with UV-detection was the analytical technique of choice because of its simplicity, robustness and wide diffusion. The DLLME extraction was performed mixing centrifuged matrix (5500 rpm, 5 minutes), Acetone (dispersive solvent) and CH2Cl2 (extraction solvent) for 1 minute; then the mixture was centrifuged (5500 rpm, 5 minutes), the extraction phase was recovered, evaporated and the analyte was re-dissolved in H2O (0,1% HCOOH). The detailed description of the method, its quantitative performance and a comparison with the MEPS results are presented. The MEPS extraction was carried out with a SGE eVol XR digital analytical syringe aspiring directly from the matrix and eluting with an Acetonitrile:H2O 90:10 both acidified with 0,1% HCOOH. The method was validated in terms of linearity, precision, accuracy and recovery, LOD, and LOQ. Good linearity was obtained both with DLLME and MEPS with correlation coefficients (R2) spanning from 0.996 to 0.9999. The limits of detection (LODs) of all analytes ranged from 1 ng/µL to 2.5 ng/µL. The recoveries in spiked beverages spanned from 31.7% to 69.7% for DLLME and from 62.3 % to 98.8 % for MEPS in all matrices spiked at the concentration of 20 ng/µL.

REFERENCES

1. US Department of Health and Human Services. Substance abuse and sexual assault: when drugs are used for rape. D.C. Crisis Center. www.drcc.org.

2. R. Jain, R. Singh, Trends in Analytical Chemistry 75 (2016) 227–237. 3. L. Magrini et al., Journal of Pharmaceutical and Biomedical Analysis 125 (2016) 48–53

Abstract

Comprehensive Two-dimensional Liquid Chromatography Coupled to Mass Spectrometry for Elucidation of the Polyphenolic Fraction

of Pistacia vera from Different Geographical Origin

Domenica Mangraviti1, Francesco Cacciola2, Katia Arena1, Francesca Rigano3, Paola Dugo1,3,4, Luigi Mondello1,3,4

1Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina – Polo Annunziata, Viale Annunziata, 98168 Messina, Italy

2Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali, University of Messina, Viale Consolare Valeria, 98165 Messina, Italy

3Chromaleont S.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina – Polo Annunziata, Viale Annunziata, 98168 Messina, Italy

4Unit of Food Science and Nutrition, Department of Medicine, University Campus Bio-Medico of Rome, Via Alvaro del Portillo, 28, 00128 Rome, Italy

Keywords: Pistacia vera, polyphenols, comprehensive two-dimensional liquid chromatography.

Pistachio (Pistacia vera L.) belongs to the Anacardiaceae family and it is a small tree species. It is native of the Middle East and Central Asia, but currently it is cultivated also in California and in some Mediterranean countries, such as Greece, and Italy. The most important pistachio producers are Iran, United States and Turkey. Besides a being a delicious nut, pistachio, due to its wholesome nutritional properties, it could be considered as a functional food. According to the results of several studies, pistachios have been proven to have various groups of valuable phytochemicals such as anthocyanins, flavan-3-ols, proanthocyanidins, flavonols, isoflavones, flavanones, stilbenes and phenolic acids, possessing excellent biological activities. The analytical techniques employed for their analysis are represented by liquid chromatography coupled to photodiode array and mass spectrometry detection. However, conventional LC can present some limits especially in terms of resolving power. A powerful alternative is represented by comprehensive two-dimensional liquid chromatography (LC×LC) where two columns of different selectivity are separated by means of a switching valve. In this contribution the polyphenolic fraction of pistachios from different geographical regions was elucidated by LC×LC incorporating a 150 mm microbore cyano column (2.7 µm dp), and 50 mm superficially porous C18 silica column (2.7 μm dp) in the first (1D) and second (2D), respectively. For boosting orthogonality a shifted 2D gradient was investigated leading to an increase in the overall peak capacity.

Abstract

Chromatographic determination of biogenic amines in wines by novel detection approaches

Luca Rivoira1, Davide Carena1,2, Mojca Zorz2, Sara Budal2, Mladen Franko2, Maria Concetta Bruzzoniti1

1Department of Chemistry, University of Torino, Via Pietro Giuria 7, 10125, Torino, Italy 2Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska 13, SI-5000 Nova Gorica, Slovenia

Keywords: biogenic amines, liquid chromatography, thermal lens spectroscopy, fluorescence, wine.

Biogenic amines (BA) are low molecular weight organic bases, involved in several biological activities of the organism. Biogenic amines play essential roles in the physiological functions of humans, however several studies show that BA could have negative toxicological effects. Their formation is due to proteolytic processes of decarboxylation of amino acids that occur in fermented foods. Since not only toxicological, but also organoleptic effects could modify the original properties of foods, biogenic amines are considered also markers of food quality. For all these reasons, biogenic amines in food and beverage matrices are extensively studied. Wine is a fermented beverage that could be affected by high concentrations of biogenic amines. Different analytical techniques have been tested for the determination of biogenic amines, such as gas-chromatography, capillary electrophoresis and liquid chromatography. This last technique is usually coupled with pre-column derivatization and fluorescence and/or UV-Vis detectors. O-phtalaldehyde (OPA) is one of the main derivatizing agent used, but its stability is questionable and might affect reproducibility of the assay. In this work, we selected naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescent agent for amines derivatization, to be tested for the first time in the analysis of wine samples. An HPLC-Fluorescence system (FL) was used to analyze five BAs (ethanolamine, histamine, methylamine, tyramine, isopenthylamine, butylamine) after NDA derivatization. The effect of reaction time and concentration of NDA on derivatization yield was studied. Moreover, the presence of tetrahydrofuran in the mobile phase was also studied, to evaluate its effect in BAs selectivity and system sensitivity. The main figures of merit of the NDA-FL method, such as linearity, limits of detection, limits of quantification and instrument reproducibility, were determined in a synthetic wine matrix. If compared to already published OPA-FL and OPA-Vis procedures, the results showed that, even if the derivatization with NDA is slightly longer (reaction time 25 min), the sensitivity and the accuracy (RSD < 3.0%) of the NDA-FL method are greatly enhanced. The NDA derivatization was also tested to quantify biogenic amines on a Rebula Slovenian wine, where two analytes (isopenthylamine and butylamine) were identified. Derivatization with NDA was compared with the one performed with OPA, obtaining good agreement between the two approaches applied on the wine sample. Finally, exploiting the maximum absorbance at 420 nm of the amines-NDA complexes, a Thermal Lens Spectroscopy (TLS) detector (with a krypton laser, 417 nm emission) was coupled to the HPLC system, studying the detection behavior of methylamine as model amine. LOQs obtained by TLS analysis (180 µg/L) were found to be only slightly higher than the ones obtained with FL detection (70 µg/L), thus confirming the potentiality of TLS detection in the determination of biogenic amines and the need to improve studies towards this direction to improve method sensitivity.

Abstract

Global Warming: Influence of Raising Temperature on Fatty Acid Composition of Muscle of Antarctic Teleost Trematomus

Bernacchii, Analysed by Gas-Chromatography Mass-Spectrometry

Cristina Truzzi, Anna Annibaldi, Matteo Antonucci, Giuseppe Scarponi and Silvia Illuminati

Department of Life and Environmental Sciences, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy

Keywords: Raising temperature, Antarctica, Trematomus bernacchii, Aquaria experiment, Muscle, Fatty acids composition, Gas chromatography-mass spectrometry

Raising temperatures have a negative influence on the physiological and biogeochemical processes of organisms, not

least on the lipid composition of tissues in phytoplankton, zooplankton and fishes1. In this context, fatty acids (FAs) composition is often used as biomarker to study the organism response to environmental stressful conditions2.

In this work, we studied the effect of seawater warming on the FAs composition of muscle of Trematomus bernacchii, an Antarctic teleost living at -1.8 °C, used as a bioindicator for environmental studies. 69 specimens were caught in Terra Nova Bay, Ross Sea, during the XXVIII Italian Antarctic expedition (austral summer 2014-15), and, after a period of acclimatization, they were put in 200-L glass aquaria at different temperatures of 0 °C, +1 °C and +2 °C. After 1, 5 and 10 days, fishes were sacrificed and muscle tissues were collected and frozen at -80 °C until analysis. The determination of muscle FAs composition was performed optimising an analytical methodology based on a fast microwave-assisted extraction of lipids from the lyophilized sample, a base-catalyzed trans-esterification of lipid extract to obtain Fatty Acid Methyl Esters (FAMEs), and a separation and identification of FAMEs by gas chromatography-mass spectrometry3. Using the optimized and validated method, a fast and accurate separation of FAMEs was performed in 43 min.

Specimens exposed at +1 °C and +2 °C showed, at any exposition time, a percentage of total lipids statistically lower than control group. Major saturated FAs, 16:0 and 18:0, were generally higher in temperature-treated specimens than the control ones. Mono-unsaturated FAs, such as 16:1n7, 18:1n9 and 18:1n7, showed in general a significant reduction with respect to the control group. Poly-unsaturated FAs 20:5n3 and 22:6n3, showed a significant increase at 1 day for all tested temperatures. These changes take place very quickly: at 1 day of exposition, specimens showed the greatest changes; at 5 days, lipid profile of exposed specimens was similar to the control group, then we can assume that fishes were trying to adapt to new environmental conditions; at 10 days of exposition, new modification on FAs composition appeared. In conclusion, we can claim that temperature and time of exposition influenced significantly the FAs composition of muscle of T. bernacchii.

REFERENCES

1. G. Kattner, W. Hagen, R.F. Lee, R. Campbell, D. Deibel, S. Falk-Petersen, M. Graeve, B.W. Hansen. Perspectives on marine zooplankton lipids. Can J Fish Aquat Sci, 2007, 64:1628-1639.

2. N.N. Fokina, L.A. Lysenko, L.A. Sukhnovskaya, et al. Biochemical response of blue mussels Mytilus edulis L. from the White Sea to rapid changes in ambient temperature. J Evol Biochem Phys, 2015, 51:378-387.

3. C. Truzzi, S. Illuminati, A. Annibaldi, M. Antonucci, G. Scarponi. Quantification of fatty acids in the muscle of Antarctic fish Trematomus bernacchii by gas chromatography-mass spectrometry: optimization of the analytical methodology Chemosphere, 2017, 173:116-123.

Abstract

Characterization of Vitamin B12–Pt(II) Conjugates by RPLC-ESI-MS

G. Venturaa, C. D. Calvanoa,b, I. Nardellaa, F. Arnesanoa,b, F. Palmisanoa,b and T. R. I. Cataldi a,b

aDipartimento di Chimica and bCentro di Ricerca Interdipartimentale SMART,

Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126 Bari (Italy)

Keywords: Cisplatin, cyanocobalamin, mass spectrometry, ESI, HPLC.

Cisplatin (cis-diamminedichloroplatinum(II), [PtCl2(NH3)2]) is one of the most effective chemotherapeutic agents used in the treatment of a variety of human solid tumors1, 2; unfortunately, nausea, vomiting and nephrotoxicity are common adverse side effects limiting its use3. Numerous attempts have been made to reduce its cytotoxicity to healthy cells: enhancing its selectivity to cancer cells, combining targeting molecules with cisplatin, would reduce potentially negative treatment effects. Vitamin B12 (cyanocobalamin, CN-Cbl)) is very important for some enzymatic processes in living cells. Indeed, rapidly proliferating cells have a high demand for vitamin B12. No cell can live without this vitamin; therefore, it can be an attractive vehicle for targeting cancer cells and for incorporating various sorts of bioactive molecules, provided that vitamin B12 is taken up by receptor-mediated endocytosis4. Here, Pt(II) drugs derivatives bearing a cyanocobalamin (CN-Cbl) unit were synthetized in aqueous solutions as anticancer therapy candidates. The reaction mixture was investigated by reversed-phase liquid chromatography-electrospray ionization linear ion trap mass spectrometry (RPLC-ESI-LIT-MS); isotopic pattern analysis, MSn mass-spectra interpretation and differential isotopic labelling were used to establish the chemical composition and to suggest the chemical structures. A coordinate bond (CoIII–CN–PtII) between monochloro-diamino-platinum(II) and CN-Cbl cyano group was formed following aqueous solution reaction, thus producing a doubly charged MS peak at m/z 810.26; a peak at m/z 811.26 was observed by using 15N labelled cisplatin [PtCl2(15NH3)2]. Furthermore, bifunctional adducts were obtained starting from the bis-aqua species (cis-[Pt(15NH3)2(H2O)2]+), where two Pt(II) coordination sites are involved in the binding to CN-Cbl via the loss of both cisplatin leaving groups. These reaction products were separated and characterized by RPLC-ESI-LIT-MS.

REFERENCES

1. B. Rosenberg, L. Vancamp, T. Krigas, Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode, Nature 1965, 205:698–699.

2. E. Wong, C. M. Giandomenico, Current status of platinum-based antitumor drugs, Chem. Rev. , 1999, 99(9):2451-66. 3. BA Baldo, N. Pham, Adverse reactions to targeted and non-targeted chemotherapeutic drugs with emphasis on

hypersensitivity responses and the invasive metastatic switch, Cancer Metastasis Rev., 2013, 32:723–761. 4. M.T. Quynh Tran, S. Sturup, I. Lambert, B. Gammelgaard, E. Furger, R. Alberto, Cellular uptake of metallated

cobalamins, Metallomics, 2016,8, 298-304.

Abstract

Liquid Chromatography-High Resolution Mass Spectrometry For The Correct Identification Of The Cyanotoxin BMAA

Iole Maria Di Gangia, Paolo Pastorea and Sara Bogiallia

a Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova

Keywords: cyanotoxins; liquid chromatography-high resolution mass spectrometry.

Since 1940’s the consumption of cycad seeds were associated with the increased incidence of amyotrophic lateral sclerosis and Parkinson-like disease in the population of the Guam isles. The neurotoxin β-N-methylamino-L-alanine (BMAA), isolated from the cycad kernels, was hereafter suspected to be involved in these neurological diseases. Actually, this amino acid was produced by a cyanobacteria living in symbiosis with the cycad roots, and was then reported as a metabolite of various cyanobacterial species, so that its presence in algae and the consequent bioaccumulation in derived-food were considered to play a relevant role in such diseases. Anyway, the widespread reports of BMAA were impaired by analytical false-positives due to the isomeric amino acid α-,γ-diaminobutyric acid (DAB)1. As a matter of fact, the use of mass spectrometric (MS)-based methods did not eliminate the possibility of an overestimation of the neurotoxin concentration, since the simultaneous presence of two isomeric hydrophilic compounds hindered the use of the conventional reversed phase chromatographic separation. A method based on liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS) able to reliably quantify BMAA was developed. Specific fragment ions were highlighted for BMAA and DAB. After the evaluation of different chromatographic strategies, a satisfactory separation of BMAA from DAB was accomplished by using a mixed cationic-reverse phase column (Figure 1). The method was validated in two different matrices, i.e. water and plasma samples, in order to propose an analytical tool for monitoring occurrence of BMAA in environment and its possible distribution in biological samples.

Figure 1: LC-MS chromatogram related to the final experimental condition for analyzing BMAA.

REFERENCES

1 E. Faassenn. Presence of neurotoxins BMAA in aquatic ecosystems: what do we really know? Toxins 2014, 6 (3), 1109-1138.

Abstract

Optimization of an ultrasound-assisted derivatization for GC/MS analysis of oxygenated organic species in atmospheric aerosol

Francesco Manarinia, José Benito Quintanab, Rosario Rodilb, Eugenia Villaverde-de-Sáab, Marco Visentina, Maria Chiara Pietrograndea

aDepartment of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17/19, I-44100 Ferrara, Italy

bIIAA - Institute of Food Analysis and Research, Department of Analytical Chemistry, University of Santiago de Compostela, R/ Constantino Candeira S/N, 15782 - Santiago de Compostela, Spain.

Keywords: Ultrasound-assisted derivatization, GC/MS method, Experimental design and response surface methodology, Atmospheric aerosol, Oxygenated organic species.

A novel ultrasound-assisted derivatization followed by GC/MS analysis was developed for the quantification of oxygenated organic species in ambient aerosol. Derivatization parameters mostly influencing the analytical response were investigated, i.e., solvent type, reagent concentration and reaction duration. Response surface methodology was used to design experiments and a quadratic model was utilized to predict the variables and establish the optimal conditions. The study was performed on standard solutions of 30 compounds representing the major classes of oxygenated compounds typically found in ambient aerosol, i.e. low molecular weight carboxylic acids, sugars and phenols. In comparison with conventional methods, the optimized procedure uses mild reaction temperature (room temperature instead of 70°C), reduces amount of silyl reagent (24 µl vs. 40 µl) and shortens derivatization times (45 min vs. 70 min), participating of the current trend of present analytical chemistry towards clean–green methods that reduce costs and decrease pollution. Once optimized, the ultrasound procedure was validated by assessing for repeatability, linearity, detection limits and derivative stability. For all oxygenated organic species, the proposed method showed a good reproducibility − as the relative standard deviations (RSDs%, n= 5) of intra-day analysis were ≤7% −, a good linearity with the correlation coefficients of calibration curves R2 ≥ 99.8, and low detection limits, ranging from 0.34 to 6.50 ng μL−1, that are suitable for its applicability in air quality monitoring. Finally, this method was successfully applied to determine thirty oxygenated organic species in three ambient PM2.5

samples collected at an urban site in Northern Italy in three different seasons.

REFERENCES

1. Pietrogrande MC, Bacco D, Chiereghin S. GC/MS analysis of water-soluble organics in atmospheric aerosol: optimization of a solvent extraction procedure for simultaneous analysis of carboxylic acids and sugars. Anal. Bioanal. Chem. 2013;405:1095-1104.

2. Maria Chiara Pietrogrande, Francesco Manarini, José Benito Quintana, Rosario Rodil, Eugenia Villaverde-de-Sáa, Marco Visentin. Analytical and Bioanalytical Chemistry. 2017. DOI: 10.1007/s00216-017-0379-6

Abstract

Teicoplanin Chiral Stationary Phases On 2.0 µm And 2.7 µm Superficially Porous Particles: Chromatographic Evaluation And Comparison With

Teicoplanin On 1.9 µm Fully Porous Particles.

Omar H. Ismaila, Michela Antonellia, Alessia Cioglia, Claudio Villania, Simona Fellettib, Martina Catanib, Alberto Cavazzinib, David S Bellc, Francesco Gasparrinia.

aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Roma, Italy

bDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Ferrara, Italy cSigma-Aldrich/Supelco, 595 North Harrison Road, Bellefonte, PA 16823

Keywords: UHPC-Tzwitt, Superficially Porous Particles CSP, Very-High Efficiency column, Ultra-fast / Ultra-High Performance chromatography

For the first time, a new generation of Chiral Stationary Phases (CSPs) for Ultra-High Performance Chromatography (UHPC) was prepared covalently bonding the teicoplanin (TE_A2) selector on Halo 2.0µm and 2.7µm (90Å, 125m2/g) Superficially Porous silica Particles (SPP) [1]. The new CSPs were compared with the already known UHPC-FPP-Titan-Tzwitt 1.9 CSP based on Fully Porous monodispersed silica Particles [2]. Columns with an internal diameter of 4.6 mm and different lengths (20, 50 and 100 mm) were packed with the three CSPs and characterized in terms of permeability, efficiency, retention and enantioselectivity under HILIC conditions. Van Deemter curves, generated using both achiral and chiral analytes, showed excellent results with more than 310000 plates/m at 1.6 mL/min (thiourea, k’= 0.6), and more than 290000 plates/m at 0.9 mL/min (2-(4-chloro-phenoxy)-propionic acid, 1st eluted enantiomer, k’= 0.8) on the SPP-Halo 2.0µm. The kinetic performance limits of the columns were investigated also through different kinetic plots, that proved the superior potential of the SPP-Halo 2.0µm and FPP-Titan 1.9µm compared to the SPP-Halo 2.7µm particles in the majority of the kinetic plots area, especially in the region of the ultra-fast/ultra-high efficiency separations. For several chiral analytes, the columns packed with SPP-Halo 2.0µm and FPP-Titan 1.9µm CSP were superior, in terms of efficiency, to the columns packed with SPP-Halo 2.7µm, proving the validity of the van Deemter analysis. Furthermore, from the thermodynamic point of view, the SPP-Halo 2.0µm have shown the best resolution power, with Rs values significantly higher than those of the SPP-Halo 2.7µm and FPP-Titan 1.9µm. Lastly Ultra-High speed – Ultra-High Performance applications performed on three 5-cm long columns, using chiral probes and different eluent flow-rate (1.0 – 6.0 mL/min) showed very high efficiency values and the FPP-Titan 1.9µm CSP, and again a lower performance for the columns packed with the SPP-Halo 2.7µm CSP. Considering the high resolution values observed, 2-cm long columns were prepared and used to explore Ultra-High speed – Ultra-High Performance separations in the seconds time regime: the enantiomers of Haloxyfop were resolved in only 3 seconds with a resolution Rs = 2 on the column packed with SPP-Halo 2.0µm CSP, using an eluent flow rate of 8.0 mL/min.

