Molecules 2009, 14, 1825-1839; doi:10.

3390/molecules14051825

OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

Phytohormones as Important Biologically Active Molecules –

Their Simple Simultaneous Detection
Vaclav Diopan 1,2, Vojtech Adam 2,3, Ladislav Havel 1 and Rene Kizek 2,*

1
Department of Plant Biology, Faculty of Agronomy, Mendel University of Agriculture and
Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mails: diopan@email.cz (V.D.),
lhavel@mendelu.cz (L.H.)
2
Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of
Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mails:
ilabo@seznam.cz (V.A.)
3
Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Mendel University
of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mail:
ilabo@seznam.cz (V.A.)

* Author to whom correspondence should be addressed; E-mail: kizek@sci.muni.cz; Tel.: +420-5-

4513-3350; Fax: +420-5-4521-2044

Received: 7 March 2009; in revised form: 8 May 2009 / Accepted: 14 May 2009 /
Published: 15 May 2009

Abstract: Phytohormones, their functions, synthesis and effects, are of great interest. To
study them in plant tissues accurate and sensitive methods are required. In the present
study we aimed at optimizing experimental conditions to separate and determine not only
plant hormones but also their metabolites, by liquid chromatography coupled with a UV-
VIS detector. The mixture we analyzed was composed of benzyladenine, kinetin, trans-
zeatin, cis-zeatin, dihydrozeatin, meta-topolin, ortho-topolin, α-naphthalene acetic acid,
indole-3-acetic acid, trans-zeatin-7-glucoside, trans-zeatin-O-glucoside, trans-zeatin-9-
riboside, meta-topolin-9-riboside and ortho-topolin-9-riboside. We measured the
calibration dependences and estimated limits of detection and quantification under the
optimal chromatographic conditions (column: Polaris C18; mobile phase: gradient starting
at 2:98 (methanol:0.001% TFA) and was increasing to 55:45 during twenty minutes, and
then decreasing for 10 min to 35:65, flow rate: 200 µL·min-1, temperature: 50 °C,
wavelength: 210 nm). The detection limits for the target molecules were estimated as tens
of ng per mL. We also studied the effect of flax extracts on the phytohormones’ signals.
Molecules 2009, 14 1826

Recovery of aliphatic and aromatic cytokinins, metabolites of cytokinins and auxins were
within the range from 87 to 105 %. The experimental conditions were tested on a mass
selective detector. In addition we analysed a commercial product used for stimulation of
roots formation in cuttings of poorly rooting plants. The determined content of
α-naphthalene acetic acid was in good agreement with that declared by the manufacturer.

Keywords: phytohormones; liquid chromatography; UV-VIS detection; flax; mass

spectrometry

Introduction

In plants, a group of chemically varied compounds called phytohormones (plant growth regulators)
regulates physiological processes. The main groups of phytohormones are auxins, cytokinins,
brassinosteroids, gibberellins, jasmonic acid and abscisic acid. Within a specific group phytohormones
are further subdivided according their chemical structures. Cytokinins induce cell division,
morphogenesis (shoot and bud initiation and formation, growth of lateral buds) and delay the
senescence of their tissues. Synthetic cytokinins are used in plant production and in cultivation
processes. Apical dominance is controlled by the concentration gradient of auxins, another group of
phytohormones [1-3]. These phytohormones are also responsible for the tropism, elongation and roots
formation. The inhibiting hormones play role in dormancy maintaining in buds and seeds and their
level increases under stress [4,5].
Except for their origonal structures, all phytohormones are present as various derivatives, e.g.
degradation metabolites, transport, (in)activated or storage forms, especially conjugates with sugars or
amino acids. Some of these derivatives often have the same biological activity as the free hormones, so
their concentrations must be taken into account for an accurate estimation of their effects [6].
Moreover, several biological effects of phytohormones are induced by cooperation of more than one
phytohormone.
Thin-layer chromatography was the first technique used for rapid separation and determination of
phytohormones [7-9]. Nowadays, high performance liquid chromatography (HPLC) coupled with
various types of detectors is the most commonly used technique for separation of phytohormones. As a
detector, UV spectrometers, mass spectrometers or electrochemical detectors are used [10-16].
Coupling of HPLC with mass spectrometry is advantageous, as shown by Prinsen et al., Novak et al.
and Chiwocha et al., who determined several phytohormones simultaneously with a detection limit
under 1 pmol [11,17-20]. Besides liquid chromatography, capillary electrophoresis was also utilized
for the analysis of phytohormones [21-24]. Papers reporting on determination of phytohormones are
typically aimed at one group of these substances, such as cytokinins, auxins and their metabolites,
however, the trend in hormone physiology goes towards hormone profiling and screening for large
numbers of compounds [7,25-27]. Therefore, the main aim of this work was to optimize conditions for
simultaneous HPLC-UV separation of fourteen phytohormones from various phytohormones groups
(Figure 1). Then a real sample of a commercial product used for stimulation of root formation in
cuttings of poorly rooting plants was studied using the optimized conditions.
Molecules 2009, 14 1827

