CITOQUININAS HPLC

8/13/2019 CITOQUININAS HPLC

1/13

Analytical methods for cytokininsPetr Tarkowski, Liya Ge, Jean Wan Hong Yong, Swee Ngin Tan

The development of sensitive analytical methods for the determination of

cytokinin levels in plant tissues is essential for elucidating the roles of

cytokinins in life science. In the past decade, more advanced analytical

systems have been applied for the rapid analyses of endogenous cytokinins.

This review primarily focuses on emerging analytical methods designed to

meet the requirements for cytokinin analyses in complex matrices with

special emphasis on high-performance liquid chromatography, gas chroma-

tography and capillary electrophoresis coupled with different detectors. We

highlight the advantages and the limitations of the techniques. As sample

preparation is a key factor in determining the success of cytokinin analyses,

we devote a section to discussing sample-extraction and clean-up techniques

prior to analysis.

2008 Published by Elsevier Ltd.

Keywords: Analysis Capillary electrophoresis; Cytokinin; Complex matrix; Gas chroma-

tography; Liquid chromatography; Mass spectrometry; Immunoassay; Sample preparation

1. Introduction

Cytokinins are a group of plant hormones

[1,2]. Certain cytokinins (e.g., kinetin and

zeatin) show significant anti-ageing, anti-

carcinogenic, and anti-thrombotic effects

[3,4]. Rapid analyses of cytokinins are

therefore of great importance to plant

physiologists and scientists from other

disciplines, especially in view of their

potential role in suppressing mammalian

tumor growth.

Chemically, most naturally occurring

cytokinins are adenine derivatives and can

be classified by the configuration of their

N6-side chain as either isoprenoid or aro-

matic (Table 1). Since plant-tissue extracts

are rather complex multi-component

mixtures, and cytokinins typically occur intrace quantities (usually at levels below

30 pmol/g of fresh weight [1,2]), suffi-

ciently sensitive and selective analytical

tools are required for determination of

their endogenous levels. Regardless of the

analytical method used, highly purified

plant samples are preferred for the analy-

ses[5].

Traditionally, quantification of cyto-

kinins has been performed using bioassays

and immunoassays. Although bioassays

are essential for the isolation of novel

compounds, they are time-consuming and

not very accurate. Immunoassay tech-

niques can be used as a sensitive, viable

alternative for determination of cytokinin

levels[6]. The two most common forms of

cytokinin immunoassay are radioimmu-

noassay (RIA) [7] and enzyme-linked

immunosorbent assay (ELISA) [8]. How-

ever, scintillation proximity assay (SPA) is

also gaining ground [9,10]. Recently,

ELISA has overtaken RIA. In addition, by

taking advantage of the highly specific

structural requirements of antibodies (Abs)

for binding, the detection of cytokinins in

plant tissues by immunolocalization offers

a powerful tool to study the distribution of

these signaling molecules[11].

Despite widespread applications, immu-

noassays sometimes suffer from problems

(e.g., cross-reactivity, imperfect validation,

and variable results for plant sample anal-

yses). Other common analytical methods

are physico-chemical techniques {gas chromatography (GC) [12], high-performance

liquid chromatography (HPLC) [13,14]

and capillary electrophoresis (CE)[15]}.

For biological samples, HPLC is still the

major tool in analyses and purification of

cytokinins.

As a complementary method, CE is also

currently available for cytokinin analyses.

In some cases, CE can have distinct

advantages over HPLC in terms of sim-

plicity, rapid method development, and

reduced cost of the operation, since packed

columns, reciprocating pumps and mobile-

phase gradient are not used[16,17].

Recent advances in instrumentation

[e.g., ultra-performance liquid chroma-

tography (UPLC)] and the range of

detectors available have enabled analytical

scientists to measure and to identify

cytokinins at trace concentrations[18,19].

This review summarizes the current

key methods for cytokinin analyses as

follows:

Petr Tarkowski

Department of Biochemistry,

Faculty of Science;and,

Laboratory of Growth

Regulators, Institute of

Experimental Botany ASCR

Palacky University, Slechtitelu

11, Olomouc, CZ-783 71

Czech Republic

Liya Ge,

Jean Wan Hong Yong,

Swee Ngin Tan*

Natural Sciences and Science

Education Academic Group,

Nanyang Technological

University, 1 Nanyang Walk,

Singapore 637616

*Corresponding author.

Fax: +65 68969432;

E-mail:

[email protected]

Trends in Analytical Chemistry, Vol. 28, No. 3, 2009 Trends

0165-9936/$ - see front matter 2008 Published by Elsevier Ltd. doi:10.1016/j.trac.2008.11.010 3230165-9936/$ - see front matter 2008 Published by Elsevier Ltd. doi:10.1016/j.trac.2008.11.010 323
mailto:%[email protected]:%[email protected]


2/13

Table 1. Structures, names and abbreviations of cytokinins

N

NN

N

HN R1

R2

1

2

3

49

8

7

56

R1 R2 R3 Compound Abbreviation

H2C CH3

CH2OR3H H trans-zeatin ZR H trans-zeatin riboside ZRG H trans-zeatin 9-glucoside Z9G H trans-zeatin 7-glucoside* Z7GH G trans-zeatin O-glucoside ZOGR G trans-zeatin riboside O-glucoside ZROG