REFERENCES

1. O.H. Ismail, M. Antonelli, A. Ciogli, G. Sestito, D.S Bell, A. Cavazzini, C. Villani, F. Gasparrini, JCA, in preparation 2. O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, JCA, 1427 (2016), 55–68

COMUNICAZIONI ORALI BIOANALITICA

Abstract

How Can We Evaluate the Binding Properties of Molecularly Imprinted Polymers without the Misleading Current Approach?

C.Baggiania, C.Giovannolia, L.Anfossia, F.Di Nardoa, and G.Spanoa

aLaboratory of Bioanalytical Chemistry, Department of Chemistry, University of Torino – Via Giuria 5 10125 Torino

Keywords: molecularly imprinted polymer, binding isotherm, binding properties, selectivity

The artificial receptors known as Molecularly Imprinted Polymers (MIPs) represent one of the most successful type of man-made materials developed to mimic the binding properties of natural receptors like antibodies and enzymes. In the recent years, many insights have been obtained concerning the thermodynamic and kinetic behaviour of these polymers, and the increased knowledge of the binding site inner working has been used to develop efficient applications in the fields of solid phase extraction, sensoristics and, more recently, immunoassay. The binding properties of a MIP are usually evalued by measuring the so-called “imprinting factor”, i.e. the ratio between the binding capacities of the imprinted and not-imprinted polymers, respectively. Selectivity is calculated in the same way, through the ratio between the binding capacities of the imprinted polymer for the competitor ligand and the template ligand, rispectively. These tasks can be accomplished directly, by measuring the bound fractions in batch rebinding experiments, or indirectly, by measuring the capacity factors in chromatographic experiments. All these approaches are based on the assumption that the underlying binding isotherms are linear, thus, the partition coefficients must be indipendent from the stoichiometric concentration of the ligands. Unfortunately, very few MIPs show linear binding behaviours. On the contrary, the most part of them show very complex binding behaviours, strongly dependent from the stoichiometric concentration of the ligands and corresponding to Langmuir-type binding isotherms. As a consequence of this, the use of the current definitions of imprinting factor and selectivity are misleading and hamper the correct calculation of the binding properties of a typical MIP. We started from the consideration that the thermodynamic and kinetic binding properties of a MIP are defined by the parameters of a binding isotherm. Thus, chosen a binding isotherm model compatible with the experimental data, it should be possible i) to calculate the imprinting factor by directly comparing the binding isotherms for an imprinted and a non-imprinted polymer; ii) to calculate the selectivity by directly comparing the binding isotherms for a ligand and its competitor. Here we discuss this approach, showing its independency from the experimental ligand concentration. Moreover, we show how the direct comparison of the binding isotherm parameters (i.e. binding site density, Bmax, and apparent binding affinity, Keq) make possible to define uniquely the imprinting factor and selectivity for a given MIP.

Abstract

Competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles

screen printed electrodes: a rapid and noninvasive screening tool for early diagnosis of bladder carcinoma

Marco Giannetto, Maria Vittoria Bianchi, Monica Mattarozzi and Maria Careri

Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma Parco Area delle Scienze 17/A, 43124 Parma

Keywords: competitive immunosensor; p53 protein; urine; bladder carcinoma p53 is an effective transcription factor, responding to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism1. Particularly, p53 is primarily involved in the inhibition of uncontrolled cell proliferation, upon activation and binding to specific DNA sequences. Concerning the Bladder Transitional Cell Carcinoma (BTCC), the findings of molecular genetics and clinical studies evidenced as abnormal p53 protein accumulation in the epithelial cells may result in its overexpression at level of extracellular fluids, with particular relevance for urine2, indicating p53 as a valuable protein biomarker. In this study, we report the development and validation of a novel disposable competitive amperometric immunosensor3 for the determination of p53 at picomolar levels, based on gold nanoparticles/carbon nanotubes modified screen-printed carbon electrodes (CNT/GNP SPCEs) directly functionalized with p53 protein.

The assay protocol requires the use of single anti-p53 mouse monoclonal antibody, (DO-7 clone) able to recognize both wildtype and mutated p53. Performance of the new competitive immunodevice are comparable with the majority of the sandwich immunosensors for determination of differently mutated p53 forms, despite these methods involve very complex and time/cost expensive nanostructured architectures. The immunosensor and the protocol of the electrochemical immunoassay were optimized by means of experimental design procedures. The device was successfully validated for the determination of p53 in untreated and undiluted urine samples, showing a limit of detection of 17 pM and a limit of quantification of 123 pM.

The developed competitive immunosensor was proved as simple, reliable and analytically robust diagnostic tool, valuable for implementation of screening and follow-up programs for urological malignancies.

REFERENCES

1. H. Jafari, R. Gharemohammadlou, A. Fakhrjou, A. Ebrahimi, K. Nejati-Koshki, M. Nadri, E. Sakhinia, Bioimpacts, 2013, 3, 135. 2. M.G. Kishore, A. Hamid, U.S. Dwivedi, V. Tandon, M. Mahmood, H. Singh, P.B. Singh, Urol. Oncol., 2006, 3, 216. 3. A. Manfredi, M. Giannetto, M. Mattarozzi, M. Costantini, C. Mucchino, M. Careri, Anal. Bioanal. Chem., 2016, 26, 7289.

 

Abstract    

TGA/Chemometrics  Approach  In  Bioanalytical  Investigations:  The  Screening  of  Sickle  Cell  Anemia    

Roberta  Risolutia,  Giuseppina  Gullifaa,  Maria  Aurora  Fabianoa,  Patrizia  Caprarib,  Carlotta  Bozzib  and  Stefano  Materazzia  

a  Department  of  Chemistry,  Sapienza  University  of  Rome,  piazzale  Aldo  Moro  5,  00185  Rome,  Italy  bDepartment  of  Haematology,  Oncology  and  Molecular  Medicine,  Istituto  Superiore  di  Sanità,  Viale  Regina  Elena  299,  00161  

Rome,  Italy  cThalassemia  Unit,  S.  Eugenio  Hospital,  piazzale  dell'Umanesimo  10,  00144  Rome,  Italy  

 

Keywords:  Sickle  Cell  Anemia,  TGA,  Chemometrics  

 

 Thermogravimetry   coupled   with   chemometrics   has   proved   to   be   a   rapid   and   cost   effective   diagnostic   tool   for   β-­‐thalassemia   screening.   This   model,   consisting   of     Partial   Least   Square-­‐Discriminant   Analysis   (PLS-­‐DA),   permitted   the  discrimination  of  thalassemic  patients  and  healthy  individuals,  using  thermogravimetric  curves  of  blood  samples.[1]  In  this  study,  the  capability  of  thermogravimetry  in  conjuction  with  a  multivariate  statistical  analysis,  was  investigated  for  the  screening  of  Sickle  Cell  Disease  (SCD),  a  hereditary  disorder  characterized  by  severe  hemolytic  anemia  with  different  clinical  manifestations.    SCD  results  from  a  mutation  in  the  sixth  codon  of  the  beta  globin  gene,  which  results  in  the  substitution  of  glutamic  acid  for  valine  and  leads  to  the  production  of  an  altered  form  of  hemoglobin,  hemoglobin  S  (HbS).  People  with  this  disorder  have   atypical   hemoglobin  molecules   called   hemoglobin   S,  which   can   distort   red   blood   cells   into   a   sickle,   or   crescent,  shape.  Systematic  screening  for  SCD  is  not  a  common  practice,  and  diagnosis  is  usually  made  when  a  severe  complication  occurs.  An  early  and  rapid  diagnosis  is  important  for  patients  in  order  to  prevent  and  trait  the  painful  episodes  that  can  occur  when  sickled  red  blood  cells,  which  are  stiff  and  inflexible,  get  stuck  in  small  blood  vessels.  Whole   blood   samples   from  patients  with   congenital   defects  were   analyzed   by   the   TG7   thermobalance   (Perkin   Elmer)  without  any  pretreatment  and   the   resulting  curves  were  compared   to   those   typical  of  healthy   individuals.  The  TG  and  DTG  curves  of  blood  samples  from  anemic  patients  were  clearly  distinct  from  those  of  healthy  individuals  as  result  of  the  different   amounts   of   water   content   and   corpuscular   fraction.   The   chemometric   approach   based   on   Principal  Components  Analysis  (PCA)  allowed  a  quick  identification  of  differences  between  healthy  and  anemic  patients  in  order  to  point   out   a  model   of   prediction   in   patients  with   heterogeneous   congenital   hematological   disorders.   Results   allow   to  consider  the  coupling  TGA/Chemometrics  as  a  promising  diagnostic  approach  to  provide  a  high-­‐throughput  and  sensitive  tool  to  obtain  an  early  detection  of  hereditary  Sickle  Cell  Desease.    References  1.   R.  Risoluti,  S.  Materazzi,  F.  Sorrentino,  L.  Maffei,  P.  Caprari.  Talanta  DOI:  10.1016/j.talanta.2016.06.037    

Abstract

Supercritical Fluid Chromatography coupled to Tandem Mass Spectrometry

for Limonoid Aglycones Detection in Citrus Essential Oils

Adriana Arigòa, Mariosimone Zoccalib, Fabio Salafiaa, Maria Luisa Calabròa, Paola Dugoa, b, c, Luigi Mondelloa, b, c

a Dipartimento di “Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali”, University of Messina, Polo Annunziata, viale

Annunziata, 98168 Messina, Italy b Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali”, University of Messina,

Polo Annunziata, viale Annunziata, 98168 Messina, Italy c Unit of Food Science and Nutrition, Department of Medicine, University Campus Bio-Medico of Rome, via Alvaro del Portillo

21, 00128 Rome

Keywords: Supercritical Fluid Chromatography, Mass Spectrometry, Limonoids, Essential Oils, Citrus

The development of fast, reliable and environmental friendly analytical techniques is currently a topic of great interest. In this regard, Supercritical Fluid Chromatography (SFC) represents a green and powerful technique, due to the use of carbon dioxide above its critical point, as the main solvent. The employment of CO2 makes this approach suitable especially for the separation of low polar compounds; however, the use of small amounts of polar co-solvents, called modifiers, can expand its applications to more polar matrices. Among the bioactive compounds of interest, limonoids are a class of metabolites naturally occurring in Citrus, both in glucosidic and aglyconic forms. Many biological properties have been related to the use of limonoids such as antiviral, antibacterial, antifungal and antioxidant properties; moreover, several recent studies refer to the anti-proliferative effects, which they have shown in many cancer cells lines, increasing their use as dietary supplements. Due to their low polar character, limonoid aglycones have been investigated mainly in RP-HPLC system, resulting in long time of analyses and high consumption of organic solvent. In this study, SFC is proposed as an analytical approach aimed to separate these hydrophobic compounds in very fast way, using only small amount of methanol as modifier. Moreover, the characterization of limonoids in several cold pressed Citrus essential oils is here reported for the first time.

The separation was carried out on a C 18 column, 250 x 4.6 mm I.D. with particle size of 5 m, providing the elution of the target compounds with less than 5% of methanol in 10 minutes. Due to the lack of available standards of limonoids, the Multiple Reaction Monitoring (MRM) parameters of each compound were optimized using the sample that showed the most abundant content; except for limonin, which was previously isolated in our laboratory (1). This work provides a qualitative and quantitative profile of limonoids contained in eleven types of Citrus essential oils that are increasingly used in the pharmaceutical, cosmetic and food products, highlighting the differences in their composition and offering further data to the characterization of Citrus species.

REFERENCES

1. M. Russo, A. Arigò, M.L. Calabrò, S. Farnetti, L. Mondello, P. Dugo, Bergamot (Citrus Bergamia Risso) as a source of nutraceuticals: limonoids and flavonoids. Journal of Functional Foods. 2015, 20:10-19.

Abstract

Identification of Plant Secondary Metabolites by HPTLC-ESI-MS and Assessment of their Antioxidant Activity by a HPTLC-DPPH

Method

Francesca Orsinia,b,c, Irena Vovkb, Vesna Glavnikb, Urška Jugb Laura De Garaa, Isabella Nicolettic, Danilo Corradinic

aUniversity Campus Bio-Medico of Rome, Via Alvaro del Portillo 21, 00128 Rome Italy; bDepartment of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; cNational Research Council, Institute of Chemical

Methodologies, Area della Ricerca di Roma 1, Via Salaria Km 29,300, I-00015 Montelibretti, Rome, Italy

Keywords: Plant secondary metabolites, HPTLC-ESI-MS, HPTLC-DPPH.

A variety of plant secondary metabolites consists of phenolic compounds with antioxidant activity. Investigations carried out during the past two-three decades have evidenced that these compounds play an important role in preventing chronic illnesses, including cancer, diabetes and coronary heart disease. In particular, the flavonoids, one of the most widespread class of phenolic compounds, have gained recent increasing attention because of their crucial function in protecting biological systems against the harmful effects of oxidative processes on macromolecules, such as polysaccharides, proteins, lipids and DNA. This communication reports the results of a study carried out to develop straightforward high performance thin-layer chromatographic (HPTLC) methods for the separation and identification of flavonoids extracted from leaves and fruits of Cyclanthera pedata Schrad. This is an ancient Peruvian plant belonging to the Cucurbitaceae family, currently known as caigua, which is grown for its edible mature fruit in many parts of Central and South America. In addition, the local folk medicine recommends the daily intake of fruits and leaves of caigua for the treatment of several diseases,including diabetes, high blood pressure and dyslipidemia (altered levels of LDL and HDL blood cholesterol). Flavonoids, and other phenolic compounds extracted from leaves and fruits of caigua, were separated by HPTLC in normal phase mode using silica plates and mobile phases consisting of a variety of organic solvents in mixtures at different volume rates. Sample detection was performed by either densitometry (absorption or fluorescence mode) or mass spectrometry (MS) with electrospray ionization (ESI), used for obtaining structural information on the separated compounds. A special surface sampling probe was used for extracting the separated analyte on-line from the HPTLC plate to the ESI-MS instrument. The identification of individual flavonoids was performed on the basis of their HPTLC retardation factor (RF) and both UV-visible and mass spectra, acquired by densitometry and ESI-MS, respectively. The antioxidant activity of each separated and identified analyte was determined by 2,2 diphenyl-1-picrylhydrazyl (DPPH) free radical bioassay, which was performed in situ on the developed HPTLC plates. The identification by HPTLC-ESI-MS of flavonoids and other phenolic compounds extracted from leaves and fruits of caigua, grown either in Slovenia or Italy, and the assay of their antioxidant activity by the HPTLC-DPPH method are reported and discussed.

Abstract

Structural Characterization of Croconaine Dyes by MALDI-Tof/Tof Mass Spectrometry Analysis

Calvano C.D.a,b, Capozzi M.A.M.a, Punzi A.a, Farinola G.M.a, Palmisano F.a,b, Cataldi T.R.I.a,b

aDipartimento di Chimica and bCentro di Ricerca Interdipartimentale SMART,

Università degli Studi di Bari Aldo Moro, via Orabona 4, 70126 Bari (Italy)

cosimadamiana.calvano@uniba.it

Keywords: croconaine, electron transfer, proton transfer, MALDI.

Croconaine dyes are appealing molecules based on the croconic acid five-member ring. They can be simply synthesized via one-pot condensation reactions between croconic acid and several electron-rich (hetero)aromatic compounds or heterocyclic methylene-active bases [1-3]. The resulting croconaines display interesting properties such as intense absorption bands in the near-infrared (NIR) region [4], high photothermal conversion efficiency, excellent chemical and photothermal stability, strong solvatochromism proving to be effective infrared absorbing dyes and promising candidates for photothermal therapy [5]. Structural investigations of these compounds are carried out by NMR spectroscopy and X-ray diffraction (XRD) [2]. Fast identification and structural characterization of croconaine dyes may be achieved by matrix assisted laser desorption ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) and tandem MS because of its inherently features such as rapid and easy sample preparation, good solubility in organic solvents, and high sensitivity. However, to our knowledge, no studies are reported on this topic. In this communication, a simple protocol for the MALDI ToF/ToF characterization of croconaines is presented. Different matrices such as protonating 2,5-dihydroxybenzoic acid, and α-cyano-4-hydroxycinnamic acid, electron-transfer (ET) secondary reaction matrices as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) and 1,5-diaminonaphthalene (DAN) and basic matrices as 9-aminoacridine (9AA) were tested. Protonating matrices originate a mix of odd molecular ions and protonated adducts alongside sodiated and potassiated species. By using 9AA, high matrix background was experienced and very low signal-to-noise ratio was observed when analyzing compounds bearing heteroatoms as bromide. Among the ET matrices, DAN was found to provide the highest ionization yield leading to the specific formation of odd-electron molecular ions M+• with a negligible fragmentation as we previously established on other highly unsaturated compounds such as chlorophylls [6] and bacteriochlorophylls [7]. Finally, MALDI MS/MS of the radical charged molecular species provide useful structural information, thus making identification very straightforward also for other croconaine derivatives.

REFERENCES

1. R. R. Avirah, K. Jyothish and D. Ramaiah, J. Org. Chem. 73 (2008) 274 2. Punzi, M.A.M. Capozzi, et al., J. Mater. Chem. C 4 (2016) 3138 3. Lynch, D.E., Hamilton D.G. Eur. J. Org. Chem. (2017) DOI: 10.1002/ejoc.201700218. 4. X. Song, J.W. Foley, Dyes. Pigments 78 (2008) 60 5. G.T. Spence, G.V. Hartland, B.D. Smith, Chem. Sci. 4 (2013) 4240 6. C.D. Calvano, G. Ventura, T.R.I. Cataldi, F. Palmisano, Anal. Bioanal. Chem. 407 (2015) 6369 7. C.D. Calvano, G. Ventura, M. Trotta, G. Bianco, T.R.I. Cataldi, F. Palmisano, J. Am. Soc. Mass Spectrom. 28 (2017) 125

Abstract

Protein Corona Sensor Array Nanosystem for Multivariate Cancer Detection

Riccardo Zenezini Chiozzia, Anna Laura Capriottia, Giulio Caraccioloa, Morteza Mahmoudib, Daniela Pozzia and Aldo Laganàa

aDepartment of Chemistry, “Sapienza” University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy bCenter for Nanomedicine, Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston,

MA 02115, United States

Keywords: proteomics, sensor array technology, nanomedicine, liposomes, lung and pancreas cancer .

It is now well accepted that the early detection of cancer substantially enhances the possibility of successful

treatment. Although recognizing the warning signs of cancers and taking prompt action may lead to early diagnosis, the majority of cancers (e.g., lung and pancreas) show symptoms only after cancer cells have already invaded the surrounding tissues and metastasized throughout the body (1).

New advances in multidisciplinary approaches to medicine (e.g., nanotechnology and proteomics) have recently introduced the exciting possibility of cancer identification at early stages 1). It is now well accepted that nanoparticles in contact with biological fluids are quickly surrounded by a selected group of adsorbed proteins that form a corona (32, 33) whose composition is strongly dependent on the physicochemical properties of the nanoparticles themselves. The overarching focus of the majority of the studies about protein corona are to delineate the adverse role of the protein corona on nanoparticle function and ranged from implications in immunogenicity, mistargeting, and unpredictable pharmacokinetics and biodistribution. Taking an orthogonal view while building on these studies we (34-36) recently introduced the concept of “disease-specific protein corona” where plasma from a single donor (as opposed to combined from multiple sources) was used in each protein corona study and we demonstrated that human plasma obtained from healthy subjects and patients with various diseases, conferred subtle differences in the protein corona profile. Importantly, these subtle differences in corona profile had enormous overlap in all these studies and lacked robustness to reproducibly assign a particular protein corona profile to a particular disease. Here, we combined nanoparticle sensor-array technology, which offers the advantage of improved accuracy while not being limited to known disease biomarkers with protein corona and developed a label-free protein corona sensor array for early detection of various human cancers, using 45 distinct human plasma obtained as part a prospective longitudinal cohort study where a subset of healthy patient later developed cancers. The sensor array consists of three different cross-reactive liposomes with various surface charges, whose protein corona profiles were measured after exposure to the plasma of a patient who have one of five cancers: lung, pancreas, myeloma, meningioma, or glioblastoma. We performed proteomic analysis on each patient in triplicate for each of the three nanoparticle sensor elements. Although no single protein corona composition is specific for any one cancer type, we hypothesized that changes in the corona composition pattern could provide a unique "fingerprint" for each type of cancer

REFERENCES

1. R. Etzioni et al., The case for early detection, Nature Reviews Cancer 2003, 3:243-252 2. M. Ferrari, Cancer nanotechnology: opportunities and challenges, Nature Reviews Cancer 2005, 5:161-171 3. M. P. Brown and K. Austin, The New Physique, Publisher City: Publisher Name, 2005, pp. 25-30.

