a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281

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Simultaneous analysis of different classes of

phytohormones in coconut (Cocos nucifera L.) water
using high-performance liquid chromatography and
liquid chromatography–tandem mass spectrometry
after solid-phase extraction

Zhen Ma a , Liya Ge a , Anna S.Y. Lee a , Jean Wan Hong Yong a ,

Swee Ngin Tan a,∗ , Eng Shi Ong b
a Natural Sciences and Science Education Academic Group, Nanyang Technological University,
1 Nanyang Walk, Singapore 637616, Singapore
b Department of Community, Occupational, and Family Medicine, Yong Loo Ling School

of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Coconut (Cocos nucifera L.) water, which contains many uncharacterized phytohormones
Received 16 November 2007 is extensively used as a growth promoting supplement in plant tissue culture. In this
Received in revised form paper, a high-performance liquid chromatography (HPLC) method was developed for the
17 January 2008 simultaneous determination of various classes phytohormones, including indole-3-acetic
Accepted 17 January 2008 acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin
Published on line 25 January 2008 (Z), N6 -benzyladenine (BA), ˛-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic
acid (2,4-D) in young coconut water (CW). The analysis was carried out using a reverse-
Keywords: phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid,
Coconut water pH adjusted to 3.2 with triethylamine (TEA)) modified by methanol, and solute detec-
Phytohormones tion made at 265 nm wavelength. The method was validated for specificity, quantification,
High-performance liquid accuracy and precision. After preconcentration of putative endogenous phytohormones
chromatography in CW using C18 solid-phase extraction (SPE) cartridges, the HPLC method was able to
Liquid chromatography–tandem screen for putative endogenous phytohormones present in CW. Finally, the identities of
mass spectrometry the putative phytohormones present in CW were further confirmed using independent liq-
uid chromatography–tandem mass spectrometry (LC–MS/MS) equipped with an electrospray
ionization (ESI) interface.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction beverage. Nowadays, CW is more widely consumed around

the world due to its beneficial health properties [1–3]. It is also
Coconut water (CW), the liquid endosperm of coconut (Cocos believed that CW may be used as an important alternative for
nucifera L.), in its natural form is a refreshing and nutritious oral rehydration and even for patient intravenous hydration in

∗
Corresponding author. Fax: +65 68969432.
E-mail address: [email protected] (S.N. Tan).
0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2008.01.045
 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281 275

Fig. 1 – Chemical structures of the eight phytohormone compounds.

remote regions [2]. Interestingly, a recent research has shown is a well recognized technique for the determination of phy-
that CW may afford protection against induction of myocardial tohormones [12,13]. However, it also requires considerable
infarction [3]. In addition, CW is widely used in the plant tissue sample purification as well as derivatization prior to GC analy-
culture industry [4–6]. The wide applications of CW should be sis. Also, there are some difficulties for the analysis some plant
due to its unique chemical composition of sugars, vitamins, hormones by GC as certain phytohormones could be thermally
minerals, proteins, amino acids and growth promoting factors labile.
[5]. Those growth promoting factors are a group of naturally High-performance liquid chromatography (HPLC) is poten-
occurring organic substances, known as phytohormones. tially an attractive approach for these polar and thermally
Phytohormones exert numerous important plant physio- labile phytohormones. Conventional UV or photodiode array
logical responses at different stages of plant development, detection (PDA) had been extensively used in HPLC for the
such as cell division, enlargement and differentiation, organ separation of phytohormones [9,14] in plant samples. Liquid
formation, seed dormancy and germination, leaf and organ chromatography combined with mass spectrometry (LC–MS)
senescence and abscission, at low concentrations [7]. Since has been proposed for the determination of either auxins
the discovery of the first phytohormone – auxin in 1926, sci- [15,16] or abscisic acid [17,18]. However, as auxins, cytokinins,
entists have reported up to several types of phytohormones, abscisic acid and gibberellins have very different plant phys-
mainly including auxins, cytokinins, abscisic acid, gibberellins iological functions and very different chemical properties;
and ethylene [8]. developing an LC–MS separation method for the simultaneous
Since phytohormones are typically present and active in analysis of four classes of phytohormones would be extremely
minor concentrations in plant tissue, the determination of challenging, if not impossible. From our knowledge, there are
phytohormone concentration in plants is extremely difficult. a few LC–MS papers dealing with the simultaneous analysis
For instance, endogenous auxin found in plants is usually of 2–3 classes of phytohormones [19–21] and relatively rare
around 1–100 ng g−1 fresh weight [9]. Apart from occurring reported work for the simultaneous analysis of four classes of
at low concentrations, many phytohormones exist in sev- phytohormones [22].
eral forms with different side groups and exist with other Therefore, as a further extension of our earlier works
endogenous organic compounds that may interfere with the on the analysis cytokinins in CW [23–25], we present here
final assay. Therefore, it is important that one chooses an the first reported work on the simultaneous separation
assay, which can detect such minimal amount. Tradition- and determination of four classes of phytohormones in
ally, enzyme- or radioimmunoassay (ELISA, RIA) were utilized CW. This work proposes the simple and sensitive HPLC
in field of the phytohormone analysis [10,11]. Immunoas- method for analyzing indole-3-acetic acid (IAA), indole-3-
say techniques, which are excellent in specificity due to the butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA),
unique ligand–antibody binding, can be used as a sensitive and zeatin (Z), N6 -benzyladenine (BA), ˛-naphthaleneacetic acid
relatively cheap alternative for estimation of phytohormone (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The devel-
levels. However, when applied to low purity extracts, quan- oped methodology was then used to screen for the presence
tification using immunoassays can be misleading due to the of endogenous phytohormones in CW after application of an
cross-reactivity of antibodies or their inhibition (or activation) efficient solid-phase extraction (SPE) method for the precon-
by potential interfering substances. Gas chromatography (GC) centration and purification of phytohormones. The presence
as well as gas chromatography–mass spectrometry (GC–MS) of putative IAA and ABA was further confirmed by LC–MS/MS
276 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281

