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Trends in Analytical Chemistry, Vol. 28, No.

3, 2009

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Analytical methods for cytokinins


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

1. Introduction
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: sweengin.tan@nie.edu.sg

Cytokinins are a group of plant hormones [1,2]. Certain cytokinins (e.g., kinetin and zeatin) show signicant anti-ageing, anticarcinogenic, 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 classied by the conguration of their N6-side chain as either isoprenoid or aromatic (Table 1). Since plant-tissue extracts are rather complex multi-component mixtures, and cytokinins typically occur in trace quantities (usually at levels below 30 pmol/g of fresh weight [1,2]), sufciently sensitive and selective analytical tools are required for determination of their endogenous levels. Regardless of the analytical method used, highly puried plant samples are preferred for the analyses [5]. Traditionally, quantication of cytokinins 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 techniques can be used as a sensitive, viable alternative for determination of cytokinin levels [6]. The two most common forms of cytokinin immunoassay are radioimmunoassay (RIA) [7] and enzyme-linked immunosorbent assay (ELISA) [8]. However, scintillation proximity assay (SPA) is also gaining ground [9,10]. Recently, ELISA has overtaken RIA. In addition, by taking advantage of the highly specic 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, immunoassays sometimes suffer from problems (e.g., cross-reactivity, imperfect validation, and variable results for plant sample analyses). Other common analytical methods are physico-chemical techniques {gas chro matography (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 purication 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 simplicity, rapid method development, and reduced cost of the operation, since packed columns, reciprocating pumps and mobilephase gradient are not used [16,17]. Recent advances in instrumentation [e.g., ultra-performance liquid chromatography (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:

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Table 1. Structures, names and abbreviations of cytokinins


HN R1

7
N

6
N1

8 9
N R2

2
N

R1

R2

R3 H H H H G G H H H H H H H G G H trans-zeatin trans-zeatin trans-zeatin trans-zeatin trans-zeatin trans-zeatin trans-zeatin

Compound riboside 9-glucoside 7-glucoside* O-glucoside riboside O-glucoside riboside-5 0 -monophosphate

Abbreviation Z ZR Z9G Z7G ZOG ZROG ZMP cZ cZR cZMP DZ DZR DZ9G DZOG DZROG DZMP iP iPR iP9G iPMP BA BAR BA9G BAMP oT oTR oT9G mT mTR mT9G pT pTR pT9G K KR KMP

CH2OR3

H2C

CH3
CH2OR3

H R G H R RP H R RP H R G H R RP H R G RP H R G RP H R G H R G

H2C

CH3 CH2OR3

cis-zeatin cis-zeatin riboside cis-zeatin riboside-5 0 -monophosphate dihydrozeatin dihydrozeatin dihydrozeatin dihydrozeatin dihydrozeatin dihydrozeatin

H2C

CH3

CH3

riboside 9-glucoside O-glucoside riboside O-glucoside riboside-5 0 -monophosphate

H2C

CH3

isopentenyladenine isopentenyladenine riboside Isopentenyladenine 9-glucoside isopentenyladenine riboside-5 0 -monophosphate benzylaminopurine benzylaminopurine riboside benzylaminopurine 9-glucoside benzylaminopurine-5 0 -monophosphate ortho-topolin ortho-topolin riboside ortho-topolin 9-glucoside meta-topolin meta-topolin riboside meta-topolin 9-glucoside para-topolin para-topolin riboside para-topolin 9-glucoside kinetin kinetin riboside kinetin riboside-5 0 -monophosphate

H2 C

HO

H2C
OH H2C

H2C

OH

H R G H R RP

H2C O

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.

