Marini Electro 2006

Roland Djang’eing’a Marini1

Carl Groom2

Francois R. Doucet2

Jalal Hawari2

Yaser Bitar3

Ulrike Holzgrabe3

Roberto Gotti4

Julie Schappler5

Serge Rudaz5

Jean-Luc Veuthey5

Roelof Mol6

Govert W. Somsen6

Gerhardus J. de Jong6

Pham Thi Thanh Ha7

Jie Zhang7

Ann Van Schepdael7

Jos Hoogmartens7

Willy Briône8

Attilio Ceccato8

Bruno Boulanger9

Debby Mangelings10

Yvan Vander Heyden10

Willy Van Ael11

Ilias Jimidar11,Matteo Pedrini12

Anne-Catherine Servais12

Marianne Fillet12

Jacques Crommen12

Eric Rozet1

Philippe Hubert1

Received November 8, 2005Revised February 15, 2006Accepted February 16, 2006

Research Article

Interlaboratory study of a NACE method forthe determination of R-timolol content inS-timolol maleate: Assessment of uncertainty

Analyses of statistical variance were applied to evaluate the precision and practicalityof a CD-based NACE assay for R-timolol after enantiomeric separation of R- andS-timolol. Data were collected in an interlaboratory study by 11 participating laboratorieslocated in Europe and North America. General qualitative method performance wasexamined using suitability descriptors (i.e. resolution, selectivity, migration times andS/N), while precision was determined by quantification of variances in the determina-tion of R-timolol at four different impurity levels in S-timolol maleate samples. Theinterlaboratory trials were designed in accordance with the ISO guideline 5725-2. Thisallowed estimating for each sample, the different variances, i.e. between-laboratory(s2

Laboratories), between-day (s2Days) and between-replicate (s2

Replicates). The variances ofrepeatability (s2

r) and reproducibility (s2R) were then calculated. The estimated uncer-

tainty, derived from the precision estimates, seems to be concentration-dependentabove a given threshold. This example of R-timolol illustrates how a laboratory canevaluate uncertainty in general.

Keywords: Interlaboratory study / Nonaqueous capillary electrophoresis / Reproduci-bility / R-Timolol / Uncertainty estimation DOI 10.1002/elps.200500832

2386 Electrophoresis 2006, 27, 2386–2399

1Laboratory of Analytical Chemistry, Institute of Pharmacy, University of Liège, Liège, Belgium2Environmental Analytical Laboratory, Biotechnology Research Institute, National Research Council, Montreal, Canada3Institute of Pharmacy and Food Chemistry, University of Würzburg, Würzburg, Germany4Laboratory of Pharmaceutical Analysis, Department of Pharmaceutical Science, Faculty of Pharmacy, University of Bologna, Bologna,Italy

5Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences – EPGL, University of Geneva, Geneva,Switzerland

6Department of Biomedical Analysis, Utrecht University, Utrecht, The Netherlands7Laboratorium voor Farmaceutische Chemie en Analyse van Geneesmiddelen, K.U.Leuven, Leuven, Belgium8Lilly Development Centre, Analytical Sciences R&D, Mont-Saint-Guibert, Belgium9Lilly Development Centre, Statistical & Mathematical Sciences, Mont-Saint-Guibert, Belgium

10Analytical Chemistry and Pharmaceutical Technology, VUB, Brussels, Belgium11Johnson & Johnson Pharmaceutical, Research and Development Analytical Development – Method Development, Beerse, Belgium12Department of Analytical Pharmaceutical Chemistry, Institute of Pharmacy, University of Liège, Liège, Belgium

1 Introduction

To meet fit-for-purpose requirements, the data obtainedfrom a given analytical method must be reported with awell-defined estimate of precision, i.e. result uncertainty,in order to facilitate result comparison and interpretation[1, 2]. The advantages of such practices are already

Correspondence: Professor Philippe Hubert, Laboratory of Analyti-cal Chemistry, Institute of Pharmacy, University of Liege, CHU, B36,B-4000 Liege 1, BelgiumE-mail: [email protected]: 132-4-3664317

Abbreviations: HDMS-�-CD, heptakis(2,3-di-O-methyl-6-sulfo)-b-CD; SST, system suitability test

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2006, 27, 2386–2399 CE and CEC 2387

described [3], and the reporting of result uncertainty isincreasingly requisite in matters related to governmentregulation, industrial production, international trade andfor a multitude of applications carried out according tofixed norms [4]. This puts chemists/analysts underincreasing pressure in order to demonstrate the quality oftheir results. The definition of uncertainty can be found inthe literature [1, 5]. In order to evaluate this performanceparameter, three strategies are frequently applieddepending on the presented situation: (i) the intralabora-tory one, which includes the ISO approach published inseveral guidelines or articles [1, 6–9] and the approachusing the validation [9–17] or the robustness [9, 18] data,(ii) the interlaboratory strategy developed by the Analyti-cal Method Committee of the Royal Chemistry Society[19] or (iii) the mixed strategies that combine dataobtained from both intra- and interlaboratory data. Theselatter procedures include that proposed by Barwick andEllison [20] and that developed by the Technical Commit-tee ISO/TS 69 [21].

