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In this study, a novel nitrogen-rich hyperbranched polymer was designed and synthesized via one-step precipitation copolymerization strategy. As possessing the lone-pair-electron-containing nitrogen atoms and positive-charged amine groups, as well as π electron-conjugated system, the prepared polymer displayed a strong tendency to adsorb protons acid, and negative-charged and conjugated compounds according to acid-base interaction, electrostatic interaction, and π-π stacking interaction. Based on these properties, a novel approach for assembling the proposed polymer coupled with high-performance liquid chromatography was successfully employed for selective enrichment and determination of auxins in plants. The extraction and desorption conditions were evaluated and the limits of detection and the limits of quantification of the proposed method were in the range of 0.15-0.29 μg L -1 and 0.49-0.98 μg L -1 for the four auxins based on the signal-to-noise ratio of 3:1 and 10:1, respectively. The recoveries of the target auxins from spiked plant samples were in the range from 85.0 to 116.3% with relative standard deviations lower than 9.6%. This study presented an inspiring thought for the construction of the versatile polymer adsorbent with highly efficient capturing of analytes from complex samples. Graphical abstract.

A new ionic liquid functionalized graphene-pipette-tip solid-phase extraction method coupled with high-performance liquid chromatography was established for the simultaneous extraction and determination of three auxins in soybean sprouts. The graphene adsorbent, with multiple adsorption mechanisms, was first synthesized by functional modification of pentafluorobenzyl imidazolium bromide ionic liquid through thiol-ene click chemistry. The ionic liquid was applied to prevent the aggregation of graphene; it also imbued graphene with the ability for π-π interactions, ionic exchange, electrostatic interactions, as well as hydrogen bonding (which is stronger than the interaction between water and analytes), by augmenting the adsorption mechanisms between the adsorbent and analytes. Under optimized conditions, linearity was achieved in the ranges 0.03-5.00 µg/g for indole-3-acetic acid and 1-naphthaleneacetic acid and 0.09-5.00 µg/g for 2,4-dichlorophenoxyacetic acid, with a detection limit of 0.004-0.026 µg/g; this adsorbent has been successfully applied for the determination of auxins in soybean sprouts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A melamine-based porous organic polymer (mPMF) was synthesized as solid phase extraction (SPE) adsorbent for inline SPE-high performance liquid chromatography (HPLC)-ultraviolet detector (UV) analysis of five phytohormones. Melamine contributed to the rich π-electron and N-containing triazine structure for mPMF, which could form π-π interaction and intermolecular hydrogen bond with COOH containing phytohormones. The synthesized mPMF adsorbent shows extremely high extraction efficiency for five target analytes (>80%) including salicylic acid, indole-3-acetic acid, abscisic acid, 2,4-dichlorophenoxyacetic acid and 1-naphthalene acetic acid. The factors affecting extraction of the target phytohormones were investigated and the optimized experimental conditions were established. The linear range was 0.2-100 μg/L with the limits of detection in the range of 0.06-0.11 μg/L for five target phytohormones. The developed method of mPMF-inline-SPE-HPLC-UV was applied for the analysis of trace target phytohormones in tomato and grape juice samples, and the recovery in the range of 83.1-116% and 87.2-121% was obtained for the spiked tomato and grape juice, respectively. This method has the advantages of high sensitivity and good reproducibility, and it has good application potential for the analysis of phytohormones in fruit samples. Moreover, inline analysis avoided the problems of sample pollution and sample loss, and provided a sample throughput of 5/h., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The methodology described here represents an improved strategy for analysis of a broad range of stress-related plant hormones including jasmonates, salicylic acid, abscisic acid, and auxin metabolites. The method conditions are optimized in order to reduce the background effect of complicated plant matrix, allow effective preconcentration and thus perform highly sensitive profiling of multiple plant hormones by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

