Analysis of abscisic acid by high-performance liquid chromatography.

Methodology for the ready analysis of abscisic acid (ABA) in plant tissues based upon application of high-performance liquid chromatography (hplc) has been developed. The method involves isolation of the acid fraction, preparation of the methyl esters with diazomethane, and hplc using a combination procedure of two columns: (1) reversed-phase C₁₈, and (2) porous silica in the absorption mode. Only isocratic elution is required so the method is readily adaptable to laboratories having limited hplc capability. Measured recoveries are 70% and the use of an internal standard allows quantification of ABA levels to 1 ng/g of tissue with minimum absolute detectable levels of ABA of 20 ng. The method is illustrated by analysis of ABA concentration in potato tubers at various times postcutting.     

Selected ion monitoring method for determination of nicotine, cotinine and deuterium-labeled analogs: absence of an isotope effect in the clearance of (S)-nicotine-3',3'-d2 in humans

A method for simultaneous determination of nicotine, its metabolite cotinine, and the stable isotope-labeled analogs nicotine-3',3'-d2 and cotinine-4',4'-d2 in human plasma has been developed. The method utilizes capillary column gas chromatography with detection by electron impact mass spectrometry and selected ion monitoring. Sensitivity is adequate for determination of nicotine and nicotine-d2 at concentrations as low as 1 ng ml-1, and cotinine and cotinine-d2 at concentrations as low as 10 ng ml-1 with good precision and accuracy. The method has been used to compare the elimination kinetics of (S)-nicotine-3',3'-d2 with natural nicotine in human subjects. Total clearance of nicotine-3',3'-d2 was virtually identical to the total clearance of natural nicotine, which validates the use of the deuterium-labeled analog in quantitative studies of nicotine metabolic disposition.     

Microextraction and Gas Chromatography/Mass Spectrometry for improved analysis of geosmin and other fungal “off” volatiles in grape juice

Geosmin is a volatile fungal metabolite with an earthy aroma produced in grape products from rotten grapes. The accumulation of geosmin in grapes is caused by the interaction of Botrytis cinerea and Penicillium expansum. Solid Phase Microextraction (SPME) has great utility for collecting volatile compounds in wine. However, contamination with earthy odours may have occurred previously in the must and novel methods are required for this commodity. In the present report, several parameters of the SPME were evaluated to optimize geosmin extraction. The method permitted quantification of geosmin and other fungal volatiles by Gas Chromatography-Mass Spectrometer (GC-MS) at very low concentrations. Limits of detection and quantification (L_D and L_Q) for geosmin were 4.7 ng L⁻¹ and 15.6 ng L⁻¹ respectively. The RSD was 4.1% and the recovery rates ranged from 115% to 134%. Uniquely, haloanisoles were analyzed by using only one internal standard (2,3,6-trichloroanisole) thus avoiding the synthesis of deuterated anisole analogues that are used as internal standard in other methods. The method was used for the analysis of grape juice samples inoculated with B. cinerea and P. expansum. Geosmin and methylisoborneol were the compounds that appeared to contribute most to earthy odours, although other fungal compounds which are claimed to cause earthy or mouldy off-odours were detected (e.g. 1-octen-3-ol and fenchol).     

Oxidation of 8'-hydroxy abscisic acid in Black Mexican Sweet maize cell suspension cultures

In a biotransformation study to prepare deuterium labelled phaseic acid (PA) from deuterated abscisic acid (ABA), the product contained fewer deuterium atoms than expected. Thus, spectroscopic data of isolated deuterated PA prepared from biotransformation of (+)-5,8',8',8'-d4-ABA in maize (Zea mays L. cv. Black Mexican Sweet) cell suspension cultures showed 83% deuterium incorporation at the 8'-exo position. Also, metabolism studies of (+)-4,5-d2-ABA in maize resulted in the isolation of deuterium labelled ABA derivatives, namely PA, dihydrophaseic acid (DPA), 4'-O-beta-D-glucopyranosylDPA, 8'-hydroxyPA, 8'-hydroxyDPA and 8'-oxoDPA, as deduced from spectroscopic methods. These combined results suggested the presence of an aldehyde intermediate which is either: (a) reduced to 8'-hydroxyABA and cyclized to PA, or (b) is hydrated and cyclized to 8'-hydroxyPA or (c) is further oxidized to the acid and cyclized to 8'-oxoPA. The chemical synthesis of this intermediate, as well as its biotransformation in maize cell cultures is presented.     

Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7'-, 8'- and 9'-hydroxy ABA in microspore derived embryos of B. napus

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.     

Uptake, Movement, Activity, and Persistence of an Abscisic Acid Analog (8′ Acetylene ABA Methyl Ester) in Marigold and Tomato

The abscisic acid (ABA) analog 8′ acetylene ABA methyl ester (PBI 429) was evaluated for its potential to alter the growth and moisture use of bedding plants during nursery production. Treating seedlings with the ABA analog as a root-dip slowed moisture use and growth of tomato seedlings under greenhouse conditions. In marigolds, comparable ABA analog treatments had no effect on growth and limited effects on plant moisture use. To determine whether these differences in response to treatment with the ABA analog were associated with differences in absorption of the analog and/or its persistence, the ABA analog was applied either as a foliar spray or root-dip, and the resulting concentrations of the ABA analog were monitored over a 10-day interval in both the roots and the leaves. In both crops, the ABA analog was detected in both leaf and root tissues irrespective of the mode of application, suggesting systemic movement of the analog. Tissue concentrations of the ABA analog were consistently lower in the foliar treatment than in the root-dip. The uptake and the retention of the ABA analog over time was similar in leaves of the two test crops, but less of the ABA analog was absorbed and retained in the roots of marigold plants than in the tomatoes. This suggests that the observed differences in responses of these two plant species to application of ABA analogs may be related to differences in retention or accumulation of ABA in the roots rather than to differences in the total amount of ABA analog absorbed or its movement and retention in the plant system. Levels of endogenous ABA were not significantly altered by application of the ABA analog.     

Changes in the ratio of cis-trans to trans-trans abscisic acid during ripening of apple fruits

Immediately after harvest, abscisic acid (ABA) extracted from fruits of the apple cultivar Golden Delicious comprised solely the cis-trans isomer. During postharvest ripening, however, trans-trans ABA accumulated and finally exceeded the level of cis-trans ABA. The two geometrical isomers were separated and identified by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. After purification by HPLC the putative trans-trans isomer yielded considerable quantities of cis-trans ABA, when irradiated with UV light. This isomerization was more rapid than the reverse reaction. The physiological significance of the accumulation of trans-trans ABA is discussed, as well as the applications of these results in the use of trans-trans ABA as an internal standard during the extraction and quantification of ABA from plant tissues.Abbreviations ABA 2-cis-4-trans abscisic acid - t-ABA 2-trans-4-trans abscisic acid - ECD electron capture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PVP water insoluble polyvinylpyrroli-done - UV ultraviolet     

Ectopic expression of a conifer <Emphasis Type="Italic">Abscisic Acid Insensitive3</Emphasis> transcription factor induces high-level synthesis of recombinant human α-<Emphasis Type="SmallCaps">l</Emphasis>-iduronidase in transgenic tobacco leaves

We are examining various plant-based systems to produce enzymes for the treatment of human lysosomal storage disorders. Constitutive expression of the gene encoding the human lysosomal enzyme, alpha-L-iduronidase (IDUA; EC 3.2.1.76) in leaves of transgenic tobacco plants resulted in low-enzyme activity, and the protein appeared to be subject to proteolysis. Toward enhancing production of this recombinant enzyme in vegetative tissues, transgenic tobacco plants were generated to co-express a CaMV35S:Chamaecyparis nootkatensis Abscisic Acid Insensitive3 (CnABI3) gene construct, along with the human gene construct. The latter contained regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene (5'-flanking, signal-peptide-encoding, and 3'-flanking regions). Ectopic synthesis of the CnABI3 protein led to the transactivation of the arcelin promoter and accordingly high activity (e.g., 25,000 pmol/min/mg total soluble protein) and levels of recombinant IDUA mRNA and protein were induced in leaves of transgenic tobacco, particularly in the presence of 150-200 microM S-(+)-ABA. Synthesis of human IDUA containing a carboxy-terminal ER retention (SEKDEL) sequence was also inducible by ABA in leaves co-transformed with the CnABI3 gene. As compared to the natural S-(+)-ABA, two persistent ABA analogues, (+)-8' acetylene ABA and (+)-8'methylene ABA, led to greater levels of beta-glucuronidase (GUS) reporter activities in leaves co-expressing the CnABI3 gene and a vicilin:GUS chimeric gene. In contrast, (+)-8' acetylene ABA and natural ABA appeared to be equally effective in stimulating the CnABI3-induced expression of an arcelin:GUS gene, and of the human IDUA gene, the latter also driven by arcelin-gene-regulatory sequences. Various stress-related treatments, particularly high concentrations of NaCl, had an even greater effect than ABA in promoting accumulation of human IDUA in co-transformed tobacco leaves. This strategy provides the means of enhancing the yields of recombinant proteins in transgenic plant vegetative tissues and potentially in cultured plant cells. The human recombinant protein can be readily induced in the presence of chemicals such as NaCl that can be added to cell cultures or even whole plants without a significant increase in production costs.     