The recent development of several capillary electropho-retic methods using acidic nonaqueous media and hep-takis(2,3-di-O-methyl-6-O-sulfo)-b-CD (HDMS-b-CD) aschiral selector has allowed the enantioseparation of basicdrugs, among them timolol [22]. The active molecule(S-timolol) is a nonselective b-adrenergic blocker used asa single enantiomer against hypertension, arrhythmiasand angina pectoris. Other medical indications are thesecondary prevention of myocardial infarcts [23, 24] andthe topical treatment of increasing intraocular pressure[25, 26]. The NACE method developed for timolol enan-tioseparation was successfully validated and applied forthe determination of R-timolol in S-timolol maleate sam-ples in the presence of pyridoxine used as internalstandard [27]. While the uncertainty of the R-timolol con-tent was reported using intralaboratory validation data[27], an examination of interlaboratory variance anduncertainty will assist in the scale up of method usage,and uncover issues related to the transfer of this methodto a wider range of users. To the best of our knowledge,no report exists on the collaborative evaluation of uncer-tainty in CE techniques using interlaboratory data. Colla-borative CE studies related to method development arereported [28–33], but no relevant conclusions regardingthe measurement uncertainty are presented.

The objective of this study was to evaluate, by means ofan interlaboratory study, the ability of the developed andvalidated NACE method to be transferred. For that pur-pose, qualitative and quantitative responses were exam-ined with regard to the performance of the method withina single laboratory, and to the method performances ofany given laboratory compared to another. The repro-

ducibility of the quantitative results, namely the content ofR-timolol, was determined according to the ISO 5725-2guidelines related to interlaboratory studies [34].

2 Materials and methods

2.1 Chemicals and reagents

Four homogeneous S-timolol maleate samples weresubjected to analysis: one (batch No. 107200204) kindlyprovided by Prosintex Industrie Chimiche Italiane (Milan,Italy) and three others (batches No. 11484, 11483 and11351) obtained from the European PharmacopoeiaSecretariat (Strasbourg, France). Chemical referencesubstance of R-timolol maleate (batch No. 11381) waskindly provided by Merck (Rahway, NJ, USA) and pyrido-xine hydrochloride by SMB Technology (Marche-En-Famenne, Belgium).

HDMS-b-CD, potassium formate, ammonium formate(1R) or (1S)-(-)-10-camphorsulphonic acid, formic acid(98–100%) and methanol were purchased from differentsuppliers depending on each laboratory. However, allmaterials satisfied the requirements prescribed for NACE(i.e. the quality requirement for methanol was liquid chro-matographic grade whereas an analytical grade of formicacid was sufficient).

2.2 Electrophoretic conditions

The CE conditions [27], applied by the different participat-ing laboratories, include the use of uncoated fused-silicacapillaries having 50 mm internal diameter and 48.5 cmlength (40 cm to the detector). The electrophoreticseparations were carried out using 30 mM HDMS-b-CD incombination with 30 mM potassium camphorsulfonate(camphorSO3

2) in methanol acidified with 0.75 M formicacid. Hydrodynamic injection was made by applying apressure of 50 mbar for a period of 8 s and UV detectionwas performed at 295 nm. The capillary was thermostatedat 157C. Under these conditions, by applying a voltage of25 kV, the last migrating peak was expected to be detect-ed at the cathodic end within 14 min.

A new capillary was conditioned at 157C with methanol for15 min. Before running the experiments, the capillary wasconditioned at 157C successively with the BGE and BGE-CD solutions for 5 min each. Between runs, the capillarywas rinsed at 157C with methanol for 2 min and condi-tioned at 157C with BGE-CD solution for 4 min. BothBGE-CD and BGE were renewed after about 70 min ofanalysis to avoid the phenomenon of buffer depletionwhich causes loss of separation efficiency. At the end of


2388 R. Djang’eing’a Marini et al. Electrophoresis 2006, 27, 2386–2399

each working day, the capillary was rinsed at 257C withmethanol for 30 min, with 20 mM ammonium formate so-lution for 20 min and finally with methanol for 30 min.Capillary wash cycles were performed at a pressure ofapproximately 1 bar.

2.3 Preparation of the solutions

Repeated vortexing and ultrasonication were specified toensure complete dissolution of the substances andhomogeneity of solutions. Prior to their use in the CEsystem, all solutions were filtered directly in suitable CEvials through filters resistant to organic solvents (i.e.polypure propylene membrane filter, 0.2 mm).

2.3.1 Solutions for CE separation

The BGE, which consisted of a camphorSO32 solution at

the required concentration, was prepared by dissolvingsimultaneously the corresponding amount of 30 mM(1R)- or (1S)-(2)-10-camphorsulfonic acid and the cor-responding amount of 30 mM potassium formate inmethanol acidified with the corresponding quantity of0.75 M formic acid. In solution, the camphorsulphonicacid is converted into the potassium salt. The use of anultrasonic bath was necessary to obtain complete dis-solution.

The BGE-CD solution was prepared by dissolving 30 mMof HDMS-b-CD in the BGE and was placed for 5 min inthe ultrasonic bath. During the CE separation, the BGEwas placed at the outlet of the capillary and the BGE-CDat the inlet.

2.3.2 Solutions for system suitability tests (SST)

To assess the suitability of the different CE systems,three reference solutions were defined: (i) a referencemixture solution (a) containing pyridoxine (5 mg/mL),S-timolol (10 mg/mL) and R-timolol (5 mg/mL), (ii) a refer-ence solution (b) containing R-timolol maleate (20 mg/mL)and pyridoxine (5 mg/mL), and (iii) a reference solution (c)which corresponds to a 0.1% solution (referred to asR-timolol maleate), prepared by diluting ten times thereference solution (b). The chemical structures of R-timolol maleate, S-timolol maleate and pyridoxine aregiven in Fig. 1.