Due to the high cost of domoic acid (DA), different carboxylic acid compounds including indole-3-acetic acid (IAA), pyrrole-2-formic acid (PFA), pyridine-2,3-dicarboxylic anhydride (PDA), trimesitinic acid (TA) and citric acid (CA) were investigated as dummy templates for the molecularly imprinted solid-phase extraction (MISPE) for selective isolation and pre-concentration of an amnesic shellfish poison (ASP), DA. The highest binding amount of the polymers towards DA was obtained when CA was used as dummy template owing to its high hydrophilicity. In addition, the "four-point" recognition site constructed by three COOH groups and a OH group in CA was also speculated to be the reason for the high binding amount of CA-MIPs and the rebinding of DA can be depend on the three COOH groups and a NH group with conformational change in the recognition process. Finally, the CA-MISPE column was chosen for DA isolation and pre-concentration and effective result was obtained with recoveries higher than 90% and relative standard deviation (RSD) less than 5% (n=3). This new polymer can be effectively applied to the monitoring and predicting the existence of trace DA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, ionic liquid-modified silica was used as sorbent for simultaneous extraction and preconcentration of 3-indole butyric acid and 3-indole acetic acid in pea plants. The effect of some parameters such as pH and ionic strength of sample solution, amount of sorbent, flow rate of aqueous sample solution and eluent solution, concentration of eluent solution, and temperature were studied for each hormone solution. Percent extraction of 3-indole butyric acid and 3-indole acetic acid was strongly affected by pH of aqueous sample solution. Ionic strength of aqueous phase and temperature showed no serious effects on extraction efficiency of studied plant hormones. Obtained breakthrough volume was 200mL for each of studied hormones. Preconcentration factor for spectroscopic and chromatographic determination of studied hormones was 100 and 4.0×10(3) respectively. Each solid sorbent phase was reusable for almost 10 times of extraction/stripping procedure. Relative standard deviations of extraction/stripping processes of 3-indole butyric acid and 3-indole acetic acid were 2.79% and 3.66% respectively. The calculated limit of detections for IBA and IAA were 9.1×10(-2)mgL(-1) and 1.6×10(-1)mgL(-1) respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phytohormones play important roles in regulating numerous plant physiological and developmental processes, even during the postharvest storage period. In order to determine the functions and changes of gibberellins acid (GA3), indoleacetic acid (IAA), abscisic acid (ABA), indolebutyric acid (IBA) and jasmonic acid (JA) in grape berries during storage, an ultrasensitive method based on direct injection online solid-phase extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. Grape berries were extracted with cold methanol. After centrifugation, the supernatants were concentrated with a vacuum centrifugal concentrator and injected into an online solid-phase extraction column. After the cleanup procedure, the analytes were determined by LC-MS/MS. The results showed that the linearity of the proposed method was 10-210 µg kg(-1) for ABA, 20-200 µg kg(-1) for IBA, 15-320 µg kg(-1) for IAA, 20-320 µg kg(-1) for GA3 and 3.0-90.0 µg kg(-1) for JA. The limits of detection of the method were 0.71, 2.79, 0.94, 0.39 and 0.57 µg kg(-1), respectively. The proposed method was successfully applied to the analysis of endogenous phytohormones in grape berries during the postharvest storage period., (© The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid-liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI-MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC-ESI-MS/MS method was effective for analysing phytohormones in fermentation samples.

Plant hormones play a crucial role in controlling plant growth and development. These groups of naturally occurring substances trigger physiological processes at very low concentrations, which mandate sensitive techniques for their quantitation. This paper describes a method to quantify endogenous (±)-2-cis-4-trans-abscisic acid, indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid. The method combines high-performance liquid chromatography (HPLC) with diode array and fluorescence detection in a single run. Hybrid tea rose 'Monferrato' matrices (leaves, petals, roots, seeds, androecium, gynoecium, and pollen) were used as references. Rose samples were separated and suspended in extracting methanol, after which (±)-2-cis-4-trans-abscisic acid and auxins were extracted by solvent extraction. Sample solutions were added first to cation solid phase extraction (SPE) cartridges and the eluates to anion SPE cartridges. The acidic hormones were bound to the last column and eluted with 5% phosphoric acid in methanol. Experimental results showed that this approach can be successfully applied to real samples and that sample preparation and total time for routine analysis can be greatly reduced.

In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (λ(ex) = 280/λ(em) = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.0625-125 μg mL(-1) (r ( 2 ) ≥ 0.998) and the limits of detection were below 0.015 μg mL(-1). The applicability of the method was confirmed by analysis of Pseudomonas putida culture supernatants.