Figure 1. Chemical formulas of the phytohormones. BA = benzyladenine, K – kinetin,

t-Z = trans-zeatin, c-Z = cis-zeatin, DHZ = dihydrozeatin, m-T = meta-topolin,
o-T = ortho-topolin, NAA = α-naphthalene acetic acid, IAA = indole-3-acetic acid,
t-Z-7-G = trans-zeatin-7-glucoside, t-Z-O-G = trans-zeatin-O-glucoside, t-ZR = trans-
zeatin-9-riboside, m-TR = meta-topolin-9-riboside and o-TR = ortho-topolin-9-riboside.
Rib = ribose, Glu = glucose.

aromatic cytokinins aliphatic cytokinins

O CH2OH CH3 CH2OH
HN CH2 HN CH2 HN CH3 HN CH2OH HN CH3
N N N N N
N N N N N

N N N N N N
N H H N H N H N H
BA K t-Z c-Z DHZ
OH HO auxins
HN CH2 HN CH2
CH2COOH
COOH
N N
N N
N
N N N N H
H H NAA IAA
m-T o-T

metabolites of cytokinins OH HO
CH2O Glu CH2OH
H 3C HN CH3 HN CH3 HN CH2
NH Glu HN CH2
N N N N N
N N N N
HOH2C N

N N N N N N
N N N N
H Rib Rib Rib
t-Z-7-G t-Z-O-G t-ZR m-TR o-TR

Results and Discussion

Due to chemical diversity of the phytohormones as a result of their substituent groups on the one
hand and due to the chemical similarity of these substances within a particular group on the other hand,
it was necessary to optimize several experimental conditions (wavelength, column, temperature,
mobile phase) for their sensitive and effective simultaneous detection.

Wavelength

The absorption spectra of the target molecules were measured over the UV range from 200 to
300 nm. The absorption maxima of the each phytohormone are different (Figure 2). If we consider
demands on simultaneous detection of these compounds, two absorption maxima, 210 nm and 270 nm,
can be used. We used z detector setting at 210 nm in the following experiments. Under this
wavelength, the absorption coefficients of t-Z, DHZ, t-ZR and zeatin glucosides are lower in
comparison with 270 nm, but similar or even higher for the other ones.
Molecules 2009, 14 1828

Figure 2. Molecular absorption UV spectra of the phytohormones. Phytohormone

concentration: 20 μg.mL-1 optical path length: 1 cm.

IAA t-Z
NAA c-Z
3 3
BA t-ZR
K DHZ
(AU)

(AU)
t-Z-O-G
Absorbance

Absorbance
t-Z-7-G
2 2
Absorbance

Absorbance
1 1

0 0

200 220 240 260 280 300 320 340 200 220 240 260 280 300 320 340
Wavelength (nm) Wavelength (nm)
4

o-TR
m-T-9-R
3
o-T
(AU)

m-T
Absorbance
Absorbance

200 220 240 260 280 300 320 340

Wavelength (nm)

Column

Low molecular weight biologically active compounds are readily separable using reversed phase
high performance liquid chromatography [28-32]. The most commonly used reversed phase is
octadecyl C18. The properties of the columns from different suppliers are not same because different
technologies are used in the preparation of the support materials and the actual packing of the columns.
In this work, C18 columns from three various suppliers (Aquasil, Phenomenex and Polaris) were tested.
Using of two columns (Aquasil and Phenomenex) under similar conditions (2:98 methanol:water, flow
rate: 200 µL·min-1, temperature: 25°C, wavelength: 210 nm) resulted in the shorter retention times,
leading to co-elution of the compounds. Therefore we analysed the phytohormones on the Polaris
column in the subsequent experiments.

Temperature

The temperature is one of the more important separation parameters. Temperature accelerates mass
transfer in the column and lowers the diffusion coefficient of the mobile phase. The separation under
higher temperatures leads to higher resolution and shorter elution times. We tested the influence of
temperature within the interval from 20 to 60 °C (in 5 °C steps, not shown). Based on the results
obtained the temperature 50 °C was chosen, because the best resolution and the shortest measurement
time were achieved. At higher temperatures co-elution of cZ with DHZ and m-TR with K occurred.
Molecules 2009, 14 1829

Mobile phase and the effect of trifluoroacetic acid

A mobile phase composed of methanol and trifluoroacetic acid (TFA) was used. In order to achieve
easily repeatable measurements no buffer components that might shorten the lifetime of the column
was used. TFA (0; 0.00040; 0.001; 0.0014; 0.002; 0.0024; 0.003%, v/v) was tested. TFA addition
affected the retention time, symmetry and peaks resolution by changing the pH level and the ionization
of the phytohormones. The changes in retention times depending on TFA concentration are shown in
Figure 3. TFA had considerable effect on the symmetry and resolution of the phytohormones’ peaks.
We assume the mechanism of action of TFA on phytohormones to be similar to that on peptides.
According these presumptions, chemically similar cytokinins should be determined with better
resolution and symmetry. We observed that the prevailing effect of an increasing TFA concentration
on retention times was their reduction, due to ion-pairing of the TFA resulting in the enhancement of
the polarity of the target molecules, which confirmed our presumption. Only NAA increased its
retention time, which probably relates with the fact that TFA inhibited ionisation and, thus, prolonged
the time of interaction of this phytohormone with the stationary phase. The highest reduction of the
retention time (by more than 7 minutes comparing values measured under null and 0.003% TFA)
occurred for m-TR and K. It clearly follows from the results obtained that the best resolution was
obtained at 0.001% TFA. All phytohormones’ peaks were well separated and symmetrical.