Table 1 – Chromatographic gradient conditions for the Table 2 – MRM transition result of phytohormones
analysis of phytohormones
Phytohormone MRM transition (m/z)
Time (min) Methanol (%) 0.1% Formic acid buffer (%)
IAA 174 → 130
0 10.0 90.0 IBA 202 → 158
5 10.0 90.0 NAA 185 → 141
10 30.0 70.0 2,4-D 219 → 161
20 30.0 70.0 Z 218 → 200
35 45.0 55.0 BA 224 → 133
50 45.0 55.0 ABA 263 → 153
50.1 95.0 5.0 GA 345 → 239
55 95.0 5.0
55.1 10.0 90.0
2.2. Solid-phase extraction

CW obtained from fresh immature coconuts (Hatyai, Thai-

assay based on its characteristic fragmentation pattern. With land) was purchased from a local supermarket. The pH of
HPLC and LC–MS/MS based methods, IAA and ABA in young CW was adjusted to 3 by adding acetic acid and subse-
CW were successfully identified and quantified. It is expected quently, filtered through filter paper to remove suspended
the presented HPLC and LC–MS methods are suitable for rou- particles. After washed sequentially with methanol–acetic
tine analysis. acid (100:1, v/v), methanol–water–acetic acid (50:50:1, v/v/v),
methanol–water–acetic acid (30:70:1, v/v/v), and finally with
water, 100 mL of filtrate was transferred to 10 C18 SPE columns
2. Experimental (J.T. Baker, Phillipsburg, NJ, USA; 500 mg, 3 mL) [26]. The
columns were washed with 10 mL water adjusted to pH 3 with
2.1. Reagents and materials acetic acid (acidified water), the putative phytohormones were
eluted with 5 mL ethanol–water–acetic acid (80:20:1, v/v/v).
The phytohormone standards: Z, BA, GA, ABA and IBA were Then the obtained eluate was evaporated at room temperature
obtained from Sigma–Aldrich (Steinheim, Germany); IAA, NAA under vacuum and finally the evaporated eluate was dissolved
and 2,4-D were obtained from PhytoTechnology Laboratories in 1 mL methanol. Prior to HPLC analysis, this reconstituted
(Shawnee Mission, USA). Fig. 1 shows the chemical structures eluate was filtered using a 0.45 ␮m Whatman glass microfiber
of the eight phytohormones. All the standards were dissolved filter [23].
in methanol with concentration ranged from 250 to 500 ␮M
and stored at temperature 0–4 ◦ C. Both methanol and ethanol 2.3. HPLC instrumental set-up and procedure
(HPLC grade) were purchased from Merck (Darmstadt, Ger-
many); triethylamine (TEA) (HPLC grade) was obtained from The analysis of phytohormones was performed using a high-
BDH (UK). Acetic acid (analytical reagent grade) was purchased performance liquid chromatography system (Waters 2695
from Fisher Scientific (Hanover Park, IL, USA). Filter paper Separations Module, Waters, USA) linked simultaneously to
(Whatman, 12.5 cm, No. 542) was used to filter the CW before a PDA system (PDA 2996 detector, Waters). Data were pro-
SPE. Ultrapure water (Milford, MA, USA) was used throughout cessed by the accompanying system software (Millennium32
the analysis. The pH value of the buffer solution was adjusted Software, Data Handling System for Windows, version 4.0).
by adding TEA and monitored using a pH meter (CORNING 440, Real samples obtained from SPE (Please see end of Section
Corning Glass Works, NY, USA). 2.2) were injected (injection volume: 10 ␮L) into a C18 reverse-

