GC-MS Metabolite Profiling Research Articles

Integrative LC-MS and GC-MS metabolic profiling unveils dynamic changes during barley malting

Malting involves complex biochemical transformations affecting sensory and quality attributes. Despite extensive research on storage carbohydrates and proteins in malting, the lack of a detailed metabolic understanding of this process limits our ability to assess and enhance malt quality. This study employed untargeted GC-MS and LC-MS metabolite profiling across six malting timepoints to identify 4980 known metabolites, 82 % of which exhibited significant changes during the malting process. Here we identified stage-dependent metabolic shifts and dynamic chemical classes and pathways between each studied stage. These results can guide the fine-tuning of malting conditions to improve malt quality for beer production and other malt-based applications. Additionally, metabolites with antimicrobial properties were identified, underscoring the interplay between barley and microbial metabolic processes during malting. Further research into these microbial metabolites and cognate microbes may lead to novel malting assessment traits for high-quality and safe malted barley.

Exploring the anticandidal potential: Evaluating leaf extract efficacy and GC–MS metabolite profiling of Chenopodium album var. album L.

IntroductionChenopodium album var. album L., renowned as lamb's quarters, is a versatile medicinal plant, revealing its efficacy in treating various ailments. This study evaluated the in vitro antifungal activity of ethyl acetate extract of C. album var. album L. MethodsChenopodium album leaf extract demonstrated antifungal potential via the Agar well-diffusion and Broth-dilution method. Active fractions, obtained through column chromatography, were rigorously tested against Candida spp. The most potent fraction underwent detailed analysis with High-Performance Thin-Layer Chromatography (HPTLC), Gas Chromatography Mass Spectrometry (GC–MS), and Fourier-Transform Infrared Spectroscopy (FT-IR), for comprehensive characterization. ResultsThe ethyl acetate extract (EAE) exhibited significant antifungal activity, with Candida albicans being the most susceptible (inhibition zone of 14.67 ± 0.58a). Fractionation of the EAE yielded a highly active F2 fraction, which showed a remarkable zone of inhibition against C. albicans (17.50 ± 1.00a). Comprehensive examinations, including HPTLC, GC–MS, and FT-IR, identified 12 compounds in the F2 fraction, and the major compounds were Furo [2,3-c] pyridine, 2,3-dihydro-2,7-dimethyl, Bis (2-ethylhexyl) phthalate, and 2,3,4,4-Tretrapropyl-1-(trimethylsilyl)-1-(trimethylsilyloxy)-1,3-diaza-2,4-diborabutane. This study marks the first report of these 12 compounds identified from the F2 fraction of C. album. ConclusionThe present study highlights the antifungal activity of EAE and its fractions from C. album var. album that could be further explored for the selection of leads that may contribute to novel drug development.

Metabolite profiling and histochemical localization of alkaloids in Hippeastrum papilio (Ravena) van Scheepen

Hippeastrum papilio (Amaryllidaceae) is a promising new source of galanthamine - an alkaloid used for the cognitive treatment of Alzheimer's disease. The biosynthesis and accumulation of alkaloids are tissue - and organ-specific. In the present study, histochemical localization of alkaloids in H. papilio's plant organs with Dragendorff's reagent, revealed their presence in all studied samples. Alkaloids were observed in vascular bundles, vacuoles, and intracellular spaces, while in other plant tissues and structures depended on the plant organ. The leaf parenchyma and the vascular bundles were indicated as alkaloid-rich structures which together with the high proportion of alkaloids in the phloem sap (49.3% of the Total Ion Current – TIC, measured by GC-MS) indicates the green tissues as a possible site of galanthamine biosynthesis. The bulbs and roots showed higher alkaloid content compared to the leaf parts. The highest alkaloid content was found in the inner bulb part. GC-MS metabolite profiling of H. papilio's root, bulb, and leaves revealed about 82 metabolites (>0.01% of TIC) in the apolar, polar, and phenolic acid fractions, including organic acids, fatty acids, sterols, sugars, amino acids, free phenolic acids, and conjugated phenolic acids. The most of organic and fatty acids were in the peak part of the root, while the outermost leaf was enriched with sterols. The outer and middle parts of the bulb had the highest amount of saccharides, while the peak part of the middle leaf had most of the amino acids, free and conjugated phenolic acids.

Antiplatelet and antithrombotic properties of methanolic leaf extract of plumbago zeylanica L.: GC-MS and HR-LCMS metabolite profiling

Antiplatelet and antithrombotic properties of methanolic leaf extract of plumbago zeylanica L.: GC-MS and HR-LCMS metabolite profiling

Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula.