RP H trans-zeatin riboside-5 0-monophosphate ZMP

CH3

CH2OR3H2C

H H cis-zeatin cZR H cis-zeatin riboside cZRRP H cis-zeatin riboside-5 0-monophosphate cZMP

H2C CH3

CH2OR3

H H dihydrozeatin DZR H dihydrozeatin riboside DZRG H dihydrozeatin9-glucoside DZ9GH G dihydrozeatinO-glucoside DZOGR G dihydrozeatin riboside O-glucoside DZROGRP H dihydrozeatin riboside-50-monophosphate DZMP

H2C CH3

CH3

H isopentenyladenine iPR isopentenyladenine riboside iPRG Isopentenyladenine 9-glucoside iP9GRP isopentenyladenine riboside-50-monophosphate iPMP

H2C H benzylaminopurine BAR benzylaminopurine riboside BARG benzylaminopurine 9-glucoside BA9GRP benzylaminopurine-5 0-monophosphate BAMP

H2C

HO

H ortho-topolin oTR ortho-topolin riboside oTRG ortho-topolin9-glucoside oT9G

H2C

OH

H meta-topolin mTR meta-topolin riboside mTRG meta-topolin 9-glucoside mT9G

H2C OH H para-topolin pT

R para-topolin riboside pTRG para-topolin 9-glucoside pT9G

H2C

O

H kinetin KR kinetin riboside KRRP kinetin riboside-50-monophosphate KMP

H, Hydrogen; R, b-D-ribofuranosyl; RP, b-D-ribofuranosyl-5 0-monophosphate; G, b-D-glucopyranosyl.*In Z7G, b-D-glucopyranosyl group is substituted at N7.

Trends Trends in Analytical Chemistry, Vol. 28, No. 3, 2009

324 http://www.elsevier.com/locate/trac


3/13

sample preparation (Section 2);

techniques for analysis, including GC, HPLC,

UPLC, CE and immunoassay (Section 3); and,

concluding remarks and the future prospects

(Section 4).

2. Sample preparation

Sample preparation is a key procedure in any modern

chemical analyses, especially for analytes present at

trace or ultra-trace levels in complex matrices. The

procedures used for cytokinin extraction and sample

preparation are therefore critically important steps in the

entire analytical process. Fig. 1 shows the comprehen-

sive procedures used for extraction, pre-concentration

and purification of cytokinins.

2.1. Extraction techniques

The qualitative and the quantitative aspects of cyto-

kinins extracted from plant tissues vary with the types of

extraction solvents and procedures employed [20]. In

order to prevent any enzymatic degradation of cyto-

kinins (e.g., conversion of cytokinin nucleotide to its

riboside), plant material should be immediately frozen or

extracted with proper solvent(s) after harvesting. Isola-

tion and identification of picomolar quantities of cyto-

kinins are often hampered by the other biological

materials present in excess in the sample.

Typically, plant tissue is frozen in liquid nitrogen and

left in Bieleskis solvent, comprising methanol/chloro-

form/water/formic acid (12/5/2/1, v/v/v/v)[21]. Hoye-rova et al. [22] compared the extraction efficiency of

three different extraction solvents: (a) 80% (v/v) metha-

nol; (b) Bieleskis solvent; and, (c) modified Bieleskis

solvent (methanol/water/formic acid; 15/4/1, v/v/v). It

was found that the modified Bieleskis solvent sufficiently

suppressed the dephosphorylation of cytokinin mono-

nucleotides and gave the highest responses for deuterated

cytokinins (used as test compounds) in plant extracts.

2.2. Liquid-liquid partition

During liquid-liquid partitioning, the plant material is

homogenized in liquid nitrogen, and cytokinins are ex-tracted with cold acetone. After centrifugation, the

supernatant is evaporated in vacuo to dryness. The

residue is dissolved in lukewarm (38C) acidified water

(pH 3.5). A triple extraction of an aqueous solution of

cytokinins with butanol saturated with acidified water

removes chlorophyll and other impurities, along with

small traces of cytokinins[23].

2.3. Solid-phase extraction

Solid-Phase Extraction (SPE) has been widely applied for

purification, extraction and isolation of many com-

pounds [16,2426]. One distinct advantage of SPE is

that high extraction recovery can usually be obtained

with a suitable sorbent and operating procedure, even

under situations when other traditional extraction

techniques have failed.

Pre-concentration of cytokinins has commonly been

achieved using SPE with C18cartridges. A more effectivebut more complex approach is to purify plant extracts by

passing them through linked columns of polyvinylpoly-

pyrrolidone powder, DEAEcellulose or DEAE-Sephacel,

and C18-SPE [5]. Fast, efficient separation of cytokinins

has been achieved using mixed-mode SPE with both

reversed-phase (RP) and ion-exchange characteristics

[24].

After C18 SPE cartridges were used as a pre-concen-

tration tool, further sample purification could be carried

out using mixed-mode cation exchanger (MCX) SPE

cartridges with good recoveries [16]. Purification of

cytokinins using MCX SPE, as compared to DEAE

Sephadex and C18SPE method, was suitable for removalof Ultraviolet (UV) absorbing contaminants with higher

recoveries of cytokinins [22].

However, due to the relatively high polarity of cyto-

kinin nucleotides leading to poor retention by C18cartridges during SPE, the use of an anion-exchange

sorbent in addition is an efficient alternative step to

separate cytokinin nucleotides from cytokinin bases

and sugar conjugates [25]. An efficient dual-step SPE

method has therefore been developed for pre-concen-

tration and purification of cytokinin nucleotides using

Oasis HLB and MAX cartridges [26].