Differences between the structural requirements for ABA 8'-hydroxylase inhibition and for ABA activity

A major catabolic enzyme of the plant hormone abscisic acid (ABA) is the cytochrome P450 monooxygenase ABA 8'-hydroxylase. For designing a specific inhibitor of this enzyme, the substrate specificity and inhibition of CYP707A3, an ABA 8'-hydroxylase from Arabidopsis thaliana, was investigated using 45 structural analogues of ABA and compared to the structural requirements for ABA activity. Substrate recognition by the enzyme strictly required the 6'-methyl groups (C-8' and C-9'), which were unnecessary for ABA activity, whereas elimination of the 3-methyl (C-6) and 1'-hydroxyl groups, which significantly affected ABA activity, had little effect on the ability of analogues to competitively inhibit the enzyme. Fluorination at C-8' and C-9' resulted in resistance to 8'-hydroxylation and competitive inhibition of the enzyme. In particular, 8',8'-difluoro-ABA and 9',9'-difluoro-ABA yielded no enzyme reaction products and strongly inhibited the enzyme (K(I) = 0.16 and 0.25 microM, respectively).     

Simultaneous determination of amphetamine and its analogs in human whole blood by gas chromatography-mass spectrometry

A sensitive and specific gas chromatography-mass spectrometry (GC-MS) method for the determination of amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA) and methylenedioxyethylamphetamine (MDEA) in whole blood was designed, using the respective pentadeuterated analogs of the analytes as internal standards (I.S.). After alkalinisation of blood samples, the amphetamines were extracted using diethyl ether, derivatized with heptafluorobutyric anhydride, then purified by successive washings with deionized water and 4% NH₄OH. Extraction recoveries were 85.2% for AM, 90.9% for MA, 76.5% for MDA, 84.1% for MDMA and 63.6% for MDEA. Chromatographic separation was performed on a non-polar 30 m×0.32 mm HP 5 MS capillary column using a temperature program. Detection was carried out in the electron-impact, selected ion-monitoring mode, using three mass-to-charge ratios for each analyte and one for each I.S. Limits of detection ranged from 0.5 to 8 ng/ml and limits of quantification were 10 ng/ml for AM, MDMA and MDEA; 20 ng/ml for MA; and 50 ng/ml for MDA. The method was linear from this limit up to 1000 ng/ml for all analytes, with good intra-assay precision and good intermediate precision and accuracy over these ranges. There was no interferences from other sympathomimetic drugs such as ephedrine, norephedrine or methoxyphenamine. This method is thus suitable for clinical and forensic toxicology, as well as for doping control.     

Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. `Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in `Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.     

Azospirillum brasilense Sp 245 produces ABA in chemically-defined culture medium and increases ABA content in arabidopsis plants

Azospirillum sp. are plant growth promoting bacteria (PGPB) that increase grain yield in cereals and other species via growth promotion and/or stress alleviation. The PGPB beneficial effects have been partially attributed to bacterial production of plant hormones, especially growth promoters like auxins, gibberellins and cytokinins. This paper reports the characterization of the stress-like plant hormone abscisic acid (ABA) by GC-EIMS in cultures of A. brasilense Sp 245 after 120 h of incubation in chemically-defined media, and chemically-defined media with moderate stress (100 mM NaCl). Chemical characterization of ABA was done by gas chromatography-electron impact mass spectrometry (GC-EIMS) and quantification by selected ion monitoring (SIM) with a stable isotope of the hormone as internal standard in the media. A. brasilense cultures produced higher amounts of ABA per ml of culture when NaCl was incorporated in the culture medium. Inoculation of Arabidopsis thaliana with A. brasilense Sp 245 enhanced two-fold the plant’s ABA content. These results contribute to explain, at least to some extent, the beneficial effects of Azospirillum sp. previously found in inoculated plants placed under adverse environmental conditions.     