Table 3 – Response characteristics of phytohormone standards using HPLC

Phytohormone Mean retention Peak Equation of R2 Linear LOQ (␮M)c
standards times areaa calibration curveb range (␮M)

mina RSD (%) Mean RSD (%)

Z 14.7 0.4 690,220 0.6 y = 24561.7x + 66889.6 0.996 10–125 3.47

GA 19.3 0.3 31,014 1.9 y = 1936.4x + 9554.7 0.994 10–125 5.33
IAA 23.3 0.3 217,288 1.6 y = 9010.6x + 16859.2 0.995 10–125 3.82
BA 31.8 0.3 1,113,763 0.3 y = 46081.8x − 51109.6 0.991 10–100 1.70
ABA 37.6 0.2 1,720,141 0.2 y = 64004.9x + 196523.7 0.999 10–125 3.06
IBA 39.6 0.2 174,150 5.0 y = 7232.1x − 13129.4 0.990 10–125 2.47
2,4-D 40.8 0.3 25,495 5.4 y = 904.5x + 3465.5 0.996 10–125 9.60
NAA 42.6 0.3 128,802 3.0 y = 5096x + 15044.5 0.992 10–125 6.28

a
The data were measured with repeated injections (n = 6) of a mixture of plant hormone standards at a concentration of 25 ␮M each.
b
In the calibration equation, x represents concentration of the analyte (␮M) and y represents the peak area (␮Au s).
c
The LOQ were estimated based on S/N = 10.
 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281 277

tion. The LC–MS elution program was similar to HPLC elution

program, which was indicated in Table 1.

2.5. Recovery study of the SPE

The extraction recoveries of eight phytohormones under SPE

conditions were also investigated. Unlabelled eight phyto-
hormone standards (0.5 ␮mol each) were added to 100 mL
coconut water, and extracted according to the same sam-
ple preparation procedure. The spiked extracted sample
was then subsequently analyzed using HPLC measure-
ment.

Fig. 2 – HPLC chromatogram of standard mixture of

phytohormones with 50 ␮M for each at 265 nm. Peak
identities: 1, Z; 2, GA; 3, IAA; 4, BA; 5, ABA; 6, IBA; 7, 2,4-D;
8, NAA. HPLC running conditions were described in
Sections 2.3 and 3.1.

phase column (Zorbax SB-C18 100 Å, 3.5 ␮m, 150 mm length,
2.1 mm diameter, Agilent Technologies, Palo Alto, CA, USA).
The initial HPLC running conditions were methanol–formic
acid buffer (10:90, v/v). The column thermostat was set at
25 ◦ C. Solvent (A) consisted of 0.1% formic acid (w/v), pH
adjusted to 3.2 with TEA; solvent (B) consisted of methanol.
The flow rate was 0.3 mL min−1 throughout the whole separa-
tion.
Optimum gradient elution program was investigated for
the separation of these eight phytohormones, which will be
discussed in Section 3.1 and presented in Table 1.