Figure 3. The dependence of reduced retention time on TFA concentration. The best
resolution was obtained at TFA concentration 0.001%. The concentrations of individual
phytohormones were chosen with respect to similar peaks height and were within the
interval from 6 to 20 μg·mL-1. The detection was carried out at 210 nm. Linear gradient
from 2:98 methanol:TFA (0 min.) up to 85:15 methanol:TFA (40 min.). Mobile phase flow
rate of 250 μL·min-1.

18 28

26
16
24
time (min)
(min)

(min)
time (min)

14 t-Z-7-G K
t-Z-O-G 22 m-T
Retentionfactor

Retentionfactor

12 t-Z 20 IAA
DZH o-T
c-Z 18
Retention

Retention

10 o-TR
t-ZR BA
m-TR 16
8 NAA
14
6
12

4 10
0.000 0.001 0.002 0.003 0.004 0.000 0.001 0.002 0.003 0.004
c TFA (%) c TFA (%)

The TFA concentration affected also the area of the phytohormones’ peaks (Figure 4). The
increasing TFA concentration decreased the response. The major decrease occurred within the interval
from 0.00040 to 0.001% TFA. A higher concentration of TFA did not decrease the signal much. In the
case of DHZ, o-T and o-TR, a sharp decrease of peak areas was observed. On the contrary, the
response of IAA increased with increasing TFA concentration. The NAA peak area did not change
much. Although TFA had adverse effect on peak area of the studied compounds, except for IAA, its
Molecules 2009, 14 1830

presence was essential for good separation. Therefore, we selected a mobile phase consisting of
methanol and 0.001% TFA for the subsequent experiments.

Figure 4. Dependence of phytohormones’ peak areas on TFA concentration.

Chromatographic conditions were the same as in Figure 3.

120 120
t-Z-7-G
t-Z-O-G 100
100 t-Z
DZH K
80 m-T
Peak area (%)

c-Z

Peak area (%)

80 t-ZR IAA
m-TR o-T
60
o-TR
60 BA
40 NAA

40
20

20 0
0.000 0.001 0.002 0.003 0.004 0.000 0.001 0.002 0.003 0.004
c TFA (%) c TFA (%)

Gradient profile and flow rate optimisation

Gradient elution is used for separation of compounds with different affinity towards a stationary
phase. Application of gradients enables separation of more complicated matrices, and eventually a
reduction of analysis time. In spite of the fact that it was possible to separate well all phytohormones of
interest isocratically, we tested a ternary gradient to improve resolution of c-Z and DHZ (tR = 12.09
and 12.27 min, respectively). The optimized gradient is shown in Table 1. Resolution of the peaks was
improved and time of analysis was shortened by about six minutes, compared to isocratic elution with
20 % MeOH.

Table 1. Gradient profile. The column was washed 80% methanol (v/v) for ten minutes and
then methanol:water:water with 0.02% TFA (2:93:5) for ten minutes prior to each analysis.
min MeOH (%) Water (%) Water with 0.02% TFA (%)
0 2 93 5
20 55 40 5
30 35 60 5

Flow rates of the mobile phase within the interval from 100 to 250 µL·min-1 was another tested
parameter, The changes in chromatograms are shown in Figure 5. It follows from the results obtained
that the most suitable flow rate for simultaneous detection of phytohormones is 200 μL·min-1. Lower
or higher flow rates resulted in overlapping signals. At lower flow rates, the K peak overlapped the
m-T peak and the IAA one overlapped the o-T one. At higher flow rates, the peaks of o-TR and BA
were overlapped (Figure 5).
Molecules 2009, 14 1831

Under the optimal chromatographic conditions (column: Polaris C18, mobile phase: Table 1, flow
rate: 200 µL·min-1, temperature: 50 °C, wavelength: 210 nm) we measured the calibration
dependences and estimated limits of detection and quantification (Table 2). The concentration interval,
in which analytical data have been obtained, was different, depending on sensitivity of the method to
certain compounds. Compounds providing the highest responses (IAA and NAA) were measured
within the interval from 0.8 to 6.25 µg·mL-1. The least detectable compound (t-Z-7-G) had an interval
from 2.8 to 22 µg·mL-1.

Figure 5. Typical HPLC-UV chromatograms of phytohormones measured at four various

flow rates: A – 100 μL·min-1, B – 150 μL·min-1, C – 200 μL·min-1 and D – 250 μL·min-1.
The best separation was achieved at 200 μL·min-1.

200 (mAU)

A
Absorbance (mAU)

Time (min)

A study of the effects of the heavy metal ions on phytohormones could provide valuable data for
processes using biological systems (plants, bacteria, fungi) for the remediation of soil and water
contaminated with toxic metals and other toxic substances. These methods are environmentally
friendly alternatives to industrially applied processes. The advantage of these approaches in
comparison with the conventional physico-chemical methods is their low price, minimum quantities of
secondary waste produced and the possibility of removing contaminants from large areas without the
need for extraction of the contaminated soil. Because of the bio-friendliness to the environment of
these remediation approaches and their aesthetic value they are well accepted by the public. Flax
seems to be one of the potential of plants suitable for remediation of heavy metals from contaminated
soil, but there is still a lack detailed information on the biological changes caused by heavy metals,
aside from well investigated plant heavy-metal-protective processes through synthesis of peptides rich
Molecules 2009, 14 1832

in cysteine moieties [33-40]. In our experiments, we studied the effect of flax extracts on the
phytohormones’ signals to use the results in subsequent biological experiments. Recovery of aliphatic
and aromatic cytokinins, metabolites of cytokinins and auxins were evaluated with MeOH extract of
flax (Linum usitatissimum L.) spiked with standards by HPLC-UV (210 nm). The extracts were
assayed blindly and the phytohormones concentration was derived from the calibration curves. The
spiking of hormones was determined as a standard measured without presence of real sample [41,42].
The results obtained are summarized in Table 2.

