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Simultaneousdeterminationofgibberellicacid,indoleaceticacidandabscisicacidinwheatextractsbysolid-phaseextractionandliquidchromatography-electrospraytandemmass...

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Talanta 76 (2008) 798–802

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Simultaneous determination of gibberellic acid, indole-3-acetic acidand abscisic acid in wheat extracts by solid-phase extraction and liquidchromatography–electrospray tandem mass spectrometry

Shengjie Houa,b, Jiang Zhub, Mingyu Dinga,∗, Guohua Lvc

a Key Lab of Bioorganic Phosphorus Chemistry & Chemical Biology, Ministry of Education,Department of Chemistry, Tsinghua University, Beijing 100084, Chinab College of Resources & Environment, Anhui Agricultural University, Hefei 230036, Chinac Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of Geographic

Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China

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Article history:Received 20 February 2008Received in revised form 7 April 2008Accepted 12 April 2008Available online 24 April 2008

Keywords:Liquid chromatography–tandem massspectrometrySolid-phase extractionGibberellic acidIndole-3-acetic acidAbscisic acid

a b s t r a c t

A liquid chromatography–neous determination of tindole-3-acetic acid (IAA)was used to concentrateThe separation was carrieformic acid (50:50, v/v) astohormones were elutedionization source was opefor quantitative measuremfor IAA and 263 → 153, 2for IAA and 0.005–10 �g mof three were 0.005 �g mrecoveries from 95.5% to

that the SPE-LC–MS/MS methophytohormones in plant sampl

1. Introduction

Phytohormones are regulators produced by plants themselves,which control the physiological processes. As a minor componentof the metabolome, phytohormones are of particular significancegiven their role in the regulation of germination, growth, repro-duction, and the protective responses of plants against stress.[1] Phytohormones are usually classified into four groups: aux-ines, gibberellines, cytokinines and inhibitors [2]. Gibberellic acid(GA3), indole-3-acetic acid (IAA) and abscisic acid (ABA) are thechief representatives of gibberellins, auxines and inhibitors, respec-tively. The quantitative analysis of the three phytohormones isoften required in agriculture and plant physiology. However, manyresearches until now have focused on a specific phytohormone orcertain group of phytohormones but ignored the others. There is

∗ Corresponding author. Tel.: +86 10 62797087; fax: +86 10 62782485.E-mail address: dingmy@mail.tsinghua.edu.cn (M. Ding).

0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.talanta.2008.04.041

em mass spectrometry (LC–MS/MS) method was developed for simulta-representative phytohormones in plant samples: gibberellic acid (GA3),abscisic acid (ABA). A solid-phase extraction (SPE) pretreatment methodurify the three phytohormones of different groups from plant samples.on a C18 reversed-phase column, using methanol/water containing 0.2%

socratic mobile phase at the flow-rate of 1.0 mL min−1, and the three phy-n 7 min. A linear ion trap mass spectrometer equipped with electrosprayin negative ion mode. Selective reaction monitoring (SRM) was employed

The SRM transitions monitored were as 345 → 239, 301 for GA3, 174 → 130r ABA. Good linearities were found within the ranges of 5–200 �g mL−1

for ABA and GA3. Their detection limits based on a signal-to-noise ratio.2 �g mL−1 and 0.003 �g mL−1 for GA3, IAA and ABA, respectively. Good

% for the three phytohormones were obtained. The results demonstratedd developed is highly effective for analyzing trace amounts of the threees.

© 2008 Elsevier B.V. All rights reserved.

little information available on the simultaneous determination ofseveral phytohormones of different groups.

It is usually rather difficult to determine phytohormones inplant tissues owing to their presence in trace amounts and to thecoexistence of a wide variety of interferents. Therefore, it is veryimportant to develop simple and reliable purification proceduresfor the accurate determination of the three phytohormones. Com-mon purification procedures such as liquid-phase extraction (LPE)[3], solid-phase extraction (SPE) [4–9], vapor-phase extraction [10]and solid-phase microextraction [11] have been employed for thepurification of phytohormones in plants. However, LPE is extremelyexpensive and adverse to the environment and human health dueto the use of large amounts of organic solvents.

Several methods have been used for the analysis of phytohor-mones. Among them, high performance liquid chromatography(HPLC) coupled to ultraviolet (UV) [5] or fluorescence [12] or coulo-metric detection [13] has been described and immunoassay [14,15]has also been employed. Mass spectrometry (MS) is the most pow-erful detector for the determination of phytohormones due to

S. Hou et al. / Talanta 76

Fig. 1. The structures of GA3, IAA and ABA.

its high sensitivity and selectivity. Gas chromatography coupledto mass spectrometry (GC–MS) [6,7,16,17] was also reported. Theshortages with GC–MS method were that not only was a priorderivatization required to enhance volatility, but also the high tem-peratures reached in GC injector and column could decompose thelabile phytohormones.