Simultaneous determination of indole-3-acetic acid and methyl indole-3-acetic acid ester in small amounts of plant tissue is essential for elucidating their mutual transformation mechanism and the in vivo function of methyl indole-3-acetic acid ester. Rapid quantification of flavonoids in the same sample is important for clarifying their roles in the transport of auxins and other phytohormones. Herein, we describe a simple method for the simultaneous determination of indole-3-acetic acid and its methyl ester in the roots of the Arabidopsis thaliana seedlings and a protocol for the rapid extraction and quantification of quercetin and kaempferol in these seedlings. High-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry was used for the detection of all the compounds. Negative data for indole-3-acetic acid and positive data for methyl indole-3-acetic acid ester were collected in two successive files with a single injection of the extracted sample. Under optimized conditions, the limit of detection for the four compounds was 2 ng/mL for indole-3-acetic acid, 0.5 ng/mL for methyl indole-3-acetic acid ester, 5 ng/mL for quercetin, and 1 ng/mL for kaempferol, respectively. Because of the high sensitivity of the assay, only 2-10 mg of the plant material was required to obtain quantitative results., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple, convenient and high selective miniaturized molecularly imprinted matrix solid-phase dispersion (mini-MI-MSPD) technique using l-tryptophan-imprinted polymers as sorbents was proposed for simultaneous extraction of four auxins (indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA)) from orange samples coupled with high performance liquid chromatography-ultraviolet detection. The molecularly imprinted polymers (MIPs) synthesized using l-tryptophan as dummy template showed high affinity and selectivity to auxins, and so they were applied as the specific dispersant of MSPD to simultaneously eliminate the effect of template leakage on quantitative analysis. Under the optimized conditions, good linearity was obtained in a range of 0.02-2.00 μg g(-1) and the average recoveries of four auxins at three spiked levels ranged from 87.6% to 104.5% with the relative standard deviation (RSD) ≤4.4%. The presented mini-MI-MSPD method combined the advantages of MSPD for allowing the extraction, dispersion and homogenization in a single step and the advantages of MIPs for high affinity and selectivity towards four auxins, which could be applied to the determination of auxins in complicated vegetal samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of five acid/alkaline phytohormones, i.e., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), gibberellic acid (GA(3)) and isopentenyladenine (2IP), in grapes was developed. After optimization, the samples were extracted with methanol containing 1% formic acid and purified by Oasis HLB SPE cartridges. The analytes were separated on a Thermo Hypersil Gold column (100 mm×2.1 mm, 3.0 μm) with water and acetonitrile, then determined with Thermo tandem quadrupole mass spectrometer operating in negative electro-spray ionization using selected reaction monitoring (SRM) mode. The established method was further validated by determining the linearity (R² ≥ 0.9990), average recovery (82.5-105.4%), sensitivity (0.05-1.00 ng mL⁻¹), precision (RSD ≤1 3.0%) and stability (RSD ≥ 82.0%). Finally, the application of the approach proposed to thirty grape samples convinced its desirable performance for rapid analysis of multiclass phytohormones, supporting its sufficient capability for multiresidue analyses or other analytical system targeting phytohormones in agriculture field., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Auxin plays an important role in cell differentiation, apical dominance, and tropism in plants. A new method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-fluorescence detection (HPLC-FLD) has been established to detect auxin. A mixture of CHCl(3) (extraction solvent) and acetone (disperser solvent) was injected quickly into a sample solution with desired salt concentration and pH value, and then a cloudy solution consisting of many dispersed fine droplets of CHCl(3) was formed. After centrifugation, the sedimented phase was withdrawn and directly analyzed by HPLC-FLD. Under optimal conditions, four auxins were baseline separated within 3.5 min, with the minimal limit of detection of 0.02 ng mL(-1) and coefficient correlations in the range of 0.9980-0.9995. This simple method was successfully applied to real sample analysis. Experimental results showed that DLLME was a high-performance and powerful preconcentration method to extract and enrich related plant auxin.