Table 2. Analytical parameters for HPLC-UV determination (n = 5).

a b c d
LOD LOQ R.S.D. Recovery
Phytohormone Regre ssion R2
(ng/mL) (ng/mL) (%) (%)
Aliphatic cytokinins
t-Z y = 91.193x - 10.217 1 60 200 4.4 96
c-Z y = 88.334x - 3.5650 0.9996 50 170 5.5 98
DHZ y = 54.361x - 10.050 0.9998 110 360 5.9 95
Aromatic cytokinins
BA y = 41.432x - 11.270 0.9988 130 430 4.6 87
K y = 82.169x - 35.783 0.9991 70 230 4.5 98
m-T y = 65.262x + 0.8680 0.9995 50 160 5.2 95
o-T y = 56.429x - 33.103 0.9935 140 460 5.5 96
Metabolites of cytokinins
t-ZR y = 85.878x - 5.3040 0.9999 60 200 5.2 92
t-ZG y = 34.709x - 6.4040 0.9999 150 500 5.5 105
t-ZOG y = 42.958x - 9.007 1 130 430 5.8 103
m-TR y = 77.946x - 47.529 0.9995 80 260 4.8 95
o-TR y = 47.491x - 13.702 0.9988 120 400 4.5 98
Auxins
IAA y = 408.970x - 136.390 0.9945 20 70 4.8 97
NAA y = 283.760x + 6.652 0.9985 20 70 3.9 99
a
Limit of Detection estimated (3 signal/noise, S/N) were calculated according to Long and Winefordner [43],
whereas N was expressed as standard deviation of noise determined in the signal domain unless stated
otherwise; b Limit of Quantification estimated as (10 S/N). [43]; c Relative Standard Deviation; d Recovery
was estimated according to protocol mentioned below.

Mass spectrometry of phytohormones

For ultrasensitive determination of target molecules HPLC is connected with modern and sensitive
detectors, most commonly with mass spectrometers. There are several various types of mass
spectrometers, including tandem mass spectrometers, which are very advantageous for analysis of
overlapping signals due to different fragmentation of mother ions with the same mass. However, these
instruments are costly. The method optimized in this study is suitable for detection of fourteen
phytohormones. Considering the fact that mobile phase did not contain any buffer, the conditions can
be applied on an instrument equipped with a mass detector. On the other hand, however, our mobile
phase contained TFA, which, like other strong ion-pairing compounds, can also degrade the signals
of target molecules [44]. Therefore, we studied the influence of TFA on the MS detection of
Molecules 2009, 14 1833

phytohormones (Figure 6). Phytohormones (0.5 µg·mL-1) were detected using flow injection analysis
with mass detection. Methanol:water (50:50, v/v) with or without addition of TFA (0.001 %, v/v) was
used as a mobile phase. We detected molecular peaks (M+H)+ in all studied phytohormones under the
above-mentioned experimental conditions. The intensity of molecular peaks was not influenced by the
presence of TFA (decrease app. 2-4 % compared to non-TFA signals). We thus assume that any
influence on the signals’ intensities due to the low TFA concentration used is negligible.

Figure 6. Mass spectra of phytohormones measured with or without addition of TFA in

mobile phase. Concentration of TFA – 0.001%, mobile phase – methanol:water (50:50,
v/v), capillary voltage 3.5 kV, drying gas temperature 250 °C, nebulizer pressure 5 MPa;
drying gas flow 6 L·min-1; cone voltage – 20 V.

Aromatic cytokinins
7.0E+05 226.30 7.0E+05 6.0E+05 6.0E+05
226.01 K 216.01 K with TFA 216.18
(M+H)+ BA with TFA (M+H)+
6.0E+05 BA 6.0E+05 5.0E+05 (M+H)+ 5.0E+05
(M+H)+

Intensity
Intensity
Intensity
Intensity

5.0E+05 5.0E+05
4.0E+05 4.0E+05
4.0E+05 4.0E+05
3.0E+05 3.0E+05 3.0E+05 3.0E+05

2.0E+05 2.0E+05 2.0E+05 2.0E+05

1.0E+05 1.0E+05
1.0E+05 1.0E+05
0 50 150 250 350 0 0 0
50 150 250 350 50 150 250 350 50 150 250 350
m/z m/z m/z m/z
242.19 242.03
4.6E+05 242.26 4.6E+05 4.8E+05 (M+H)+ 4.6E+05 242.11
o-T o-T w ith TFA (M+H)+ m-T m-T with TFA (M+H)+
(M+H)+
3.8E+05 3.8E+05 4.0E+05 3.8E+05
Intensity
Intensity

Intensity

Intensity

3.0E+05 3.0E+05 3.2E+05 3.0E+05

2.2E+05 2.2E+05 2.4E+05 2.2E+05

1.4E+05 1.4E+05 1.6E+05 1.4E+05

0.6E+05 0.6E+05 0.8E+05 0.6E+05

0 50 150 250 350 0 0 50 150 250 350 0
50 150 250 350 50 150 250 350
m/z m/z m/z m/z