In recent years, liquid chromatography–electrospray ioniza-tion tandem mass spectrometry (LC–ESI-MS/MS) has been appliedto the determination of IAA [3,8,9,18] and ABA [3,9,18–20] inplant samples, but the determination of GA3 [9] in LC–ESI-MS/MShas been reported rarely. In this study, the validity of LC–UV todetermine the three phytohormones was tested. This detectionmethod was not sensitive and was not able to separate targetanalytes from co-extractives. In LC–ESI-MS/MS, however, the inter-ference from co-extractives was reduced dramatically by usingselected reaction monitoring (SRM). This method was specificand sensitive and did not require chemical derivatization of ana-lytes.

In the present study, the main objectives were to simplify the

procedures of extraction and purification and to develop a rapidand sensitive detection method for the determination of the threeacidic phytohormones. The conditions of SPE have been optimized.The efficiency of two different extraction solutions for the threephytohormones has also been discussed. In addition, to illustratethe validity of the present method, different plant materials havebeen used.

2. Experimental

2.1. Chemicals and reagents

GA3, IAA and ABA were purchased from Sigma (St. Louis, MO,USA). The structures of the three phytohormones are shown inFig. 1. Purified water from a Milli-Q system from Millipore Corp.(Bedford, MA, USA) and analytical reagent grade chemicals wereused. Benzoic acid was used as internal standard (ISTD). Stock solu-tions of the three phytohormones and ISTD (1 g L−1) were preparedwith 20% methanol in water containing 0.1% formic acid and main-tained at 4 ◦C in a refrigerator. The four stock solutions were thenfurther diluted by 20% methanol in water containing 0.1% formic

(2008) 798–802 799

acid to yield appropriate working solutions. C18 SPE cartridges(3 mL, 500 mg) were obtained from Varian (Palo Alto, CA, USA).

2.2. Plant materials

Plant samples were obtained by cultivating wheat seeds in circu-lar tray covered with clean papers for 4, 6 and 13 days, respectively.A constant dampness with tap water at 25 ◦C and a controlled lightcondition of 16 h light and 8 h darkness were kept. The young wheatplants for 4, 6 and 13 days were at the growth stages of bud, one leafand two leaves, respectively. The wheat plants at different growthstages were halved into overground tissues and roots for analysis.

2.3. Chromatographic and mass procedures

The experiment was performed on a Finnigan LC–MS/MS sys-tem (Thermo Electron, San Jose, CA, USA) consisting of a surveryorautosampler, a surveyor MS pump and a Finnigan LTQ linear iontrap mass spectrometer equipped with an ESI source that was oper-ated in negative mode. The data acquisition software used wasXcalibur. The LC separation was carried out by an HiQ Sil C18 col-umn (250 mm × 4.6 mm i.d., 5 �m). The three phytohormones wereeluted isocratically with methanol/water containing 0.2% formicacid (50:50, v/v) at the flow-rate of 1.0 mL min−1. The injector vol-ume selected was 25 �L. LC–MS/MS conditions were as follows: ESIspray voltage, 4 kV; sheath gas flow-rate, 70 arb; auxiliary gas flow-rate, 20 arb; capillary voltage, −38 V; capillary temperature, 350 ◦Cand tube lens, 95 V. The SRM mode was used for the determinationof the three phytohormones. GA3, IAA and ABA were monitored atm/z transitions of 345 → 239, 301; 174 → 130 and 263 → 153, 219,respectively. The optimized collision energies for GA3, IAA and ABAwere 21, 28 and 20 eV, respectively. Selected ion monitoring (SIM)mode was used for the determination of ISTD. ISTD was monitoredat m/z 121.

2.4. Extraction and purification procedures

Plant samples were snipped and ground to fine powder inthe presence of liquid nitrogen. Subsequently, 2 g powdered plantmaterials were extracted at 4 ◦C for 12 h with either 20 mL deion-ized water or 20 mL 80% methanol [3,19]. In both cases, before anyextraction was performed, 100 �g ISTD was added. The extract wascentrifuged at 4 ◦C at 3000 rpm for 15 min. When water was usedas an extractant, the supernatant was passed directly through a C18

cartridge preconditioned with 3 mL deionized water, followed by3 mL methanol. When 80% methanol was used as an extractant,the methanol was evaporated in vacuum at room temperature,and then the aqueous residue was adjusted to 20 mL with waterbefore passed through a preconditioned C18 cartridge. Both car-tridges were washed with 1 mL 20% methanol containing 0.1% (v/v)formic acid and the retained phytohormones were eluted with 1 mL80% methanol.