The reference mixture solution (a) was used in a first SSTto identify the different peaks in the electropherogram aswell as to test the CE system’s enantioresolution. Indeed,the three analyte concentrations were selected to allowpeak identification on the basis of peak height. Under the

Figure 1. Chemical structures of timolol maleate (A) andpyridoxine (B).

prescribed CE conditions, the enantiomeric resolutionwas not allowed to be less than 5.5. The resolution be-tween the internal standard and S-timolol had also to bereported. Its value had to be at least 9. In case of anunsuitable test result, i.e. the enantioresolution below 5.5,the protocol stipulated to adjust the voltage. A decreasemay improve the enantioseparation. In all cases, themigration times of analytes and the selectivity (ratio ofmigration times) between consecutive peaks had to bereported. The resolution (Rs) was calculated according tothe European Pharmacopoeia [34]:

RsAB ¼ 1:18�tM;B � tM;A� �

wAð0:5Þ þwBð0:5Þ� �

" #

(1)

with tM,A and tM,B, the migration times of the peaks corre-sponding to the first (A) and second (B) analyte, respec-tively, and wA(0.5) and wB(0.5), their corresponding peakwidths at half of the signal height, respectively.

In a second SST, the reference solution (c) was used tocheck the sensitivity of the detector by determining theS/N. That ratio was calculated according to the EuropeanPharmacopoeia [35] specification:

S=N ¼ 2Hhn

(2)

where H is the height corresponding to the R-timololpeak in the electropherogram obtained with the refer-ence solution (c) (= 0.1% solution) and hn is the absolutevalue of the largest noise fluctuation from the baseline inthe same electropherogram and observed over a dis-tance equal to 20 times the width at half height of thepeak obtained with reference solution (b) and situatedequally around the place where R-timolol was found. Ifthe ratio is at least 10, then the 0.1% concentrationlevel is considered to be above the quantitation limitand all solutions containing the analyzed substance atconcentrations above that level can be quantified. If not,



the detector should be checked or changed. Since thereference solution (c) contained the internal standard, theS/N value related to that analyte also had to be reported.

In the third SST, the repeatability of the injection wasevaluated by injecting six times the reference solution (b).The repeatability of the migration times for each analytewas also evaluated as well as the relative migration timewhich is the ratio of R-timolol migration time over that ofthe internal standard.

2.3.3 Solutions for quantitation

2.3.3.1 Internal standard solution

The internal standard solution was prepared by dissolvingabout 12.5 mg of pyridoxine hydrochloride in 25.0 mLmethanol. Then the solution was diluted 50-fold with thesame solvent to obtain the stock solution of the internalstandard (10 mg/mL).

2.3.3.2 Reference solution

A stock solution of R-timolol was prepared by dissolving in10.0 mL methanol an accurately weighed amount of ap-proximately 20 mg of R-timolol maleate. This stock solu-tion was then diluted 100-fold to obtain a reference solu-tion representing 1.0% (20 mg/mL) of impurity level andcontaining the internal standard at 5 mg/mL. This referencesolution was used for the determination of R-timolol ma-leate content in the different S-timolol maleate samples.

2.3.3.3 Test solutions

Four different test samples (A, B, C and D) containingR- and S-timolol were provided in powder form. Twoseparate independent solutions for each sample were thenprepared to contain 2.0 mg/mL S-timolol and 5 mg/mLpyridoxine internal standard. Each solution was then an-alysed under identical conditions as in the repeatabilitytrials. The test solution was prepared by dissolving to10.0 mL methanol an accurately weighed amount of ap-proximately 20 mg of the test sample and adding thecorresponding volume of the internal standard stock so-lution to obtain 5 mg/mL.

2.4 Instrumentation

In the framework of this collaborative study, instrumentsfrom different manufacturers must meet the minimumrequirements to run the NACE method. They must there-fore ensure sufficient cooling to maintain a capillary at

157C, and be fitted with uncoated fused-silica capillarieswith internal diameter, total length and length to detectorof 50 mm, 48.5 cm and 40 cm, respectively. However, forBeckman equipments, the total length was adaptedaccording to the capillary handling device (50.2 cm).

Three laboratories used P/ACE MDQ systems fromBeckman Coulter (Fullerton, USA) and eight used HP3DCEones from Agilent Technologies (Waldbronn, Germany);however, for the latter, the data managers were different.The capillaries were from different suppliers such as BGBAnalytik, Polymicro technologies, Beckman Coulter andComposite Metal Services.

The statistical calculations were performed using the JMPsoftware version 5.1 for Windows (SAS Institute, Cary,NC, USA).

2.5 Setup of the study

The study included 11 laboratories from pharmaceuticalcompanies, private and governmental institutes as well asfrom universities, located in Europe and in North America.The number of laboratories for the study fitted with therequirements of the ISO guideline 5725-2 [34]. The setup ofthe study is shown in Fig. 2. Each laboratory had to exe-cute the whole analysis on two different days (c = 2). Foreach day, the four S-timolol maleate samples had to beanalysed twice meaning that two sample solutions have tobe prepared independently and analyzed (g = 2). The con-tent of R-timolol was determined by comparison to thereference solution. Commonly, in interlaboratory studies,e.g. in [36, 37], only the top and bottom levels of theexperimental design are performed, i.e. different labora-tories and within each laboratory two replicates. In thisstudy, the setup represents a more elaborate collaborativestudy since it allows evaluation of the intermediate preci-sion time-dependence for each laboratory, which is thesum of the repeatability and interday (s2

Interday) variances.This can be done only if the contribution of each variancecomponent (laboratory, time and replicate) is estimated.

Figure 2. Setup of the collaborative study for eachsample: r = 8 laboratories, c = 2 days and g = 2 replicatemeasurements.



2.6 Statistical analysis of the results

Prior to any further analysis, the data issued from the studywere first submitted to a critical examination for outliers andstragglers. Two approaches defined by the ISO guideline[34] were used, namely graphical consistency techniquesand numerical outlier tests. Both within- and between-lab-oratoryvariabilities were evaluated. Mandel’sk statistic wasapplied for the graphical evaluation of the within-laboratoryvariability, while the Cochran’s test was applied as anumerical test. Mandel’s h statistic and Grubbs’ tests wereapplied for graphical and numerical evaluations of the be-tween-laboratory variability, respectively.