Introduction: The overuse of petrochemical-based synthetic fertilisers has caused detrimental effects to soil, water supplies, foods and animal health. This, in addition to increased awareness of organic farming, has generated considerable interest in the evaluation of renewable biofertilisers., Objective: The three objectives of the current research were: (1) to evaluate and optimise a solid phase extraction procedure for extraction of three plant hormones, IAA, GA(3) and ABA from two model biofertilisers produced from coconut shells and pineapple peels; (2) to develop an HPLC analysis procedure for the simultaneous separation and quantification of three plant hormones (IAA, GA(3) and ABA); and (3) to evaluate the changes in three plant hormones levels at four different fermentation time periods and varying number of general bacteria, lactic acid bacteria and yeast., Result: An optimised procedure for sample preparation, separation and simultaneous analysis of three plant hormones [indole-3-acetic acid (IAA), gibberellic acid (GA(3)) and abscisic acid (ABA)] produced in liquid biofertilisers was developed. This method involves sample cleanup using a Sep-pack OasisMAX cartridge containing mixed-mode anion-exchange and reverse-phase sorbents that provided optimum recovery of 85.6, 91.9 and 94.3%, respectively, for the three hormones, IAA, GA(3), and ABA. Baseline separation of three hormones was achieved using mobile phase consisting of 1% acetic acid and acetonitrile (75:25, v/v) at pH 4.0. The amounts of hormones produced in liquid biofertilisers were influenced by fruit types, fermentation time and total number of general bacteria, lactic acid bacteria and yeasts. The quantities of three plant hormones produced during fermentation correlated well with the total number of microorganisms present in the liquid biofertilisers., Conclusion: A simple and rapid sample preparation procedure followed by RP-HPLC with UV detection was optimised and developed for simultaneous quantification and identification of three plant hormones namely, IAA, GA(3) and ABA in the liquid biofertilisers. This procedure allows quantification of the three plant hormones in their natural states without any prior derivatisation step. The results presented illustrate that the contents of the three plant hormones depended on the type of fruit wastes, fermentation time and the number of microorganisms found in liquid biofertilisers. This method can be extended to determine the quantity of three hormones in other matrices. This assay procedure will aid in the development of liquid biofertilisers, a valuable alternative fertilisers to promote plant growth. This process will help farmers to reduce production cost and pollution problems.

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants., (Copyright 2009 Elsevier Inc. All rights reserved.)

A novel method for determination of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in an extract from mung bean sprouts using high performance liquid chromatography (HPLC) with chemiluminescence (CL) detection is described. The method is based on the CL reaction of auxin (indole-3-acetic acid and indole-3-butyric acid) with acidic potassium permanganate (KMnO(4)) and tris(2,2'-bipyridyl)ruthenium(II), which was immobilized on the cationic ion-exchange resin. The chromatographic separation was performed on a Nucleosil RP-C18 column (i.d.: 250 mm x 4.6 mm, particle size: 5 microm, pore size: 100) with an isocratic mobile phase consisting of methanol-water-acetic acid (45:55:1, v/v/v). At a flow rate of 1.0 mL min(-1), the total run time was 20 min. Under the optimal conditions, the linear ranges were 5.0x10(-8) to 5.0x10(-6)g mL(-1) and 5.0x10(-7) to 1.0x10(-5)g mL(-1) for IAA and IBA, respectively. The detection limits were 2.0x10(-8)g mL(-1) and 2.0x10(-7)g mL(-1) for IAA and IBA, respectively. The relative standard deviation (RSD) of intra-day were 3.1% and 2.3% (n=11) for 2x10(-6)g mL(-1) IAA and 2x10(-6)g mL(-1) IBA; The relative standard deviations of inter-day precision were 6.9% and 4.9% for 2x10(-6)g mL(-1) IAA and 2x10(-6)g mL(-1) IBA. The proposed method had been successfully applied to the determination of auxin in mung bean sprouts.