These SPE conditions were optimized after a deep study, shownin Section 3, the same for optimization of chromatographic condi-tions and SRM detection.

3. Results and discussion

3.1. Optimization of LC–ESI-MS/MS conditions

In the study, the full mass spectra of the three phytohormonesshowed that [M−H]− ions were the most intensive at m/z 345for GA3, m/z 174 for IAA, m/z 263 for ABA, and m/z 121 for ISTD,so they were chosen as precursor ions for the phytohormones. Inthe product ions spectra (Fig. 2, 2a for GA3, 2b for IAA and 2c for

800 S. Hou et al. / Talanta 76 (2008) 798–802

Fig. 2. Negative ion MS/MS spectra from LC–ESI-MS/MS analysis of GA3 (2a), IAA(2b) and ABA (2c).

ABA), [M−H–COO–COO–H2O]− ion (m/z 239) and [M−H–COO]−

ion (m/z 301) for GA3, [M−H–COO]− ion (m/z 130) for IAA and[M−H–C6H6O2]− ion (m/z 153) and [M−H–COO]− ion (m/z 219) forABA were selected in SRM acquisition respectively, because theydisplayed better intensities.

The three phytohormones were separated using a C18 col-umn with water/methanol isocratic elution. With the addition offormic acid, chromatographic separation was improved greatlywith sharper peak shape and better peak symmetry of thethree phytohormones, because formic acid converted the threeacidic phytohormones from the anionic forms to the neutralones. A typical LC–ESI-MS/MS chromatogram of a mixed stan-dard solution containing 50 �g mL−1 GA3, IAA and ABA underSRM conditions is shown in Fig. 3. GA3, IAA, ABA and ISTDwere eluted at 4.14 ± 0.04 min, 5.21 ± 0.03 min, 6.76 ± 0.05 min and6.07 ± 0.05 min, respectively. In the GA3 standard chromatogram, aminor peak occurred at 3.70 min, which could be the GA3 isomeric

Fig. 3. Typical LC–ESI-MS/MS chromatogram of a mixed standard solution contain-ing 50 �g mL−1 GA3, IAA, ABA and ISTD: (3a) GA3, 345 → 239, 301 transition; (3b)IAA, 174 → 130 transition; (3c) ISTD, 121; (3d) ABA, 263 → 153, 219 transition.

compound. In addition, benzoic acid instead of labeled isotope wasused as internal standard. It is noteworthy that benzoic acid asinternal standard not only produced the same effect but made theexperiment simpler, compared with the labeled isotope.

3.2. Optimization of the procedures of extraction and purification

A series of methanol solutions of different concentrations weretested to determine the suitable medium for loading and washingsample extract on C18 cartridges. Four mixed standard solutionsof the three phytohormones (20 �g mL−1) and ISTD (100 �g mL−1)were obtained by dissolving their standards into 10%, 20%, 30%and 40% methanol in water containing 0.1% formic acid, respec-tively. Subsequently, 1 mL of the four mixed standard solutions wasloaded onto four preconditioned C18 cartridges, respectively. Theconcentration of the acidified methanol containing 0.1% formic acidfor washing (1 mL volume) was the same as that for loading. Thephytohormones were eluted by 1mL 80% methanol. For ABA, IAAand ISTD, 30% of methanol solution containing 0.1% formic acidwas the highest concentration that allowed for loading and wash-ing with their complete retentions on the cartridge. However, 20%methanol solution containing 0.1% formic acid was required for

complete retention of GA3, so 20% methanol containing 0.1% formicacid was used for sample loading and washing. In order to elutecompletely the three phytohormones from C18 cartridge and elim-inate the concomitant interferents as much as possible, a series ofmethanol solutions of different concentrations in water and theacidified (0.1% formic acid) ones of corresponding concentrationswere employed. In our study, 1 mL mixed standard solution of thethree phytohormones (20 �g mL−1) and ISTD (100 �g mL−1) in 20%methanol containing 0.1% formic acid was loaded respectively ontoten preconditioned C18 cartridges and then washed with 1mL 20%methanol containing 0.1% formic acid. At last, the eluting operationwas carried out using 1mL of 60%, 70%, 80%, 90% and 100% methanolsolutions and the acidified (0.1% formic acid) ones of correspondingconcentration, respectively. The results showed that non-acidifiedmethanol solutions had stronger eluting abilities than the acidified(0.1% formic acid) ones, which could be because the acidified (0.1%formic acid) methanol solutions had converted the phytohormonesfrom the anionic forms to the neutral ones. As the phytohormonesin the neutral forms tended to retain on C18 cartridge, the acidified(0.1% formic acid) methanol solutions were excluded in our exper-iment. Furthermore, 70% methanol was required to elute GA3 and

nta 76 (2008) 798–802 801

coefficient Detection limit (�g mL−1) Precision (R.S.D., %)

Retention time Peak area

0.005 0.73 2.42.2 0.96 4.70.003 0.4 2.9

).