Abstract

Mitochondrial Proteome Modifications due to eIF6 Depletion by UHPLC-QTOF MS/MS with SWATH-MS Acquisition

Elisa Robottia, Simona Martinottia, Marcello Manfredia,b, Daniela Brinac, Alessandra Scagliolac, Elia Ranzatoa, Stefano Biffoc,d, Fabio Gosettia and Emilio Marengoa

a University of Piemonte Orientale, Department of Science and Technological Innovation, viale T. Michel 11, 15121 Alessandria, Italy; b ISALIT srl, Via Bovio 6, 28100, Novara; c Istituto Nazionale Genetica molecolare “Romeo ed Enrica Invernizzi”, Via Sforza

28, 20122, Milano, Italy; d University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milano, Italy

Keywords: eIF6, proteomics, UHPLC, High-res MS

Eukaryotic Initiation Factor 6 (eIF6) is an initiation factor that binds 60S ribosomal subunits and has an anti-association property, by hampering 60S premature joining to 40S. In general, eIF6 is rate limiting for tumour onset and progression. eIF6 haploinsufficient cells are normal, but not efficiently transformed in vitro. Mitochondria are the main compartments of energy production, and some lines of evidence have shown that mitochondrial alterations contribute to the development of metabolic syndrome. To this aim, we analysed, by uHPLC-QTOF-MS/MS exploiting the SWATH-MS (Sequential Window Acquisition of all Theoretical fragment ion spectra) acquisition, the expression of mitochondrial proteome of AML-12 (non-tumourigenic murine liver hepatocytes) cell line, where eIF6 was down-regulated by shRNA, and of three different tissues from wild type and +/- mice for eIF6 (liver, muscle and brain). The SWATH-MS acquisition method is a high throughput label-free method for protein quantitation that combines the traditional shotgun proteomics with the quantitative accuracy and reproducibility of selected reaction monitoring (SRM). We found that depletion of eIF6 by shRNA induces profound and varied impact on mitochondrial proteome, impairing the energy production, steering the metabolism toward the up-regulation of aerobic glycolysis and the inhibition of oxidative phosphorylation.

Abstract

Three-Dimensional Graphene/Fe3O4 Nanocomposite Based Dispersive Magnetic Solid Phase Extraction Coupled With UHPLC-PDA For Simultaneous Determination Of NSAIDs In Human Plasma

Vincenzo Ferronea, Lorenzo Di Marcoa, Maura Carluccib, Valeria Ettorrea, Antonella Fontanaa, Giuseppe Carlucci

aDipartimento di Farmacia - bDipartimento di Scienze Mediche Orali e Biotecnologiche - Università degli Studi “G. d’Annunzio” Chieti - Pescara - via dei Vestini 66100 Chieti - Italy

Keywords: Magnetic solid phase extraction, UHPLC-PDA method

Non-steroidal anti-inflammatory drugs (NSAIDs) are a well-established class of drugs that have long been used for the blockage of pain and inflammation in both acute and chronic pain. Although there are no reports of toxic effects of NSAIDs used at therapeutic levels, side effects such as ulcers in the stomach if taken in over dose may occur without warning symptoms, and they may cause death. At present, the development of green, efficient, cost-effectiveness and miniaturized methods has become the trend in analytical chemistry. In recent years, magnetic solid phase extraction (MSPE) has been developed for extraction and preconcentration of organic pollutants, heavy metals and drugs due to its high recovery and ease of operation. Graphene (G), an emerging carbon material, has gained a lot of importance in analytical chemistry in which it has been used as sorbents in sample extraction due to its ultra-high surface area, its hydrophobicity as well as the possibility of establishing π-π interaction thanks to its delocalized electrons. However, graphene is hard to be separated from aqueous solution [1]. The possibility to combine the magnetic materials with graphene has become a research hotspot. The combination of Fe3O4-graphene makes an efficient adsorbent with high adsorption capacity of graphene and separation convenience of magnetic materials[2]. The magnetic G (G/ Fe3O4) are usually synthesized by the in-situ chemical co-precipitation of Fe2+ and Fe3+ in an alkaline solution in the presence of G. Because the G/ Fe3O4 is dispersed into the aqueous solution, the adsorption equilibrium can be achieved in short time (1-10 min) even if large amount of sample (200-300mL) and small amount of adsorbent (15-40mg) are used. The present study focused on the synthesis of a graphene based magnetic nanocomposite (G/ Fe3O4) as a newly designed materials for MSPE and investigated its performance for adsorption of NSAIDs in human plasma and urine. Several parameters affecting the extraction technique such as adsorption conditions, pH of the sample solution, amount of G/ Fe3O4, salt concentration and desorption solvent were investigated in detail. The results showed that the proposed method was easier and more sensitive compared to the existing method for the determination of NSAIDs

REFERENCES

1. M.L. Castillo.Garcia, M.P. Aguilar-Caballos, A.Gomez-Hens . Nanomaterials as tools in chromatographic methods. TrAC 85 (2016) 203-220

2. J. Tian, J. Xu, F. Zhu, T. Lu, C. Su, G.Ouyang. Application of nanomaterials in sample preparation. J.Chromatogr.A 1300 (2013) 2-16

Abstract

Dual Signal Readout for Sensitive Detection Of Fluorescence Immunochromatographic Test Strip

Laura Anfossia, Fabio Di Nardoa, Irina Y. Goryachevab, Valentina V. Goftmanb, Cristina Giovannolia, Giulia Spanoa, and Claudio Baggiania

aDepartment of Chemistry, University of Turin. Via Giuria, 5, I-10125 Turin, Italy bDepartment of General and Inorganic Chemistry, Chemistry Institute, Saratov State University, Astrakhanskaya 83, R-410012

Saratov, Russia

Keywords: Lateral Flow Immunoassay, fluorescence quenching, FRET, Quantum Dots.

Nowadays, lateral flow immunochromatographic assays are becoming increasingly popular as a diagnostic tool for point-of-care (POC) test based on their simplicity, specificity, and sensitivity. The gold nanoparticles (GNPs) based ICTS with colorimetric readout enables a quick spectrum screening but suffers from non-quantitative performance; although ICTS with fluorescence readout (FICTS) allows quantitative detection, its sensitivity still deserves more efforts and attentions. Among fluorescent probes, Quantum Dots (QDs) have attracted the interest of the biosensing community due to their unique luminescent properties; such as high quantum yields, size-tunable fluorescence, broad absorption spectra, narrow and symmetric emission spectra, and high resistance to photobleaching. Recently, some ICTSs exploiting QDs have been reported [1]. We, also, developed an ICTS employing QDs as fluorescent probes for detecting fumonisins in maize and compared the figures of merits of the QD-based ICT to those achieved by using traditional GNP probes. The FICTS allowed for the rapid and accurate quantitation of a relevant food contaminant. The use of QDs compared to GNPs allowed us improving sensitivity and precision of the ICTS significantly, thus consolidating QDs as promising labels to be applied in ICTS for small molecule detection. More interestingly, QDs fluorescence is quenched by resonant energy transfer to nanoparticles of suitable materials when these are kept at a convenient distance, i.e.: the distance achieved by antibody-antigen complex formation [2]. Quenching of QD emission also occurs due to the high extinction coefficient of GNPs through the inner filter effect. Therefore, the combined use of QDs and GNPs allows designing ICTS with a dual readout: colorimetric and quenching fluorescence. In particular, the quenching fluorescence modal allows for increasing sensitivity of competitive ICTS, where the QD-labeled antigen is placed on the analytical membrane to form the Test line, and the GNP-linked antibody flows across the membrane. A negative sample switches-off the signal because of the binding of GNP-labeled antibodies to the QD-labeled antigen. Instead, sample positivity turns-on the QD luminescence on the Test line, allowing for a direct and sensitive interpretation of the test result. The presentation will discuss the feasibility of the proposed approach by applying the quenching FICTS to fumonisins determination and comparing performances to the GNP-based colorimetric assay.

REFERENCES

1. Huang X, Aguilar ZP, Xu H, Lai W, Xiong Y. Biosens Bioelectron., 2016, 75:166-80. 2. Anfossi L, Calza P, Sordello F, Giovannoli C, Di Nardo F, Passini C, Cerruti M, Goryacheva IY, Speranskaya ES, Baggiani C. Anal Bioanal

Chem., 2014, 406:4841-9.

Abstract

Recent Strategies for Multiplex Immunochromatographic Strip Test Based on Noble Metal Nanoparticles

Fabio Di Nardoa, Laura Anfossia, Cristina Giovannolia, Giulia Spanoa, Claudio Baggiania

aLaboratory of Bioanalytical Chemistry, University of Torino, Via Pietro Giuria 5, 10125 - Torino

Keywords: Immunochromatographic Strip Test – Multicolor labels for immunoassay – Multiplex analyses.

Standard instrumental methods have high accuracy and precision, and are usually used for quantitative analysis. However, they require expensive equipment and professional expertise, and may take hours or days to yield result, limiting their application. Therefore, the trend is to use, whenever possible, screening methods that are easier, cheaper and faster than standard analytical methods. Some screening systems required unskilled personnel and no infrastructure; therefore, they can also be used directly on-site. Moreover, low-cost screening systems are extremely useful in developing countries that cannot afford standard analytical instrumentation to perform analyses. The Immunochromatographic Strip Test (ICST), also known as Lateral Flow Immunoassay, or simply strip test, is one of the most successful analytical format for screening analysis. ICST is the most rapid, simplest and cost-effective screening method and can be used directly at the point-of-need. From the first and the most representative of these tests, that is the at-home Pregnancy Test, nowadays strip tests are widely applied to detect a huge variety of analytes in many different fields1. The typical ICST configuration consists of a membrane and several functional pads that are connected to each other and assembled on an adhesive backing card. ICST devices involve immunoassays in which the sample and a suitable labeled probe flow by capillary forces along an analytical membrane that contains immobilized bioreagents, dispensed in specific area called Test line and Control line. The occurring of immunoreactions leads to the development of detectable bands in correspondence of the Test and Control lines, due to the accumulation of the label in such zones. Over time, the ICST has become easier to use and faster, with improved analytical performances. However, conventional ICSTs can only detect one target molecule at one time. In order to improve the detection efficiency and achieve high throughput detection, lots of efforts have been made on the development of multiplex ICSTs. The application of multiplex analytes detection can significantly improve efficiency of testing in terms of both cost and time in comparison to performing multiple single tests, promising to be an exceptional methodology improvement for advanced decision-making2. The ICST technique offers easy implementation of multiplex analysis and the most direct way to develop a multiplex ICST is to dispense two or more Test lines on the membrane. In addition, the use of different colored labels can facilitate the simultaneous detection of multiple analytes on a single device. In this communication, the recent multicolour multiplexing strategies studied and applied by our research group will be discussed.

REFERENCES

1. J. Li, J. Macdonald, Biosensors and Bioelectronics 83 (2016) 177-192. 2. L. Anfossi, C. Giovannoli, C. Baggiani, Current Opinion in Biotechnology 37 (2016) 120-126.

Abstract

Paper-based (bio)sensors for the detection of chemical warfare agents

Fabiana Arduinia , Stefano Cintia, Noemi Colozzaa, Danila Mosconea, Giuseppe Palleschia

aDepartment of Chemical Science and Technologies, Tor Vergata University, Via della Ricerca Scientifica, 00133, Rome, Italy

Keywords: printed electrode, filter paper, enzyme inhibition-based bioassay.

The availability of analytical tools for fast and in situ detection of warfare agents is a huge issue for the management

of suspected areas. Sensor technology allows an easy development of sensors to be integrated in a miniaturised device for in field analysis. To date, several biosensors are reported in literature for nerve agents' detection; for instance, we have developed a miniaturised biosensor for Sarin gas detection [1], which was also embedded in a lab-on a chip for easily monitoring of contaminated areas [2].

Herein, we reported the first example of a fully-integrated paper-based electrochemical biosensor able to detect nerve agents exploiting screen-printing technology to print the electrochemical cell on paper as well as wax printing for paper based microfluidics. The use of paper reduces the costs of fabrication as well as the impact of the environment. In addition, the paper-based sensor after the measurement can be easily incinerated, improving the sustainability in the waste management. This biosensor is based on the use of butyrylthiocholine as a substrate being able to provide an electroactive by-product, thiocholine. We exploited a nanocomposite constituted of carbon black (CB)/PB nanoparticles for thiocholine detection since it is able to electrocatalyse the oxidization of thiol-containing compounds at low applied potential with high sensitivity. Furthermore, the porosity of the paper was exploited to load the required reagents for the measurements, making this device reagent free. This biosensor was challenged towards paraoxon (nerve agent simulant), achieving a detection limit of 3 μg/L [3]. For the mustard agent detection, we have developed a bioassay based on the choline oxidase inhibition, using bis(2-chloroethyl)amine and 2-chloroethyl ethyl sulfide as simulants [4]. Herein, we present the preliminary results obtained in the development of a paper-based, ready to use device for mustard agents, without requiring any treatment in the case of liquid samples.

REFERENCES

1. F. Arduini, F. Ricci, A. Amine, D. Moscone, G. Palleschi, Anal. Bioanal. Chem. 2007, 388,1049-1057. 2. F. Arduini. D. Neagu, S. Dall’Oglio, D. Moscone, G. Palleschi, Electroanalysis 2012, 24, 581 – 590. 3. S. Cinti, C. Minotti, D. Moscone, G. Palleschi, F. Arduini, Biosensors and Bioelectronics, 2017, 93, 46-51. 4. F. Arduini, V. Scognamiglio, C. Covaia, A. Amine, D. Moscone, G. Palleschi, Sensors 2015, 15, 4353-4367.

Abstract

Smartphone–based reflectance biosensor for rapid detection of oxidase substrates using confined “wafer-like” multilayer paper-

based enzymatic assays.

Calabria Da, Caliceti Ca,b, Zangheri Mb, Mirasoli Ma,b, Simoni Pc, Roda Aa,b

aCentro Interdipartimentale di Ricerca Industriale Energia e Ambiente, Alma Mater Studiorum, Università di Bologna, Bologna, Italia.

bDipartimento di Chimica "Giacomo Ciamician", Alma Mater Studiorum, Università di Bologna, Bologna, Italia e Istituto Nazionale Biostrutture e Biosistemi (INBB), Roma, Italia.

cDipartimento di Scienze Mediche e Chirurgiche – (DIMEC), Alma Mater Studiorum, Università di Bologna, Bologna, Italia.

Keywords: Optical biosensor, paper-based enzymatic assay, reflectometric measurements, smartphone-based point-of-care biosensor.

The advance in complementary metal oxide semiconductor (CMOS) camera technology makes the smartphone suitable as a detector for reflectance measurements that is the most popular, simple, inexpensive and straightforward method to detect paper color-based assays (1). Despite the increasing interest of recent years in the development of color change paper-based biosensors, they still suffer from a poor detectability and reproducibility related to inhomogeneity of color development, attributed to the mobility of enzymes and reagents towards the edge of detection zone when the sample is applied onto paper surface. In this work, we propose a smartphone paper-based biosensor, in which all the reagents necessary to complete the analysis are co-entrapped on paper in a “wafer”-like bilayer film of polyelectrolytes (Poly (allyl amine hydrochloride/poly (sodium 4-styrene sulfonate)) (2). The sequential adsorption of oppositely charged polyelectrolytes by layer-by-layer (LbL) deposition technique was exploited to obtain self-assembled polyelectrolyte multilayer thin films. The main advantages of this method are the possibility to control the structure and the total film thickness on a molecular level and the efficient selective entrapment of biomolecules such as enzymes exploiting non-covalent bonding and preserving biological activity in their microenvironment. The smartphone-based device was realized using a 3D printing low-cost technology. Its components are a semi-cover accessory attached to the smartphone, a polydimethylsiloxane (PDMS) light diffuser placed in front of the smartphone flash LED to improve the image quality, a mini dark box and a disposable analytical cartridge containing the bioactive paper-based support necessary for the analysis. The biosensor was developed exploiting coupled enzyme reactions for quantifying oxidase substrates such as L-lactate in oral fluid, which is considered a biomarker of poor tissue perfusion in sports performance evaluation. The developed method is sensitive, rapid and it allows detecting L-lactate in the relevant physiological range, with a limit of detection of 0.1 mmol L−1. The extreme simplicity of assay execution (no reagents need to be added) together with the high versatility (the assay is HRP-based color change detection of H2O2 produced by an analyte-specific oxidase), make our approach generally applied for all oxidases.

REFERENCES

1. Hu, J. et al. Biosensors and Bioelectronics, (2014) 54, 585-597. 2. Calabria, D. et al. Biosensors and Bioelectronics, (2017) 94, 124-130.

Abstract

A Versatile Aptasensor Material for Lab-on-Chip Applications

F. Costantinia,d, N. Lovecchiob, M. Nardecchiab,d, C. Manettic, G. de Cesareb, A. Nascettid, D. Caputob

a Department of Chemistry, Sapienza University of Rome, p.le A. Moro 5, 00186 Rome, Italy. bDepartment of Information Engineering, Electronics and Telecommunications, Sapienza University of Rome, via

Eudossiana 18, 00184 Rome, Italy. dDepartment of Environmental Biology, Sapienza University of Rome, p.le A. Moro 5, 00186 Rome, Italy.

cSchool of Aerospace Engineering, Sapienza University of Rome, via Salaria 851/881, 00138 Rome, Italy

Keywords: Aptasensor, polymer brushes, Lab-on-chip, Ochratoxin A.

The use of aptamers instead of antibodies, as recognition and detection molecules, has been shown advantageous. The integration of aptamers in Lab-on-Chip (LoC) results in miniaturized devices for clinical diagnostics, environmental analysis and food safety assessment. In this framework, the main issue is the development of a suitable aptasensor material, which can be integrated into microfluidic channels and that, after the recognition between the aptamer and the target molecule, promotes a detectable signal with a low limit of detection. Here, we present the development of an aptasensor material based on the immobilization of an aptamer into poly 2-(hydroxyethyl methacylate) (PHEMA) polymer brushes. The layer of PHEMA is grown in microfluidic channels that are optically coupled with an array of amorphous silicon photosensors for monitoring fluorescence. As proof of principle, we selected an aptamer having high affinity for the mycotoxin Ochratoxin A (OTA). OTA has been recognized toxic for different organs and its early detection in food and beverages, using a LoC in the production site, reduces risks for human and animal health. The aptamer is immobilized into the PHEMA brushes grown into a polydimethylsiloxane (PDMS) microfluidic channel by atom transfer radical polymerization (ATRP). Afterwards PHEMA is modified in order to immobilize, through the formation of amide bonds, the OTA-aptamer bearing an amino group at the 3’-end. Subsequently, the aptasensors is treated with a solution of [Ru(phen)2(dppz)]2+ as DNA intercalating dye (Fig. 1a), whose fluorescence is poor in aqueous solutions but intense when intercalated into the DNA. The functionalized chip is coupled with amorphous silicon photosensors (Fig 1b). When the food sample is flowed into the channel, OTA molecules, if present, bind the aptamers, which in turn release the intercalated [Ru(phen)2(dppz)]2+

(Fig. 1a). As a consequence, the dye fluorescence and the photosensor current decrease proportionally to the OTA concentration. The LoC has been tested with OTA standard solutions showing sensitivity down to 10 ppb. This result suggests that the developed aptasensors material can be applied into LoCs for a wide range of applications.

Figura 1: a) Schematic representation of [Ru(phen)2(dppz)]2+ DNA intercalating dye upon interaction with OTA and b) LoC system for the detection of OTA.

a b

Abstract

Prototype of an optical sensor for oxygen measurements in oenological matrices

Denis Badoccoa, Nicola Trivellinb, Diego Barbisanc, Angelo Cenedesed and Paolo Pastorea

aUniversity of Padova Dipartimento di Scienze Chimiche, via Marzolo 1 bUniversity of Padova - Department of Information Engineering and LightCube s.r.l., Via Gradenigo 6/B

cLightCube s.r.l. dUniversity of Padova - Department of Information Engineeringe, Via Gradenigo 6/B

Keywords: PtTPP, polysulfone polymer, optical sensor, oxygen and oenological matrices

The control and optimization of oxygen content in wine matrices is becoming more and more important in wine production in order to guarantee its best quality. It is well known that in a first phase O2 is necessary to facilitate the development and activity of yeasts, to favor the combination of anthocyanins and the color stabilization, and to help reducing the astringency of red wines. In a second phase, however, during maturation, O2 can severely deteriorate the organoleptic characteristics of wine. The main oenological practices foresee the addition of remarkable amounts of oxygen and for this reason, instruments to be used in cellars are needed to control the amount of dissolved oxygen in wine in all its production steps.

In this work, a new economic prototype of optical sensor for oxygen measurements in oenological matrices was developed. It is based on the sampling of the light emission of 5,10,15,20-Tetraphenyl-21H,23H-porphyrin platinum (II) (PtTPP) dissolved in a polysulfone polymer membrane. The experimental parameter used for calibrating the sensor is the life-time of the PtTPP luminescence. That parameter was obtained by fitting of the light emission decay profile produced by stimulation of the membrane with short pulses at 390 nm. The membrane guarantees a linear behavior of the Stern-Volmer equation and long-lasting signal stability1,2. Studies on the behavior of the sensor were performed in various environments such as air, water, synthetic wine, and in real red and white wine samples at different temperatures. The sensor was suitably designed to work in food matrices and to optimize the signal to noise ratio, while keeping a cheap cost. The sensor was then tested for a month inside a 500 hL wine stainless-steel tank in the first fermentation phase. In particular, two equal sensors were placed into the wine at 0.5 and 2.5 m of depth, respectively. The oxygen content was measured during this period and resulted lower than 1%.