Two-dimensional liquid chromatography (2D-LC) is rapidly gaining popularity for the analysis of very complex mixtures, including proteomic and metabolomic samples. It provides an effective strategy for separating such samples, because the resolving power of 2D-LC is far superior to that of traditional single-dimension separations. The present work focuses on the development of data analysis methods for the extremely large data sets, on the order of 10 million data points, generated by 2D-LC with diode-array detection (DAD). Specifically, we have applied and adapted chemometric methods to the analysis of maize seedling digests, focusing on compounds related to the biosynthetic pathways of indole-3-acetic acid, the primary growth regulator in plants. The chemometric techniques of window target testing factor analysis (WTTFA), along with parallel factor analysis - alternating least squares (PARAFAC-ALS) were used to analyze 2D-LC-DAD chromatograms of a sample composed of 26 indolic standards, 2 extracts of mutant orange pericarp maize seedlings, 2 extracts of wild-type maize seedlings, and a blank sample. The indolic compounds studied belonged to six spectrally unique groups, and WTTFA was able to specifically identify the presence or absence of any of the 26 indolic standards in the mutant and wild-type samples. A PARAFAC-ALS algorithm and an ALS algorithm with flexible constraints were successfully applied to resolve the spectrally rank deficient data and to demonstrate the quantitative potential of multivariate curve resolution methods. Using this procedure, 95 total peaks were resolved in the data set analyzed. Of those 95 peaks, 45 were found in both the mutant and wild-type maize samples, 16 peaks were unique to the mutant maize samples, 13 peaks were unique to the wild-type maize samples, and 15 peaks were unique to the standard chromatograms. Of the 26 standards included in the data set, several indole acetic acid conjugates were identified and quantified in the maize samples at levels of approximately 0.3-2 microg/g plant material.

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.

A method for separation and purification of plant hormones auxin and abscisic acid based on mixed mode reversed-phase anion-exchange solid phase extraction and two-dimensional HPLC was developed. Two-dimensional HPLC in "heart cutting" mode was very efficient in the purification of these two hormones. Its purification power is high enough to allow reliable on-line quantification of both hormones even with non-selective detectors.

The plant growth promoting bacteria are closely associated to plant. The bacteria are used to adhering to plant rhizoplane, promoting plant growth by fixing nitrogen from atmosphere, secreting stimulating substances or producing antagonistic to plant pathogens. It was indicated that the biological nitrogen fixation played an important role in plant growth promoting function. In fact, it was verified recently by overall research that IAA does it. Therefore research of IAA production and biosynthetic pathway of plant growth-promoting bacteria is much more important. The various ways of IAA production indicated the strong or weak promoting function of bacterium to plants in general. The purpose of this paper is to determine whether IAA exists in cultured medium of Klebsiella axytoca SG-11 and biosynthetic pathway of IAA, in order to opimize cultural conditions for IAA production. Klebsiella axytoca SG-11 is a plant growth promoting bacterium, isolated from rice rhizoplane, which can fix nitrogen. The supernatant of SG-11 cultured medium determined by HPLC showed that 47.4 mg/L of IAA existed in LB medium and 1.2 mg/L of IAA, in basal medium. IAA in metabolite was identified by GC/MS as well. The intermediate determination of tryptamine, indole-3-acetamide, tryptophol and indole-3-acetonitrile indirectly indicated that IAA was biosynthesized in a pathway of indole-3-pyruvic acid. Meanwhile, tryptophol in metabolite of SG-11 was verified by GC/MS. The direct intermediates of indole-3-pyruvic acid and indole-3-acetaldehyde in the pathway can not be determined, because both are unstable under normal condition. As reversible conversion existed between indole-3 pyruvic aldehyde and tryptophol, the presence of tryptophol also proved the pathway of indole-3-pyruvic acid in the synthesis of IAA by Klebsiella axytoca SG-11. The results laid basis for further research of plant growth-promoting function of the bacterium.

The development of a robust method to analyse the content of tryptophan and of indole-3-acetic acid at the microgram per litre level in must and wine is necessary in order to study the formation of 2-aminoacetophenone and of other indole compounds causing the 'untypical ageing off-flavour'. The present paper discusses the development and validation of a reversed-phase high-performance liquid chromatography method with fluorescence detection for the analysis of indole-3-acetic acid, tryptophan, tryptophol, indole and skatole in must and white wine. The required selectivity and sensitivity was gained through the solid-phase extraction on a polystyrene-based polymer column.

A simple and sensitive method using high performance liquid chromatography with fluorometric detection has been developed for the identification and quantitation of the endogeneous tryptamine metabolites, indoleacetic acid (IAA) and tryptophol (TOL) in the normal mouse brain. The limits of sensitivity are 5pg for both IAA and TOL. The extract procedure from the brain is only to deproteinize samples. The mean concentrations of IAA and TOL in the mouse brain are 8.99 +/- 0.31 ng/g and 3.56 +/- 0.21 ng/g respectively. The effects of pargyline and tryptamine on the levels of IAA and TOL were also studied.