Different Grubbs’ tests [34] were used for the numericalevaluation: G1 for one small suspected outlier, Gr for onelarge suspected outlier, G1,2 for two small suspected out-liers and Gr,r 2 1 for two large suspected outliers. A G testfor a polar pair (one low and one high result) was also used[38]. The different formulas applied in Mandel’s, Cochranand Grubbs’ tests are detailed elsewhere [36, 37, 39].

After the test for outliers, the mean squares between-laboratories (MSLaboratories), between-days (MSDays) andbetween-replicates (MSReplicates) were calculated byapplying the ANOVA indicated in Table 1. The laboratoriesare ordered in an arbitrary way. From the mean-squaresdata, the repeatability (s2

r) and the between-laboratory(s2

Laboratories) variances as well as the time-dependentintermediate precision (s2

T) were calculated [34].

3 Results and discussion

3.1 System suitability checking and qualitativeresponses

Before undertaking the study, all laboratories wererequested to maintain their instruments as described inCE user’s guides. Improper vial prepuncher and electrode

maintenance in particular were observed to have negativeeffects on separation efficiency. The maintenance proce-dure was completed prior to operation for each day of thestudy.

A detailed protocol was elaborated for the execution ofthe study. First, a training round was organised to allowlaboratories to get familiarised with the method and toverify the adequacy of the protocol. It involved the evalu-ation of some qualitative (resolution and selectivity of dif-ferent peak analytes, their migration times and S/N) andquantitative (content of R-timolol in one S-timolol maleatesample) aspects. Afterwards, on the basis of the reportedresults, the laboratories were allowed to perform the finalstudy. In the latter, for the quantitative aspect, the contentof R-timolol was determined independently in four differ-ent S-timolol maleate samples containing the R-enantio-mer at four different impurity concentration levels.

The training revealed several problems with laboratoriesusing Beckman equipment: no peak observed within14 min for some laboratories, low current and even loss ofcurrent in some cases. A closer analysis of these situa-tions indicated that the problems might be due to thecooling system of the capillary. In the Agilent equipments,an air cooling system maintains the capillary at low tem-peratures whereas the Beckman equipment uses a cool-ing liquid system which seems to be more efficient [33,40]. In this NACE method, a temperature of 157C wasprescribed for analytical separation. With the Beckmanequipment, a very efficient capillary cooling seems tooccur; thus one observes a lower current, as it is propor-tional to the temperature and consequently the peakanalytes will have slower migration than Agilent equip-ment [33]. Another more dramatic outcome, that canoccur related to the cooling system, is the fact that thetemperature attained causes a crystallisation and a pre-cipitation of the BGE-CD inside the capillary or at its endsleading to the absence of current. This situation was fre-

Table 1. ANOVA components

Sources of variability Mean squares Estimated variances

Laboratories MSLaboratories ¼cgP

�xi � �xð Þ2

r � 1s2

Lab ¼MSLab �MSDays

cg

Days MSDays ¼gPP

�xij � �xi� �2

rðc� 1Þ s2Days ¼

MSDays �MSReplicates

g

Replicates MSReplicates ¼PPP

ðxijk � �xijÞ2

rcðg� 1Þ s2Replicates = MSReplicates

g = Number of replicates per day.c = Number of days per laboratory.r = Number of laboratories.



quently reported by the laboratories using Beckmanequipments. However, two of them reported some resultsabout the reference mixture solution (a). The migrationtimes of pyridoxine, S-timolol and R-timolol obtained withthat solution were 13.3, 16.4 and 18.8 min, respectively,for the first laboratory and 12.4, 13.0 and 14.7 min,respectively, for the second one. Due to problems reportedabove, those laboratories were not able to continue toprovide sufficient results. On the other hand, the labora-tories using Agilent equipment did not encounter suchproblems and reported sufficient results for data analysis.

On the basis of this training, only the laboratories usingAgilent equipment were allowed to continue the inter-laboratory study. Since their number (r = 8) was still inagreement with the ISO norms, we were able to continuethe study in the framework of the reproducibility evalua-tion. The study started with the evaluation of the SSTs.

In the first and the second SSTs, the adequacy of the CEsystem had to be checked by running the reference solu-tions (a) and (c). If the prescribed requirements wereachieved, then the repeatability SST was performed andthe R-timolol quantifications were made as outlined inFig. 3. A typical electropherogram of the reference mix-ture solution (a) is presented in Fig. 4. The electrophoreticseparation was suitably and efficiently achieved by alllaboratories with corresponding currents ranging from 14to 20 mA. It can be noticed that, according to the differ-ence in peak heights, all laboratories identified the threepeaks and their migration order. Results of the SSTs aresummarised in Table 2. All laboratories achieved therequirement concerning enantioresolution since all values

were above 5.5. The expected resolution between pyr-idoxine and S-timolol (Rs1 2 2 = 9) was also achieved byalmost all laboratories except laboratory 7 whichobtained a lower value (Rs1 2 2 = 5.3). In addition, thatlaboratory had low migration times for all peaks com-pared to other laboratories although the reported currentwas acceptable. No particular problem was reported bythat laboratory which, presented satisfactory results dur-ing the training round. Since the results obtained on thesecond day were better, it was suspected that insufficientcapillary conditioning may have been the problem [33, 41,42]. Laboratory 8 performed the analysis by applying aseparation voltage below 25 kV. Indeed, using 25 kV dur-ing the training round, the resolution of the enantiomericpeak pair was sufficient, but that for S-timolol and thepyridoxine was not (Rs1 2 2 = 7.5). A tailing of the parentpeak that is S-timolol, was also observed. Therefore, avalue of 23.5 kV allowed to improve that resolution(Rs1 2 2 = 10.7). The parent peak became more symme-trical. Moreover, the tailing of the parent peak wasdecreased in such a way that the detection, integrationand quantitation of the R-timolol peak in the S-timololmaleate samples were allowed. This phenomenon mightbe due to the capillary batch-to-batch differences [41]which are known to affect the reproducibility of the capil-lary electrophoretic separations.