We thank Smart Future s.r.l. and the project "WOW: DEPLOYMENT OF WSAN TECHNOLOGY FOR MONITORING

OXYGEN IN WINE PRODUCTS" financed by the Veneto Region ex LR 5/2001 - ex LR 9/2007.

REFERENCES

1. D. Badocco, A. Mondin, P. Pastore, S. Voltolina, S. Gross, Anal. Chim. Acta 627, 2008, 239. 2. D. Badocco, P. Pastore, Anal. Chem. 80, 2008, 2091

Abstract

Separation And Characterisation Of Exosomal Subpopulations Through Flow Field-Flow Fractionation

Valentina Marassia, Barbara Rodaa, Andrea Zattonia, Pierluigi Reschigliana

a Department of Chemistry “G. Ciamician”, Via Selmi 2, 40126 Bologna, Italy

Keywords: Exosomes, Hollow-Fiber Flow Field-Flow Fractionation, Characterization, Separation, microvesicles.

In the course of their life span, cells release a multitude of different vesicles in the extracellular matrix, constitutively and/or upon stimulation, for intercellular communication. These micro- and nanovesicles can carry signals either inside or on their membrane. In particular, exosomes are small membrane vesicles (30-100 nm in diameter) secreted by many cell types as a consequence of the fusion of multivesicular late endosomes/lysosomes with the plasma membrane.

However, the precise biological functions of exosomes are not fully understood and are dependent on their cell of origin: it is now believed that they are involved in a wide range of physiological functions such as intercellular communication, membrane exchange between cells, and as an alternative to lysosomal degradation1 2. They exert complex immunomodulatory effects on target cells, acting both as antigen-presenting vesicles and as a vehicle for proteins, coding and non-coding RNA3, and DNA fragments.

In the past few years these extracellular vesicles have gained huge interest from the scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is the accurate size characterization and quantification of exosomes because of the lack of reliable characterization/isolation techniques. Indeed, ultracentrifugation, density gradient centrifugation and cytofluorimetry present many limitations. In our work, we focused on the added value of an FFF-based step to size-separate, characterize, and quantify exosomal subpopulations by hollow fiber flow field-flow fractionation (HF5), coupled to a multidetection system (UV, Fluorescence and MALS). The use of a miniaturized FlFFF device is a key step to avoid dilution while improving performance.

This promising platform provides information about the subpopulations of the exosomal and –more in general- vesicular samples and can be exploited to screen samples from different in-vitro sources, from whole medium to pre-fractionated samples, in order to select the most informative and diagnostic population, and narrow down the particle fractions containing the desired information (DNA/RNA content, marker expression upon labeling). While FFF provides separation, the in-line detectors describe the size, the surface properties and the absorption/emission profile of each fraction; the detected populations can be collected for lipidomic/proteomic characterization.

Within this context, the analytical platform used can achieve 1. A quick screening and characterization of the different vesicular populations in a biological sample and 2. A step of simplification and selection for further biological tests.

REFERENCES

1. F. Prattichizzo, A. Giuliani, V. De Nigris, et al., Diabetes Obes. Metab, 2016, pp 855–867 2. S. Ohno, G.P. Drummen, M. Kuroda, Int. J. Mol. Sci., 27, 2016, pp. 172. 3. Guescini, M., Genedani, S., Stocchi, V. et al. J Neural Transm, 2010, pp 117

Abstract

Effect of Surfactants on the Binding Properties and Selectivity of Molecularly Imprinted Polymers

C.Giovannolia, L.Anfossia, F.Di Nardoa, G.Spanoa and C.Baggiania

aLaboratory of Bioanalytical Chemistry, Department of Chemistry, University of Torino – Via Giuria 5 10125 Torino

Keywords: molecularly imprinted polymer, surfactant, binding properties, selectivity

The artificial receptors known as Molecularly Imprinted Polymers (MIPs) represent one of the most successful type of man-made materials developed to mimic the binding properties of natural receptors like antibodies and enzymes. One of the most significant application concerns the use of MIPs as substitutes of natural antibodies in immunoaffinity extraction, in the so-called Molecularly Imprinted Solid Phase Extraction (MISPE). In this technique, the target analyte contained in the sample is loaded onto an imprinted solid phase, and after a washing step, it is recovered to be quantified. Of consequence, to enhance the MIP’s selectivity and to assure high recoveries of the analyte it is of paramount relevance to minimize non-specific interactions between MIP and the sample matrix. Such non-specific interactions are generally based on weak hydrophobic forces between the surface of the MIP and the less hydrophylic components of the sample matrix. Consequently, additives able to interfere with such interactions should be able to significantly reduce any non-specific binding effect. Among the possible additives, surface active agents represent an interesting classes of substances as they are cheap, easily available and compatible with the organic solvents commonly used in MISPE technique. Here we report a study about the effect of several surfactants on the binding properties and the selectivity of a trichlorophenoxyacetic acid-imprinted polymer. HPLC columns packed with imprinted and non-imprinted polymers were eluted with mixtures of water/acetonitrile (containing acetic acid 2% v/v) added with variable amounts of three different surfactants: the anionic sodium dodecylsulphate (SDS, 0-0.2% w/v), the cationic cetyltrimethylammonium bromide (CTAB, 0-0.2% w/v) and the non-ionic polyoxyethylene-(20)-sorbitan monolaurate (Tween 20, 0-2.5% w/v). The binding ability of the polymers towards the template 2,4,5-T and two related ligands 2,4-D and 4-M was evalued by measuring the capacity factors of these ligands and calculating the normalized imprinting factor: NIF= (kMIP – kNIP) / kMIP) The relative selectivity was measured in the same experimental conditions as: α = (ktemplate – kligand) / kligand

From the experimental results it is possible to observe that surfactants are able to increase the imprinting effect by decreasing the non-specific binding. This effect is less marked with the non-ionic surfactant respect to the two ionic ones. This indicates that surfactants act mainly by inhibiting the ionic pair interaction between the acidic ligands and the basic functional monomer 4-vinylpyridine. About selectivity, all the surfactants show no influence, as any increase of selectivity observed can be directly related only to the amount of water present in the mobile phase.

Abstract

Nanosized materials in amperometric sensing

Chiara Zanardi, Laura Pigani, Fabio Terzi, Barbara Zanfrognini, Stefano Ruggeri, Giulio Maccaferri and Renato Seeber

Dipartimento di Scienze Chimiche e Geologiche, Università di Modena e Reggio Emilia, via G. Campi 103, 41125 Modena

Keywords: electrochemistry, electrochemical sensors, nanostructured surfaces, electrocatalysis.

Nanosized materials are nowadays attracting the interest of the scientific and technological communities. Thanks to peculiar and tunable physico-chemical properties, they are also widely used as electroactive coatings in amperometric sensing.1,2 In particular, the nanosized dimensions allow the formation of nanostructured surfaces showing high electrochemical area and electrocatalytic properties with respect to chemical species of interest. Furthermore, the presence of highly reactive atoms and/or functional groups at edges and vertexes of the nanostructure are at the basis of the peculiar reactivity of the material. The talk aims at discussing the main advantages afforded by the use of nanosized materials in the development of amperometric sensors, as inferred by our most recent activity. In particular, Au nanoparticles (AuNPs) for the detection of carbohydrates in food matrices will be considered. Beside the use of conventional electrode coatings, different solid electrodes3,4 and paper based electrode systems5 will be also taken into account for the stable deposition of AuNPs. The advantages afforded by these last systems for the selective determination of carbohydrate and for the independent determination of glucose and fructose in the same matrix will be discussed.

REFERENCES

1. R. Seeber, F. Terzi and C. Zanardi, Functional materials in amperometric sensing polymeric, inorganic, and nanocomposite materials for modified electrodes. F. Scholz (Ed). Springer-Verlag, Berlin, 2014. 2. F. Terzi and C. Zanardi, Nanosized material in amperometric sensors. In: L. Moretto, K. Kalcher (Eds) Environmental analysis with electrochemical sensors and biosensors. Springer, New York, 2015. 3. J. Rafael Crespo-Rosa, C. Zanardi, M. ElKaoutit, F. Terzi, R. Seeber and I. Naranjo-Rodriguez, Electroanalytical applications of a graphite–Au nanoparticles composite included in a sonogel matrix, Electrochim. Acta 122 (2014) 310-3125. 4. F. Terzi, J. Pelliciari, B. Zanfrognini, L. Pigani, C. Zanardi, R. Seeber, Behaviour of Ti electrode in the amperometric determination of high concentrations of strong oxidising species, Electrochem. Comm. 34 (2013) 138–141. 5. F. Terzi, B. Zanfrognini, S. Ruggeri, N. Dossi, G. Morais Casagrande, E. Piccin, Amperometric paper sensor based on Cu nanoparticles for the determination of carbohydrates Sens. Actuat. B 245 (2017) 352-358.

Abstract

Evaluation of Sample Preparation Methods for the Determination of PBDE in foodstuff by Immuno-assay-based Screening Methods

Ilaria Palchettia, Francesca Bettazzia, Sara Romanellia, Alessandra Cincinellia, Tania Martellinia, Roberta Galarinib, Weilin L. Shelverc

a Dipartimento di Chimica Ugo Schiff, Università degli Studi di Firenze, Sesto Fiorentino (Fi), Italy; b Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Italy; c USDA-ARS Biosciences Research

Laboratory, 1605 Albrecht Boulevard, Fargo, ND 58102, USA; ilaria.palchetti@unifi.it

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants used worldwide in electric and electronic equipment, plastics, and textiles. Because PBDEs are not covalently bound to materials to which they are added, they can make their way into the environment and foods. In order to improve the knowledge about contamination patterns and levels and to evaluate human and wildlife exposure, in March 2014 the European Commission issued a Recommendation in which member states are requested to monitor brominated flame retardants in food. The PBDE analysis is usually carried out by gas chromatography coupled to various mass spectrometric detectors applying the isotope dilution calibration. These techniques permit to achieve low detection limits (1-2 part per trillion) with high selectivity and accuracy, but they require a lengthy procedure, and well-trained personnel. Therefore, this kind of analysis is mainly suitable as confirmatory method, which requires high confidence in the identification and accurate quantitation of analytes even with the associated high costs and low throughput. The purpose of the present study1 was to develop a convenient sample preparation approach combined with immune-assay-based screening methods for the analysis of seafood. Different PBDE extraction approaches were evaluated for mussels (Mytilus galloprovincialis), which are water-filtering organisms and have been often used to detect persistent organic pollutants (POPs) in the marine environment. Mussels are a widely used indicator species, because they possess well-documented feeding habits, stationary condition and are integral parts of the food chain. Moreover, shellfish farming is a common industry along European coasts and mussels are one among the primary types of shellfish consumed in Europe. Traditional methods for food sample preparation are generally labor-intensive, time consuming, and based on organic solvent extraction. In this study, an alternate sample preparation method was evaluated. QuEChERS extraction is a simple and cost saving procedure widely used in pesticide multiresidue analysis and recently reported to be suitable for PBDEs. A simple, cost-effective, rapid sample preparation method (QuEChERS-like extraction followed by solid-phase purification) was optimized and coupled with either colorimetric or electrochemical immunoassays. Several mussel samples collected from Adriatic coast and a Certified Reference Material (2974a - freeze-dried mussels) were tested. The samples were investigated by colorimetric and electrochemical immunoassays as well as by GC-MS. In comparison to GC-MS results, 106 and 102 % relative accuracy were obtained for the colorimetric and electrochemical immunoassays, respectively.

Acknowledgements: This work was supported by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) in the framework of PRIN 2012 (grant no.20128ZZS2H)

REFERENCES

1. S. Romanelli, F. Bettazzi, T. Martellini , W. L. Shelver , A. Cincinelli, R. Galarini, I. Palchetti, Evaluation of a QuEChERS-like extraction approach for the determination of PBDEs in mussels by immuno-assay-based screening methods, Talanta, 2017, 170: 540-545

Abstract

Microextraction techniques coupled to different carbon black based electrochemical detection strategy:

application to carbamates analysis

Flavio Della Pellea, b, Louis Vázquezc, Michele Del Carloa, Manuel Sergia, Alberto Escarpab, Dario Compagnonea

aFaculty of Bioscience and Technology for Food, Agriculture and Environment University of Teramo 64023, Teramo, Italy bDepartment of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental

- , Madrid, Spain cInstitute of Materials Science of Madrid (CSIC) C/Sor Juana Inés de la Cruz Nº 3, Cantoblanco, 28049 Madrid, Spain

Keywords: carbamates, electrochemical, nanomaterials, carbon black, microextraction In this work two strategies for rapid and sensitive determination of phenyl carbamate pesticides in environmental and food samples are proposed. They consists of the following steps: (a) Enrichment and clean-up of the analytes using microextraction devices (µ-SPE and MEPS) based procedure; (b) alkaline hydrolysis of the carbamate to form phenol derivatives; and (c) electrochemical detection (in the microfluidic chip MCs strategy this step is preceded by an analytes rapid separation) exploiting the carbon black nanoparticles (CBNPs) properties. The final aim is the development of two analytical methods: one for the total phenyl carbamates quantification and another one for the carbamates separation and quantitative identification. In the first method the sample has been extracted with a microextraction by packed sorbent (MEPS) and the detection has been performed with a drop casted carbon black modified screen printed electrode. In the second method the sample has been extracted with a C18 microtip (µ-SPE), the separation has been performed in a microfluidic chips and a lab made press-transferred CBNPs transducer as been employed for the electrochemical sensing [1-3]. In both the strategies an efficient and rapid cleanup based on microextraction has been achieved with a good enrichment factor together with a low matrix effect. Furthermore, this extraction strategy gave reduction of the amount of solvent required and possibility of automation of the procedure [1, 4]. In both the approaches, the realized CBNPs-based device (electrode fabricated by drop casting and press transfer) were characterized by electrochemical, spectroscopic and microscopic techniques. CBNPs amount was a critical parameter during the modification/fabrication process and had a deep influence on their electrochemical performance. Furthermore, a good reproducibility (RSDs < 10%, n=5) in the fabrication of CBNP-based transducers was demonstrated. Also in this case carbon black has shown to be a useful nanomaterial for electrochemical sensing, because of the high surface area (increases faradaic currents of electrochemical reactions) and at the same time insignificant fouling of nanomaterial-based electrodes. They also exhibit an apparent electrocatalytic activity lowering the redox potentials of the studied compounds. These results revealed the analytical potential of this carbon nanomaterial in electrochemical sensing. The exploitation of the CB coupled to microfluidics and microextraction procedures for real sample analysis represents a viable tool for the analysis of complex real samples, for on-site environmental monitoring, and for rapid diagnosis.

REFERENCES

1. F. Della Pelle, M. Del Carlo, M. Sergi, D. Compagnone, A Escarpa, Press-transferred carbon black nanoparticles on board of microfluidic chips for rapid and sensitive amperometric determination of phenyl carbamate pesticides in environmental samples, Microchimica Acta, 2016, 183 (12)pp. 3143-3149. 2. F. Della Pelle, L. Vázquez, M. Del Carlo, M. Sergi, D. Compagnone, A Escarpa, Press-Printed Conductive Carbon Black Nanoparticle Films for Molecular Detection at the Microscale, Chemistry - A European Journal, 2016, 22 (36) pp. 12761-12766. 3. F. Della Pelle, R. Di Battista, L. Vázquez, F.J. Palomares, M. Del Carlo, M. Sergi, D. Compagnone, A Escarpa, Press-transferred carbon black nanoparticles for class-selective antioxidant electrochemical detection, Applied Materials Today, 2017, 9 pp. 29-36. 4. F. Di Ottavio, F. Della Pelle, C. Montesano, R. Scarpone, A. Escarpa, D. Compagnone, M. Sergi. Determination of Pesticides in Wheat Flour Using Microextraction on Packed Sorbent Coupled to Ultra-High Performance Liquid Chromatography and Tandem Mass Spectrometry, Food Analytical Methods, 2017, 10 (6) pp. 1699-1708.

Abstract

Environmental Odor Pollution. A GC-MS/O Study with OdorPrep Sampling Approach

Ivano Battaglia,a Enrico Davoli,b Gianluigi de Gennaro,c Alessia Di Gilioc and Jolanda Palmisanic

a LabService Analytica srl, Via Emilia 51/C, Anzola dell'Emilia, 40011 Bologna, Italy, e-mail: bativ@labservice.it b Environmental Health Sciences Department IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri" Via La Masa 19, 20156

Milano, Italy cDepartment of Biology, University of Bari, via Orabona 4, 70126 Bari, Italy

Keywords: OdorPrep, sampling, odor pollution.

Nowadays, odor emissions are an environmental pollution problem, therefore during the last decades scientific activity in this research field has been focused on the identification of the odor active compounds and on their correlation with the human perception [1]. Conventional approaches coupling the sampling on fiber or adsorbent substrates with Gas Chromatography Mass Spectrometry (GC/MS) have been widely developed in order to characterize odorants [2-4]. A notable improvement in odor identification was gained with the development of GC-MS with olfactometric detection (GC-MS/O) that allows to associate the identification and quantification of odorants with their human perception [5,6]. Local regional normatives describe GC/MS approaches for odor characterization [4], while standardized new sampling devices, OdorPrep, are currently under development under EU H2020 R&I programme ref. G.A. nr. 756865. This study shows the potentialities of an innovative methodological approach to identify odor active compounds responsible of odor annoyance coming from different industrial activities such as landfills, wastewater treatment plants and refineries. When the odor nuisance is perceived by population and when the VOCs concentration, measured by a single sensor or a network system, exceed the threshold value the prototype OdorPrep (LabService s.r.l.) starts to collect air in Nalophan bags. The qualitative characterization of samples can then be carried out by AirServer- Thermal Desorber (TD)-GC/MS-O. The application of the aforementioned methodology during the nuisance events allows to overcome the limitations of the conventional approaches related to the lack of instrumental sensitivity and to identify in a more accurate way the chemical compounds contributing to the annoyance. The innovative and automatic monitoring approach, proposed in this study, is proven to be a useful tool to collect real time information about the emission sources and their impacts on the urban settlement. Moreover, the application of the aforementioned methodology during the nuisance events allows to overcome the limitations of the conventional approaches related to the lack of instrumental sensitivity and to identify in a more accurate way the chemical compounds contributing to the annoyance.

REFERENCES

[1] Yuwono, A.S., Lammers, P.S. Int. Agric. Eng. J. 6, 1–33 (2004). [2] Davoli, E., Gangai, M.L., Morselli, L., Tonelli, D. Chemosphere 51, 357–368 (2003). [3] Dincer, F.; Odabasi, M.; Muezzinoglu, A. J. Chromatogr. A, 1122, 222–229 (2006). [4] Regione Lombardia: Linee guida relative alla costruzione e all’esercizio degli impianti di produzione di compost – Revoca della d.g.r.

16 luglio 1999, n. 44263, Allegato D (2003). [5] Friedrich, J.E., Acree, T.E. Int. Dairy J. 8, 235–241(1998). [6] Lo, Y.C.M., Koziel, J.A., Cai, L., Hoff, S.J., Jenks, W.S., Xin, H. J. Environ. Qual. 37, 521–534 (2008). [7] EN 13725: Air Quality—Determination of Odor Concentration by Dynamic Olfactometry; Committee for European Normalization

(CEN), Brussels, Belgium, 2003.

COMUNICAZIONI POSTER SCIENZA DELLE SEPARAZIONI

Abstract

New Generation Of Teicoplanin-Based Zwitterionic, 2 Micron, Superficially Porous, Chiral Stationary Phase: a Study Of Large Library Of N-FMOC

Amino acids Through Ultra-High Performance Chromatography.

Michela Antonelli*,a, Omar H. Ismaila, Alessia Cioglia, Giulia Mazzoccantia, Walter Cabrib, Antonio Riccib, Alberto Cavazzinic, Martina Catanic, David S Belld, Claudio Villania and Francesco Gasparrinia.

aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Roma, Italy

bFresenius Kabi, Via San Leonardo 23, 45010, Villadose (RO), Italy cDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Ferrara, Italy

dSigma-Aldrich/Supelco, 595 North Harrison Road, Bellefonte, PA 16823

*antonellimichela92@gmail.com

Keywords: Zwitterionic Teicoplanin-based CSP, Superficially Porous Particles CSP, UHPC-SPP-Halo-Tzwitt 2.0, Ultra-High Performance Chromatography, N-FMOC-amino acids separations.