2.4. Immunoaffinity purification

Immunoaffinity purification methods, based on anti-

body-antigen (Ab-Ag) interactions, can provide selective

sample enrichment, and thus greatly enhance limits of

detection (LODs) of trace analyses [27], so, when an

immunoaffinity approach is used as the purification step

before final analysis, highly purified cytokinin prepara-

tions containing only traces of other UV-absorbing

material can be obtained.

Several papers have reported on the immunoextrac-

tion of cytokinins using monoclonal and polyclonal

antibodies (mAbs and pAbs) [27,28]. The set-up for asuitable affinity system requires an appropriate Ab, and

consideration of matrix factors that are not specific to

phytohormone analysis [28]. Generic cytokinin mAbs

are frequently used in this respect [13,27]. Immunoaf-

finity columns purify analytes according to structural

similarities, so they hold promise for the discovery of new

cytokinins [28]. Immunoextraction has higher selectiv-

ity than conventional SPE, but low throughput. An

efficient, off-line, batch-immunoextraction method was

developed for the purification of new cytokinins and

their ribosides[27].


http://www.elsevier.com/locate/trac 325


4/13

Octadecyl C18Cartridge

Wash by acidified

water (pH 3.0)

Elution by ethanol;

water: acetic acid(80:20:1)

Flow through

(Some CKnucleotides may be

lost here)

Elution by 0.35 M

NH4OH in 60%

CH3OH

CK bases,

ribosides,

glucosides

Oasis MCX Cartridge

Evaporate and

redissolve 1 M HCOOH

Elution by 0.35 M

NH4OHCK nucleotides

(not full complement)

Wash by CH3OH

IAA, ABA

Oasis H

Elut

Eva

redissolv

Oasis M

Wash by 1 M

NH4OH

Extraction (Bieleskis solution or

modified Bieleskis solution)

-20C

PolyclarVT Cartridge

DEAE-Sephacel Cartridge

Plant materials

Flow-through fraction

Flow-through fraction

CK bases, ribosides,glucosides

Elution by 1 M

NH4HCO3

CK nucleotides

Immuno-affinity Column Chromatography

CK

mononucleotides

CK

dinucleotides

CK

trinucleotides

Dephosphorylation by treatment with phosphatase

Hydrolysis glucosides

by -glucosidaseMonoQ Column

Remove CH3OH, pH adjust to 1.8

4C

Remove CH3OH, pH adjust to 3.025C

W

N

Figure 1. Procedures used for extraction, pre-concentration and purification of cytokinins.

326

http://www.elsevier.

com/locate/trac


5/13

3. Techniques for analyses of cytokinins

3.1. Gas Chromatography

GC-based methods provide high resolution and low

LODs, but they are labor-intensive and costly. GC has

been used for the analyses of cytokinins since the early

1970s [20,29]. As cytokinins are not volatile com-pounds, they have to be derivatized to increase their

volatility and also to improve their thermal stability prior

to GC analysis. Derivatization methods have some

inherent technical problems (e.g., hydrolysis of the

derivatives, multiple product formation, and limited

volatility[30,31]). However, GC with mass spectrometry

(GC-MS) was a reliable, specific means for identification

and quantification of cytokinins, until the recent intro-

duction of using a combination of HPLC-MS. Structures

of almost all naturally-occurring cytokinins were eluci-

dated by GC-MS before the 1990s [20,29].

3.1.1. Derivatization methods. Derivatization procedures

for GC-MS analyses have been well described: silylation

(trimethylsilyl) with N-methyl-n(trimethylsilyl)trifluoro-

acetamide in 50% pyridine containing 1% trimethyl-cholorsilane for hours at room temperature, or a shorter

time at 80C. This approach gives good results with an

LOD of about 10 lg of anhydrous cytokinins [29].

Complications have been associated with derivatiza-

tion of cytokinins for GC-MS analysis using trimethylsilyl

[20,32], permethyl [33], t-butyldimethylsilyl [34],

trifluoroacetyl [35] and acetyl [12] derivatives. Penta-

fluorobenzyl derivatives of cytokinin free bases for

negative-ion MS were reported by Hocart et al. [36]. A

Figure 2. HPLC chromatograms of standard mixtures of (A) 20 and (B) 18 cytokinins (Adapted from [13,39], with permission).




6/13


7/13

novel method based on N-methyl-N-(tert-butyldimethyl-

silyl)trifluoroacetamide as a derivatization reagent in a

comprehensive chemical derivatization protocol for the

GC-MS analysis of cytokinins and other phytohormones

has also been reported [37].

3.1.2. Chromatographic conditions and detection. The

separation conditions have not changed much since the

1970s, although currently fused-silica capillary columns

are used instead of packed glass columns. The separation

power of capillary GC and the selectivity of MS detection

make GC-MS a powerful technique for cytokinin

analyses[29]. The main advantage of using the GC-MS-

based identification, compared with the other soft-ioni-

zation techniques, is differentiation of the various sugar

moieties. Besides MS detection, the use of two other

detectors, namely flame-ionization detector (FID) and

electron-capture detector (ECD), has been reported

[12,33]. The main drawback of FID is limited sensitivitythat makes it inapplicable in the trace analysis of cyto-

kinins. Even though ECD is quite sensitive, halogenated

derivatives are required in order to obtain satisfactory

LODs.