The detectability of the CE system was checked by eval-uating the S/N values for R-timolol and pyridoxine. Alllaboratories achieved the prescribed requirements for R-timolol (Table 2). However, for pyridoxine, S/N valueswere never above 10. Particularly, laboratory 5 presenteda strikingly low value (S/N = 2). Moreover, the ratio of

Figure 3. Setup of the injection scheme and determined responses.



Table 2. Results of SST-1 and SST-2 for day 1

Laboratoryno.

Migration time (min) Resolution (Rs) Selectivity (a) S/N

Peak 1 Peak 2 Peak 3 Rs 1–2 Rs 2–3 a 1–2 a 2–3 S/N peak 1 S/N peak 3

1 10.2 11.2 12.5 8.6 10.6 1.12 1.10 5 102 9.2 10.2 11.1 10.0 10.0 1.10 1.10 6 113 10.0 11.0 12.0 9.3 9.3 1.10 1.09 4 114 10.7 12.5 13.5 11.9 5.7 1.17 1.08 8 225 10.9 11.5 12.5 9.2 6.5 1.13 1.09 2 156 10.0 11.5 12.4 12.9 6.8 1.07 1.15 7 167 (day 1) 8.2 8.7 9.6 5.3 8.6 1.07 1.11 5 117 (day 2) 9.3 10.2 11.0 8.9 7.9 1.10 1.08 5 108 10.4 11.6 12.9 10.7 10.0 1.11 1.11 5 15

(1) = Pyridoxine, (2) = S-timolol, (3) = R-timolol.

Figure 4. Typical electropherogram of a reference mix-ture solution obtained by carrying out the CE separationusing a BGE containing 30 mM HDMS-b-CD and apply-ing a voltage of 25 kV (for other conditions, see Section 2).Peaks and concentrations: (1) pyridoxine (5 mg/mL),(2) S-timolol (10 mg/mL) and (3) R-timolol (5 mg/mL).

S/NR-timolol over S/Npyridoxine was about 7.5 for that labora-tory while in general it was between 1.8 and 3.0. Thisclearly indicates for laboratory 5 an increased uncertaintywhen quantifying R-timolol near the LOQ (0.1%) [27].

In the third SST, the precision of the CE systems wasexamined in terms of variations in migration times andareas for pyridoxine and R-timolol peaks. Results aresummarised in Table 3. The RSD values of the migrationtimes (tM’s) were not above 1% in all laboratories exceptfor laboratories 3 and 7 (for pyridoxine and R-timolol). Animprovement was observed in the second day data (notshown). However, when considering the relative migrationtimes (tM, R-timolol/tM, Pyridoxine), RSD values were below 1%for all laboratories. Considering the 2 days, the RSD

values for the relative migration times were below 1.2%,except for laboratory 5 (RSD = 2%). RSD value for theoverall study was 2.2% (N = 96 runs), demonstrating verystable relative migration times for the NACE methodunder the repeatability, time-different intermediate preci-sion and reproducibility conditions.

Concerning the peak areas, high variabilities wereobserved for both pyridoxine and R-timolol peaks withhigher RSD values (i.e. 12.1% for laboratory 3) whichwere not really improved by correcting the peak area withthe corresponding peak migration time. However, accep-table values (RSD below 3%) could be achieved by atleast 4 laboratories for the ratio of corrected peak areas ofR-timolol over that of pyridoxine (Table 3). In the light ofthese observations and those reported in the S/N data,the variations observed for peak areas seem to be affect-ed by sources other than the corresponding migrationtimes (i.e. conditions of UV source, data acquisition rate,etc.). For the other four laboratories, the RSD values werehigher for the results calculated with internal standard thanwithout. Higher RSD values for the pyridoxine peak areathan for the timolol peak were also noticed. This may havean influence on the quantitative results.

3.2 Quantitative responses

3.2.1 Content of R-timolol

It can be observed from typical electropherograms of testsample solutions A–D (Fig. 5) that in these conditions,only one impurity peak corresponding to R-timolol couldbe observed.

The content of R-timolol in those samples was deter-mined by comparing the normalised ratio of R-timolol



Table 3. Results of SST-3; repeatability (n = 6 injections)

Labora-toryNo.

Pyridoxine R-Timolol RMTa)

RSD(%)

Ratiob) (R-Timolol/pyridoxine)

M.T. Area Corr. area M.T. Area Corr. area

Mean RSD(%)

Mean RSD(%)

Mean RSD(%)

Mean RSD(%)

Mean RSD(%)

Mean RSD(%)

Mean RSD (%)(day 1/day 2)

1 10.3 0.3 10.85 1.1 1.05 1.3 12.6 0.4 25.71 1.9 2.04 2.1 0.1 1.935 1.1/1.92 9.3 0.9 8.06 4.8 0.87 4.9 11.1 1.0 23.12 0.5 2.08 1.2 0.2 2.400 4.4/6.23 9.9 1.9 8.64 12.1 0.87 12.7 11.7 1.8 28.80 5.3 2.47 6.4 0.7 2.842 8.2/6.24 10.6 0.8 8.52 6.5 0.80 7.2 13.1 0.9 25.52 3.1 1.95 3.8 0.3 2.425 5.2/2.75 10.3 0.2 8.03 3.9 0.78 3.9 12.5 0.2 28.17 4.0 2.26 4.1 0.3 2.894 6.3/3.56 9.8 0.3 7.28 1.6 0.74 1.6 12.2 0.3 24.98 1.8 2.05 1.9 0.1 2.773 1.8/1.67 8.3 2.1 11.13 1.2 1.34 1.6 9.8 2.2 26.41 0.9 2.70 2.6 0.2 2.008 2.1/2.78 10.4 0.6 5.69 3.4 0.55 3.3 12.8 0.5 20.63 2.8 1.61 2.4 0.2 2.9539 2.1/2.6

M.T = migration time (in minutes).Corr. = corrected.a) Relative migration times (MTR-timolol/MTPyridoxine).b) Ratio of corrected areas.