An innovative teicoplanin-based zwitterionic Chiral Stationary Phase (CSP) [1] was prepared bonding the selector onto Fused-Core® silica particles (SPPs) Halo 2.0 µm and columns of 150 x 4.6, 100 x 4.6 and 50 x 4.6 mm, L x I.D., were packed. In the first part of work, the UHPC-SPP-Halo-Tzwitt 2.0 µm columns were evaluated through van Deemter analysis under HILIC conditions, allowing very high efficiency values both for achiral and chiral employed probes. The best value, in achiral set-up, was 311 000 theoretical plates/m corresponding to a reduced plate of h= 1.6 (/) for thiourea (k’: 0.6). The main part of work has been focused on the fast separations of a family of N-FMOC derivatized Amino Acids. In the screening of optimal elution conditions, all samples were analyzed in HILIC (ACN/H2O 85:15 + 20 mM HCOONH4) and RP (MeOH/H2O 85:15 + 20 mM HCOONH4) modes. Under HILIC condition, all samples were resolved on the 5-cm long UHPC-SPP-Halo-Tzwitt 2.0 µm column, which provided fast analyses, in most of the cases, under 60 seconds, maintaining a baseline separation. As general observed trend, HILIC mode ensures the best kinetic performances (higher theoretical plate/m) while the RP elution highlights the thermodynamic performance (higher alpha values). In this contest, a remarkable result in N/m was a gain of 23% and 57% for the first and second enantiomer, respectively, of the probe N-FMOC-D,L-Ser-(tBu)-OH, moving from RP to HILIC conditions (first eluted enantiomer: 225 000 plates/m; second eluted enantiomer: 192 000 plates/m). On the other hand, using the RP condition, an increased enantioselectivity of about 40-60% in comparison to that observed in HILIC mode, was achieved: i.e. the α values were 1.80 in RP and 1.21 in HILIC for the same analyte N-FMOC-D,L-Ser-(tBu)-OH. In addition, the RP mode emphasizes also the chemoselectivity of selector. In line with obtained results, the HILIC mode seems to be an enabling solution for fast analyses and, at the same time, good enantioseparations. Whereas, in order to highlight the enantioselectivity/enantioresolution of CSP, as frequently needed in the enantiomeric excess determination, the RP represents the optimal elution condition.

REFERENCES

[1] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, JCA, 1427 (2016), 55–68

Abstract

Characterization of Polypeptide-drug Conjugates in Aqueous Solution: Asymmetric Flow Field-Flow Fractionation Approach

Juan J. Arroyoa, Valentina Marassib, c, María J. Vicenta, Andrea Zattonib, c

aPolymer Therapeutics Lab. Príncipe Felipe Research Institute, Av. Eduardo Primo Yúfera 3, 46012, Valencia, Spain

bDepartment of Chemistry “G. Ciamician”, Via Selmi 2, 40126 Bologna, Italy

cbyFlow Srl, Via Caduti della Via Fani 11/b, 40127 Bologna, Italy

Keywords: Polypeptides, Flow Field-Flow Fractionation, Drug Delivery System, Protein Corona

Polypeptides have accomplished great impact within the field of nanomedicine thanks to their attractive properties for drug delivery, such as their intrinsic biocompatibility, their multivalence, the presence of functional groups in their sequence that provide specific site for direct attachments or the charge-induced interactions with different biomolecules.1 Covalently conjugated drugs or imaging agents introduce new spatial and charged motifs that modify drastically the electrostatic equilibrium of the whole molecule and this has a complex effect on polypeptide size and conformation.2 Among separative techniques for nanodispersed analytes, Asymmetric Field Flow-Field Fractionation (AF4) is increasingly employed as a mature separation method able to size-sort and isolate nanostructures of great diversity of natures.3 When coupled with online uncorrelated detection methods as MALLS, DLS, absorbance and fluorescence spectrophotometry, AF4 offers a multidimensional analytical platform for nanomaterial analysis providing measurements of size, populations distribution according to size and identification of aggregation phenomena.4 Different families of polyglutamate-conjugates have been systematically analyzed by AF4 looking for a direct correlation between its molecular structure and its spectroscopic properties and elution profile. Several relevant effects as the influence of polypeptide chain length, conjugated hydrophobic and/or fluorescent moieties or percentage of modification have been also investigated. Moreover, the formation of protein corona in the presence of blood proteins is one of the most outstanding effect at the nano-bio-interface. So additionally, a selection of the most interesting nanoconjugates are being systematically studied by AF4 under physiological blood serum conditions trying to reveal the formation of this protein complex, the subsequent modification of size and physico-chemical conformation in solution, and how these changes drive biological fate. Understanding the formation of protein corona is a key factor to get feedback for the proper design and engineering of polypeptide-conjugates, searching for the best therapeutic effect.

REFERENCES

1. Duro-Castano, A.; Conejos-Sanchez, I.; Vicent, M. J., Peptide-Based Polymer Therapeutics. Polymers 2014, 6 (2), 515-551. 2. Zagorodko, O.; Arroyo-Crespo, J. J.; Nebot, V. J.; Vicent, M. J., Polypeptide-Based Conjugates as Therapeutics: Opportunities and Challenges. Macromolecular Bioscience 2017, 17 (1), 22. 3. Marassi, V.; Casolari, S.; Roda, B.; Zattoni, A.; Reschiglian, P.; Panzavolta, S.; Tofail, S. A. M.; Ortelli, S.; Delpivo, C.; Blosi, M.; Costa, A. L., Hollow-fiber flow field-flow fractionation and multi-angle light scattering investigation of the size, shape and metal-release of silver nanoparticles in aqueous medium for nano-risk assessment. Journal of Pharmaceutical and Biomedical Analysis 2015, 106, 92-99. 4. Reschiglian, P.; Rambaldi, D. C.; Zattoni, A., Flow field-flow fractionation with multiangle light scattering detection for the analysis and characterization of functional nanoparticles. Analytical and Bioanalytical Chemistry 2011, 399 (1), 197-203.

Abstract

Quality by Design for the optimization of a fast HPLC-DAD method for the analysis of cannabinoids in magistral preparations

Claudia Caprinia, Benedetta Pasquinia, Serena Orlandinia, Roberto Barontic, Massimo Del Bubbab, Sandra Furlanettoa

Department of Chemistry “U. Schiff”, University of Florence, aVia U. Schiff 6, bVia della Lastruccia 3-13, 50019 Sesto Fiorentino, Florence, Italy

c Clinical Toxicology and Anti-Doping Laboratory, Azienda USL Toscana centro Via di San Salvi, 12 - 50135 Florence, Italy

Keywords: Cannabinoids, Cannabis sativa, HPLC, Magistral Preparations, Plant Products, Quality by Design.

Cannabis sativa contains a large variety of different chemical compounds, among which cannabinoids are generally accepted as the main medicinal ingredients. Already more than 70 different cannabinoids have been identified so far, anyway, among the major cannabinoids present in Cannabis sativa Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) are the compounds that possess the most relevant psychoactive properties. A fast and simple HPLC method for the quantitation of these two cannabinoids in magistral oil preparations has been developed and validated following the Quality by Design (QbD) principles (1). Risk assessment and Design of Experiments represent fundamental tools of this new systematic approach. QbD leads to the establishment of the Design Space (DS), namely the multidimensional space of combination and interaction of variables that have been demonstrated to provide quality. A scouting system was set-up to test different types of organic solvents. A mixture of acetonitrile and phosphate buffer led to good selectivity and fast time of analysis. The Critical Quality Attributes (CQAs), representative of the quality of the chromatogram, were resolution and analysis time. The Critical Process Parameters (CPPs) were selected using a fishbone diagram and were related to the characteristics of both the mobile phase and the column: flow, temperature, pH of the mobile phase. In the Response Surface Methodology, a quadratic polynomial model was postulated to link CQAs to CPPs. The models were calculated by means of Doehlert Design and were graphically represented by contour plots. The search for the global optimum zone was performed by the sweet spot plots, which were analysed in order to identify the zones where the multidimensional combinations of the CPPs values allowed the desired values for both the CQAs to be obtained (resolution value ≥0.85, analysis time ≤6 min). The DS was identified by a risk of failure map and was defined on the basis of the calculated models and on the basis of Monte Carlo simulations. The final conditions selected for the analysis were (with the related DS range): chromatographic column, Poroshell® 120 SB-C18 (150 mm x 2.1 mm i.d., particle diameter 2.7 µm, Agilent Technologies); type of organic eluent, acetonitrile/phosphate buffer (75:25 v/v); flow, 0.38 mL min-1 (0.31-0.39 mL min-1); temperature, 53 °C (44-57 °C); pH of the mobile phase, 3.45 (3.10-3.50). The baseline separation of the analytes was obtained in less than 5 min. In order to validate the DS, additional points at the extremities of the DS were tested, selected by a Plackett-Burman matrix, verifying the agreement between measured and predicted values for the CQAs. The same type of matrix was successively used for method robustness testing. Finally, the method was applied to a real sample of magistral oil preparation.

REFERENCES

1. S. Orlandini, S. Pinzauti, S. Furlanetto, Application of quality by design to the development of analytical separation methods, Anal. Bioanal. Chem. 2013, 405:443-450.

Abstract

Teicoplanin-based Zwitterionic Chiral Stationary Phases: Moving From Sub 2-micron Fully Porous Particles To 2-micron Superficially Porous Ones.

What Can We Expect In The Matter Of Performance Gain?

Michela De Martinoa, Omar H. Ismaila, Alessia Cioglia, Michela Antonellia, Simona Fellettib, Martina Catanib, David S Bellc, Alberto Cavazzinib, Claudio Villania, and Francesco Gasparrinia.

aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Roma, Italy

bDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Ferrara, Italy cSigma-Aldrich/Supelco, 595 North Harrison Road, Bellefonte, PA 16823

Keywords: UHPC-FPP-Titan-Tzwitt 1.9, UHPC-SPP-Halo-Tzwitt 2.0-2.7, Ultra-High Performance CSPs, Ultra-fast/Ultra-High performance analysis.

In this study we introduce the first example of teicoplanin-based zwitterionic chiral stationary phases (CSPs) [1] prepared on superficially porous silica particles (SPPs). Two different CSPs were produced by using particles with 2.0 and 2.7µm diameter (fused-core® particles). The synthetic strategy used for the preparation of these CSPs is the same as previously developed for the functionalization of 1.9 µm fully porous particles (FPPs) of narrow particle size distribution (Titan-120 1.9µm) [1,2], which has been proven to be highly reproducible and efficient. Aim of this work is to compare the kinetic and thermodynamic performance of SPP and fully porous particles (FPP) teicoplanin-based zwitterionic CSPs. Columns with a length of 20, 50 and 100 mm, all 4.6 mm ID, were packed in house

with both 2.0 µm SPPs and 1.9 µm FPPs. SPP (2.7 µm) particles were packed into a 1504.6 mm column also. Kinetic performance of columns was evaluated through the analysis of van Deemter curves in HILIC conditions. The thermodynamics of teicoplanin-based CSP was investigated by employing N-derivatized amino acids, aryloxy acids, pharmaceutical and agro-chemical compounds as probes. As expected, in light of the smaller specific surface areas (m2/g) of fused-core particles with respect to FPPs, columns packed with the former kind of material were characterized, in most of the cases, by lower retention factors. On the other hand, selectivity was found to be essentially independent on particle morphology. Concerning resolution/column-length ratio, columns packed with 2.0µm SPPs and 1.9µm FPPs outperformed those made of 2.7 µm SPPs due to improved efficiency.

REFERENCES

1. O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, JCA, 1427 (2016), 55–68 2. O.H. Ismail, M. Antonelli, A. Ciogli, G. Sestito, D.S Bell, A. Cavazzini, C. Villani, F. Gasparrini, JCA, in preparation

Abstract

Profiling the Glucosinolates content of Cauliflower by HPLC-HRMS

Francesca Ferraris, Giorgia La Barbera, Riccardo Zenezini Chiozzi, Aldo Laganà

Dipartimento di Chimica, Sapienza Universita di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy francesca.ferraris@uniroma1.it

Keywords: Glucosinolates, HPLC, HRMS, metabolomics.

Brassicaceae are considered a functional food due to them high content of bioactive compounds, mainly glucosinolates. In fact, several studies point out Brassica species prevent some type of cancers, activity connected to the high concentration of glucosinolates in those vegetables. It is known that Glucosinolates are hydrolyzed by endogenous thioglucosidases, called Myrosinases, to produce a wide range of degradation products (isothiocyanates, nitriles, epithiocyanates, oxazolidine- 2-thiones, and thiocyanates) with diverse biological activities (1,2). Frequent consumption of high-GLS-content vegetables is associated with a lowered risk of cancer and cardiovascular disease. During the processing of these vegetables, an amount of byproducts is produced. Regarding the byproduct proportion, leaves constitute about 50% of the total; the rest is mainly stem. These residues are responsible for important environmental problems in the industries (3) and diminishing their environmental impact has been the subject of an increasing concern in recent years. However, just a few studies has been done on the waist products of cauliflower’s. Nevertheless the waste still represent a good source of nutraceuticals that can further be subjected to isolation processes. The aim of this study is to asses different kind of glucosinolates extraction methods, comparing the 80°C extraction against the RT one. Subsequently the purpose is to set up the best chromatographic/mass spectrometric condition in order to characterize the whole glucosinolates composition of cauliflower by-products.

REFERENCES

1. D. J. Kliebenstein, J. Kroymann, P. Brown, A. Figuth, D. Pedersen, J. Gershenzon, T. Mitchell-Olds, Plant Physiol. 2001, 126: 811. 2. B.A. Halkier, J. Gershenzon, Annu. Rev. Plant Biol. 2006, 57: 303. 3. F. Bonilla, M. Mayen, J. Merida, M. Medina, Food Chem. 1999; 66: 209.

Abstract

Taking The Efficiency Of Chiral Super Critical Fluid Chromatography To The Limit: How To Achieve The Full Potential Of Sub 2-micron

Whelk-O1 Fully Porous Chiral Columns

Omar H. Ismaila, Gioacchino L. Losaccoa, Giulia Mazzoccantia, Alessia Cioglia, Claudio Villania, Alberto Cavazzinib, Martina Catanib, Scott Andersonc, Francesco Gasparrinia

aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Roma, Italy bDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Ferrara, Italy

cRegis Technologies, Inc., 8210 Austin Avenue, Morton Grove, IL 60053, USA

*email : omar.ismail@uniroma1.it

Keywords: UHPC-Whelk-O1-1.8µm; UHPSFC; Instrumental optimization; Reduced extra-column variance.

The full potential of Super-Critical Fluid Chromatography (SFC) towards ultrafast high-efficient chiral separations has been hindered by instrumental limitations, basically the extra-column volume of commercial equipments and the maximum operative flow-rate. On the contrary, the improvement in the technology of chiral particle preparation has led to the production of different kinds of very high efficient sub-2µm particles that often were employed in SFC under their potential due to instrumental constraints. In this work, the kinetic performance of a 1.8µm fully porous Whelk-O1 CSP has been evaluated on an Acquity Waters UPC2: firstly in its standard configuration (Vextra: 70µL) and then after some instrumental modifications (Vextra: 8 µL). Columns of different geometry have been employed, with length of 100, 75 and 50 mm and internal diameter of 4.6 and 3.0 mm. By using trans-stilbene oxide as probe (Eluent: CO2/MeOH 80:20; column: 50x4.6 mm), it was found that with the standard instrumental configuration an efficiency of 248,000 theoretical plates per meter (N/m) at the maximum flow-rate (4.0 mL/min) was observed for the first enantiomer (k’= 0.9). Under these conditions, apparently, the minimum of the van Deemter curve could not be reached. On the other hand, for the second eluted enantiomer (k’= 2.4), roughly 280,000 N/m were obtained at the optimal flow-rate of 3.7 mL/min. The lower efficiency of the first enantiomer in comparison to the second one, can be explained by considering the effect of the extra-column band broadening (vide infra). The same behavior was clearly evident also on the 100x3.0 mm column. In this case, the first enantiomer (k’= 0.8) showed only 129,000 N/m against the 193,000 N/m reached by the second one (k’= 2.1). To reduce the extra-column variance, a modified low-dispersion configuration was realized using a VICI 200 nL external injector, column and pre-column in house external heater, 0.100 mm ID Viper capillaries to connect the column and a 3µL flow cell. The extra-column volume was reduced to 8 µL, with 2.4 µL2 extra-column variance (calculated with the second statistical moment at 2.5 mL/min). Almost 300,000 and 290,000 N/m were reached, respectively for the first and the second enantiomer, with the 50x4.6 mm ID column. Still more significant was the improvement of performance obtained with the 3.0 mm column, where for the first enantiomer an increase of efficiency of almost 90% at 2.0 mL/min was observed.

Abstract

A New PAHs and BC Monitor for Automatic Routine Monitoring of Urban Ambient Aerosol

Francesco Manarinia,b , Mara Russoa, Claudio Carbonec, Enrico Cozzanic, Stefano Zampollid, Antonella Poggic,d, Sandro Fuzzic,e, Stefano Decesarie and Maria Chiara

Pietrograndea,b

aDepartment of Chemical and Pharmaceutical Sciences, Via Fossato di Mortara 17/19 - 44121 Ferrara, bTerra&AcquaTech, University of Ferrara, Via Fossato di Mortara 17/19 - 44121 Ferrara,

cProambiente S.c.r.l., CNR Research Area, Via P. Gobetti, 101 - 40129 Bologna dInstitute of Microelectronics and Microsystems, Italian National Research Council, Via P. Gobetti, 101 - 40129 Bologna eInstitute of Atmospheric Sciences and Climate, Italian National Research Council, Via P. Gobetti, 101 - 40129 Bologna

Keywords: GC/MS, polycyclic aromatic hydrocarbons, urban aerosol sources, Micro-Electro-Mechanical-Systems

This work concerns the development of a low cost, automatic and small-sized instrument to monitor near-real time

particulate black carbon (BC) and polycyclic aromatic hydrocarbons (PAHs) in urban ambient air, as relevant markers of the combustion sources (e.g. traffic, residential heating), exerting relevant effects on global warming and human health.

This new monitor device reflects the most recent innovative trend for air quality monitoring: the strengthening of the traditional monitoring networks with low cost, small-sized and automatic sensors, able to produce high flows of real time data and send them to the administrations or directly to citizens.

The innovative character of the proposed device lies in the Micro-Electro-Mechanical-Systems (MEMs) fabrication technology used to realize the sensing device capable to overcome the high cost of the current sampling procedures and off-line laboratory analyses.

Silicon micro-components with nanoliter volume micro chambers for forced flow are the basis of a portable micro system providing the integration of all the measurement chain for automatic PAHs quantitative analysis: Aerosol Sampling; Thermal Desorption; Gas Chromatography System with photoionization detector (PID).

Here we present the concept of the instrument and some preliminary results obtained on standard mixtures of 16 PAHs of environmental interest at concentrations comparable to real ambient conditions. In addition, some real samples collected during rush hours at Bologna in winter 2017.

In this work Gas Chromatography-Mass Spectrometry analyses were performed as standard method for PAH analysis2 and the equivalence with IPA/BC-Monitor system was evaluated as prerequisite to validate the analytical performance of the chemical methods. This work was supported by the “Programma Operativo Regionale Fondo Europeo Sviluppo Regionale (POR-FESR)” 2014-2020 http://www.regione.emilia-romagna.it/fesr/por-fesr/.

REFERENCES

1. A. Scorzoni, D. Caputo, G. Petrucci, P. Placidi, S. Zampolli, G. de Cesare, M. Tavernelli, A. Nascetti, Sensors and Actuators A-Physical, 2015, 229, pp. 203-210

2. M.C. Pietrogrande, M.G. Perrone, G. Sangiorgi, L. Ferrero, E. Bolzacchini, Talanta 2014, 120, pp. 283–288.

Abstract

Chemo-enantioselective Separation Of Naturally Occurring Cannabinoids Through ICCA Method Under eUHPSFC Conditions

By Using The New eUHPC-Whelk-O1 sub-2 µm CSPs

Giulia Mazzoccanti*,a, Omar H. Ismaila, Ilaria D’Acquaricaa, Michela de Martinoa, Claudio Villania and Francesco Gasparrinia

1Dipartimento di Chimica e Tecnologie del Farmaco - Sapienza Università di Roma, p.le Aldo Moro 5, 00185 Roma, Italy

*giulia.mazzoccanti@uniroma1.it

Keywords: Whelk-O1 sub-2 µm CSPs, naturally occurring cannabinoids, ICCA method, extreme e.e.