Flavonoids from Andrographis lineata

Three flavonoids, 5,7,2',3',4'-pentamethoxyflavone (1), 2'-hydroxy-2,4',6'-tri methoxychalcone (2) and dihydroskullcapflavone I (3), together with 17,19,20-trihydroxy-5beta, 8alpha H, 9beta H,10alpha-labd-13-en-16,15-olactone (4), a known diterpenoid and six known flavonoids, 5-hydroxy-7,8-dimethoxyflavanone (5), 5-hydroxy-7,8,2',3',4'-pentamethoxyflavone (6), 5,2'-dihydroxy-7-methoxyflavanone (7), 5,2'-dihydroxy-7,8-dimethoxyflavone (8), 5,2'-dihydroxy-7-methoxyflavone (9) and 5,2'-dihydroxy-7-methoxyflavone 2'-O-beta-D-glucopyranoside (10) were isolated from the whole plant of Andrographis lineata. The structures of these compounds were elucidated on the basis of spectral and chemical studies.     

Quantitative analysis of levamisole in porcine tissues by high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry

This work presents the development and the validation of an LC–MS–MS method with atmospheric pressure chemical ionization for the quantitative determination of levamisole, an anthelmintic for veterinary use, in porcine tissue samples. A liquid–liquid back extraction procedure using hexane–isoamylalcohol (95:5, v/v) as extraction solvent was followed by a solid-phase extraction procedure using an SCX column to clean up the tissue samples. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher^® 60 RP-select B (5 μm) column using a mixture of 0.1 M ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometer was operated in MS–MS full scanning mode. The method was validated for the analysis of various porcine tissues: muscle, kidney, liver, fat and skin plus fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r>0.99 and goodness of fit <10%). Limits of quantification of 5.0 ng/g were obtained for the analysis of levamisole in muscle, kidney, fat and skin plus fat tissues, and of 50.0 ng/g for liver analysis, which correspond in all cases to half the MRLs (maximum residue limits). Limits of detection ranged between 2 and 4 ng/g tissue. The within-day and between-day precisions (RSD, %) and the results for accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of levamisole in tissue samples from pigs medicated via drinking water. Moreover the product ion spectra of the levamisole peak in spiked and incurred tissue samples were in close agreement (based on ion ratio measurements) with those of standard solutions, indicating the worthiness of the described method for pure qualitative purposes.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

A sensitive and specific stable isotope assay for warfarin and its metabolites

A capillary gas chromatographic mass spectrometric method for the quantification of warfarin and its known metabolites from microsomal incubations is described. Deuterium labelled 4', 6-, 7- and 8-hydroxy warfarins are used as internal standards and the method has detection limits of 1 ng ml-1 with 20 ng ml-1 being the lower limit for accurate quantification.     

Sensitive determination of trimetazidine in spiked human plasma by HPLC with fluorescence detection after pre-column derivatization with 9-fluorenylmethyl chloroformate

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 degrees C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm x 4.6 mm, i.d., 5 microm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5-200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5+/-1.3% (n=6; RSD=2.1%).     

Biosynthesis of abscisic acid by the non-mevalonate pathway in plants, and by the mevalonate pathway in fungi

The biosynthetic pathways to abscisic acid (ABA) were investigated by feeding [1-(13)C]-D-glucose to cuttings from young tulip tree shoots and to two ABA-producing phytopathogenic fungi. 13C-NMR spectra of the ABA samples isolated showed that the carbons at 1, 5, 6, 4', 7' and 9' of ABA from the tulip tree were labeled with 13C, while the carbons at 2, 4, 6, 1', 3', 5', 7', 8' and 9' of ABA from the fungi were labeled with 13C. The former corresponds to C-1 and -5 of isopentenyl pyrophosphate, and the latter to C-2, -4 and -5 of isopentenyl pyrophosphate. This finding reveals that ABA was biosynthesized by the non-mevalonate pathway in the plant, and by the mevalonate pathway in the fungi. 13C-Labeled beta-carotene from the tulip tree showed that the positions of the labeled carbons were the same as those of ABA, being consistent with the biosynthesis of ABA via carotenoids. Lipiferolide of the tulip tree was also biosynthesized by the non-mevalonate pathway.