3.2. High-performance liquid chromatography

HPLC is a particularly suitable chromatographic tech-

nique for cytokinin analyses, as cytokinins exhibit gra-

dations in polarity and are readily detected by UV

absorbance [38]. HPLC enables rapid, high-resolution

purification of cytokinins from plant extracts prior to

analysis by MS, immunoassay, or bioassay. HPLC anal-

ysis alone can also provide reliable identification of

cytokinins in plant extracts. However, as absorbance at a

single UV wavelength is inadequate for this purpose, the

most widely used procedure for the quantifying cyto-

kinins is isotope-dilution MS, especially with LC-Elec-

trospray Ionization-MS (LC-ESI-MS)[13,14].

3.2.1. Chromatographic conditions and separation. Cyto-

kinin-free bases and their sugar conjugates are relatively

hydrophobic compounds that behave like weak bases, so

they are well separated on RP columns under acidic

conditions [20]. However, the more ionic cytokinin

nucleotides are not so well separated by RP-HPLC.Typically, the nucleotides are converted to ribosides with

alkaline phosphatase [14], or derivatized [25] to lower

their polarity, before RP-HPLC separation.

Nowadays, separation and analyses of cytokinins by

HPLC are carried out using RP-C18 or C8 columns

[13,39,40]. The volatile eluent additives (e.g., acetic/

formic acid and their ammonium salts) are usually

added to solvents containing aqueous methanol/

acetonitrile [39,40]. To achieve good separation, gradi-

ent elution by increasing content of organic modifier is

often used [3941]. For preparative purification ofPropionylatedoTOG,

2MeSoTOG

andBA9G

Chenopodium

rubrumcells

SCXSPEandC

18

SPE.

Alkalinephosphatase

treatmentofnucleotides.

CapillaryLCcolumn

(150

0.3mmp

ackedwith

4lm

Symmetry

ODS

packingmaterial)

50%

aqueousACN,1%

glycerol,1%

formicacid

05min,20

lL/min;

545min,4

.5lL/min

Isocratic

frit-FAB

D

oublefocusing

m

agneticsector

[47]

pT,mT,pTR,oT,mTR,BA,

MeoT,MemT,oTR,BAR,MeoTR

andMemTR

Arabidopsistha

liana

and

Populus

canadensis

leaves

Octadecyl-silicaSPE,

DEAE-SephadexSPE,C18

SPE,andIACAlkaline

phosphatasetreatmentof

nucleotides

SymmetryC

18column

(3.5lm,

150mm

2.1mm)

A:MeOH;B:0.1%

formic

acidadjustedtopH2.9

withammonium

0.25mL/min

(25%

effluent

wasintroducedintoESI

source)

Gradient05min,

3015%

A;525min,

1540%

A;

2530min,40%

A

ESI

Si

nglequadrupole

[48]

PropionylatedpT,mT,pTR,oT,

mTR,BA,MeoT,MemT,oTR,

BARandMeoTR

CapillaryLCco

lumn

(150

0.3mm

packedwith4lm

SymmetryODS

packingmateria

l)

55%

aqueousACN,1%

glycerol,1%

formicacid

05min,20lL/m

in;

565min,4.0lL

/min

Isocratic

frit-FAB

Doublefocusing

magneticsector

Z,ZR,Z7G,Z9G,DZ,DZR,iP,

iPR,iP9G,ZOG

andZROG

Macadamia

integrifolia

C18

SPEAlkaline

phosphatasetreatmentof

nucleotides

C18

column(3l

m,

20mm

2.1mm

)Guard

column(C18,5lm,

4mm

2.0mm)

A:10mM

ammonium

acetate;B:350mLMeOH,

100mLACN,50mL,

10mM

ammonium

acetate

0.1mL/min

Gradient03min,

510%

B;327min,

1043%

B;

2730min,4380%

B;3033min,

80100%

B

API

Q

-TOF

[49]

2MeSoTOG,6-[2-(

b-D-glucopyranosyloxy)benzylam

ino]-2-methylthiopurine;MeoT,ortho-methoxytopolin;M

emT,meta-methoxytopolin.




8/13

Table 3. Optimal separation conditions for cytokinins using different CE approaches[15,53]

Analytes Sample matrix Samplepreparation

Mode Buffer Capillarydimensions

Separationvoltage

In

iP, iPR, Z, ZR,DZ,DZR, BA andBAR.

STD sugar beet SPE CZE 150 mMphosphoricacid, pH 1.8

77 cm (effectivelength61 cm) 75 lm

20 kV 10

STD, tobacco MEKC 20 mM SDS,50 mM borate,pH 9.2

10

BA, BA9G, BAR,mTR, oTR, KR, ZR,DZR, iP and iPR.

STD NS CD-modifiedCZE

100 mMphosphate-Tris(pH 2.5) bufferwith 25 mM c-CD

47 cm (effectivelength 40 cm)50 lm.

20 kV N

BA, K, and otherplant hormonesincluding ABA,IAA, NAA, GA and2,4-D.

STD, transgenictobacco flower

LLE MEKC 50 mM boratecontaining50 mM SDS,pH 8.0

48.5 cm (40 cmeffectivelength) 50 lm.