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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 comprehensive procedures used for extraction, pre-concentration and purication of cytokinins. 2.1. Extraction techniques The qualitative and the quantitative aspects of cytokinins extracted from plant tissues vary with the types of extraction solvents and procedures employed [20]. In order to prevent any enzymatic degradation of cytokinins (e.g., conversion of cytokinin nucleotide to its riboside), plant material should be immediately frozen or extracted with proper solvent(s) after harvesting. Isolation and identication of picomolar quantities of cytokinins 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/chloroform/water/formic acid (12/5/2/1, v/v/v/v) [21]. Hoye et al. [22] compared the extraction efciency of rova three different extraction solvents: (a) 80% (v/v) methanol; (b) Bieleskis solvent; and, (c) modied Bieleskis solvent (methanol/water/formic acid; 15/4/1, v/v/v). It was found that the modied Bieleskis solvent sufciently suppressed the dephosphorylation of cytokinin mononucleotides 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 extracted with cold acetone. After centrifugation, the supernatant is evaporated in vacuo to dryness. The residue is dissolved in lukewarm (38C) acidied water (pH 3.5). A triple extraction of an aqueous solution of cytokinins with butanol saturated with acidied 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 purication, 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 C18 cartridges. A more effective but more complex approach is to purify plant extracts by passing them through linked columns of polyvinylpolypyrrolidone powder, DEAEcellulose or DEAE-Sephacel, and C18-SPE [5]. Fast, efcient 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-concentration tool, further sample purication could be carried out using mixed-mode cation exchanger (MCX) SPE cartridges with good recoveries [16]. Purication of cytokinins using MCX SPE, as compared to DEAE Sephadex and C18 SPE method, was suitable for removal of Ultraviolet (UV) absorbing contaminants with higher recoveries of cytokinins [22]. However, due to the relatively high polarity of cytokinin nucleotides leading to poor retention by C18 cartridges during SPE, the use of an anion-exchange sorbent in addition is an efcient alternative step to separate cytokinin nucleotides from cytokinin bases and sugar conjugates [25]. An efcient dual-step SPE method has therefore been developed for pre-concentration and purication of cytokinin nucleotides using Oasis HLB and MAX cartridges [26]. 2.4. Immunoafnity purication Immunoafnity purication methods, based on antibody-antigen (Ab-Ag) interactions, can provide selective sample enrichment, and thus greatly enhance limits of detection (LODs) of trace analyses [27], so, when an immunoafnity approach is used as the purication step before nal analysis, highly puried cytokinin preparations containing only traces of other UV-absorbing material can be obtained. Several papers have reported on the immunoextraction of cytokinins using monoclonal and polyclonal antibodies (mAbs and pAbs) [27,28]. The set-up for a suitable afnity system requires an appropriate Ab, and consideration of matrix factors that are not specic to phytohormone analysis [28]. Generic cytokinin mAbs are frequently used in this respect [13,27]. Immunoafnity columns purify analytes according to structural similarities, so they hold promise for the discovery of new cytokinins [28]. Immunoextraction has higher selectivity than conventional SPE, but low throughput. An efcient, off-line, batch-immunoextraction method was developed for the purication of new cytokinins and their ribosides [27].

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Plant materials Extraction (Bieleskis solution or modified Bieleskis solution) -20C Remove CH3OH, pH adjust to 1.8 4C PolyclarVT Cartridge Elution by 60% CH3OH DEAE-Sephacel Cartridge Evaporate and redissolve 1 M NH4OH Flow through (Some CK nucleotides may be lost here) Hydrolysis glucosides Evaporate and redissolve 1 M HCOOH by -glucosidase MonoQ Column Wash by 1 M Wash by 0.2 M NH4OH in 80% NH4OH CH3OH Oasis MCX Cartridge CK CK CK mononucleotides dinucleotides trinucleotides Dephosphorylation by treatment with phosphatase Elution by 2% Wash by acidified water (pH 3.0) Elution by ethanol; water: acetic acid (80:20:1) Flow-through fraction CK bases, ribosides, glucosides Elution by 1 M NH4HCO3 CK nucleotides Oasis MAX Cartridge Remove CH3OH, pH adjust to 3.0 25C Oasis HLB Cartridge

Flow-through fraction Octadecyl C18 Cartridge

Wash by acidified water (pH 1.8)

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HCOOH in 50% CH3OH CK nucleotides

Wash by CH3OH IAA, ABA

Elution by 0.35 M NH4OH CK nucleotides (not full complement)

Elution by 0.35 M NH4OH in 60% CH3OH CK bases, ribosides, glucosides

Immuno-affinity Column Chromatography

CK: cytokinin

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

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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 compounds, 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, specic means for identication and quantication of cytokinins, until the recent introduction of using a combination of HPLC-MS. Structures

of almost all naturally-occurring cytokinins were elucidated 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)triuoroacetamide in 50% pyridine containing 1% trimethylcholorsilane 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 derivatization of cytokinins for GC-MS analysis using trimethylsilyl [20,32], permethyl [33], t-butyldimethylsilyl [34], triuoroacetyl [35] and acetyl [12] derivatives. Pentauorobenzyl 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).