2.4. LC–MS/MS conditions

LC/MSD (Model 1100 Series, Agilent Technologies) coupling

with an electrospray ionization (ESI) interface was used in
scan mode for standards. ESI–MS analysis was performed
in the negative mode, and the ion trap was scanned at
m/z 50–400 in full scan mode. The maximum accumula-
tion time for the ion trap was set at 200 ms and the target
count was set at 100,000. The actual accumulation time
was controlled by ion charge control (ICC), which was used
to prevent ion saturation in the ion trap. The nebulizer
gas pressure, drying gas flow rate, drying gas temperature
and capillary voltage for the ESI source were set at 30 psi,
8 L min−1 , 350 ◦ C, and 3500 V, respectively. Other instrument
parameters were optimized for generating the highest sig-
nal intensities. The whole gradient program is as same as
HPLC’s.
The multiple reaction monitoring (MRM) modes were used
to monitor the transitions from the precursor ions to the most
abundant product ions. Table 2 showed the MRM transition Fig. 3 – HPLC chromatograms of (A) CW extracts; (B) spiked
results of different phytohormones. The data were processed CW extracts by IAA; and (C) spiked CW extracts by ABA at
using LC/MSD Chemstation software. The same C18 reverse- 265 nm. The concentration of IAA and ABA were
phase column was employed here as HPLC with an injection 122.0 × 10−3 ␮M and 55.0 × 10−3 ␮M, respectively. Note:
volume of 10 ␮L. The column thermostat was set at 40 ◦ C. Sol- Injection volume was 10 ␮L. CW extracts were purified
vent (A) consisted of ultrapure water with 0.1% formic acid earlier using both C18 SPE column (Section 2.2). HPLC
and solvent (B) consisted of methanol with 0.1% formic acid. running condition and peak identities were the same as
The flow rate was 0.3 mL min−1 throughout the whole separa- described in Fig. 2.
278 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281

Table 4 – Precision of eight phytohormones for retention time (tR ) and peak area (pa ) expressed as RSDs (%)
Plant hormones Intra-day variabilitya Inter-day variabilityb

RSDs (tR c %) RSDs (pa d %) RSDs (tR %) RSDs (pa %)

Z 0.26 1.49 0.51 1.74

GA 0.31 2.37 1.01 2.68
IAA 0.46 2.44 1.95 2.27
BA 0.50 4.54 1.37 4.13
ABA 0.38 3.31 1.25 3.17
IBA 0.34 3.44 1.46 2.76
2,4-D 0.34 0.99 1.42 7.13
NAA 0.31 3.13 1.20 1.78

a
The intra-day variation was determined by analyzing in triplicate the same standard mixtures (25 ␮M for each phytohormones) dissolved in
methanol solution for six times within 1 day.
b
The inter-day variation was investigated by analyzing in triplicate the same standards mixtures (25 ␮M for each phytohormones) for consec-
utive 3 days and repeated it 1 month later.
c
The retention time.
d
The peak area.

cally for 10 min, before switching to a linear gradient towards

3. Results and discussion a methanol–formic acid buffer (45:55, v/v) in 35 min and
finally isocratically at methanol–formic acid buffer (45:55, v/v)
3.1. Development of HPLC–PDA method for 15 min (please refer to Table 1 for the gradient elution
program). The sample was injected while a weaker mobile
Different gradient elution programs were tested to find the phase was being applied to the system. The strength of the
optimum separation conditions for the eight phytohormones, mobile phase was later increased by raising the organic sol-
namely IAA, IBA, ABA, GA, Z, BA, NAA and 2,4-D. With vent fraction, which subsequently resulted in elution of the
respect to separation efficiency and shorter analysis time, retained components. Under the optimum separation condi-
the following gradient elution approach was eventually used tions, all eight compounds were successfully separated within
as the preferred method: the column was initialized iso- 45 min. After each analysis, column was washed with 95:5
cratically with methanol–formic acid buffer (10:90, v/v) for methanol–formic acid buffer for 5 min, and then formic acid
5 min, then a linear gradient to methanol–formic acid buffer buffer–methanol 90:10 for ca. 30 min is used for re-equilibrated
(30:70, v/v) in 5 min, which was later maintained isocrati- after this procedure.

Fig. 4 – LC–MS/MS chromatograms of (A1) IAA standard (total ion chromatogram), (B1) putative IAA in CW (MRM: 174 → 130);
and MS/MS spectrum of (A2) IAA standard, (B2) putative IAA. indicates the precursor ion. Note: Injection volume was 2 ␮L.
CW extracts were purified earlier using both C18 SPE column (Section 2.2). LC running conditions was described in Section
3.4. Conditions for the MS were as follows: drying gas (nitrogen) temperature was 350 ◦ C; drying gas flow was 8.0 L min−1 ;
nebulizing gas (nitrogen) pressure was 30.0 psi.
 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281 279

Table 5 – Recoveries of eight phytohormones for SPE procedure

Plant hormones Estimated concentration Recovery (%) Estimated original
(×10−3 ␮M) concentration (×10−3 ␮M)

IAA 122.0 81 150.6

IBA n.d. 82 Below 3.0
NAA n.d. 67 Below 9.4
2,4-D n.d. 90 Below 10.7
Z n.d. 78 Below 4.4
BA n.d. 95 Below 1.8
ABA 55.0 84 65.5
GA n.d. 72 Below 7.4

n.d.: not detected.