15 kV 55

Z, DZ, ZOG,DZOG, mTR,iP and BA

STD, coconutwater Dual-stepSPE MEKC Combinationof 10 mMphosphate and10 mM boratebuffercontaining50 mM SDS,pH 10.4


15 kV 5p0

oT, mT, pT, oTR,mTR and pTR

STD, coconutwater

Dual-stepSPE

MEKC 20 mM boricacid and50 mM SDS,pH 8.0, with anextra 20% (v/v)MeOH added


15 kV 5p0

K and KR STD, coconutwater

Dual-stepSPE

CZE 100 mMammoniumphosphatebuffer, pH 2.5

40 cm (effectivelength 30 cm)76 lm

15 kV 5p0

330

http://www.elsevier.

com/locate/trac


9/13

iP, DZ, Z, BA, K, oT,DZOG, ZOG, DZR,ZR, oTR and KR

STD, coconutwater

Dual-stepSPE

CZE 25 mMammoniumformate/formicacid buffer(pH 3.4) and3% ACN (v/v)

65 cm 50 lm 25 kV Osastin

DZMP, iPMP,cZMP, ZMP, BAMPand KMP

STD, coconutwater

dual-stepSPE

CZE 25 mMammoniumformate/formicacid buffer(pH 3.8) and2% MeOH (v/v)

57 cm 50 lm Gradientseparationvoltage (25 kVfor 32 min, andthen lineargradient to30 kV in 5 min,finally 30 kV toend ofseparation)

Osastin

oT, mT, pT, oTR,mTR, pTR, oT9G,mT9G, pT9G, ZR,cZR, Z and cZ

STD, bananapulp

Dual-stepSPE

Partialfilling-MEKC

50 mMammoniumformate/ammoniumhydroxide atpH 9.0.

Micellarsolution with70 mM ALSand 10%MeOH wasinjected for90 s at 50 mbarbefore thesample and120 s at50 mbar afterthe sample

100 cm 50 lm 20 kV Osastin

STD, Standard; NS, not stated; GA, Gibberellic acid; ABA, Abscisic acid; IAA, Indole-3-acetic acid; NAA, a-naphthalene acetic acid; 2,4-D, 2,4-di

http://www

.elsevier.com/locate/trac

331


10/13

cytokinins, a column size of 150 10 mm i.d. can be a

good compromise between cost and sample-loading

capacity [20]. HPLC columns with i.d. ranging from

conventional (4.6 mm), narrow-bore (2.1 mm), micro

(1 mm) to capillary columns (0.3 mm)[3941]are used

for cytokinin analyses.Fig. 2shows representative HPLC

chromatograms of cytokinins.As the best performance of early ESI interfaces was at

flow rates of around 0.55 lL/min[42], the process was

compatible with chromatographic miniaturization, so

narrow-bore LC coupled to MS was used as a relatively

new method for the analyses of cytokinins [13].

Coupling capillary LC to ESI-MS significantly improves

mass sensitivity[43]. However, it needs to be taken into

account that down-scaling decreases injection volume,

loading capacity and dynamic range. The problem of

restricted injection volume, which is critical in analyzing

biological samples, could be overcome by using both on-

pre-column and on-chromatographic column trace

enrichment. Sample loading is performed undernon-eluting conditions using a pre-column or directly

(analytical column), which is followed by backflush

(pre-column) or direct gradient elution (analytical

column). Prinsen et al. [44] comprehensively compared

important analytical parameters (sensitivity, linearity

range, robustness, sample throughput) of conventional,

micro and capillary LC combined with tandem MS (MS2)

detection.

3.2.2. Detection. As cytokinins exhibit strong UV

absorbances between 220 nm and 300 nm, UV detection

is suitable for their quantification [20]. Coincidentally,

the UV-visible (UV-VIS) absorbance detector is the most

widely used detector for HPLC, so HPLC-UV is widely

used to separate and detect cytokinins. For example,

HPLC fractions collected after chromatographic separa-

tion are analyzed by bioassays, immunoassays or con-

verted to volatile derivatives for GC analysis. However,

using this non-specific UV-absorbance method for

detection requires significantly higher amounts of sam-

ple that needs extensive purification.

The LC-MS approach offers a new tool to detect,

quantify and characterize cytokinins in plant-tissue

extracts at biologically meaningful levels. Furtherimprovement in LC systems as well as mass analyzers

may overcome the low detectability of cytokinins.

Different ionization techniques were used for MS analy-

ses of cytokinins in combination with RP-HPLC,

including thermospray (TS) [45], fast atom bombard-

ment (FAB) [4648], atmospheric pressure ionization

(API) [19,49], and ESI [13,14,18,19,43,44,48,50].

Although use of frit-FAB MS has been reported [46

48], ESI-MS is currently the most common LC-MS

method in cytokinin analyses. Compared to frit-FAB LC-

MS, ESI-MS has a fairly high sensitivity and is associated

with lower background [13,14,18,19,43,44,48,50]. In

1997, the first application of LC-ESI-MS2 with multiple

reaction monitoring (MRM) for cytokinin determination

was reported as a fast method for the quantification of

16 different cytokinins with an LOD of 1 pmol [50].

Subsequently, improved gradient elution together with a

capillary column provided an LOD at low-fmol levels[44].

It is obvious that the main advantage of the LC-MS

approach over that of GC-MS is elimination of the

derivatization step. However, in order to increase

sensitivity, pre-column derivatization for LC-MS cyto-

kinin analyses was used to give stronger quasi-

molecular ion currents and to obtain more spectral

information [14,43,4648]. Table 2 presents a sum-

mary of some representative LC-MS methods for cyto-

kinin analyses.