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Table 2. Typical LC-MS analytical methods for the determination of cytokinins


Analytes Sample matrix Sample preparation Column Z9G, DZ9G, Z, DZ, cZ, mT9G, ZR, DZ, cZR, mT, oT9G, mTR, BA9G, iP9G, oT, oTR, BA, iP, BAR and iPR Cytokinin autonomous tobacco BY-2 cell, fully expanded leaves of Populus canadensis Moench., cv Robusta Arabidopsis thaliana wild-type variant Colombia plants Two ion-exchange chromatography steps, SCX SPE, DEAE Sephadex SPE, C18 SPE and IAC Symmetry C18 column (5 lm, 150 mm 2.1 mm) LC conditions Mobile phase A: 15 mM formic acid adjusted to pH 4.0 by ammonium hydroxide; B: MeOH Flow rate 250 lL/min (50% efuent was introduced into ESI source) Elution Gradient 025 min, 1050% B; 2530 min, 50% B Source ESI MS conditions Analyzer Single quadrupole [13] Ref.

Propionylated iP, Z, DZ, ZR, iP, ZMP, iPMP, ZOG, Z7G, Z9G, ZROG, DZR

C18 SPE and MCX SPE

10 1 mm BetaMax Neutral dropin guard cartridge with 5-lm particles

A: 3% formic acid; B: 3% formic acid in ACN

013 min, 10 lL/min; 13.117 min, 40 lL/min; 17.119 min, 20 lL/min; 20 min, 10 lL/min

Gradient 02 min, 5% B; 210 min, 555% B; 1012 min, 55% B; 12.113 min, 80% B; 13.117 min, 100% B; 17.120 min, 5% B Gradient 09 min, 3064% A Gradient 015 min, 700% A

ESI

Triple quadrupole

[14]

6-(2-hydroxy-3methoxybenzylamino) purine riboside, 6-(2,4dimethoxybenzylamino) purine riboside and other 6benzyladenosine derivatives Z, Z, Z9G, ZOG, ZROG, ZMP, DZ, DZR, DZ9G, DZOG, DZROG, DZMP, iP, iPR, iP9G, iPMP, BA, BAR, BA9G, BAMP, mT, and oT Z, DZ, ZR, DZR, Z9G, DZ9G, iP and iPR

Arabidopsis thaliana, A. tumefaciens

C18 SPE, IAC for A. thaliana Octadecyl-silica SPE, DEAE-Sephadex SPE, C18 SPE, and Immunoextraction for A. tumefaciens DEAE-Sephadex SPE, octadecyl-silica SPE and IAC

C18 column (1.7 lm; 150 mm 2.1 mm) Symmetry C18; 5 lm; 0.3 150 mm (Waters)

A: MeOH; B: 5 mM formic acid A: 5 mM formic acid and 2% MeOH; B: MeOH with 0.05% formic acid.

0.25 mL/min 5 lL/min

ESI ESI

Triple quadrupole Q-TOF

[18]

Physcomitrella patens

BEH C18 (1.7 lm; 150 mm 2.1 mm) column

A: 15 mM ammonium formate (pH 4.0); B: MeOH

0.25 mL/min

Gradient 010 min, 9050% B

API

Triple quadrupole

[19]

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Transgenic homozygote and hemizygote as well as wild-type Nicotiana tabacum species, and cauliower samples Pea roots

DEAE-cellulose SPE and C18 SPE or IAC.

LUNA C8 column (5 lm, 15 cm 1.0 mm)

A: 10 mM ammonium acetate; B: MeOH

60 lL/min

Gradient 020 min, 1080% B

ESI

Triple quadrupole

[40]

Propionylated iPMP, iPR, ZMP, cZMP and cZR

SCX SPE, DEAE-Sephadex SPE, C18 SPE, and IAC. Alkaline phosphatase treatment of nucleotides. Strong cation-exchange cartridge (SCX) DEAESephadex anion exchanger C18 cartridge IAC

Symmetry C18 column (5 lm, 150 mm 0.3 mm)

A: 15 mM ammonium formate (pH 4.0); B: MeOH

5 lL/min

Gradient 025 min, 1050% A

ESI

Q-TOF

[43]

Derivatized Z, iP, ZR, iP, ZMP, iPMP, ZOG, Z7G, Z9G and ZROG

Arabidopsis thaliana

Capillary LC column (150 0.3 mm packed with 4 lm Symmetry ODS packing material)

A: 98% water, 1% formic acid and 1% glycerol; B: 97% ACN, 1% water, 1% formic acid and 1% glycerol

4 lL/min

Gradient 1012 min, 2050% B; 1240 min, 50% B

frit-FAB

Double focusing magnetic sector

[46]