Some naturally occurring cannabinoids (or phytocannabinoids) present stereogenic elements, beside cannabigerol (CBG) and cannabinol (CBN) which are achiral. (–)-Cannabidivarin [(–)-CBDV], (–)-cannabidiol [(–)-CBD], (–)-Δ9-trans-tetrahydrocannabinol [(–)-Δ9-trans-THC], and (–)-trans-Δ8-tetrahydrocannabinol [(–)-trans-Δ8-THC], have two chirality centres. Another fascinating chiral phytocannabinoid is cannabichromene (CBC), that presents only one chirality centre, and it is typically reported as a racemate [1,2]. The research on cannabinoids has focused almost exclusively on THC, CBD, CBC, and CBG content, while not investigating the stereochemical feature of such molecules and the relative enantiomeric purity (namely, enantiomeric excess determination). We decided to investigate the composition of different cannabis plant extracts by using chiral stationary phases (CSPs) in enantioselective “e”Ultra High Performance Supercritical Fluid Chromatography (eUHPSFC). Analyses were performed using “e”Ultra High Performance Chromatography eUHPC-Whelk-O1 1.8 µm CSPs [2] developed in our laboratory. The totally synthetic CSP Whelk-O1 allowed to resolve the racemic compound cannabichromene (rac-CBC) and the synthetic racemic (±)-Δ9-tetrahydrocannabinol. Excellent separation in terms of chemo- and enantio-selectivity, with a high resolution was obtained on all cannabinoids and on their acid precursors in isocratic conditions, in less than 12 minutes. The problem we faced was that vegetable extracts are highly enriched and complex mixtures and often the minor enantiomer, or the racemate, are not available as reference samples. Having the possibility of column switching between Whelk-O1 CSPs with opposite configuration, we used the so-called “inverted chirality column approach” (ICCA) [3]. This approach allows to achieve the identification and the accurate quantitation of the minor enantiomer in the trace analysis of natural products (extreme e.e.), and to prove the potential enantioselectivity of the chromatographic system by generating a “simulated virtual racemate” of those natural chiral compounds present as single enantiomer (such as (–)-CBDV and (–)-CBD) . Herein, we successfully applied, for the first time, a genuine ICCA method by using, as the inverted chirality columns, the (S,S)- and (R,R)-Whelk-O1 sub-2 µm CSPs, and under eUHPSFC conditions.

REFERENCES

1. M. ElSohly, et al. Life Sciences, 78 (2005) 539. 2. L. O. Hanuˇs, et al. Nat. Prod. Rep., 33 (2016) 1357. 3. D. Kotoni, et al. Anal. Chem., 84 (2012) 6805. 4. E. Badaloni, et al. Anal.Chem., 79 (2007) 6013.

Abstract

Applications of a Quality-by-Design Approach to the Development of RP-HPLC Methods for the Analysis of Phenolic Compounds in Samples Extracted from Edible Plants and Food of Plant Origin

Isabella Nicolettia, Imre Molnárb and Danilo Corradinia

aNational Research Council, Institute of Chemical Methodologies, Area della Ricerca di Roma 1, Via Salaria Km 29,300, I-00015 Monterotondo, Rome, Italy; bMolnár-Institute for Applied Chromatography, Schneegloeckchenstrasse 47, 10407 Berlin,

Germany

Keywords: RP-HPLC, Quality by Design, plant secondary metabolites, phenolic compounds.

Plant secondary metabolites are organic compounds produced besides the primary biosynthetic and metabolic routes, occurring as “non-nutritive” compounds in edible plants and in food of plant origin. Most of these phytochemicals have found to play important roles in disease prevention and health-promoting effects, in addition to confer specific sensory characteristics. The technique of choice for their qualitative and quantitative analysis is reversed phase high performance liquid chromatography (RP-HPLC) with a variety of detection systems. This poster reports and discusses the results of our recent studies carried out to investigate a variety of factors that influence chromatographic behavior of plant secondary metabolites, mainly phenolic compounds, in RP-HPLC. We have investigated the dependence of the retention behavior of a variety of biomolecules in RP-HPLC on the experimental parameters, such as flow rate, column length and internal diameter, dwell volume, column temperature, isocratic and gradient elution mode, variation of the organic solvent concentration in gradient elution mode (gradient shape and duration). The influence of the considered parameters on the chromatographic behavior of the selected compounds has been evaluated in the framework of solvophobic theory, using a chromatographic modeling software that allows the development of RP-HPLC methods according to a Quality by Design (QbD) criteria, with the result of decreasing the number of experiments requested for method development and increasing flexibility in routine operations. A limited number of experiments have been carried out to generate a Design Space (i.e. a multidimensional combination and interaction of input variables), describing the influence of the mobile phase composition and column temperature on the separation selectivity of mixtures of standard phenolic compounds. It is shown that the resulting three dimensional model allows to evaluate the influence of the selected experimental parameters on the resolution of all components of the standard mixtures, displaying how the simulated chromatograms looks like for any set of conditions within the three dimensional model. Practical applications of the investigated approach to the analysis of secondary metabolites in samples extracted from edible plants and processed food of plant origin are then discussed.

Abstract

Dispersive Liquid-Liquid (Micro)Extraction Followed by GC-MS/IT Analysis for Determining Phthalates in Alcoholic and Non-

Alcoholic Beverages

Ivan Notardonatoa, Mario Vincenzo Russoa and Pasquale Avinoa,b

aDiAAA, University of Molise, via De Sanctis, 86100 Campobasso bDIT, INAIL, via Roberto Ferruzzi 38/40, 00133 Roma

Keywords: Phthalates; Contaminants; Endocrine Disruptors; DLLME; Extraction; GC-MS.

Phthalates (PAEs) [1] are chemical plasticizers that have been widely used since the 1950s to soften plastics that would otherwise be brittle and crack when bent: nowadays, they are largely used in polymers to enhance the flexibility and extensibility of the materials. From a chemical point of view phthalates are compounds primarily used to make polyvinyl chloride (PVC) or vinyl flexible and pliant: for this reason they are plasticizers often used in the production of many types of plastics, certain inks, paints, and other products. Worldwide production of PAEs is approximately 6 million tons per year: with a wide range of physical and chemical properties, phthalates are used in a multitude of consumer and industrial products that demand high performance, long-lasting wear and durability [2]. They can be employed in a variety of applications even if the phthalates are not necessarily interchangeable. Because these compounds are reported to act as endocrine disruptors, and exposure to high levels can cause harmful effects in the human reproductive system, their determination in food and beverages is fundamental. The PAEs levels in foods are highly variable and suggest contamination events, which may occur in the source or original collection of the food, in the packaging or processing prior to market, or in the preparation leading to consumption. Phthalates, especially DEHP, have been reported at high concentrations in some foods. Additional evidences that food and food packaging are important sources of DEHP contamination come from observational human epidemiologic and panel studies. The beverage matrix is very complex: in fact, it ranges from baby beverages (e.g., fruit juices) and milk to soft drinks (e.g., soda, tonic water, cola) and drinks at very different alcoholic grade (this varies between 3-55 alc vol-1 in popular drinks such as beer, wine, whisky, vodka). Among the different and traditional methods used for this determination, this group has developed a long experience in extraction and determination of toxicologically interesting compounds. The same group studied extensively the SPE application with XAD-2 or a Dispersive Liquid Liquid (Micro)Extraction (DLLME) method and relative analysis by means of GC-MS. The DLLME approach reduces the volume of organic solvent required and simultaneously improves the extraction efficiency without the use of a dispersive solvent. This communication reviews the application of the dispersive extraction method in alcoholic matrices (beer, wine and liquors with a alcohol content ranges between 4 and 40 % alcohol by volume) and soft drinks (soda, cola, tonic, sprite, 7up), basically all the beverages. Use of a vortex is essential for dispersing the extracting solvent. The authors have investigated and optimized different parameters, like the extraction solvents, the number of repeated extraction steps and the isolation technique. Using the optimum analytical condition, very good LODs (≥ 0.022 µg L-1) and LOQs (≥ 0.075 µg L-1) are achieved with recoveries ranging between 85 % and 100.5 % and enrichment factors from 220-to 300-fold.

REFERENCES

1. M.V. Russo, P. Avino, L. Perugini and I. Notardonato, Extraction and GC-MS analysis of phthalate esters in food matrices: a review, RCS Adv. 2015, 5:37023-37043.

2. M.V. Russo and P. Avino, A Critical Review on the Chromatographic Detection of Phthalates in Different Matrices: Problems and Solutions, New York: Nova Science Publishers, 2015, pp. 29-60.

Abstract

Study On New Psychoactive Substance (NPS) Metabolite Pathway By Means Of UHPLC-HRMS: The Case Of MT-45

Camilla Montesanoa, Gabriele Vannutellia, Federico Fantia, Flaminia Vincentia, Roberta Curinia, Adolfo Gregorib, Anna Rita Tognac, Matteo Martid, Isabella Canazzad, Manuel

Sergie

aSapienza University of Rome, Department of Chemistry, 00185 Rome, Italy; bCarabinieri, Department of Scientific Investigation (RIS), 00191 Rome, Italy;

cSapienza University of Rome, Department of Physiology and Pharmacology Vittorio Erspamer, 00185 Rome, Italy; dUniversity of Ferrara, Department of Life Sciences and Biotechnology (SVeB), 44100 Ferrara, Italy;

eUniversity of Teramo, Faculty of Bioscience and Technology for Food, Agriculture and Environment, 64100 Teramo, Italy

Keywords: MT-45, LC–HRMS, metabolites, in silico prediction, in vivo studies

The use of new psychoactive substances (NPS), associated with dissociate mental states and psychedelic sensations, has been largely reported and monitored worldwide since the late nineties (1). However, because of the lack of legal restrictions to their marketing, these drugs are easily available. Pharmacological effects of many NPS are still unknown and the difficulty in detecting the parent compound in urine highlights the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. The aim of this work was to ascertain the chemical structures of the metabolites of MT-45, in order to allow further pharmacological studies and to better assess the health implications of the abuse of this substance (2). MT-45 is a synthetic opioid firstly developed in the early 1970s by the Japanese company Dainippon Pharmaceutical Co. Ltd, it has a pharmacological activity comparable to morphine and it has been involved in intoxications and fatalities reported in Europe and in USA (3). It was recently subjected to control measures, but until now, the metabolic pathways of the substance were still unknown (4). MT-45 metabolites were firstly predicted in silico, then the drug was incubated with rat hepatocytes and the obtained metabolites were identified by LC–HRMS, with retention times, mass shift between theoretical mass and observed mass (<10 ppm), peak abundance and fragmentation pattern. The presence of all metabolites was confirmed by in vivo experiments in urine samples of CD-1 male mice; in these samples, hydroxy-MT-45 glucuronide and di-hydroxy-MT-45 glucuronide are the most abundant metabolites. In this work using rat hepatocytes and LC–HRMS, 14 novel phase I and II MT-45 metabolites were identified: products of monohydroxylation, dihydroxylation, and N-dealkylation; glucuronide conjugation of mono- and dihydroxylated metabolites. The knowledge of phase I and II MT-45 metabolite structure is then crucial to develop analytical methods to identify MT-45 consumption in clinical and forensic testing.

REFERENCES

1. M. P. Brown and K. Austin, The New Physique, Publisher City: Publisher Name, 2005, pp. 25-30. 1. L.A. King and A.T. Kicman, A brief history of ‘new psychoactive substances’, Chichester, Drug Testing and Analysis, 2010, pp. 401-403. 2. D. Papsun, A. Krywanczyk, J.C. Vose, E.A. Bundock, B.K. Logan, Journal of Analytical Toxicology, 2016, pp. 313-317. 3. European Monitoring Centre for Drugs and Drugs Addiction, 2015, Avaible at

http://www.emcdda.europa.eu/attachements.cfm/att_233323_EN_MT-45%20Risk%20Assessment%20Report.pdf. 4. World Health Organization, 2015, Avaible at http://www.who.int/medicines/access/controlled-substances/5.1_MT-45_PR1.pdf?ua=1

Abstract

Photocatalytic Degradation of Contaminants in Aqueous Matrix by Heterogeneous Sodium Decatungstate

Claudia Stevanin1, Alessandra Molinari1, Nicola Marchetti1, Luisa Pasti1 Laboratorio Terra&AcquaTech

1University of Ferrara, Department of Chemistry and Pharmaceutical Sciences, via L. Borsari, 46, Ferrara claudia.stevanin@unife.it

Keywords: Environmental contaminants, Hydroxyl radicals, Photocatalysis, Decatungstate anion

In view of the rising world industrialization paralleled by the increment of world population, the re-use of wastewaters will be necessarily an increasing practice, bringing potential health risks due to the presence of highly toxic organic pollutants. In particular, the contaminants of emerging concerns (CECs), which include pharmaceuticals, were proved challenging for conventional wastewaters and recycled water depuration treatments, which are only partially effective. This work is focused on photocatalytic method for removal and photodegradation of organic contaminants from water. The photoactive species is the decatungstate anion, having light absorption properties similar to those of TiO2. An important advantage in the use of the polyoxoanion with respect to TiO2 is that it can be immobilized on different solid supports with tunable and desired characteristics, such as hydrophobicity, pore size and distribution, surface areas, etc. Generally, a good compromise between support and drug characteristics can be reached in order to warrant an efficient approach of the organic molecule to the immobilized photoactive species and therefore increase the efficiency of degradation process. The target molecules were selected for being some of the most representative contaminants: atenolol and propranolol (b-blockers), levofloxacin, trimethoprim and sulphametoxazol (antibiotics), carbamazepine (anti-depressant). Pharmaceuticals are contaminants of emerging concern (CECs) frequently detected at low concentrations in natural water and can cause adverse effect in biota, such as antibiotic resistance. Therefore, their removal efficiency from wastewaters must be improved in order to reduce their introduction in the environment. The main aim of this work is twice: i) exploitation of the adsorption ability of the solid support that transfers the pollutant from water to a new solid phase that contains also the photocatalyst; ii) estimation of the degradation efficiency of the pollutant concentrated in the solid matrix by the photocatalytic activity of decatungstate. Different kinds of solid supports have been employed: silica particles modified with aminopropyl-silane groups and microporous silicas, ion-exchange resins. The photodegradation process is studied at pH values similar to that of natural waters. EPR spin trapping technique points out that also immobilized decatungstate suspended in water is able to photo-generate OH• radicals, which start the degradative pathway. The degradation by-products were investigated by HPLC-MS that gives evidence that degradation process is mediated by OH• radicals. Heterogeneous photocatalysts are stable and can be recycled several times without a significant loss of efficiency, thus opening the possibility of developing new solid materials with interesting photocatalytic performance.

Abstract

Liquid Chromatography–tandem Mass Spectrometry Method For The Determination Of Vitamin K Homologues In Human Milk After Overnight Cold Saponification

Alessandra Gentilia, Alfredo Micchelia, Pierpaolo Tomaia, Maria Elisabetta Baldassarreb, Roberta Curinia and Virginia Pèrez-Fernàndeza

a Department of Chemistry, Faculty of Mathematical, Physical and Natural Science, “Sapienza” University of Rome, Piazzale Aldo Moro 5, P.O. Box 34, Posta 62, 00185 Rome, Italy

b Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Piazza Giulio Cesare 11, 70124 Bari,

Italy

Keywords: Vitamin K; Phylloquinone; Menaquinone-4; Menquinone-7; Vitamin speciation; Human milk; Overnight cold saponification; HPLC–MS; Food composition; Food analysis.

Human milk is the only source of vitamin K for exclusively breastfed neonates. This vitamin is crucial both for blood coagulation (vitamin K1) and for the normal neurological and skeletal development of the foetus and newborn (vitamin K2). Since vitamin K is ubiquitous in foods, deficiency is not common in adults, but at birth the plasma levels of this vitamin are low due to the limited placental transfer and hepatic storage capacity of the foetus. In light of the importance of this valuable micronutrient, a non-invasive method for verifying that exclusively breastfed infants are receiving an adequate supply of the vitamin is clearly a topic of great significance. In spite of this, the determination of the several vitamin K homologues in human milk has still not been completely elucidated. This work presents an HPLC–MS/MS method for the simultaneous determination of the main forms of vitamin K (K1, MK-4, and MK-7) in human milk using a simple, rapid and inexpensive extraction procedure. Overnight cold saponification and extraction of the analytes with hexane provided yields above 75%. This procedure, combined with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), made it possible to achieve limits of detection (LODs) below 0.8 ng/mL. After a complete validation study, the method was applied to measure vitamin K congeners in several human milk samples; results showed vitamin K1 concentrations comparable with those reported in the literature. In addition, this is the first study performed for the determination of MK-4 and MK-7 in the maternal milk of Italian women.

COMUNICAZIONI POSTER BIOANALITICA

Abstract

Application of Bioanalytical Techniques in the Investigation of Binding Sites in COT1, a Zinc Transporter in Candida Albicans

Denise Bellotti,a Magdalena Rowińska-Żyrekb and Maurizio Remellia

a Department of Chemical and Pharmaceutical Sciences, University of Ferrara, via L. Borsari 46, 44121 Ferrara, Italy; e-mail: rmm@unife.it

a Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wrocław, Poland

Keywords: Analytical Chemistry, Mass Spectrometry, Drug Development, Bioanalytical, Spectroscopy, COT1, Zinc.

The development of innovative and performing analytical techniques is crucial in order to better understand the role of xenobiotics and biotics in biological systems. The study of interesting biomolecules is nowadays a key issue that connects different scientific fields, proceeding through medicine, physics, chemistry, engineering and even mathematics and represents the most powerful mean to find and design effective, pathogen-specific therapeutics.

In recent years, bacteria and fungi have developed alarming drug resistance attitudes, becoming extremely dangerous for the patients’ subsistence. In particular, the dramatic increase of invasive mycoses, due to opportunistic fungal pathogens [1], represents both a serious threat and a challenging scientific issue.

One highly strict dominating factor appoints the biggest obstacle to find efficient pathogen-specific drugs that will not produce unwanted side-effects in patients: both fungi and humans share essential metabolic pathways, as they belong to Eukaryota domain. In order to develop a highly specific antifungal drug, it is crucial to focus on differences between human and pathogen metabolism, such as the transport system of zinc.

In our work, we focus on the zinc uptake and vacuole storage controlled by COT1, a transmembrane protein composed of 199 amino acid residues, located in the vacuolar, mitochondrial and cell membrane. Its main function is the transport of cobalt and zinc ions and its structure remains unsolved [2]. We used Phyre2 [3] to simulate and analyze the predicted, highly probable structure. It occurs that the most probable Zn(II) binding sites of COT1 are located at the C-terminal region, in the extracellular, or in the case of vacuolar transporter, in cytoplasmic part of the protein. We also decided to investigate Cu(II) binding, since Cu(II) is a good reference metal for Zn(II) as well as a necessary nutrient for C. Albicans, a fungal pathogen containing COT1.

In the present work, the binding affinity of Zn(II) and Cu(II) towards three peptides (Ac-FHEHGHSHSHGSGGGGGG-NH2, Ac-SHSHSHSHS-NH2 and Ac-FHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS -NH2), models for the COT1 metal binding sites, were characterized exploiting different analytical techniques.

High-resolution mass spectra were obtained using a time-of-flight mass analyzer (TOF) with an electrospray ionization (ESI) source spectrometer. ESI-TOFs are popular in bioanalytical chemistry, since these kinds of instruments can be routinely used to get accurate mass measurements of small molecules, peptides or even intact proteins. Furthermore, mass spectra provide useful information about stoichiometry of complexes at a given pH. Stability constants for proton, Zn(II) and Cu(II) complexes were calculated from pH/metric titrations. Finally, spectroscopic techniques, such as UV-Vis, Circular Dichroism and Electron Paramagnetic Resonance, provided information about structure and coordination modes of the investigated peptides.

REFERENCES

[1] P. Carver et al., Ann. Pharmacother. 2015, 49825-49837. [2] D. Conklin, J. McMaster, M. Culbertson, C. Kung, Mol. Cell Biol., 1992, 12(9), 3678- 3688. [3] L. A. Kelley et al., Nature Protocols, 2015, 10, 845-858.

Abstract

Sustainable recovery of antocyanins from red grape pomace and assessment of their antioxidant, anti-inflammatory and

antitumoral activities with predictive human cell-based bioassays

Caliceti Ca b, Calabria Db, Guardigli Ma, b, Porru Ea, Tacchini Mc, Bernardi T c, Massi Ad, Sacchetti Gc, Mirasoli Ma b and Roda Aa,b

a Dipartimento di Chimica "Giacomo Ciamician", Alma Mater Studiorum, Università di Bologna, Bologna, Italia e Istituto Nazionale Biostrutture e Biosistemi (INBB), Roma, Italia. b Centro Interdipartimentale di Ricerca Industriale Energia e Ambiente, Alma Mater Studiorum, Università di Bologna, Bologna, Italia. c Dipartimento di Scienze della Vita e Biotecnologie, Università di Ferrara, Ferrara, Italia. d Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Ferrara, Italia.

Keywords: red grape pomace; antocyanins, human cell- based biosensors; safety; biological effects.