Aliphatic cytokinins
220.35 220.35
6.0E+05 cZ 220.31 6.0E+05 cZ with TFA (M+H)+ 6.0E+05 tZ (M+H)+ 6.0E+05 tZ with TFA 220.36
(M+H)+ (M+H)+
5.0E+05 5.0E+05 5.0E+05 5.0E+05
Intensity
Intensity

Intensity
Intensity

4.0E+05 4.0E+05 4.0E+05 4.0E+05

3.0E+05 3.0E+05 3.0E+05 3.0E+05

2.0E+05 2.0E+05 2.0E+05 2.0E+05

1.0E+05 1.0E+05 1.0E+05 1.0E+05

0 50 150 250 350 0 50 150 m/z 250 350 0 50 150 m/z 250 350 0 50 150 250 350
m/z m/z

222.15 222.18
5.5E+05 DHZ (M+H)+ 5.5E+05 DHZ with TFA (M+H)+
4.5E+05 4.5E+05
Intensity

Intensity

3.5E+05 3.5E+05

2.5E+05 2.5E+05

1.5E+05 1.5E+05

0.5E+05 0.5E+05

0 50 150 250 350 0 50 150 250 350

m/z m/z
Molecules 2009, 14 1834

Figure 6. Cont.

Metabolites of cytokinins
373.34 373.36 373.37
6.5E+05 o-TR 6.5E+05 o-TR with TFA (M+H)+
6.4E+05 m-TR (M+H)+ 6.4E+05 m-TR with TFA 373.36
(M+H)+ (M+H)+
5.5E+05 5.5E+05 5.4E+05 5.4E+05

Intensity
Intensity

Intensity

Intensity
4.5E+05 4.5E+05 4.4E+05 4.4E+05

3.5E+05 3.5E+05 3.4E+05 3.4E+05

2.5E+05 2.5E+05 2.4E+05 2.4E+05

1.5E+05 1.5E+05 1.4E+05 1.4E+05

5.0E+05 5.0E+05 0.4E+05 0.4E+05

0 0 50 150 m/z 250 350 0 50 150 250 350 0
50 150 m/z 250 350 50 150 m/z 250 350
m/z

382.36 382.36
5.7E+05 5.7E+05 tZ-O-G with TFA
tZ-O-G (M+H)+ (M+H)+

4.7E+05 4.7E+05

Intensity
Intensity

3.7E+05 3.7E+05

2.7E+05 2.7E+05

1.7E+05 1.7E+05

0.7E+05 0.7E+05
0 50 150 m/z 250 350 0 50 150 m/z 250 350

352.41 382.33 382.33

352.41 5.6E+05 5.2E+05 5.2E+05
5.6E+05 tZR tZR with TFA (M+H)+ (M+H)+ tZ-7-G with TFA (M+H)+
(M+H)+ tZ-7-G
4.6E+05 4.2E+05 4.2E+05
4.6E+05
Intensity

Intensity
Intensity
Intensity

3.6E+05 3.2E+05 3.2E+05

3.6E+05
2.6E+05 2.2E+05 2.2E+05
2.6E+05

1.6E+05 1.2E+05 1.2E+05

1.6E+05
0.6E+05 0.2E+05 0.2E+05
0.6E+05
0 0 50 150 m/z 250 350 0
0 50 150 250 350 50 150 m/z 250 350 50 150 m/z 250 350
m/z

Auxins
176.04 176.06 187.20 187.22
5.5E+05 IAA 5.5E+05 IAA 3.7E+05 NAA 3.7E+05 NAA
(M+H)+ (M+H)+ (M+H)+ (M+H)+
with TFA with TFA
4.5E+05 4.5E+05
Intensity

Intensity

Intensity
Intensity

2.7E+05 2.7E+05
3.5E+05 3.5E+05

2.5E+05 2.5E+05 1.7E+05 1.7E+05

1.5E+05 1.5E+05

0.5E+05 0.5E+05 0.7E+05 0.7E+05

0 50 150 250 350 0 50 150 250 350 0 50 150 250 350 0 50 150 250 350
m/z m/z m/z m/z

Determination of phytohormones in growth stimulator

The proposed chromatographic method with UV detection was employed for analysis of the
commercial product Gelastim A (Czech Republic) containing IAA, indole-3-butyric acid (IBA) and
NAA, used for the stimulation of roots formation in cuttings of poorly rooting plants. According to the
manufacturer, the product contains 9.5 mg·L-1 of α-naphthalene acetic acid (NAA). In addition, the
mixture also contains 8-hydroxyquinoline sulphate (150 mg.L-1) for disinfecting the wounded surfaces
of cuttings. HPLC measurement of the sample was carried out without any pre-treatment of the
Gelastim A. The sample was diluted eight-fold with a mixture of methanol and water (1:1) prior to
injection on the column. We determined two major signals in the chromatogram (Figure 7A). For
identification of the compounds, measurement with standard addition of the growth regulators IAA,
IBA (Sigma-Aldrich, USA) and NAA was carried out (Figure 7B). This addition demonstrated that the
terminal signal in the chromatogram belongs to NAA, which was confirmed by a mass detector.
Molecules 2009, 14 1835

According to calibration curve, we determined the NAA content as 9.3 ± 0.3 mg·L-1. This value is in
good agreement with that declared by the manufacturer (9·5 mg.L-1).