3.3. Ultra-performance liquid chromatographyUPLC instrumentation can provide liquid flow at pres-

sures of up to 1000 bar, and features columns packed

with 1.7-lm particulate packing materials, so UPLC

extends beyond the chromatographic limits of conven-

tional HPLC instrumentation. UPLC can achieve higher

resolutions, lower sensitivities and more rapid separa-

tions [18,19].

UPLC-MS provides significant advantages concerning

selectivity, sensitivity and speed, and is undoubtedly a

suitable system for the study of cytokinins [18,19,51].

Schwartzenberg et al. successfully applied an efficient

UPLC-MS2 method to establish the profile of 40 cyto-

kinins found within bryophyte Physcomitrella patens[18]. Dolezal et al. also applied UPLC-MS2 to isolate new

cytotoxic members of aromatic cytokinins present

endogenously in extracts of Arabidopsis thaliana and

Agrobacterium tumefaciens [19]. Recently, Novak et al.

developed an efficient UPLC-MS2 method for cytokinin

profiling in plant tissues, which is almost four-fold faster

than the standard HPLC analysis[51].

3.4. Capillary electrophoresis

CE is particularly suitable for the analyses of cytokinins,

due to its speed, high resolving power, and minimal

requirements for sample and buffer [1517]. Althoughsome successful examples have been reported, the LOD of

CE is somewhat higher that the LODs of HPLC and GC,

which is a consequence of lower amounts of sample in-

jected during analysis and also in having a shorter

optical path length, so most CE applications in cytokinin

analyses require sensitivity to be enhanced by using

more specific detection systems (e.g., MS) and on-line or

off-line sample pre-concentration to increase sample-

solute concentration [26,52]. Ge et al. [15] compre-

hensively reviewed information pertaining to this aspect

of cytokinin analyses.




11/13


12/13

3.5. Immunoassays

Prior to the introduction of hyphenated techniques

(e.g., LC-MS or GC-MS), immunoassay techniques were

the methods of choice for trace analysis of phytohor-

mones due to their low LODs [610]. Several quanti-

tative immunological methods were developed to detect

cytokinins. Despite having some problems associatedwith immunological methods, a few reports have

shown that Abs are useful tools to detect trace cyto-

kinins in plant tissues, and even at the ultra-structural

level of cells. Immunolocalization of endogenous cyto-

kinins provides complementary insights into their

involvement at the cellular level [11].

Immunoassay techniques (e.g., RIA, ELISA and SPA)

can be used as sensitive, viable options for the deter-

mination of cytokinins [610]. RIAs are very sensitive

methods, which can detect nmol or pmol of molecules

and have provided useful information on the

biochemical processes dealing with ligand-receptor

systems [7,54]. Due to the strong cross-reactions, pAbsfor RIA cannot be used directly on individual

cytokinins in crude plant extracts, so substances

interfering with RIA should be removed from the ex-

tracts [54].

Compared with RIA, ELISA is less expensive and

easier to set up; moreover, the problems associated

with the disposal of radioactive waste can be avoided.

For the detection of cytokinins, avidin-biotin amplified

ELISA, immunoaffinity purification and immunocyto-

chemical techniques have been developed [8,55].

ELISA detects cytokinins in the fmol range.

Hapten-homologous and hapten-heterologous competi-tive ELISAs were developed for detecting endogenous

cytokinin levels in crude plant extracts without

intense purification so they needed less plant extract

[55]. ELISAs are still widely used for cytokinin anal-

yses.

SPA is a novel radioisotopic technique, applicable to

assays involving ligand-Ab binding, which eliminates

the need to separate free and bound ligand, and to use

scintillation fluid as required in conventional RIA [9,10].

First described by Wang et al. [9], SPA is a sensitive

assay for the quantification of cytokinins as free bases at

concentration of less than 0.02 ng. Yong et al. used the

SPA method to measure the distribution of cytokinins in

cotton leaves and xylem sap[10]. Unfortunately, SPA is

not yet widely used by the research community for the

routine analysis of cytokinins.

A disadvantage of the cytokinin immunoassays,

compared with LC-MS, is that they estimate the com-

bined content of similar groups (free bases, ribosides, 9-

glucosides and nucleotides) of cytokinins, due to their

lower specificity, rather than specific cytokinins [610].

However, the effective range and the sensitivity of

immunoassays are similar to those reported for LC-MS.

4. Conclusions and perspectives

We have addressed recent trends in separation and

determination of cytokinins. Since free cytokinins pres-

ent in plants are at extremely low levels, we also

summarized the comprehensive sample-preparation

steps prior to cytokinin analyses.Each method that can be used for cytokinin analyses

has its own sensitivity and selectivity. Most recently

published papers on cytokinins were based on

applications of GC, LC, and CE. Recent literature also

indicated that MS2 combined with GC [12], LC[13,14]

or CE [15] would provide more convincing and satis-

factory results for cytokinin analyses in most cases.

From these results, we conclude that MS has rapidly

become a highly sensitive, selective tool for cytokinin

analyses. Its use with GC, LC or CE ensures more

reliable detection, identity confirmation and quantifi-

cation of cytokinins, as well as screening for potentially

novel cytokinins.As an established classical approach, immunoassay

(especially ELISA) is still commonly used as a sensitive,

viable method for the determination of endogenous

cytokinins [611]. Furthermore, immuno-chemical

staining using appropriate Abs appears to be the only

means of getting information about endogenous cyto-

kinin distribution at cellular and sub-cellular levels

[11].