Recently, there has been great social and environmental interest for the efficient reuse of agricultural industry residues. In fact, agriculture and food processing industry generate a huge amount of waste streams, as well as co- and by-products, that are often not properly taken care of, both in environmental and economic terms. The present work aimed at the recovery and characterization of polyphenolic compounds extracted from red grape pomace (Vitis vinifera L.), a winemaking by-product. Polyphenolic compounds of dried red pomace were recovered by different extraction methods (ultrasonic-assisted extraction UAE, Naviglio®-assisted extraction NAV and supercritical fluids technique SFE). A significantly amount of anthocyanins, above all malvidin-3-O-glucoside, was recovered from dried red pomace with UAE and NAV extractions. The best extracts contained high amounts of anthocyanins were explored to determine antioxidant and anti-cancer activities. We developed an in vitro chemiluminescent bioassay to determine xanthine oxidase (XO) activity, one of the major sources of ROS in living endothelial cells. Intracellular XO activity was measured in 5 × 103 human endothelial cells in less than 20 min with a luminol/catalyst-based chemiluminescence assay with a limit of detection of 0.4 μU/mL. Moreover, the IC50 value of oxypurinol, the active metabolite of the standard inhibitor drug allopurinol was determined; oxypurinol addition (ranging from 5.0 to 0.5 μM) caused a linear decrease in XO activity, with an IC50 of 1.0 ± 0.5 μM (1). The extracts derived from UAE and NAV were measured (ranging from 10 to 0.0 ug/ml), obtaining an IC50 of 0,86±0,07 µg/mL and 1,27±0,08 µg/mL, respectively. Then, we investigated the antitumoral properties of both extracts in two highly predictive human cell models, leukemia and breast cancer, using a spectrophotometric approach with a new tetrazolium salt (WST-8) in respect to a standard cell counting method. WST-8 is a highly soluble tetrazolium salt in water, reduced to yellow formazan by the activity of intracellular dehydrogenase. The amount of formazan generated in the cells is directly proportional to the number of living cells (λmax450 nm). After 72h of incubation, UAE and NAV extracts (ranging from 50 to 0,1 µg/mL) exhibited a GI50 (50% growth inhibition) in human breast cancer cells of 28.2 ± 1.2 µg/mL and 16.1 ± 0.9 µg/mL, respectively, while in leukemia cells 6.9 ± 0.1 µg/mL and 6.9 ± 0.1 µg/mL and 4.0 ± 0.2 µg/mL, respectively. The doxorubicin antineoplastic drug (ranging from 10 to 0,1 μM) was used as standard, showing a GI50 of 0.13 ± 0.07 µM in breast cancer cells and 0.76 ± 0.09 µM in leukemia cells. The results support the possibility of exploiting the extracts coming from grape processing by-products as ingredients for functional and innovative products in the nutraceutical, pharmaceutical or cosmetic fields. POR FESR 14-20 Asse 1, Azione 1.2.2 - Progetto VALSOVIT (CUP F72I16000010009)

REFERENCES

1. C. Caliceti, D. Calabria and A. Roda, Analytical and Bioanalytical Chemistry, 2016;408(30):8755-8760.

Abstract

Magnetic Solid Phase Extraction Followed by Liquid Chromatography - Tandem Mass Spectrometry for UV Filter

Determination in Surface Water

Chiara Cavaliere, Francesca Ferraris, Carmela Maria Montone, Susy Piovesana, and Aldo Laganà

Dipartimento di Chimica, Università di Roma “La Sapienza”, p.le Aldo Moro 5, 00178 Rome

Keywords: dispersive solid phase extraction, LC-MS/MS, magnetic graphitized carbon black, UV filters, water samples.

UV filters are ingredients of personal care products, such as sunscreens, cosmetics, shampoos etc., to protect skin and hair from the effects of UV sunlight. Furthermore, some UV absorbing substances are also present in several plastic products to prevent yellowing and degradation of polymers and pigments [1]. Due to their widespread use, UV filters have been found in environmental waters [2] and biota [3]. According to Regulation 1223/2009 of the European Parliament, 25 chemical UV filters, including benzimidazole, p-aminobenzoic acid and derivatives, dibenzoylmethane derivatives, cinnamates, crylenes, salicylates, triazines, benzophenones, camphor derivatives, are allowed, within certain limits, in cosmetic products. Benzophenones have been detected at μg L-1 levels in some surface water samples, and have shown developmental toxicity and estrogenic or anti-androgenic activity in animal models [4]. Generally, gas chromatography and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) are used for the simultaneous analysis of these compounds and their transformation products. Regarding the extraction from aqueous samples, the classical solid phase extraction (SPE) is still the most widespread technique [4]. Among the new extraction techniques, magnetic (m)SPE represent a very attractive alternative. The main advantage of magnetic materials over classical sorbents is that the separation process can be carried out directly in sample solution, from where the magnetic material can be collected and separated using a magnetic field, thus eliminating tedious filtration or centrifugation [5]. Magnetic particles are usually grafted or coated with organic or inorganic layers, in order to enhance their stability and selectivity towards the target analytes. In the present work, we prepared and characterized a new magnetic material based on graphitized carbon black (mGCB) for the extraction of UV filters from surface water samples. After magnetization, GCB surface area was reduced to 55.0 ± 0.5 m2 g-1 and the pore volume to 0.30 cm3 g-1 after magnetization; the structure was still mesoporous and the Fe3O4 content was 55%. After a screening test of recovery (RE) and matrix effect (ME) of fifty compounds, ten UV filters belonging to the chemical classes of benzophenones (seven) and p-aminobenzoic acids (three) were selected, and a method for determining these compounds in surface water by mGCB extraction followed by LC-MS/MS analysis was validated and applied to water samples collected in the area of Rome (Italy) from January to February 2016, from the Bracciano lake (four samples) and Tiber river (five samples). Handling fifty mL of water sample, REs >82% and MEs ranging 82-115% were obtained. To the best of our knowledge, mGCB stable in aqueous medium has never been prepared, characterized and used to extract pollutants from environmental waters.

REFERENCES 1. A P. Gago-Ferrero, M.S. Díaz-Cruz, D. Barceló, Liquid chromatography-tandem mass spectrometry for the multi-residue analysis

of organic UV filters and their transformation products in the aquatic environment, Anal Method. 2013, 5:355–366. 2. S.D. Richardson, T.A. Ternes, Water Analysis: Emerging Contaminants and Current Issues, Anal. Chem. 2014, 86:2813–2848. 3. P. Gago-Ferrero, M.S. Díaz-Cruz, D. Barceló, An overview of UV-absorbing compounds (organic UV filters) in aquatic biota ,

Anal. Bioanal. Chem. 2012, 404:2597-2610.D 4. S. Ramos, V. Homem, A. Alves, L. Santos, Advances in analytical methods and occurrence of organic UV-filters in the

environment - A review, Sci. Total Environm. 2015, 526:278–311. 5. A. Ríos, M. Zougagh, M. Bouri, Magnetic (nano)materials as an useful tool for sample preparation in analytical methods. A

review, Anal. Methods 2013, 5:4558–4573.

Abstract

A Novel, Rapid Response Bioluminescent Yeast Bioreporter Assay For Endocrine Disruptors

L. Cevenini1, A. Lopreside1, M.M. Calabretta1, E. Michelini1,2, A. Roda1,2.

1Department of Chemistry “G. Ciamician”, University of Bologna Via Selmi, 2, Bologna, Italy 2INBB, Istituto Nazionale di Biostrutture e Biosistemi, Viale Medaglie d'Oro 305, Roma, Italy.

Keywords: Bioluminescence, Whole-cell biosensors, Endocrine disruptors.

Living cells (bacteria, yeast and mammalian cell lines) have been widely exploited for biosensing as they provide useful information about the bioavailability of target analyte (1). Nonetheless, one of the main drawback of cell-biosensors based on reporter gene technology is the long assay time (typical several hours) due to the slow production of the reporter enzyme upon induction and its accumulation to obtain a sensitive detection.

Here we report the development of a novel bioluminescent yeast-based whole-cell biosensor which provide a fast (15 min) and sensitive detection of estrogenic compounds, in 96-well microplate format.

In particular, S.cerevisiae cells were genetically engineered to express the human estrogen receptor (hER) and the Nanoluc luciferase in response to compounds able to activate the estrogen signalling pathway. Nanoluc luciferase was used as BL reporter since it represents the brightest luciferase available to date (150-fold brighter than firefly luciferase), emitting at 460 nm, using the coelenterazine analog furimazine as BL substrate (2). In addition, its small size (only 19KDa) and the absence of post-translational modifications and disulphide bonds, enable rapid synthesis and folding of the reporter thus reducing response time. The Nanoluc luciferase coding sequence was further optimized to increase its expression efficiency in yeast cells, and the use of a destabilized variant, allowed to reduce the background expression obtaining a rapid response upon induction by target analyte.

The estrogen bioassay is performed in 96-well plates by incubating 90µL of yeast culture (1,5x106 cells/well) with 10µL of sample at room temperature (25°C) for 15 minutes, (compared with 2.5h of previously published yeast estrogen assays) making it the fastest BL yeast biosensor developed to date. Luminescence measurements are then performed with a luminometer without washing steps, by simple addition of 50µL of a custom BL substrate containing 10µM furimazine in a yeast-specific lysis buffer (Y-PER), optimized to provide a glow-type emission.

The developed yeast bioreporters respond to 17β-estradiol (selected as model analyte) with a limit of detection of 0.03 nM and an EC50 of 1.5 nM, with just 15 min incubation time, making it the fastest BL yeast biosensor developed to date. The biosensor response to estrogenic compounds and endocrine disruptors (e.g. 17α-ethynylestradiol, estrone, diethylstilbestrol, bisphenol A) is reported. In the perspective to apply the developed yeast bioreporters for actual on-field analysis, we also integrated yeast cells with a smartphone-controlled GoPro Hero5 camera as light sensor. To this end a 3D printed adaptor was fabricated to hold the GoPro camera and to house a custom 3D printed multi-well cartridge containing immobilized yeast bioreporters. This configuration combines the use of standardized sensitive detector with smartphone functionalities (i.e. connectivity, data elaboration, geo-tagging and the possibility to develop custom App), resulting in a versatile, user-friendly biosensing platform. Preliminary results obtained with the integrated device are presented.

REFERENCES

1. L. Cevenini, M.M. Calabretta, A. Lopreside, G. Tarantino, A. Tassoni, M. Ferri, A. Roda, E. Michelini. Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity. Anal Bioanal Chem. 2016, 408:8859-8868.

2. C.G. England, E.B. Ehlerding, W. Cai. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjug Chem. 2016.27:1175-87.

Abstract

A Color Encoded Strip Test for Multiplexed Analysis

Fabio Di Nardoa, Laura Anfossia, Cristina Giovannolia, Giulia Spanoa, Claudio Baggiania

aLaboratory of Bioanalytical Chemistry, University of Torino, Via Pietro Giuria 5, 10125 - Torino

Keywords: Multiplex strip test – Multicolor gold Nanoparticles – Color encoded analysis.

In the last decades, the Immunochromatographic Strip Test (ICST), also known as Lateral Flow Immunoassay, has emerged as a leading technique in point-of-need tests. The eminent role of ICSTs is due to the their simplicity, rapidness and low cost, which explain their great commercial success. Nowadays, the new trend in ICST technique is the multiplexing, i.e. the simultaneous detection of more than one analyte in a single assay. Multiplexing capacity is critical for improving the efficiency of testing while reducing its cost and it represents a hot topic in ICST. In a recent work we developed a multicolor ICST based on gold Nanoparticles (GNPs) for the simultaneous determination of aflatoxin B1 and fumonisins in maize flour samples1. We exploited the tunable surface plasmon resonance band of GNPs to obtain two different colored label (red and blue). The multiplex ICST was obtained by dispensing two Test lines on the nitrocellulose membrane and conjugating the multicolor GNPs to polyclonal antibodies that bind to specific targets, and thus use the resulting GNPs color to distinguish between two mycotoxins. The use of several Test lines on the nitrocellulose membrane is the most direct way to develop a multiplexed ICST. However, this strategy has some limitations when the number of lines increases, e.g. the enlargement of the device and the delay in which the liquid sample reaches the different Test lines through the capillary forces. To minimize these limitations, we applied a different strategy in order to obtain the multiplexed detection in a single test line, which can facilitate miniaturization by increasing the number of targeted antigens in a given strip. Moreover, this detection configuration could reduce strip test dimensions and simplify device design, potentially reducing material costs. We dispensed a single Test line with a mixture of two different antigens and we employed the previously mentioned multicolored GNPs as labels. In this way we obtained a color encoded strip test, in which the Test line can assume different coloring resulting from the combination of red and blue GNPs depending whether one analyte or both are present in the sample. The color encoded multiplex capability of the assay was illustrated using the simultaneous detection of aflatoxin B1 and fumonisins as model system and the results will be presented in this communication.

REFERENCES

1. F. Di Nardo, C. Baggiani, C. Giovannoli, G. Spano, L. Anfossi, Microchimica Acta, 184 (2017), 1295-1304.

Abstract

Amperometric Genosensor Based on PNA Probes Implemented on Carbon Nanotubes-Modified Screen Printed Electrodes

Simone Fortunatia, Marco Giannetto, Andrea Rozzi, Monica Mattarozzi, Alex Manicardi, Roberto Corradini, Maria Careri

aDipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma

Keywords: Genosensors, PNA, Carbon Nanotubes, Transgenic Soy Nucleic acid-based biosensors (genosensors) have received great attention in the past decade, nucleic acids being promising molecular probes due to the ease of functional modification, the specificity for base pairing, and the predictability for intermolecular or intramolecular interactions. In a research program dealing with the development of innovative sensors as powerful analytical tools for assessing food safety (1,2), we combined performance of DNA-mimic probes based on Peptide Nucleic Acids (PNA) with the enhancing properties of carbon nanotubes (CNTs) as binding substrates on carbon screen printed electrodes (CSPEs). The findings of our previous studies (3), focused on the use of the same PNA probes on all-gold SPEs, evidenced best results using a non-competitive approach, based on the binding of a target DNA ascribable to “Roundup-Ready (RR)” transgenic soy by a PNA-Capture Probe (CP)-functionalized sensor, followed by the hybridization with a properly synthesized PNA-Reporter Probe (RP). The latter bears a biotin tag, capable of strong interactions with a streptavidin-alkaline phosphatase conjugate, which converts a substrate into an electroactive species. In this work, we experimented the use of CNT-modified electrodic platforms in order to reach higher loading capability of CP, if compared to gold substrates combined with Self-Assembled Monolayers from mercaptoundecanoic acid as linkers. Another crucial aspect deals with the nature of the RP, since we noticed the formation of CP/Target-DNA/RP adduct when using PNAs both for capture and reporter probes. To overcome this limit, we moved to DNA-based RPs with the same oligonucleotidic sequence, obtaining encouraging results in terms of signal inhibition associated to hybridation of the target DNA up to nanomolar scale. Further studies currently ongoing are focused on the comparison of such inhibitive approach with the use of a sandwich-type assay based on the use of a longer DNA target, showing hetero-complementarity with CP and RP. Further improvements in terms of sensitivity enhancement could be also reached by conjugation of RPs with molecular nanomaterials such as dendrimers. The performance of polyamidoamine (PAMAM) dendrimers were already demonstrated in our previous studies concerning the development of competitive (4) and noncompetitive (5) immunosensors. These strategies will be further investigated to optimize the DNA assay protocols aimed at specific recognition of transgenic material at trace levels for GMO food labelling purposes.

REFERENCES

1. A. Manfredi, M. Giannetto, M. Mattarozzi, M. Costantini, C. Mucchino, M. Careri, Anal Bioanal Chem (2016) 7289–7298 2. M. Giannetto, E. Umiltà, M. Careri, Anal. Chim. Acta 806 (2014) 197–203 3. S. Fortunati, MsSCI thesis, 2016 4. M. Giannetto, E. Maiolini, E.N. Ferri, S. Girotti, G. Mori, M. Careri, Anal Bioanal Chem (2013) 737–743 5. M. Giannetto, L. Mori, G. Mori, M. Careri, A. Mangia, Sens. Actuators, B (2011) 185–192

Abstract

Peptides Functionalized ZnO Nanoparticles Array as New Tool for Gas Sensors

S. Gaggiottia, F. Della Pellea, C. Di Natalebb, S. Qakalc, E. Iwuohac, D. Compagnonea

aFaculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64023 Teramo, Italy bDepartment of Electronic Engineering, University of Tor Vergata, 00133 Rome, Ital

cSensorLab, Department of Chemistry, University of Western Cape, Robert Sobukwe Road, Bellville, Cape Town 7535, South Africa

Keywords: gas sensor; ZnO-nanoparticles; peptides; molecular modeling Abstract The purpose of this work is the evaluation of the ZnONPs functionalized with different peptides in a quartz crystal microbalance array format, and the comparison with AuNPs modified with the same peptides. 8 quartz crystal microbalance (QMC) E-nose developed at University of Tor Vergata was used for the purpose placed [1]. Among the different approaches, sensing via the well-known quartz crystal microbalance (QCM) sensors appears still an interesting choice considering cost, sensitivity, ease of handling and robustness. In addition, since analysis is carried out at room temperature, it is possible the use of organic compounds as sensing elements [2]. ZnONPs were characterised with Transmission Electron Microscopy (TEM) and Scanning Electron Microscope (SEM). Furthermore, the absorption spectra of the nanoparticles have been studied by UV/Vis (Ultraviolet-Visible Spectroscopy) and FTIR (Fourier Transform Infrared Spectroscopy). The AuNPs and the ZnNPs have been modified with different peptide sequences built to maximize the signal information. The different array sensors outputs have been evaluated, with different solvents. The signal obtained were normalized in order to remove the effect of the concentration in the head space and to compare different QCM. In order to investigate the ability to work with high humidity content samples performances in pure water and in samples with high water content were also compared. The experimental data indicated similar behaviour of the arrays demonstrating that binding and orientation of the different peptides occurs in a similar way for the 2 nanomaterials used. Different behaviour have been observed in the presence of water; in this case the ZnONPs-peptides array does not exhibit any drift in the response as observed for AuNPs peptide array.

REFERENCES

1. Pizzoni, D., et al., Selection of peptide ligands for piezoelectric peptide based gas sensors arrays using a virtual screening approach. Biosensors and Bioelectronics, 2014. 52: p. 247-254.

2. Mascini, M., et al., Tailoring gas sensor arrays via the design of short peptides sequences as binding elements. Biosensors and Bioelectronics, 2017. 93: p. 161-169.

Abstract

Sensing toxicants in Marine waters makes Sense using biosensors

K. Petropoulosa, L. Michelia, L. Fabiania, G.Volpad, G.Palleschia

a Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma “Tor Vergata”, via della Ricerca Scientifica, 00133 Rome,

Italy laura.micheli@uniroma2.it@uniroma2.it

Keywords: domoic acid, okadaic acid, saxitoxin, ELIME

Sensing toxicants in Marine waters makes Sense using biosensors (SMS) will deliver a novel automated networked system that will enable real-time in situ monitoring of marine water chemical and ecological status in coastal areas by the detection of a series of contaminants regulated by the Marine Strategy Framework Directive (MSFD). SMS will design a multi-modular apparatus that will host in a single unit—the Main Box (MB)—a Sampling Module and an Analysis Module. The former will contain sample collection and treatment components, whereas the latter will include four biosensor sub-modules that will enable detection and measurement of algal toxins and their associated algal species; several hazardous compounds (tributyltin, diuron and pentaBDPE); sulphonamides and a series of standard water quality parameters. The MB will be equipped with a communication module for real-time data transfer to a control center, where data processing will take place, enabling alarm functionality to Health Warning Systems, whenever some critical value exceeds a pre-defined threshold. It will be placed on a floating platform or buoy positioned in loco at defined locations. SMS will also develop a Specific Marine Pollution Metric that will combine real-time data of pollutant concentrations and water quality parameters, to produce a quantitative assessment of marine water quality. In this framework the Analytical Chemistry laboratory aimed to develop a novel automated networked system for in situ monitoring of Okadaic acid (OA), Domoic acid (DA) and STX (STX) using a colometric assay based on the use of magnetic beads. OA is a lipophilic marine toxin produced by Dinophysis and Prorocentrum, and is responsible for causing diarrheic shellfish poisoning (DSP) to humans after ingestion of contaminated shellfish. So, an early detection of OA, directly in marine water, is an important aspect for public safety and natural environment. The mechanism of action of OA is based on the inhibition of protein phosphatase type 2A (PP2A). The degree of inhibition of the PP2A enzyme can be used as a measure of toxin concentration. DA is a naturally occurring neurotoxin produced by several species of marine diatoms from the genus Pseudo-nitzschia and is responsible for causing a human intoxication syndrome known as amnesic shellfish poisoning (ASP), characterized by severe gastrointestinal and neurological disorders. For this reason, its measurement in seawater could be an early alert for potential toxin accumulation in marine organisms. STX is one of the most lethal non-protein toxins (LD50 = 9 µg Kg-1) and is the only marine natural product that has been declared chemical weapon. STX has the ability to bio-accumulate up trophic levels. Ingestion of infected marine organisms, by humans, induces a lethal disease known as Paralytic Shellfish Poisoning (PSP) that is currently without antidote or detoxification pathway. Acknowledgements. Special thanks go to SMS Project -Sensing toxicants in Marine waters makes Sense using biosensors -

GRANT AGREEMENT N° 613844

Abstract

Development of an Integrated Analytical Platform for Detecting Biomarkers of Extant Life in Planetary Explorations

Mara Mirasolia, Martina Zangheria, Angela Volpeb, Domenico Caputoc, Augusto Nascettid, Giampiero De Cesarec and Aldo Rodaa

aDepartment of Chemistry “Giacomo Ciamician”, Alma Mater Studiorum - University of Bologna, Via Selmi 2, 40126 Bologna, Italy

bItalian Space Agency, Via del Politecnico snc 00133 Rome, Italy cDepartment of Information, Electronics and Communication Engineering, Sapienza University of Rome, Via Eudossiana 18,

00184 Rome, Italy dSchool of Aerospace Engineering, Sapienza University of Rome, Via Salaria 851/881, 00138 Rome, Italy

Keywords: Chemiluminescence, Lab-on-chip, thin film photosensors, immunoassay

One of the main research objectives in the field of exobiology is the search for organic molecules in extraterrestrial environments and the identification of their biological or non-biological origin. The availability of integrated and self-standing analytical devices to be employed in unmanned planetary exploration missions enables in-situ analysis of material samples in search of organic molecules, amino acids, nucleic acids, polysaccharides and other molecular systems characteristic of organized biological systems. An important example is given by the development of the Life Marker Chip (LMC) [1]. Lab-on-chip (LOC) devices offer favorable characteristics for such application, in terms of reduced size and weight, amenability to automation, very low sample and reagent consumption, reduced analysis time and, often, superior achievable performances in terms of limits-of-detection. Thus, LOC devices are extremely suitable for space missions and are under investigation in view of future planetary exploration. Herein, we report about the development of an integrated analytical platform for the execution of a competitive chemiluminescence immunoassay for the detection of Adenosine triphosphate (ATP), which is commonly accepted as a high ranking biomarker of extant life in extraterrestrial environments. Chemiluminescence detection was chosen, as it provides high detectability and sensitivity in bioassays and because of its suitability for miniaturized analytical devices, as it avoids the need for external radiation sources and complex optical systems combining filters and lenses [2]. The design of a portable device will be described, which exploits a microfluidic network based on capillary forces, integrated with an array of thin film hydrogenated amorphous silicon (a-Si:H) photosensors for the detection of the emitted photons [3].