(NRT) and the normalized ratio of R-timolol from referencesolution (a) (NRR) using the following equation:

Content of R-timolol ð%Þ ¼ NRTNRR

� 1% (3)

The ratio of R-timolol in the test or reference samples wasnormalised as follows:

Normalized ratio ¼ ratio of test or reference sample � 20weighed mass mgð Þ (4)

The ratio of test or reference samples was obtained usingthe following calculation:

Ratio ¼ corrected area of R-timolol in test or reference samplecorrected area of pyridoxine in test or reference sample

(5)

the corrected area being obtained by dividing the peakarea over the peak migration time.

The results presented in Table 4 show that all the labora-tories were able to quantify R-timolol in all samples, evenin sample D which contained a R-timolol content near theLOQ (0.1%) [27]. The reported contents were in the rangeof 0.49–0.78, 0.26–0.41, 0.19–0.34 and 0.06–0.20% forsamples A, B, C and D, respectively. The RSD values insample A (RSD value of 12.4%), sample B (RSD value11.7%) and sample C (RSD value 15.5%) were lower thanthat obtained in sample D (RSD of 34.9%).

3.2.2 Critical examination of data

The within-consistency evaluation was carried out usingMandel’s k formula which allowed construction of the cor-responding plot, presented in Fig. 6. In order to evaluatewhether a problem exists in a certain laboratory or with acertain sample, the k values were plotted per laboratoryand per sample. As illustrated in Fig. 6A, laboratory 2showed some large k values suggesting a large intrala-boratory variance compared to the other participants. Forthat laboratory, the largest variability was attributed tosample D. In contrast, laboratory 1 presented small k va-lues suggesting that the variability observed in this labora-tory was very low. From Fig. 6B, it can be deduced that theproblems of within-laboratory variability were not related tothe S-timolol sample. Cochran’s test was used to evaluatewhether the variability observed in laboratory 2 for sam-ple D was numerically inconsistent. If a calculated Cochranvalue is found to be above the critical 5% limit (C = 0.68)and below the critical 1% limit (C = 0.79), this indicates astraggler, while a value above the critical 1% limit indicatesan outlier. The calculated Cochran-value for sample D inlaboratory 2 was close (C = 0.65) to but below the critical5% limit (C = 0.68). Therefore neither outliers nor stragglerswere found in the data. No analytical problem during theexecution of the study was reported by laboratory 2 either.The Cochran-values for the other samples were below thecritical 5% limit (C = 0.24, 0.41 and 0.37, for samples A, Band C, respectively). It can be concluded that the within-laboratory variabilities observed for the four samples aresimilar for the participants.



Table 4. Contents (in%) of R-timolol in test samples A, B, C and D

Laboratoryno.

Sample A Sample B Sample C Sample D

Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

1 0.63 0.64 0.40 0.39 0.34 0.33 0.19a) 0.20a)

0.63 0.64 0.40 0.40 0.34 0.33 0.19a) 0.20a)

2 0.64 0.78 0.35 0.37 0.24 0.31 0.09 0.180.69 0.66 0.30 0.41 0.26 0.23 0.07 0.11

3 0.71 0.76 0.33 0.33 0.25 0.27 0.15 0.140.61 0.65 0.36 0.32 0.24 0.26 0.11 0.12

4 0.69 0.60 0.34 0.31 0.24 0.24 0.09 0.100.70 0.60 0.34 0.33 0.26 0.26 0.14 0.11

5 0.51 0.61 0.30 0.37 0.27 0.28 0.10 0.100.65 0.67 0.35 0.35 0.27 0.30 0.10 0.10

6 0.57 0.50 0.31 0.29 0.23 0.22 0.10 0.080.61 0.49 0.30 0.26 0.24 0.22 0.06 0.07

7 0.66 0.69 0.37 0.36 0.29 0.33 0.09 0.090.64 0.71 0.35 0.32 0.28 0.27 0.09 0.08

8 0.51 0.50 0.30 0.35 0.23 0.20 0.10 0.100.51 0.51 0.28 0.28 0.24 0.19 0.09 0.08

General mean 0.62 0.34 0.26 0.11

a) Straggler for between-lab variability.

To evaluate graphically the between-laboratory variability,Mandel’s h plots (Fig. 7) were used. In a normal result, theh values are randomly distributed around zero. Eitherpositive or negative distribution of all (or most) h results isan indication of an abnormal situation. As illustrated inFig. 7A, laboratories 2–5 and 7 have a random distributionof h values around zero, whereas laboratory 1 showedonly positive values and laboratories 6 and 8 only nega-tive. Again the variability was not related to the samples(Fig. 7B). This suggests the occurrence of a systematicerror in these laboratories. An explanation related to thisobservation might be the differences in data-samplingrate of the detectors (a factor not evaluated during meth-od development), which can affect the low R-timololcontent estimation even in the presence of an internalstandard [41, 43]. The numerical Grubb’s tests were usedto detect statistically outlying values. The results pre-sented in Table 5 indicate the presence of a straggler inlaboratory 1 for sample D. The stragglers were marked inTable 4 but were maintained for the determination ofuncertainty.