Figure 7. Chromatogram A shows analysis of fivefold diluted gelastim A. Chromatogram

B shows analysis of the eightfold diluted gelastin A with standard addition of IAA and
NAA (0.5 µg to one mL of the diluted sample). Chromatographic conditions were as
follows: column: Polaris C18, mobile phase: Table 1, flow rate: 200 µL·min-1, temperature:
50 °C, wavelength: 210 nm, sample injection: 20 µL.

400
3

B
1 2
300
U (mV)(mAU)

3
Absorbance

200 1: IAA
2: IBA
3: NAA
A 2
100

0 10 20 30 40

time (min)

Experimental

Chemicals

Phytohormones (trans-zeatin, cis-zeatin, dihydrozeatin, trans-zeatin-7-glucoside, trans-zeatin-9-

riboside, trans-zeatin-O-glucoside, meta-topolin, meta-topolin-9-riboside, ortho-topolin, ortho-topolin-
9-riboside, α-naphthalene acetic acid) purchased from Olchemim (Olomouc, Czech Republic) and
indole-3-acetic acid, kinetin and 6-N-benzyladenine purchased from Sigma-Aldrich (St. Louis, USA)
were of 98 % purity prior to HPLC analyses. Ultrapure Milli-Q (18 MΏ) water and methanol from
Merck (Darmstadt, Germany) and trifluoroacetic acid (Sigma-Aldrich) were used as mobile phase.
Molecules 2009, 14 1836

The stock solutions of phytohormones (0.2 - 2 mg·mL-1) were prepared using methanol-water 50/50
(w/w) and stored in the dark at -20°C. The working solutions were prepared daily by diluting of the
stock solutions. Stock o-TR solution was prepared daily due to its instability in methanol.

High performance liquid chromatography with UV-VIS/MS detection (HPLC/UV/VIS/MS)

To separate the target molecules three reversed phase columns were used: Phenomenex C18,
250 × 2.1 mm, particle size 5 μm, MetaChem Polaris C18, 250 × 2.1 mm, particle size 5 μm and
Thermoelectron Aquasil C18 250 × 2.1 mm, particle size 5 μm. Chromatographic pump Rheos (Flux
Instruments, Switzerland) connected with a HTS PAL (CTC Analytics, Sweden) autosampler was
used. The instrument was controlled by Xcalibur software. UV-VIS detector SpectraSYSTEM UV
2000 (Thermo separation products Inc.) and mass detector Finnigan AQA (ThermoQuest) were used
for detection of target molecules. The samples were injected by autosampler. Other chromatographic
parameters were optimized and are shown in the “Results and Discussion” section.

Plants

Flax (Linum usitatissimum L.) hybrid Viola was used in our experiments. Flax kernels were
germinated on wet cellulose in Petri dishes at 23 ± 2 °C in dark. Germination was carried out in
Versatile Environmental Test Chamber (MLR-350 H, Sanyo, Japan) for seven days with 12 h long
daylight per day (maximal light intensity was about 100 μE.m-2s-1) at a temperature 21.5 – 22.5 °C and
humidity 45 – 55 %. Cellulose was wetted twice a day to avoid its dessication. At the end of seven day
long cultivation the seedlings were harvested and used in the subsequent experiments.

Sample preparation

The harvested flax plants (app. 1 g of fresh weight) were extracted with 10 mL of methanol.
The extract was purified with two solid-phase-extraction columns (WAT020515, Waters, USA). The
extract was diluted eight times and spiked with phytohormones standards prior to evaluation of
recovery.

Descriptive statistics

Data were processed using Microsoft Excel® (USA). Results are expressed as mean ± standard
deviation (S.D.), unless stated otherwise.

Acknowledgements

Financial support from DP 40/AF IGA MZLU and 1M06030 is greatly acknowledged.

References and Notes

1. Swarup, K.; Benkova, E.; Swarup, R.; Casimiro, I.; Peret, B.; Yang, Y.; Parry, G.; Nielsen, E.; De
Smet, I.; Vanneste, S.; Levesque, M.P.; Carrier, D.; James, N.; Calvo, V.; Ljung, K.; Kramer, E.;
Roberts, R.; Graham, N.; Marillonnet, S.; Patel, K.; Jones, J.D.G.; Taylor, C.G.; Schachtman,
Molecules 2009, 14 1837