REFERENCES

1. M. R. Sims, D. C. Cullen, C. S. Rix, A. Buckley, M. Derveni, D. Evans, N. Holm, Planetary and Space Science, 2012, 72, pp. 129-137. 2. M. Mirasoli, M. Guardigli, E. Michelini, A. Roda, Journal of Pharmaceutical and Biomedical Analysis, 2014, 87, pp. 36-52. 3. M. Mirasoli, A. Nascetti, D. Caputo, M. Zangheri, R. Scipinotti, L. Cevenini, A. Roda, Analytical and Bioanalytical Chemistry, 2014, 406,

pp. 5645-5656. Authors acknowledge the Italian Space Agency (ASI) for financial support to the project PLEIADES (Planetary Life Explorer with Integrated Analytical Detection and Embedded Sensors) 2015-037-R.0

Abstract

Modified Sonogel Carbon Electrode as Amperometric Sensors for Simultaneous Determination of Dihydroxybenzene Isomers

Laura Pigani, Fabio Terzi, Renato Seeber, Chiara Zanardi

Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Modena e Reggio Emilia, Via G. Campi, 103 – 41125 Modena

Keywords: dihydroxybenzene isomers; catechol; hydroquinone; caffeic acid; sonogel carbon electrode; carbon black.

The isomers of dihydroxybenzene, such as catechol (CC) and hydroquinone (HQ), are used in many fields, namely as pharmaceuticals, fine chemicals, cosmetics, and in food industries. As a consequence of their wide use, their presence in the environment as pollutants can easily occur. Due to their high toxicity and low degradability their determination in different matrices through a simple and fast analytical method is therefore particularly urgent. Electrochemical methods 3exhibit several advantages with respect to other techniques, including low cost of the relevant instrumentation, simple handling, fast response, high sensitivity, selectivity and stability, and the possibility of simultaneous detection of HQ and CC. Several carbon-based electrodes, including carbon nanotubes (CNT)-modified electrode [1] and graphene-based electrode [2] have been investigated for the electrochemical determination of dihydroxybenzene isomers. In the present communication we present the preliminary results obtained by using a sonogel carbon (SNGC) electrode modified with carbon black (SNGC_CB) for the same purpose. Carbon black substitutes in part for graphite as the conductive and sensitive component in the composite electrode material. SNGC electrodes [3] are characterized by robustness, coupled to reduced dimensions and good electrochemical efficiency, particularly as to sensitivity and reproducibility of the sensor and of the consequent electrochemical responses. Moreover, through the inclusion of different components in the graphite phase, such as metal nanoparticles [4], electrocatalytic processes can be activated. In this context, CB is particularly appealing due to its excellent conductive and electrocatalytic properties; the advantages of the use of CB as the sensing material have been widely demonstrated [5]. In this work, SNGC_CB electrodes have been characterized by SEM and cyclic voltammetry, showing good reproducibility and repeatability of the responses. SNGC_CB electrodes have shown improved efficiency with respect to SNGC electrodes in CC and HQ individual and simultaneous detection in different buffer solutions, exhibiting wide linear ranges of the signals and low limit of detection. Moreover, the behaviour of SNGC_CB electrodes with respect the detection of caffeic acid (CA), viz. a molecule containing a catechol group, widely diffused in plants and fruits as well as in the derived beverages, has also been investigated, leading to satisfactory results. Preliminary tests performed in matrices rich of natural antioxidants species, such as different wines, suggest that, under proper conditions, SNGC_CB electrodes can be successfully used for the detection of the principal families of anthocyanins.

REFERENCES

1. H. Zhang, J.S. Zhao, H.T. Liu, R.M. Liu, H.sh. Wang, J.F. Liu, Electrochemical determination of diphenols and their mixtures at the multiwall carbon nanotubes/poly (3-methylthiophene) modified glassy carbon electrode, Microchim. Acta 169 (2010) 277-282.

2. H.J. Du, J.S. Ye, J.Q. Zhang, X.D. Huang, C.Z. Yu, A voltammetric sensor based on graphene-modified electrode for simultaneous determination of catechol and hydroquinone, J. Electroanal. Chem. 650 (2011) 209–213.

3. L.M. Cubillana-Aguilera, J.M. Palacios-Santander, I. Naranjo-Rodríguez, J.L. Hidalgo-Hidalgo-De-Cisneros, Study of the influence of the graphite powder particle size on the structure of the Sonogel-Carbon materials, J. Sol-Gel Sci. Techn. 40 (2006) 55-64.

4. F. Arduini, C. Zanardi, S. Cinti, F. Terzi, D. Moscone, G. Palleschi, R. Seeber, Effective electrochemical sensor based on screen-printed electrodes modified with a carbon black-Au nanoparticles composite, Sens. Actuat. B Chem., 212 (2015), 536–543.

5. D. Talarico, F. Arduini, A. Constantino, M. Del Carlo, D. Compagnone, D. Moscone, G. Palleschi. Electrochem. Comm. 60 (2015) 78–82.

Abstract

DNA-based Nanoswitches Allosterically regulated By Biological Inputs For Controlled Drug Release Applications

Marianna Rossetti, Simona Ranallo, Andrea Idili, Giuseppe Palleschi, Alessandro Porchetta and Francesco Ricci

Dipartimento di Scienze e Tecnologie Chimiche, Università degli Studi di Roma “Tor Vergata”, Via della Ricerca Scientifica 1 – 00133 Roma

Keywords: DNA-based nanoswitches, allostery, biological targets, drug release, molecular cargo

Here we demonstrate the rational design of a new class of DNA-based nanoswitches that are allosterically regulated by specific biological targets, antibodies and transcription factors, and are able to load and release a molecular cargo (i.e. doxorubicin) in a controlled fashion. In our first model system we rationally designed a stem-loop DNA-nanoswitch that adopts two mutually exclusive conformations: a “Load” conformation containing a doxorubicin-intercalating domain and a “Release” conformation containing a duplex portion recognized by a specific transcription-factor (here Tata Binding Protein). The binding of the transcription factor pushes this conformational equilibrium towards the “Release” state thus leading to doxorubicin release from the nanoswitch. In our second model system we designed a similar stem-loop DNA-nanoswitch for which conformational change and subsequent doxorubicin release can be triggered by a specific antibody. Our approach augments the current tool kit of smart drug release mechanisms regulated by different biological inputs.

REFERENCES

1. M. Rossetti, S. Ranallo. A. Idili, G. Palleschi, A. Porchetta, F. Ricci. Chem. Sci. 2017, 8, 914-920.

Abstract

Non Conventional Approaches to Amperometric Sensing

Fabio Terzia, Stefano Ruggeria, Barbara Zanfrogninia, Elia Corsia, Laura Pigania, Chiara

Zanardia, Nicolò Dossib, Giulio Maccaferria, Renato Seebera

aDipartimento di Scienze Chimiche e Geologiche, Università di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy

bDipartimento di Scienze degli Alimenti, Università di Udine, Via Cotonificio 108, 33100, Udine, Italy

Keywords: electroanalysis, titanium, titanium nanoparticles, copper nanoparticles, copper alloys, paper electrodes

Novel amperometric sensors based on materials poorly exploited in electroanalysis have been developed and tested. In particular, pristine Ti, Au nanoparticles/Ti bimetallic surfaces and Cu alloys have been employed. The sensing systems consisting of these materials have been used for the determination of species of interest in the frame of industrial process control: different chemical species, such as hydrogen peroxide in detergents and treated wastewater, as well as heavy metals in solutions from recycling processes of electronic waste and batteries, have been investigated. The proposed systems could find application in on-line analysis, complementing and, sometimes, even replacing chemical analyses carried out by conventional laboratory techniques. In addition, disposable sensing systems based on paper modified with conductive materials, such as Cu nanoparticles, graphite or polypyrrole, have been tested by us. This

approach extends the applicability of the bulk cited materials and of paper‑based sensors to industrial problems such as the determination of carbohydrates in soft drink production or of ammonia gas in soil fertilization and refrigeration systems.

REFERENCES

1. F. Terzi, J. Pelliciari, B. Zanfrognini, L. Pigani, C. Zanardi, R. Seeber, Electrochem. Comm. 34 (2013) 138-141. 2. F. Terzi, B. Zanfrognini, S. Ruggeri, G. Maccaferri, L. Pigani, C. Zanardi, R. Seeber, Anal. Bioanal. Chem. 407 (2015) 983-990. 3. F. Terzi, B. Zanfrognini, S. Ruggeri, N. Dossi, Electrochim. Acta 188 (2016) 262-­268. 4. F. Terzi, B. Zanfrognini, N. Dossi, S. Ruggeri, G. Maccaferri, Electrochim. Acta 188 (2016) 327-335. 5. F. Terzi, B. Zanfrognini, S. Ruggeri, N. Dossi, G. Casagrande, E. Piccin, Sens. Act. B, 245 (2017) 352-358.

LISTA PARTECIPANTI

COGNOME NOME AFFILIAZIONE INDIRIZZO EMAIL Abdi Bellau Mohamed Adami Giampiero Agostini Alessandro Ancillotti Claudia Anfossi Laura Annibaldi Anna Antonelli Michela Arduini Fabiana Arigò Adriana Arroyo Crespo Juan José Avino Pasquale Badocco Denis Baggiani Claudio Barbieri Marica Battaglia Ivano Bellotti Denise Bergantin Caterina Bettazzi Francesca Bianchi Federica Bogialli Sara Brandolini Vincenzo Bruzzoniti Maria Concetta Calabria Donato Caliceti Cristiana Calvano Cosima Damiana Cappiello Achille Caprini Claudia Capriotti Anna Laura Careri Marinella Carlucci Giuseppe Cataldi Tommaso Catani Martina Cavaliere Chiara Cavazzini Alberto Cevenini Luca Chenet Tatiana Cinti Stefano Compagnone Dario Contado Catia Corradini Danilo Costa Valentina Costantini Francesca De Martino Michela Del Bubba Massimo Della Pelle Flavio Di Gangi Iole Maria Di Nardo Fabio Di Nardo Fabio Dugo Giovanni Dugo Paola Fanali Chiara Felletti Simona Ferraris Francesca Ferrone Vincenzo

Università degli Studi di Ferrara Università degli Studi di Trieste FKV Università degli Studi di Firenze Università degli Studi di Torino Università Politecnica delle Marche Università degli Studi di Roma “La Sapienza” Università degli Studi di Roma “Tor Vergata” Università degli Studi di Messina Príncipe Felipe Research Center - Valencia INAIL Università degli Studi di Padova Università degli Studi di Torino Agilent Technologies LabService Università degli Studi di Ferrara Università degli Studi di Ferrara Università degli Studi di Firenze Università degli Studi di Parma Università degli Studi di Padova Università degli Studi di Ferrara Università degli Studi di Torino Università degli Studi di Bologna Università degli Studi di Bologna Università degli Studi di Bari Università degli studi di Urbino Università degli Studi di Firenze Università degli Studi di Roma “La Sapienza” Università degli Studi di Parma Università degli Studi “G. d’Annunzio” Chieti-Pescara Università degli Studi di Bari "Aldo Moro" Università degli Studi di Ferrara Università degli Studi di Roma “La Sapienza” Università degli Studi di Ferrara Università degli Studi di Bologna Università degli Studi di Ferrara Università degli Studi di Roma “Tor Vergata” Università degli Studi di Teramo Università degli Studi di Ferrara Istituto di Metodologie Chimiche del CNR Università degli Studi di Ferrara Università degli Studi di Roma “La Sapienza” Università degli Studi di Roma “La Sapienza” Università degli Studi di Firenze Università degli Studi di Teramo Università degli Studi di Padova Università degli Studi di Torino Università degli Studi di Torino Università degli Studi di Messina Università degli Studi di Messina Università Campus Bio-Medico di Roma Università degli Studi di Ferrara Università degli Studi di Roma “La Sapienza” Università degli Studi “G. d’Annunzio” Chieti-Pescara

mohamedlamin.abdibellau@unife.it gadami@units.it a.agostini@fkv.it claudia.ancillotti@unifi.it laura.anfossi@unito.it a.annibaldi@univpm.it antonellimichela92@gmail.com fabiana.arduini@uniroma2.it aarigo@unime.it jjarroyoc@gmail.com p.avino@inail.it denis.badocco@unipd.it claudio.baggiani@unito.it marica_barbieri@agilent.com bativ@labservice.it denis.bellotti@student.unife.it caterina.bergantin@unife.it francesca.bettazzi@unifi.it federica.bianchi@unipr.it sara.bogialli@unipd.it bnv@unife.it mariaconcetta.bruzzoniti@unito.it donato.calabria2@unibo.it cristiana.caliceti@unibo.it cosimadamiana.calvano@uniba.it achille.cappiello@uniurb.it claudia.caprini@unifi.it annalaura.capriotti@uniroma1.it maria.careri@unipr.it giuseppe.carlucci@unich.it tommaso.cataldi@uniba.it ctnmtn@unife.it chiara.cavaliere@uniroma1.it cvz@unife.it luca.cevenini5@unibo.it tatiana.chenet@unife.i stefano.cinti@uniroma2.it dcompagnone@unite.it catia.contado@unife.it danilo.corradini@cnr.it valentina.costa@unife.it francesca.costantini@uniroma1.it michela.demartino16@gmail.com delbubba@unifi.it Flaviodellapelle@gmail.com iolemaria.digangi@unipd.it fabio.dinardo@unito.it fabio.dinardo@unito.it giodugo@unime.it pdugo@unime.it c.fanali@unicampus.it fllsmn1@unife.it francesca.ferraris@uniroma1.it vincenzo.ferrone@unich.it

Fortunati Simone Frapiccini Emanuela Gaggiotti Sara Gasparrini Francesco Giannetto Marco Giovannoli Cristina Guerrini Alessandra Ismail Omar Habib La Barbera Giorgia Laganà Aldo Maietti Annalisa Manarini Francesco Mangia Alessandro Mangraviti Domenica Marassi Valentina Marchetti Nicola Mazzoccanti Giulia Micalizzi Giuseppe Micheli Laura Mirasoli Mara Mondello Luigi Montone Carmela Maria Moscone Danila Nicoletti Isabella Notardonato Ivan Orsini Francesca Oteri Marianna Pagnoni Antonella Palleschi Giuseppe Pantò Sebastiano Pasquini benedetta Pasti Luisa Pastore Paolo Piergiovanni Maurizio Pietrogrande Chiara Pigani Laura Piovesana Susy Piparo Marco Remelli Maurizio Risoluti Roberta Rivoira Luca Robotti Elisa Roda Aldo Rossetti Marianna Russo Marina Schepis Antonino Sciarrone Danilo Seeber Renato Sergi Manuel Stevanin Claudia Tacchini Massimo Tedeschi Paola Termopoli Veronica Tomai Pierpaolo Trifirò Ferruccio Tripodo Giusy Truzzi Cristina

Università degli Studi di Parma CNR ISMAR Ancona Università degli Studi di Teramo Università degli Studi di Roma “La Sapienza” Università degli Studi di Parma Università degli Studi di Torino Università degli Studi di Ferrara Università degli Studi di Roma “La Sapienza” Università degli Studi di Roma “La Sapienza” Università degli Studi di Roma “La Sapienza” Università degli Studi di Ferrara Università degli Studi di Ferrara Università degli Studi di Messina Università degli Studi di Bologna Università degli Studi di Ferrara Università degli Studi di Roma “La Sapienza” Università degli Studi di Messina Università degli Studi di Roma “Tor Vergata” Università degli Studi di Bologna Università degli Studi di Messina Università degli Studi di Roma “La Sapienza” Università degli Studi di Roma “Tor Vergata” Istituto di Metodologie Chimiche del CNR Università degli Studi del Molise Università Campus Bio-Medico di Roma Università degli Studi di Messina Università degli Studi di Ferrara Università degli Studi di Roma “Tor Vergata” LECO Università degli Studi di Firenze Università degli Studi di Ferrara Università degli Studi di Padova Università degli Studi di Urbino "Carlo Bo" Università degli Studi di Ferrara Università degli Studi di Modena e Reggio Emilia Università degli Studi di Roma “La Sapienza” Università degli Studi di Messina Università degli Studi di Ferrara Università degli Studi di Roma “La Sapienza” Università degli Studi di Torino Università del Piemonte Orientale Università degli Studi di Bologna Università degli Studi di Roma “Tor Vergata” Università Campus Bio-Medico di Roma Università degli Studi di Messina Università degli Studi di Messina/DANI Università degli Studi di Modena e Reggio Emilia Università degli Studi di Teramo Università degli Studi di Ferrara Università degli Studi di Ferrara Università degli Studi di Ferrara Università degli Studi di Urbino "Carlo Bo" Università degli Studi di Roma “La Sapienza” Università degli Studi di Bologna C.I.R. Università campus Bio-Medico Università Politecnica delle Marche

simone.fortunati@studenti.unipr.it emanuela.frapiccini@an.ismar.cnr.it sgaggiotti@unite.it francesco.gasparrini@uniroma1.it marco.giannetto@unipr.it cristina.giovannoli@unito.it grrlsn@unife.it omar.ismail@uniroma.it giorgia.labarbera@uniroma1.it aldo.lagana@uniroma1.it mttnls@unife.it mnrfnc@unife.it dmangraviti@unime.it valentina.marassi2@unibo.it mrcncl@unife.it giulia.mazzoccanti@uniroma1.it gmicalizzie@unime.it laura.micheli@uniroma2.it mara.mirasoli@unibo.it lmondello@unime.it carmelamaria.montone@uniroma1.it moscone@uniroma2.it isabella.nicoletti@cnr.it ivan.notardonato@unimol.it f.orsini@unicampus.it moteri@unime.it pgn@unife.it palleschi@uniroma2.it sebastiano_panto@leco.com benedetta.pasquini@unifi.it luisa.pasti@unife.it paolo.pastore@unipd.it m.piergiovanni1@campus.uniurb.it mpc@unife.it laura.pigani@unimore.it susy.piovesana@uniroma1.it mpiparo@unime.it rmm@unife.it roberta.risoluti@uniroma1.it luca.rivoira@unito.it elisa.robotti@uniupo.it aldo.roda@unibo.it marianna.rossetti@uniroma2.it marina.russo@unicampus.it aschepis@unime.it dsciarrone@unime.it renato.seeber@unimore.it msergi@unite.it claudia.stevanin@unife.it tccmsm@unife.it tdspla@unife.it veronica.termopoli@uniurb.it pierpaolo.tomai@uniroma1.it ferruccio.trifiro@unibo.it g.tripodo@unicampus.it c.truzzi@univpm.it

Vecchietti Davide Ventura Giovanni Vincenti Flaminia Volders Filip Zanardi Chiara Zangheri Martina Zenezini Chiozzi Riccardo

Shimadzu Università degli studi di Bari "Aldo Moro" Università degli Studi di Roma “La Sapienza” Elementar Università degli Studi di Modena e Reggio Emilia Università degli Studi di Bologna Università degli Studi di Roma “La Sapienza”

dgvecchietti@shimadzu.it giovanni.ventura@uniba.it flavincenti@gmail.com volders@elementar.de chiara.zanardi@unimore.it martina.zangheri2@unibo.it riccardo.zenezini@uniroma1.it