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Double focusing magnetic sector

Single quadrupole

novel method based on N-methyl-N-(tert-butyldimethylsilyl)triuoroacetamide 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-MSbased identication, compared with the other soft-ionization techniques, is differentiation of the various sugar moieties. Besides MS detection, the use of two other detectors, namely ame-ionization detector (FID) and electron-capture detector (ECD), has been reported [12,33]. The main drawback of FID is limited sensitivity that makes it inapplicable in the trace analysis of cytokinins. Even though ECD is quite sensitive, halogenated derivatives are required in order to obtain satisfactory LODs.
2MeSoTOG, 6-[2-(b-D-glucopyranosyloxy)benzylamino]-2-methylthiopurine; MeoT, ortho-methoxytopolin; MemT, meta-methoxytopolin.

[47]

[48]

frit-FAB

Gradient 05 min, 3015% A; 525 min, 1540% A; 2530 min, 40% A

0.25 mL/min (25% efuent was introduced into ESI source)

05 min, 20 lL/min; 545 min, 4.5 lL/min

A: 10 mM ammonium acetate; B: 350 mL MeOH, 100 mL ACN, 50 mL, 10 mM ammonium acetate

A: MeOH; B: 0.1% formic acid adjusted to pH 2.9 with ammonium

50% aqueous ACN, 1% glycerol, 1% formic acid

0.1 mL/min

frit-FAB

Gradient 03 min, 510% B; 327 min, 1043% B; 2730 min, 4380% B; 3033 min, 80100% B

Isocratic

Double focusing magnetic sector

API

ESI

Q-TOF

[49]

Octadecyl-silica SPE, DEAE-Sephadex SPE, C18 SPE, and IAC Alkaline phosphatase treatment of nucleotides 55% aqueous ACN, 1% glycerol, 1% formic acid

SCX SPE and C18 SPE. Alkaline phosphatase treatment of nucleotides.

C18 SPE Alkaline phosphatase treatment of nucleotides

3.2. High-performance liquid chromatography HPLC is a particularly suitable chromatographic technique for cytokinin analyses, as cytokinins exhibit gradations in polarity and are readily detected by UV absorbance [38]. HPLC enables rapid, high-resolution purication of cytokinins from plant extracts prior to analysis by MS, immunoassay, or bioassay. HPLC analysis alone can also provide reliable identication 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 cytokinins is isotope-dilution MS, especially with LC-Electrospray Ionization-MS (LC-ESI-MS) [13,14]. 3.2.1. Chromatographic conditions and separation. Cytokinin-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, gradient elution by increasing content of organic modier is often used [3941]. For preparative purication of

Capillary LC column (150 0.3 mm packed with 4 lm Symmetry ODS packing material)

Isocratic

Arabidopsis thaliana and Populus canadensis leaves

Chenopodium rubrum cells

Capillary LC column (150 0.3 mm packed with 4 lm Symmetry ODS packing material) Propionylated pT, mT, pTR, oT, mTR, BA, MeoT, MemT, oTR, BAR and MeoTR

pT, mT, pTR, oT, mTR, BA, MeoT, MemT, oTR, BAR, MeoTR and MemTR

Propionylated oTOG, 2MeSoTOG and BA9G

Z, ZR, Z7G, Z9G, DZ, DZR, iP, iPR, iP9G, ZOG and ZROG

Macadamia integrifolia

C18 column (3 lm, 20 mm 2.1 mm) Guard column (C18, 5 lm, 4 mm 2.0 mm)

Symmetry C18 column (3.5 lm, 150 mm 2.1 mm)

05 min, 20 lL/min; 565 min, 4.0 lL/min

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Table 3. Optimal separation conditions for cytokinins using different CE approaches [15,53] Analytes iP, iPR, Z, ZR, DZ,DZR, BA and BAR. Sample matrix STD sugar beet Sample preparation SPE Mode CZE Buffer 150 mM phosphoric acid, pH 1.8 20 mM SDS, 50 mM borate, pH 9.2 100 mM phosphate-Tris (pH 2.5) buffer with 25 mM cCD 50 mM borate containing 50 mM SDS, pH 8.0 Capillary dimensions 77 cm (effective length 61 cm) 75 lm Separation voltage 20 kV Injection 10 mbar, 0.05 min 10 mbar, 0.1 min Detection UV 265 nm Reference assay NS

STD, tobacco

MEKC

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

STD

NS

CDmodied CZE

47 cm (effective length 40 cm) 50 lm.