D.P.; May, S.; Sandberg, G.; Benfey, P.; Friml, J.; Kerr, I.; Beeckman, T.; Laplaze, L.; Bennett,
M.J. The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 2008, 10,
946-954.
2. Dhonukshe, P.; Tanaka, H.; Goh, T.; Ebine, K.; Mahonen, A.P.; Prasad, K.; Blilou, I.; Geldner,
N.; Xu, J.; Uemura, T.; Chory, J.; Ueda, T.; Nakano, A.; Scheres, B.; Friml, J. Generation of cell
polarity in plants links endocytosis, auxin distribution and cell fate decisions. Nature 2008, 456,
962-975.
3. De Smet, I.; Vassileva, V.; De Rybel, B.; Levesque, M.P.; Grunewald, W.; Van Damme, D.; Van
Noorden, G.; Naudts, M.; Van Isterdael, G.; De Clercq, R.; Wang, J.Y.; Meuli, N.; Vanneste, S.;
Friml, J.; Hilson, P.; Jurgens, G.; Ingram, G.C.; Inze, D.; Benfey, P.N.; Beeckman, T. Receptor-
like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science 2008, 322,
594-597.
4. Barendse, G.W.M.; Peeters, T.J.M. Multiple hormonal-control in plants. Acta Bot. Neerl. 1995,
44, 3-17.
5. Weyers, J.D.B.; Paterson, N.W. Plant hormones and the control of physiological processes. New
Phytol. 2001, 152, 375-407.
6. Sakakibara, H. Cytokinins: Activity, biosynthesis, and translocation. Annu. Rev. Plant Biol. 2006,
57, 431-449.
7. Tarkowski, P.; Dolezal, K.; Strnad, M. Analytical methods in cytokinin research. Chem. Listy.
2004, 98, 834-841.
8. Karadeniz, A.; Topcuoglu, S.F.; Inan, S. Auxin, gibberellin, cytokinin and abscisic acid
production in some bacteria. World J. Microbiol. Biotechnol. 2006, 22, 1061-1064.
9. Tuomi, T.; Rosenqvist, H. Detection of abscisic, gibberellic and indole-3-acetic-acid from plants
and microbes. Plant Physiol. Biochem. 1995, 33, 725-734.
10. Gomez-Cadenas, A.; Pozo, O.J.; Garcia-Augustin, P.; Sancho, J.V. Direct analysis of abscisic
acid in crude plant extracts by liquid chromatography-electrospray/tandem mass spectrometry.
Phytochem. Anal. 2002, 13, 228-234.
11. Prinsen, E.; Redig, P.; Vandongen, W.; Esmans, E.L.; Vanonckelen, H.A. Quantitative-analysis of
cytokinins by electrospray tandem mass-spectrometry. Rapid Commun. Mass Spectrom. 1995, 9,
948-953.
12. Ge, L.; Yong, J.W.H.; Goh, N.K.; Chia, L.S.; Tan, S.N.; Ong, E.S. Identification of kinetin and
kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid
chromatography-tandem mass spectrometry, high performance liquid chromatography and
capillary electrophoresis. J. Chromatogr. B. 2005, 829, 26-34.
13. Hahn, H. High-performance liquid-chromatography and its use in cytokinin determination in
Agrobacterium-tumefaciens B6. Plant Cell Physiol. 1976, 17, 1053-1058.
14. Lebuhn, M.; Hartmann, A. Method for the determination of indole-3-acetic-acid and related-
compounds of L-tryptophan catabolism in soils. J. Chromatogr. 1993, 629, 255-266.
15. Nordstrom, A.; Tarkowski, P.; Tarkowska, D.; Dolezal, K.; Astot, C.; Sandberg, G.; Moritz, T.
Derivatization for LC electrospray ionization-MS: A tool for improving reversed-phase separation
and ESI responses of bases, ribosides, and intact nucleotides. Anal. Chem. 2004, 76, 2869-2877.
Molecules 2009, 14 1838

16. Hernandez, P.; Paton, F.; Hernandez, L. Voltammetric study of kinetin in a carbon fiber
microelectrode. Determination by adsorptive stripping in apple extracts. Electroanalysis 1997, 9,
1372-1374.
17. Novak, O.; Tarkowski, P.; Tarkowska, D.; Dolezal, K.; Lenobel, R.; Strnad, M. Quantitative
analysis of cytokinins in plants by liquid chromatography-single-quadrupole mass spectrometry.
Anal. Chim. Acta 2003, 480, 207-218.
18. Chiwocha, S.; Rouault, G.; Abrams, S.; von Aderkas, P. Parasitism of seed of Douglas fir
(Pseudotsuga menziesii) by the seed chalcid, Megastigmus spermotrophus, and its influence on
seed hormone physiology. Sex. Plant Reprod. 2007, 20, 19-25.
19. Chiwocha, S.D.S.; Abrams, S.R.; Ambrose, S.J.; Cutler, A.J.; Loewen, M.; Ross, A.R.S.;
Kermode, A.R. A method for profiling classes of plant hormones and their metabolites using
liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone
regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant J. 2003, 35, 405-417.
20. Chiwocha, S.D.S.; Cutler, A.J.; Abrams, S.R.; Ambrose, S.J.; Yang, J.; Ross, A.R.S.; Kermode,
A.R. The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and
gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and
germination. Plant J. 2005, 42, 35-48.
21. Ge, L.Y.; Tan, S.; Yong, J.W.H.; Tan, S.N. CE for cytokinin analyses: A review. Electrophoresis
2006, 27, 4779-4791.
22. Zheng, B.; Yang, H.X.; He, J.L. Quantitative analysis of plant hormones with capillary
electrophoresis. Chin. J. Anal. Chem. 1999, 27, 704-707.
23. Liu, J.; Li, S.F.Y. Separation and determination of auxins by capillary electrophoresis. J. Liq.
Chromatogr. Relat. Technol. 1996, 19, 1697-1713.
24. Olsson, J.C.; Andersson, P.E.; Karlberg, B.; Nordstrom, A.C. Determination of plant indoles by
capillary electrophoresis with amperometric detection. J. Chromatogr. A. 1996, 755, 289-298.
25. Hoyerova, K.; Gaudinova, A.; Malbeck, J.; Dobrev, P.I.; Kocabek, T.; Solcova, B.; Travnickova,
A.; Kaminek, M. Efficiency of different methods of extraction and purification of cytokinins.
Phytochemistry 2006, 67, 1151-1159.
26. Lopez-Carbonell, M.; Gabasa, M.; Jáuregui, O. Enhanced determination of abscisic acid (ABA)
and abscisic acid glucose ester (ABA-GE) in Cistus albidus plants by liquid chromatography-
mass spectrometry in tandem mode. Plant Physiol. Biochem. 2009, 47, 256-261.
27. Owen, S.J.; Abrams, S.R. Measurement of plant hormones by liquid chromatography-mass
spectrometry. Methods Mol. Biol. 2009, 495, 39-51.
28. Gazdik, Z.; Reznicek, V.; Adam, V.; Zitka, O.; Jurikova, T.; Krska, B.; Matuskovic, J.; Plsek, J.;
Saloun, J.; Horna, A.; Kizek, R. Use of liquid chromatography with electrochemical detection for
the determination of antioxidants in less common fruits. Molecules 2008, 13, 2823-2836.
29. Petrlova, J.; Mikelova, R.; Stejskal, K.; Kleckerova, A.; Zitka, O.; Petrek, J.; Havel, L.; Zehnalek,
J.; Adam, V.; Trnkova, L.; Kizek, R. Simultaneous determination of eight biologically active thiol
compounds using gradient elution-liquid chromatography with Coul-Array detection. J. Sep. Sci.
2005, 29, 1166-1173.
30. Potesil, D.; Petrlova, J.; Adam, V.; Vacek, J.; Klejdus, B.; Zehnalek, J.; Trnkova, L.; Havel, L.;
Kizek, R. Simultaneous femtomole determination of cysteine, reduced and oxidized glutathione,
Molecules 2009, 14 1839