We anticipate that LC-MS2 will continue to play an

important role in cytokinin analyses in the near future.

Next to excellent sensitivity, this technique can provide

structural information based on the fragmentationpatterns. Unlike the well-established GC-MS method, LC-

MS2 can analyze cytokinins without derivatization.

The recently developed UPLC technique, using a

combination of higher pressure and small diameter

particles as column packing, could also be a useful, high-

throughput approach for the routine analyses of cyto-

kinins. However, to date, UPLC has been used only to a

limited extent as a separation technique to analyze

cytokinins[18,19,51].

As the basic separation principles of CE differ from

those of HPLC and the other chromatographic tech-

niques, it could be an attractive complementary tech-

nique in analyses of cytokinins. MS detection has been

used in conjunction with CE to determine different

cytokinins in biological matrices[15]. In order to screen

numerous samples, on-line sample pretreatment (pre-

concentration and removal of interfering substances),

and CE separation in microchip formats require further

development.

We anticipate that the development of analytical

methods will enable us to unravel some of the mysteries

concerning cytokinins in plants as well as their beneficial

effects in medicine[14].




13/13

References[1] D.W.S. Mok, M.C. Mok, Annu. Rev. Plant Physiol. Plant Mol. Biol.

52 (2001) 89.

[2] H. Sakakibara, Annu. Rev. Plant Biol. 57 (2006) 431.

[3] K. Vermeulen, M. Strnad, V. Krystof, L. Havlcek, A. Van der Aa,

M. Lenjou, G. Nijs, I. Rodrigus, B. Stockman, H. Van Onckelen,

D.R. Van Bockstaele, Z.N. Berneman, Leukemia 16 (2002) 299.

[4] S.I.S. Rattan, L. Sodagam, Rejuvenation Res. 8 (2005) 46.

[5] G.A. Tucker, J.A. Roberts (Editors), Plant Hormone Protocols,

Humana Press Inc., Totowa, NJ, USA, 2000.

[6] R.O. Morris, D.G. Blevins, J.T. Dietrich, R.C. Durley, S.B. Gelvin,

J. Gray, N.G. Hommes, M. Kaminek, L.J. Mathews, R. Meilan,

T.M. Reinbott, L. Sayavedrasoto, Aust. J. Plant Physiol. 20 (1993)

621.

[7] A. Grayling, D.E. Hanke, Phytochemistry 31 (1992) 1863.

[8] R. Maldiney, B. Leroux, I. Sabbagh, B. Sotta, L. Sossountzov,

E.A. Miginiac, J. Immunol. Methods 90 (1986) 151.

[9] J. Wang, D.S. Letham, E. Taverner, J. Badenoch-Jones, C.H. Hocart,

Physiol. Plant. 95 (1995) 91.

[10] J.W.H. Yong, S.C. Wong, D.S. Letham, C.H. Hocart, G.D. Farquhar,

Plant Physiol. 124 (2000) 767.

[11] E. Casanova, A.E. Valdes, B. Fernandez, L. Moysset, M.I. Trillas,

J. Plant Physiol. 161 (2004) 95.

[12] P.O. Bjorkman, E. Tillberg, Phytochem. Anal. 7 (1996) 57.

[13] O. Novak, P. Tarkowski, D. Tarkowska, K. Dolezal, R. Lenobel,

M. Strnad, Anal. Chim. Acta 480 (2003) 207.

[14] A. Nordstrom, P. Tarkowski, D. Tarkowska, K. Dolezal, C. Astot,

G. Sandberg, T. Moritz, Anal. Chem. 76 (2004) 2869.

[15] L. Ge, J.W.H. Yong, S.N. Tan, E.S. Ong, Electrophoresis 27 (2006)

4779.

[16] L. Ge, J.W.H. Yong, S.N. Tan, X.H. Yang, E.S. Ong, J. Chromatogr.,

A 1048 (2004) 119.

[17] L. Ge, J.W.H. Yong, S.N. Tan, X.H. Yang, E.S. Ong, Electrophoresis

26 (2005) 1768.

[18] K. Dolezal, I. Popa, E. Hauserova, L. Spchal, K. Chakrabarty,

O. Novak, V. Krystof, J. Voller, J. Holub, M. Strnad, Bioorg.

Med. Chem. 15 (2007) 3737.

[19] K. von Schwartzenberg, M.F. Nunez, H. Blaschke, P.I. Dobrev,O. Novak, V. Motyka, M. Strnad, Plant Physiol. 145 (2007)

786.

[20] R. Horgan, I.M. Scott, in: L. Rivier, A. Crozier (Editors), Principles

and Practice of Plant Hormone Analysis, Academic Press, London,

UK, 1987, Chapter 5, p. 303.

[21] R.L. Bieleski, Anal. Biochem. 9 (1964) 431.

[22] K. Hoyerova, A. Gaudinova, J. Malbeck, P.I. Dobrev, T. Kocabek,

B. Solcova, A. Travnckova, M. Kam nek, Phytochemistry 67

(2006) 1151.

[23] R. Horgan, Analytical procedures for cytokinins, in: J.R. Hillman

(Editor), Isolation of Plant Growth Substances, Society for

Experimental Biology, Seminar Series 4, Cambridge University

Press, Cambridge, UK, 1978, p. 97.