Considering the difference UV absorbance of different in triplicate for consecutive 3 days and repeated it 1 month
phytohormones, 265 nm was chosen as the compromised later. The RSDs of the retention time (tR ) and peak area (pa )
optimum detecting condition with the programmable PDA were taken as the measure of precision. The results were given
system. Fig. 2 showed the well resolved HPLC chromatogram in Table 4.
of the eight phytohormone standards mixture with 50 ␮M
for each standard concentration at 265 nm wavelength. The
3.3. Analyses of putative phytohormones in CW using
HPLC–PDA method also allowed for the identification of
HPLC
the eight phytohormones through the comparison of their
retention times and of their UV spectra with those of the stan-
Under the optimum operating conditions, HPLC method was
dards.
applied for the determination of phytohormones in CW. Typ-
Manipulation of the buffer pH value is one of the key strate-
ical chromatogram for the sample after SPE was shown in
gies used for optimizing the separation of analytes in HPLC,
Fig. 3(A). On the basis of retention time as well as the results
since it determines the degree of ionization of each individual
obtained from using the standard addition method (Fig. 3 (B)
analyte. The effect of buffer pH on the separation of eight phy-
and (C)), the presence of IAA and ABA in the CW were success-
tohormones was investigated within the range of 3.0–4.0 for
fully identified. The estimate concentrations of IAA and ABA in
the formic acid buffer (0.1%) as the separation buffer. Finally,
the CW were 122.0 × 10−3 ␮M and 55.0 × 10−3 ␮M, respectively,
the optimum pH condition was at 3.2 and the eight analytes
disregarding the loss during the extraction and purification
can be separated well.
steps. Fig. 3(A) showed certain possible interferences from real
matrix for the analytes of interest. The identity of the analytes
3.2. HPLC method evaluation
of interest for other future application(s), if detected near to
certain interfering peak(s), could be independently confirmed
Fig. 2 showed the chromatograms of the eight phytohor-
by LC–MS experiments.
mones standards mixture obtained by HPLC under optimum
conditions within 45 min. The reproducibility of the reten-
tion time of eight phytohormones under optimum HPLC 3.4. LC–ESI–MS measurements
conditions was investigated by doing repeated injections
(n = 6) of a mixture of the standards at a concentration of The main aim of the LC–MS/MS investigation was to further
25 ␮M. The relative standard deviations (RSDs) of the reten- confirm the identities of the putative phytohormones found in
tion times for all analytes were in the range of 0.2–0.4%. CW using HPLC analysis. The optimized HPLC method (Table 1)
A linear correlation was found between the concentra- was adopted for LC–MS/MS experiment. However, since the
tion and the peak area for all analytes in the range of TEA may affect the MS intensity, it was omitted in LC–MS/MS
10–125 ␮M except BA (10–100 ␮M); typically R2 values were analysis. Typically, with the absence of TEA in the buffer, there
in the range of 0.990–0.999; the limits of quantification was a very slight effect on retention times, the peak shapes
(LOQs) were in the range of 1.70–9.60 ␮M. Three indepen- and resolutions, respectively.
dent injections were carried out for each calibration point. Based on the findings of product ion scan of IAA standard
The reproducibility of peak areas obtained for all the phy- (Fig. 4(A1) and (A2)), the following ion transition was used for
tohormones with RSDs values (n = 6) was in the range of the final MRM detection: m/z 174 → m/z 130. Fig. 4(B1) and (B2)
0.2–5.4%. All the response characteristics of the phytohor- illustrated that IAA was detected without significant interfer-
mone standards using HPLC analysis are summarized in ences from the plant matrix using LC–MS/MS with MRM mode.
Table 3. Similarly, the presence of ABA was confirmed in CW based on
Intra- and inter-day variations were chosen to determine LC–MS/MS results (Fig. 5). From the results of the LC–MS/MS
the precision of the HPLC method. The intra-day variation was investigation, IAA and ABA were unequivocally found to be
determined by analyzing in triplicate the same standard mix- present in the CW sample, based on similar retention times
tures dissolved in methanol solution for six times within 1 day. with LC analysis and their MRM mass transition of IAA and
While for inter-day variability test, the solution was examined ABA.
280 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281

Fig. 5 – LC–MS/MS chromatograms of (A1) ABA standard (total ion chromatogram), (B1) putative ABA in CW (MRM:
263 → 153); and MS/MS spectrum of (A2) ABA standard, and (B2) putative ABA. The LC-MS running conditions were the
same as Fig. 4.

