Post on 04-Jun-2018
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:
sweengin.tan@nie.edu.sg
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:%06sweengin.tan@nie.edu.sgmailto:%06sweengin.tan@nie.edu.sg8/13/2019 CITOQUININAS HPLC
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
8/13/2019 CITOQUININAS HPLC
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].
Trends in Analytical Chemistry, Vol. 28, No. 3, 2009 Trends
http://www.elsevier.com/locate/trac 325
8/13/2019 CITOQUININAS HPLC
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
8/13/2019 CITOQUININAS HPLC
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).
Trends in Analytical Chemistry, Vol. 28, No. 3, 2009 Trends
http://www.elsevier.com/locate/trac 327
8/13/2019 CITOQUININAS HPLC
6/13
8/13/2019 CITOQUININAS HPLC
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.
Trends in Analytical Chemistry, Vol. 28, No. 3, 2009 Trends
http://www.elsevier.com/locate/trac 329
8/13/2019 CITOQUININAS HPLC
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
57 cm (effectivelength 47 cm)76 lm.
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
60 cm (effectivelength 50 cm)76 lm.
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
8/13/2019 CITOQUININAS HPLC
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
8/13/2019 CITOQUININAS HPLC
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.
Trends Trends in Analytical Chemistry, Vol. 28, No. 3, 2009
332 http://www.elsevier.com/locate/trac
8/13/2019 CITOQUININAS HPLC
11/13
8/13/2019 CITOQUININAS HPLC
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].
Trends Trends in Analytical Chemistry, Vol. 28, No. 3, 2009
334 http://www.elsevier.com/locate/trac
8/13/2019 CITOQUININAS HPLC
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.
Trends in Analytical Chemistry, Vol. 28, No. 3, 2009 Trends
http://www elsevier com/locate/trac 335