3.2.3 Calculation of the variance estimates

From the ANOVA equations (Table 1), the mean squaresfor the laboratories (MSLaboratories), days (MSDays) andreplicates (MSReplicates) were calculated. The estimatedvariance for laboratories (s2

Laboratories), days (s2Days) and

replicates (s2Replicates) were also determined. According to

ISO norms [33], calculations of repeatability (s2r), and re-

producibility (s2R) estimates are performed using the fol-

lowing equations:

s2r = s2

Replicates (6)

s2R = s2

Replicates 1 s2Laboratories (7)

In the present study and taking into account the setup ofthe study in Fig. 2, the analytical method execution wasperformed in 2 days allowing calculation of the estimatedvariance for replicates (s2

Replicates) and laboratories(s2

Laboratories) as well as for intermediate precision (s2T):

s2r = s2

Replicates (8)

s2T = s2

Days 1 s2Replicates (9)

s2R = s2

T 1s2Laboratories = s2

Replicates 1 s2Days 1 s2

Laboratories (10)

The results of the calculations which were done by meansof JMP software are presented in Table 6. It can be notedthat the contribution to the total variability observed in theR-timolol content results for all samples is mainly due to thecomponent “laboratory” (57, 53, 74 and 72% for sam-ples A, B, C and D, respectively). This indicates that thedeviations for the R-timolol content observed from onelaboratory to another are thehighest ones. According to theset-up of Fig. 2, this result was expected since the condi-



Figure 5. Typical electropherograms of test samplesolution A (A), test sample solution B (B), test samplesolution C (C) and test sample solution D (D) obtained bycarrying out the CE separation using a BGE containing30 mM HDMS-b-CD and applying a voltage of 25 kV (forother conditions, see Section 2). Peaks and concentra-tions (1) pyridoxine (5 mg/mL), (2) S-timolol (about 2.0 mg/mL) and (3) R-timolol; (A) = 12.4, (B) = 6.8, (C) = 5.2 and(D) = 2.2 mg/mL).

tions applied in one laboratory differ from that applied inanother, i.e. different equipments, operators and labora-tory conditions. However, the variability contribution ofthe component “day” was the lowest (12, 17, 3 and 5%for samples A, B, C and D, respectively), whereas that ofcomponent “replicate” was of second importance. Thispattern, which was not expected according to the set-upof Fig. 2, indicates that the contents of R-timolol in a lab-oratory were not much deviating from 1 day to another incomparison to the deviation of values observed between-laboratories. The fact that the between-day variance islower than the within-day variance is a good indication ofthe stability and reliability of the NACE method in a labo-ratory. On the other hand, the variance between-repli-cates was found to be illogically of second importance.Some values, such as those observed in the laboratories3 and 5 for sample A, are explaining this tendency whichwas already reported in a previous study [39]. Accordingto Horwitz [44], it is often expected to have the reproduc-ibility variance about two to four times higher than therepeatability variance. This means that the between-laboratories variance is similar in magnitude or larger thanthe repeatability variance. The ratio values were 3.2, 3.3,4.4 and 4.3 for samples A, B, C and D, respectively (seeTable 6). This situation is often observed when dealingwith impurities at very low concentrations as it is the casehere [37]. The expectations were satisfied in samples Aand B. According to ISO norms [34], one has to checkwhether there is a relationship between RSD and con-centration. In this study, the relationship between uncer-tainty and concentration was checked.

The reproducibility of variance allowed the calculation ofthe standard uncertainty �u�x using the equation:

�u�x ¼ffiffiffiffiffis2

R

q(11)

The standard uncertainty was ux = sR = 0.0799, 0.0407,0.0422 and 0.0410 for R-timolol content in samples A, B,C and D, respectively. The expanded uncertainty Ux

defines an interval around the measurement result whichcontains the unknown true value with a defined prob-ability level. Ux is obtained by multiplying the standarduncertainty ux by a coverage factor k. In this study, a valueof k = 2 [1, 3, 19] was considered, meaning that the truevalue is expected to be within the interval x 6 2ux with aprobability of 95%. The values of Ux were 0.1597,0.08142, 0.08446 and 0.08206 for R-timolol content insamples A, B, C and D, respectively. The nominal contentof R-timolol in those samples was 0.62, 0.34, 0.26 and0.11%, respectively (Table 4). Uncertainty was found tobe constant for samples B, C and D, while it was higherfor sample A. Indeed, this indicates that the uncertaintyseems to be affected not only by R-timolol content but



Figure 6. Plots of Mandel’sk statistics grouped per labora-tory (A) and per sample (B) forthe graphical evaluation ofwithin-laboratory consistency.

Figure 7. Plots of Mandel’sh statistics grouped per labora-tory (A) and per sample (B) forthe graphical evaluation of be-tween-laboratory consistency.



Table 5. Results of the Grubbs’ tests

Grubbs’tests

Calculated values Critical values r = 8

Sample A0.66%

Sample B0.35%

Sample C0.29%

Sample D0.10% 5% 1%

G1 1.78 1.85 1.88 1.31 2.13 2.27Gr 2.08 1.90 1.98 2.16 2.13 2.27G1,2 0.81 0.80 0.81 0.89 0.11 0.06Gr,r 2 1 0.75 0.78 0.73 0.68 0.11 0.06G 9.93 9.04 0.84 8.19 64.5 74.9Outliers – – – Straggler in

laboratory 1

– = No outliers nor stragglers.