S. Hou et al. / Tala

Table 1Calibration curves and other quantitative data for GA3, IAA and ABA

Analyte Calibration curvea Range (�g mL−1) Correlation

GA3 Y = 0.0316X + 0.0045 0.005–200 0.9904IAA Y = 0.0009X + 0.0044 5.00–200 0.995ABA Y = 0.0756X + 0.0087 0.005–200 0.9956

a Y: peak area ratio of standard and internal standard; X: concentration (�g ml−1

80% methanol to elute IAA and ABA and ISTD completely. Therefore,80% methanol was selected as the eluting medium in the experi-ment.

In order to investigate the efficiency of different extractants,20 mL water and 20 mL 80% methanol were compared. We affirmedthat no significant difference was found between water and 80%methanol (data not shown). So pure water was used as extrac-tant to extract the phytohormones from the wheat samples in theexperiment.

3.3. Method evaluation

The linearity of each analyte was evaluated by using a series ofstandard solutions, and each standard solution was measured intriplicate. The calibration curves were constructed based on peakarea ratios of the analytes to ISTD versus concentrations of ana-lyte standards. Table 1 is the summary of calibration curves, linearranges and detection limits of the three phytohormones. Good lin-earities were found in the ranges of 5–200 �g mL−1 for IAA and0.005–10 �g mL−1 for ABA and GA3. The detection limits based on asignal-to-noise ratio of three were 0.005 �g mL−1, 2.2 �g mL−1 and0.003 �g mL−1 for GA3, IAA and ABA, respectively, The detection

limit of IAA was higher than that of ABA or GA3.

The precisions of LC–ESI-MS/MS detection were examined byfive repeated injections of a mixed standard solution containing20 �g mL−1 GA3, IAA and ABA. The relative standard deviations(RSDs) of retention times and peak areas were all under 4.7% forGA3, IAA and ABA. In order to examine the recovery of the devel-oped method, the standards of the three phytohormones at twodifferent concentration levels were spiked to samples and thenextracted by the optimized extraction method. The recoveries aresummarized in Table 2. Good recoveries from 95.5% to 102.4% wereobtained. The stability of the three phytohormones was investi-gated by analyzing a wheat sample maintained at 4 ◦C for 2 days.The three phytohormones were shown to be stable at 4 ◦C within 2days.

3.4. Quantitative analysis of plant samples

The LC–ESI-MS/MS method developed in our study was suc-cessfully applied to analyze GA3, IAA and ABA in wheat samples. Atypical LC–ESI-MS/MS chromatogram of a wheat sample is shownin Fig. 4. The analytical results of GA3, IAA and ABA at the differ-

Table 2Average recoveries of GA3, IAA and ABA in wheat samples (n = 3)

Analyte Added (�g mL−1) Recovery (%)Mean

GA3 2 97.35 98.5

IAA 2 97.75 102.4

ABA 0.02 95.50.05 99

Fig. 4. Typical LC–ESI-MS–MS chromatogram of a wheat sample: (4a) GA3,345 → 239, 301 transition; (4b) IAA, 174 → 130 transition; (4c) ISTD, 121; (4d) ABA,263 → 153, 219 transition.

Table 3Analytical results of GA3, IAA and ABA in wheat samples (n = 3)

Analyte Mean (R.S.D., %)

GA3 (�g g−1) IAA (�g g−1) ABA (�g g−1)

Overground tissue (bud) 1.924a (1.21) 3.124a (2.56) 0.012a (3.37)Overground tissue (one leaf) 0.817b (3.77) 2.974a (5.67) 0.008b (6.43)Overground tissue (two leaves) 0.679c (2.44) 3.289b (1.60) 0.009b (3.40)

Roots (bud) 0.733a (2.16) N.D.a 0.010a (4.58)Roots (one leaf) 0.224b (3.41) N.D. 0.005b (5.03)Roots (two leaves) 0.013c (4.45) N.D. 0.005b (5.29)