20 kV

NS

UV 200 nm

NS

BA, K, and other plant hormones including ABA, IAA, NAA, GA and 2,4-D. Z, DZ, ZOG, DZOG, mTR, iP and BA

STD, transgenic tobacco ower

LLE

MEKC

48.5 cm (40 cm effective length) 50 lm.

15 kV

5 s at 50 mbar

UV 210 nm

NS

STD, coconut water

Dual-step SPE

MEKC

Combination of 10 mM phosphate and 10 mM borate buffer containing 50 mM SDS, pH 10.4 20 mM boric acid and 50 mM SDS, pH 8.0, with an extra 20% (v/v) MeOH added 100 mM ammonium phosphate buffer, pH 2.5

57 cm (effective length 47 cm) 76 lm.

15 kV

5 s under a pressure of 0.5 psi

UV 254 nm

HPLC, LCMS

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oT, mT, pT, oTR, mTR and pTR

STD, coconut water

Dual-step SPE

MEKC

60 cm (effective length 50 cm) 76 lm.

15 kV

5 s under a pressure of 0.5 psi

UV 269 nm

HPLC, LCMS2

K and KR

STD, coconut water

Dual-step SPE

CZE

40 cm (effective length 30 cm) 76 lm

15 kV

5 s under a pressure of 0.5 psi

UV 269 nm

HPLC, LCMS2

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

STD, coconut water

Dual-step SPE

CZE

25 mM ammonium formate/formic acid buffer (pH 3.4) and 3% ACN (v/v) 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% MeOH (v/v)

65 cm 50 lm

25 kV

On-line sample stacking injections

MS, MS2

NS

DZMP, iPMP, cZMP, ZMP, BAMP and KMP

STD, coconut water

dual-step SPE

CZE

57 cm 50 lm

Gradient separation voltage (25 kV for 32 min, and then linear gradient to 30 kV in 5 min, nally 30 kV to end of separation) 20 kV

On-line sample stacking injections

MS2

NS

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

STD, banana pulp

Dual-step SPE

Partial llingMEKC

50 mM ammonium formate/ ammonium hydroxide at pH 9.0. Micellar solution with 70 mM ALS and 10% MeOH was injected for 90 s at 50 mbar before the sample and 120 s at 50 mbar after the sample

100 cm 50 lm

On-line sample stacking injections

MS, MS2

NS

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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-dichlorophenoxyacetic acid.

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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. 2 shows representative HPLC chromatograms of cytokinins. As the best performance of early ESI interfaces was at ow 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 signicantly 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 onpre-column and on-chromatographic column trace enrichment. Sample loading is performed under non-eluting conditions using a pre-column or directly (analytical column), which is followed by backush (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 quantication [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 separation are analyzed by bioassays, immunoassays or converted to volatile derivatives for GC analysis. However, using this non-specic UV-absorbance method for detection requires signicantly higher amounts of sample that needs extensive purication. The LC-MS approach offers a new tool to detect, quantify and characterize cytokinins in plant-tissue extracts at biologically meaningful levels. Further improvement in LC systems as well as mass analyzers may overcome the low detectability of cytokinins. Different ionization techniques were used for MS analyses of cytokinins in combination with RP-HPLC, including thermospray (TS) [45], fast atom bombardment (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 LCMS, ESI-MS has a fairly high sensitivity and is associated

with lower background [13,14,18,19,43,44,48,50]. In 1997, the rst application of LC-ESI-MS2 with multiple reaction monitoring (MRM) for cytokinin determination was reported as a fast method for the quantication 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 cytokinin analyses was used to give stronger quasimolecular ion currents and to obtain more spectral information [14,43,4648]. Table 2 presents a summary of some representative LC-MS methods for cytokinin analyses. 3.3. Ultra-performance liquid chromatography UPLC instrumentation can provide liquid ow at pressures of up to 1000 bar, and features columns packed with 1.7-lm particulate packing materials, so UPLC extends beyond the chromatographic limits of conventional HPLC instrumentation. UPLC can achieve higher resolutions, lower sensitivities and more rapid separations [18,19]. UPLC-MS provides signicant 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 efcient UPLC-MS2 method to establish the prole of 40 cytokinins 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 efcient UPLC-MS2 method for cytokinin proling 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]. Although some 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 injected 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 specic detection systems (e.g., MS) and on-line or off-line sample pre-concentration to increase samplesolute concentration [26,52]. Ge et al. [15] comprehensively reviewed information pertaining to this aspect of cytokinin analyses.