Table 6. Estimation of the variance components

Sources of variability Sample A 0.66% Sample B 0.35% Sample C 0.29% Sample D 0.10%

Variancecomponent

Laboratories (s2Laboratories)

Days (s2Days)

Replicates (s2Replicates)

36.1161024

7.6461024

20.0461024

8.7661024

2.8161024

5.0161024

13.2761024

0.4761024

4.0961024

12.0961024

0.8161024

3.9461024

Repeatability variance (s2r)

Reproducibility variance (s2R)

Ratio reproducibility/repeatability

20.0461024

63.7861024

3.2

5.0161024

16.5761024

3.3

4.0961024

17.8361024

4.4

3.9461024

16.8361024

4.3

also up to a certain level by other uncertainty contribu-tions. In a collaborative study applying an LC method andanalyzing similar S-timolol maleate samples to those ofthis study [39], the uncertainty was found to be con-centration dependent as observed for samples A and B.In addition, the uncertainty values found were very similarin magnitude to those obtained using the present NACEmethod. However, in our previous work [27], it was foundthat the uncertainty of the R-timolol content obtainedfrom validation data of only one laboratory was con-centration dependent.

Finally, for a single result x obtained when applying theNACE method, the result for R-timolol content in sampleswith concentrations similar to sample A (i.e. about 0.62%)is expected to be x 6 0.16% while for samples containingR-timolol at about half concentration (i.e. 0.34% in sam-ple B), it is expected to be about half (x 6 0.081%); i.e.95% of the reported values will be within 0.500–0.819%and 0.265–0.428%, respectively. The result for R-timololcontent in samples with concentrations less than 0.35%,i.e. similar to samples C and D (contents 0.26 and 0.11%,respectively), the result is expected to be x 6 0.08% forboth, i.e. 95% of the reported values will be within 0.206–0.375% and 0.018–0.182%, respectively. The measureduncertainty was found to be rather high compared to the

contents of R-timolol (Table 4). This situation was notabnormal as far as the impurities are concerned. In aprevious interlaboratory study dealing also with impuritiesand applying an LC method [39], similar situation wasobserved. As can be seen in Table 4, the measurementsfulfiled this expectation since measurements outside ofthese ranges occurred only in 1 out of 32 times for theR-timolol concentration of samples A and B, and 2 out of32 times for sample C. However, for sample D (R-timololimpurity content near the LOQ) measurements outsidethe expected range of variability occurred 4 out of32 times.

It can be concluded that the method is very useful todetermine R-timolol impurity in S-timolol maleate sam-ples but at concentrations above 0.3%. The method isstill useful when considering the maximum toleratedcontent (1.0%) of R-timolol in S-timolol maleate asreported in the European Pharmacopoeia [35].

If a laboratory wants to evaluate its uncertainty from threereplicates under repeatability conditions when determin-ing the content of R-timolol in a S-timolol maleate samplewith a concentration similar to sample A (0.62%), thestandard uncertainty of the mean result will be



u�x ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffis2

replicates

3þ s2

T þ s2laboratories

s

¼ 0:0710 (12)

Then, the expanded uncertainty will be Ux = 0.142 andthe mean result written as x 6 0.14%. On the other hand,if the same laboratory performs three replicate analyses in3 days, then, the standard uncertainty of the mean resultbecomes

u�x ¼


replicates

3� 3þ s2

T

3þ s2

laboratories

s

¼ 0:0639 (13)

with an expanded uncertainty of Ux = 0.128. The meanresult will be written as x 6 0.13%.

On the other hand, when two laboratories perform theanalysis in triplicate under repeatability conditions, thestandard uncertainty of the mean result will be

u�x ¼


replicates

3� 2þ s2

T þs2

laboratories

2

s

¼ 0:0539 (14)

The expanded uncertainty is reported as Ux = 0.108 andthe mean result as x 6 0.11%. It can be remarked that, inthe three cases, the observed uncertainties were notconsiderably improved.

In the same manner, the standard and the expandeduncertainties can be calculated for R-timolol content insamples with concentration similar to B, C and D.

4 Concluding remarks

An interlaboratory uncertainty assessment was success-fully applied to a CD based NACE method for the enan-tiomeric separation and determination of R-timololimpurity in S-timolol maleate samples. As far as we know,it is the first time that an interlaboratory study has beencompletely achieved in CE allowing to report uncertaintymeasurement results. The method was successfullytransferred between equipments from the same manu-facturer but not to other instruments. The qualitative per-formance and the suitability of the method were judged tobe acceptable when analysis were conducted using rela-tive migration times and normalised peak areas. In aninterlaboratory study, on the 11 participating laboratories,only eight laboratories reported the contents of R-timololat four different levels of impurity in S-timolol samples.The estimated uncertainty of R-timolol seemed to beconcentration dependent at high enantiomeric impuritycontents, i.e. above a given concentration, while at lowimpurity contents, such a dependence was not observed.However, the method is very useful to determine R-timolol

impurity in S-timolol maleate samples but at concentra-tions above 0.3%. The method is still useful when con-sidering the maximum tolerated content (1.0%) of R-timolol in the S-timolol maleate as reported in the Euro-pean Pharmacopoeia.

The authors wish to thank the Belgian Government (ThePrime Minister Services – Federal Office for Scientific,Technical and Cultural Affairs, Standardisation Pro-gramme) for the financial support of this project (No.NM/12/23). Research grants from the Belgium National Fundfor Scientific Research (FNRS) to A.-C. Servais andM. Fillet are gratefully acknowledged. Many thanks arealso due to FNRS and to the Léon Fredericq foundationfor financial support. A research grant from the WalloonRegion and the European Social Fund to E. Rozet isgratefully acknowledged (First Europe Objective 3 projectno. 215269). The authors are also grateful to the EuropeanPharmacopoeia Secretariat, Prosintex Industrie ChimicheItaliane (Milan, Italy) and Merck for kindly providing thesamples.

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