Column means followed by the same letters are not significantly different at the 95%confidence level as determined by the Duncan Test.

a N.D.: not detected.

ent growth stages of the wheat samples are summarized in Table 3.The results indicated that the developed method was suitable forthe determination of the three phytohormones in plant samples.Furthermore, The Duncan Test, a statistical method, was used toanalyze the variation of the three phytohormones at the differentgrowth stages of the wheat samples. With the growth of wheat, thecontent of GA3 in overground tissues and roots all decreased gradu-ally. The content of ABA in overground tissues and roots decreasedfrom the bud stage to one-leaf stage, but it had no obvious variationfrom one-leaf stage to two-leaf stage. The content of IAA in over-ground tissues had no significant difference from the bud stage toone-leaf stage and increased from one-leaf stage to two-leaf stage,but it was not detected in roots.

4. Conclusion

In this study, we have developed an LC–ESI-MS/MS method todetermine GA3, IAA and ABA simultaneously and also simplifiedthe procedures of extracting and purifying plant samples for the

802 S. Hou et al. / Talanta 76

analysis. In addition, an SPE method developed in the experimentcould remove the interferents effectively and enhance the determi-nation sensitivity. The LC–ESI-MS/MS method has been successfullyapplied to determine the three phytohormones in wheat samples atdifferent growth stages. The satisfactory results have demonstratedthat this method may be suitable for the determination of the threephytohormones in agriculture and plant physiology.

References

[1] P.J. Davies, in: P.J. Davies (Ed.), Plant Hormones: Physiology, Biochemistry andMolecular Biology, second ed., Kluwer, Dordrecht, Netherlands, 1995, pp. 1–12.

[2] P. Hernandez, M. Dabrio-Ramos, F. Paton, Y. Ballesteros, L. Hernandez, Talanta44 (1997) 1783.

[3] A. Durgbanshi, V. Arbona, O. Pozo, O. Miersch, J.V. Sancho, A. Gomez-Cadenas,J. Agric. Food Chem. 53 (2005) 8437.

[4] R. Zhou, T.M. Squires, S.J. Ambrose, S.R. Abrams, A.R.S. Ross, A.J. Cutler, J. Chro-matogr. A 1010 (2003) 75.

[5] P.I. Dobrev, L. Havlıcek, M. Vagner, J. Malbeck, M. Kamınek, J. Chromatogr. A1075 (2005) 159.

[

[

(2008) 798–802

[6] J. Rolcık, J. Recinska, P. Bartak, M. Strnad, E. Prinsen, J. Sep. Sci. 28 (2005)1370.

[7] A. Muller, P. Duchting, E.W. Weiler, Planta 216 (2002) 44.[8] E. Prinsen, W.V. Dongen, E.L. Esmans, H.A.V. Onckelen, J. Mass Spectrom. 32

(1997) 12.[9] Z. Ma, L. Ge, A.S.Y. Lee, J.W.H. Yong, S.N. Tan, E.S. Ong, Anal. Chim. Acta 610

(2008) 274.[10] E.A. Schmelz, J. Engelberth, J.H. Tumlinson, A. Block, H.T. Alborn, Plant J. 39

(2004) 790.[11] H.T. Liu, Y.F. Li, T.G. Luan, C.Y. Lan, W.S. Shu, Chromatographia 66 (2007) 515.12] A. Crozier, K. Loeferski, J.B. Zaerr, B.O. Morris, Planta 150 (1980) 366.

[13] M. Wright, P. Doherty, J. Plant Growth Reg. 4 (1985) 91.[14] R. Maldiney, B. Leroux, I. Sabbagh, B. Sotta, L. Sossountzov, E. Miginiac, J.

Immunol. Methods 90 (1986) 151.[15] B. Fernandez, M.L. Centeno, I. Feito, R. Sanchez-Tames, A. Rodriguez, Phytochem.

Anal. 6 (1995) 49.[16] A. Champault, C. R. Acad. Sci. D Nat. 280 (1975) 591.[17] E.A. Schmelz, J. Engelberth, H.T. Alborn, P. O’Donnell, M. Sammons, H. Toshima,

J.H. Tumlinson, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 10552.[18] J. Cao, S.J. Murch, R. O’Brien, P.K. Saxena, J. Chromatogr. A 1134 (2006) 333.[19] A. Gomez-Cadenas, O.J. Pozo, P. Garcıa-Augustın, J.V. Sancho, Phytochem. Anal.

13 (2002) 228.20] M. Lopez-Carbonell, O. Jauregui, Plant Physiol. Biochem. 43 (2005) 407.