[24] P.I. Dobrev, M. Kamnek, J. Chromatogr., A 950 (2002) 21.

[25] K. Takei, T. Yamaya, H. Sakakibara, J. Plant Res. 116 (2003)265.

[26] L. Ge, J.W.H. Yong, S.N. Tan, X.H. Yang, E.S. Ong, J. Chromatogr.,

A 1133 (2006) 322.

[27] E. Hauserova, J. Swaczynova, K. Dolezal, R. Lenobel, I. Popa,

M. Hajduch, D. Vydra, K. Fuksova, M. Strnad, J. Chromatogr.,

A 1100 (2005) 116.

[28] B. Nicander, U. Stahl, P.O. Bjorkman, E. Tillberg, Planta 189

(1993) 312.

[29] G. Teller, in: D.W.S. Mok, M.C. Mok (Editors), Cytokinins:

Chemistry, Activity and Function, CRC Press, Boca Raton, FL,

USA, 1994.

[30] E.J. Trione, G.M. Banowetz, B.B. Krygier, J.M. Kathrein,

L.A. Sayavedra-Soto, Anal. Biochem. 162 (1987) 301.

[31] L.M.S. Palni, S.A.B. Tay,J.K. MacLeod, in: H.F.Linkens,J.F. Jackson

(Editors), Modern Methods of Plant Analysis, New Series, Vol. 3,

Springer-Verlag, Berlin, Germany, 1987.

[32] B.H.Most, J.C.Williams, K.J.Parker,J. Chromatogr. 38 (1968) 136.

[33] J.K. MacLeod, R.E. Summons, D.S. Letham, J. Org. Chem. 41

(1976) 3959.

[34] C.H. Hocart, O.C. Wong, D.S. Letham, S.A.B. Tay, J.K. MacLeod,

Anal. Biochem. 153 (1986) 85.

[35] M. Ludewig, K. Dorffling, W.A. Konig, J. Chromatogr., A 243

(1982) 93.

[36] C.H. Hocart, J. Wang, D.S. Letham, J. Chromatogr., A 811 (1998)

246.

[37] C. Birkemeyer, A. Kolasa, J. Kopka, J. Chromatogr., A 993 (2003)

89.

[38] C. Chen, in: H.F. Linskens, J.F. Jackson (Editors), High Perfor-

mance Liquid Chromatography in Plant Sciences, Springer, Berlin,

Germany, 1987, p. 23.

[39] L. Ge, J.W.H. Yong, N.K. Goh, L.S. Chia, S.N. Tan, X.H. Yang,

E.S. Ong, J. Chromatogr., B 829 (2005) 26.

[40] J.A. Van Rhijn, H.H. Heskamp, E. Davelaar, W. Jordi, M.S. Leloux,

U.A.T. Brinkman, J. Chromatogr., A 929 (2001) 31.

[41] B. Fernandez, M. Centeno,I. Feito, R. Sanchez-Tames, A. Rodriguez,

Phytochem. Anal. 6 (1995) 49.

[42] J. Abian, A.J. Oosterkamp, E. Gelp, J. Mass Spectrom. 34 (1999)

244.

[43] W.A. Stirk, O. Novak, K. Vaclavkova, P. Tarkowski, M. Strnad,

J. van Staden, Planta 227 (2008) 1279.

[44] E. Prinsen, W. Van Dongen, E.L. Esmans, H.A. Van Onckelen,

J. Chromatogr., A 826 (1998) 25.

[45] E. Prinsen, P. Redig, M. Strnad, I. Gals, W. van Dongen, H. van

Onckelen, in: K.M.A. Gartland, M. Davey (Editors), Methods in

Molecular Biology, Agrobacterium Protocols, Humanae Press,

New Jersey, USA, 1995, p. 245.

[46] C. Astot, K. Dolezal, T. Moritz, G. Sandberg, J. Mass Spectrom. 33

(1998) 892.

[47] K.Dolezal,C.Astot, J. Hanus, J. Holub, W.Peters,E. Beck,M. Strnad,

G. Sandberg, Plant Growth Regul. 36 (2002) 181.

[48] D. Tarkowska, K. Dolezal, P. Tarkowski, C. Astot, J. Holub,

K. Fuksova, T. Schmulling, G. Sandberg, M. Strnad, Physiol.

Plant 117 (2003) 579.

[49] A.T. Fletcher, J.C. Mader, J. Plant Growth Regul. 26 (2007) 351.

[50] E. Prinsen, W. van Dongen, E. Esmans, H. van Onckelen, J. Mass

Spectrom. 32 (1997) 12.

[51] O. Novak, E. Hauserova, P. Amakorova, K. Dolezal, M. Strnad,

Phytochemistry 69 (2008) 2214.

[52] L. Ge, J.W.H. Yong, S.N. Tan, E.S. Ong, Electrophoresis 27 (2006)

2171.

[53] L. Ge, J.W.H. Yong, S.N. Tan, L. Hua, E.S. Ong, Electrophoresis 29

(2008) 2024.

[54] N.C. Cook, D.U. Bellstedt, G. Jacobs, Scientia Hort. 87 (2001) 53.

[55] A. Szekacs, G. Hegedus, I. Tobias, M. Pogany, B. Barna, Anal.

Chim. Acta 421 (2000) 135.


http://www elsevier com/locate/trac 335

Documents

CITOQUININAS HPLC