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mT9G mTR pT9G oTR ZR pTRoT9G mT pT cZR oT cZ Z

m/z 404 m/z 374 m/z 352 m/z 242 m/z 220

15

16

17

18

19

20 Time [min]

21

22

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Figure 3. Selected ion mass electropherograms of (A) a standard mixture of 12 cytokinins obtained by CZE-MS; and, (B) a standard mixture of 13 cytokinins obtained by partial lling-MEKC-MS underoptimized conditions (Adapted from [52,53], with permission).

3.4.1. Electrophoretic conditions and separation. As is well known, without changes in instrumental hardware, CE separations can be carried out using several operation modes. The different CE modes used for the separation of cytokinins are capillary-zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Table 3 outlines the optimal electrophoretic conditions for the analyses of cytokinins in various biological samples. Electrolyte composition is extremely important in order to obtain a good CE separation. Since a non-volatile buffer in ESI-MS at a relatively high concentration results in signicant loss of electrospray efciency and produces ion-source contamination, volatile buffer systems (e.g., ammonium formate) are generally preferred for cytokinin analyses when using CZE-MS [26,52]. CZE is more suited for direct separation of charged cytokinin-nucleotide compounds without any derivatization step, when compared to HPLC [26]. Furthermore, due to its experimental simplicity and its practicality with less puried sample, CZE has been applied for the determination of the physicochemical constants of cytokinins and their analogs [15]. MEKC has better separation selectivity than CZE, since surfactant is added to buffer solution to form micelles. For ionic cytokinins, MEKC separations are based on both degree of ionization and hydrophobicity. MEKC results are consistent with results obtained using HPLC or LC-MS for the determination of cytokinins in coconut water [16,17]. The optimized MEKC method developed for topolin and topolin riboside isomers took less than half the time of a typical HPLC run [17]. The shortened analysis time is an

advantage of CE, which could be further exploited as a routine analytical tool for cytokinins. 3.4.2. Detection. The small volumes of sample injections make detection a signicant challenge in CE. So far, UV and MS detectors have been used for the determination of cytokinins. As the standard detector on many commercial CE instruments, the UV detector is widely used for cytokinin analyses. In addition to UV detection, MS has been carried out on-line coupled with CE, which provides unsurpassed opportunities in identication and structural elucidation of cytokinins. Currently, ESI is the most common interface between CE and MS, as it can produce ions directly from liquids at atmospheric pressure, and with high sensitivity and selectivity. The rst CZE-MS method for the analysis of 12 cytokinins was developed and applied to screen for cytokinins in coconut water [52]. A CZE-MS2 method was also developed for analyzing cytokinin nucleotides without sample derivatization [26]. More recently, a new method based on MEKC directly coupled to ESI-MS was developed for the simultaneous separation and determination of 13 structurally similar cytokinins, including geometric and positional isomers [53]. In the MEKC-MS method, a partial lling technique was used to prevent the micelles from reaching the MS as this is detrimental to its signal. Fig. 3 shows typical CE-MS electropherograms of cytokinins.

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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 phytohormones due to their low LODs [610]. Several quantitative immunological methods were developed to detect cytokinins. Despite having some problems associated with immunological methods, a few reports have shown that Abs are useful tools to detect trace cytokinins in plant tissues, and even at the ultra-structural level of cells. Immunolocalization of endogenous cytokinins 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 determination 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, pAbs for RIA cannot be used directly on individual cytokinins in crude plant extracts, so substances interfering with RIA should be removed from the extracts [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 amplied ELISA, immunoafnity purication and immunocytochemical techniques have been developed [8,55]. ELISA detects cytokinins in the fmol range. Hapten-homologous and hapten-heterologous competitive ELISAs were developed for detecting endogenous cytokinin levels in crude plant extracts without intense purication so they needed less plant extract [55]. ELISAs are still widely used for cytokinin analyses. 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 uid as required in conventional RIA [9,10]. First described by Wang et al. [9], SPA is a sensitive assay for the quantication 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 combined content of similar groups (free bases, ribosides, 9glucosides and nucleotides) of cytokinins, due to their lower specicity, rather than specic 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 present 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 satisfactory 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 conrmation and quantication 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 cytokinin 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 fragmentation patterns. Unlike the well-established GC-MS method, LCMS2 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, highthroughput approach for the routine analyses of cytokinins. 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 techniques, it could be an attractive complementary technique 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 (preconcentration 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 benecial effects in medicine [14].

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