and phytochelatin in maize (Zea mays L.) kernels using high-performance liquid chromatography
with electrochemical detection. J. Chromatogr. A. 2004, 1084, 134-144.
31. Klejdus, B.; Petrlova, J.; Potesil, D.; Adam, V.; Mikelova, R.; Vacek, J.; Kizek, R.; Kuban, V.
Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by
high-performance liquid chromatography coupled with diode array detection. Anal. Chim. Acta.
2003, 520, 57-67.
32. Klejdus, B.; Vacek, J.; Adam, V.; Zehnalek, J.; Kizek, R.; Trnkova, L.; Kuban, V. Determination
of isoflavones in soybean food and human urine using liquid chromatography with
electrochemical detection. J. Chromatogr. B. 2004, 806, 101-111.
33. Grill, E.; Winnacker, E.L.; Zenk, M.H. Phytochelatins - the principal heavy-metal complexing
peptides of higher-plants. Science 1985, 230, 674-676.
34. Zenk, M.H. Heavy metal detoxification in higher plants - A review. Gene. 1995, 179, 21-30.
35. Cobbett, C.; Goldsbrough, P. Phytochelatins and metallothioneins: Roles in heavy metal
detoxification and homeostasis. Annu. Rev. Plant Biol. 2002, 53, 159-182.
36. Hall, J.L. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 2002,
53, 1-11.
37. Babula, P.; Adam, V.; Opatrilova, R.; Zehnalek, J.; Havel, L.; Kizek, R. Uncommon heavy
metals, metalloids and their plant toxicity: a review. Environ. Chem. Lett. 2008, 6, 189-213.
38. Diopan, V.; Shestivska, V.; Adam, V.; Macek, T.; Mackova, M.; Havel, L.; Kizek, R.
Determination of content of metallothionein and low molecular mass stress peptides in transgenic
tobacco plants. Plant Cell Tissue Organ Cult. 2007, 94, 291-298.
39. Supalkova, V.; Huska, D.; Diopan, V.; Hanustiak, P.; Zitka, O.; Stejskal, K.; Baloun, J.; Pikula, J.;
Havel, L.; Zehnalek, J.; Adam, V.; Trnkova, L.; Beklova, M.; Kizek, R. Electroanalysis of plant
thiols. Sensors 2007, 7, 932-959.
40. Klejdus, B.; Zehnalek, J.; Adam, V.; Petrek, J.; Kizek, R.; Vacek, J.; Trnkova, L.; Rozik, R.;
Havel, L.; Kuban, V. Sub-picomole high-performance liquid chromatographic/mass spectrometric
determination of glutathione in the maize (Zea mays L.) kernels exposed to cadmium. Anal. Chim.
Acta. 2003, 520, 117-124.
41. Causon, R. Validation of chromatographic methods in biomedical analysis - Viewpoint and
discussion. J. Chromatogr. B. 1996, 689, 175-180.
42. Bugianesi, R.; Serafini, M.; Simone, F.; Wu, D.Y.; Meydani, S.; Ferro-Luzzi, A.; Azzini, E.;
Maiani, G. High-performance liquid chromatography with coulometric electrode array detector
for the determination of quercetin levels in cells of the immune system. Anal. Biochem. 2000,
284, 296-300.
43. Long, G.L.; Winefordner, J.D. Limit of detection. Anal. Chem. 1983, 55, A712-A724.
44. Annesley, T.M. Ion suppression in mass spectrometry. Clin. Chem. 2003, 49, 1041-1044.

Sample Availability: Samples of the compounds of interest are available from the authors.

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