3.5. Recovery study of SPE procedure The effectiveness of this new analytical HPLC method was
evaluated by screening for the putative phytohormones in CW
We investigated on the recovery of the SPE procedure by com- after preconcentration, extraction and purification using SPE.
paring the difference of the obtained analyte concentration The presence of phytohormones in CW, first detected by HPLC,
in extracted spiked sample and the extracted blank sample was further confirmed independently by LC–MS/MS experi-
with the concentration of the spiked analytes in the same ments. The results from the HPLC and LC–MS/MS indicated
volume of blank solution, i.e. Ultrapure water. The concentra- the presence of IAA and ABA in CW samples. The presence
tion in extracted spiked sample and extracted blank sample of ABA in CW would explain why sometimes CW does not
were calculated based on calibration curves. If one depicts the work well as a growth supplement additive, due to the inher-
concentration of spiked analyte in the same volume of blank ent inhibitory properties of ABA on cell growth (via the direct
solution as C1 , the concentration in extracted spiked sample inhibition of cell cycle control at the G1–S point). The impli-
as C2 , and the concentration in extracted blank sample as C3 , cations of the results would be suggestive that ABA should
the recovery values can be calculated as follows: first be removed from CW, before it is being used as a growth
C2 − C3 supplement in tissue culture industry. Alternatively, the occur-
%Recovery = × 100 rence of ABA in CW is an indication that the parent coconut
C1
trees bearing these fruits were under some form of environ-
As estimated in Table 5, the recovery yields of the eight
mental stress (e.g. water deficit, nitrogen deprivation) prior to
different plant hormones ranged from 67 to 95%, which was
these fruits being harvested for sale [27].
considered reasonably high for SPE procedure.
In the current analysis, however, cytokinins were not
detected (Z and BA), despite the known properties of CW as
4. Conclusions an importance source of isoprenoid and aromatic cytokinins
[23–25]. This could be due the types of cytokinins present in
This study reported on the development and evaluation of the CW – other forms of cytokinins may be present instead
novel and relatively simple HPLC and LC–MS/MS methods for of those tested in this project. Furthermore, CW variability
the simultaneous analysis of various classes phytohormones may be a major factor in hindering cross-comparison with past
(Z, GA, IAA, BA, ABA, IBA, 2,4-D and NAA) with high detection studies as this study uses Thai coconuts instead of Malaysian
sensitivity. The separation of the eight standards, including coconuts that were used by Ge et al. [23].
different classes of phytohormones, was completed within 1 h More work on the other phytohormones like brassinos-
with well resolved peaks. Running time was shortened and teroids, jasmonates, and other forms of cytokinins and auxins
separation of ABA, IBA and NAA were optimized by increasing should be done to further characterize the CW. Meanwhile,
the strength of the organic solvent. The optimum separation capillary electrophoresis (CE) work can also be explored to
conditions developed in this project will definitely be useful develop a shorter analysis time and higher resolving power for
for future studies, as typically combinations of such phyto- the analysis. Also, IAA precursor, tryptophan (Trp – an amino
hormones in a single analysis are not normally reported in acid), and IAA conjugates can be quantified as well to indicate
the literature. the potential IAA pools in CW. Finally, with the capability of
 a n a l y t i c a c h i m i c a a c t a 6 1 0 ( 2 0 0 8 ) 274–281 281

the HPLC method to detect phytohormones, it may be possible [10] E. Watanabe, Y. Tsuda, S. Watanabe, S. Ito, M. Hayashi, T.
to further apply the method to analyze different kinds of com- Watanabe, Y. Yuasa, H. Nakazana, Anal. Chim. Acta 424
plex plant samples (e.g. non-liquid endosperm of the coconut (2000) 149.
[11] H.F. Linskens, J.F. Jackson, Modern Methods of Plant
seed, banana, etc.) of interest.
Analysis, Immunology in Plant Sciences, vol.2, Springer,
Berlin Heidelberg, New York, 1986, p. 1.
[12] E. Epstein, J.D. Cohen, J. Chromatogr. 209 (1981) 413.
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Technological University (NTU), Singapore and especially for Chromatogr. A 1075 (2005) 159.
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[16] J. Chamarro, A. Östin, G. Sandberg, Phytochemistry 57 (2001)
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