In vitro fermentation strategies with fecal inocula are considered cost-effective methods to gain mechanistic insights into fecal microbiota community dynamics. However, all in vitro approaches have their limitations due to inherent differences with respect to the in vivo situation mimicked, introducing possible biases into the results obtained. Here, we aimed to systematically optimize in vitro fermentation conditions to minimize drift from the initial inoculum, limit growth of opportunistic colonizers, and maximize the effect of added fiber products (here pectin) when compared to basal medium fermentations. We evaluated the impact of varying starting cell density and medium nutrient concentration on these three outcomes, as well as the effect of inoculation with fresh vs. stored fecal samples. By combining GC-MS metabolite profiling and 16 s rRNA gene-based amplicon sequencing, we established that starting cell densities below 1010 cells/ml opened up growth opportunities for members the Enterobacteriaceae family. This effect was exacerbated when using fecal samples that were stored frozen at -80°C. Overgrowth of Enterobacteriaceae resulted in lowered alpha-diversity and larger community drift, possibly confounding results obtained from fermentations in such conditions. Higher medium nutrient concentrations were identified as an additional factor contributing to inoculum community preservation, although the use of a less nutrient dense medium increased the impact of fiber product addition on the obtained metabolite profiles. Overall, our microbiome observations indicated that starting cell densities of 1010 cells/ml limited opportunities for exponential growth, suppressing in vitro community biases, whilst metabolome incubations should preferably be carried out in a diluted medium to maximize the impact of fermentable substrates.

Cytotoxic and antimicrobial effects of selected Egyptian Asteraceae species as well as GC-MS metabolite profiling of Senecio cruentus lipophilic fraction

Cytotoxic and antimicrobial effects of selected Egyptian Asteraceae species as well as GC-MS metabolite profiling of Senecio cruentus lipophilic fraction

In vitro and in silico Screening of Bioactive Compounds from Jasminum subtriplinerve Blume as α-glucosidase Inhibitor

In vitro and in silico Screening of Bioactive Compounds from Jasminum subtriplinerve Blume as α-glucosidase Inhibitor

High-Throughput Profiling of Metabolic Phenotypes Using High-Resolution GC-MS.

Metabolite profiling provides insights into the metabolic signatures, which themselves are considered as phonotypes closely related to the agronomic and phenotypic traits such as yield, nutritional values, stress resistance, and nutrient use efficiency. GC-MS is a sensitive and high-throughput analytical platform and has been proved to be a vital tool for the analysis of primary metabolism to provide an overview of cellular and organismal metabolic status. The potential of GC-MS metabolite profiling as a tool for detecting metabolic changes in plants grown in a high-throughput plant phenotyping platform was explored. In this chapter, we describe an integrated workflow of semi-targeted GC-high-resolution (HR)-time-of-flight (TOF)-MS metabolomics with both the analytical and computational steps, focusing mainly on the sample preparation, GC-HR-TOF-MS analysis part, and data analysis for plant phenotyping efforts.

SPME and solvent-based GC–MS metabolite profiling of Egyptian marketed Saussurea costus (Falc.) Lipsch. concerning its anticancer activity

BackgroundSaussurea costus Falc with synonymous Aucklandia costus, S. lappa, and A. lappa, is an ancient perennial plant native to Himalayan region. PurposeS. costus possesses an ethnopharmacological background for treating diarrhea, tenesmus, dyspepsia, vomiting, inflammation, and age-related diseases because it contains many chemical compounds with different biological potentials. Study designIn the current study, we investigated the phytochemical constituents of S. costus and its anticancer potential against human colon (HCT116) and hepatic (HepG2) cancer cell lines. MethodsIn the current study, we identified 122 different chemical compounds using a gas chromatography-mass spectrometry (GC–MS) method and investigated its extract's in vitro anticancer activity. ResultsDehydrocostus lactone, β-caryophyllene oxide, anethole, costunolide, β-sitosterol, and γ-bisabolene were recognized by GC–MS to have an anticancer potential as stated in published studies. Vanillosmin, santolinatriene and β-Sitosterol were identified for the first time in costus oil. Therefore, in the current study, S. costus extract induced apoptotic and anti-angiogenic effects against HCT116 and HepG2 cancer cell lines using in vitro and chorioallantoic membrane (CAM) models. Furthermore, S. costus extract has successfully potentiated the anticancer effect of doxorubicin (Dox), the common chemotherapeutic agent for different cancer type treatment. ConclusionWe can consider S. costus extract as a promising agent for human colon and hepatic cancers either alone or combined with Dox.

Bioprospection and secondary metabolites profiling of marine Streptomyces levis strain KS46

The quest for novel broad spectrum bioactive compounds is needed continuously because of the rapid advent of pathogenic multi drug resistant organisms. Actinomycetes, isolated from unexplored habitats can be a solution of this problem. The motive of this research work was isolation of actinomycetes having potential antimicrobial activities from unexplored regions of Devbag and Tilmati beach. The isolated actinomycetes were screened against pathogenic microbes for antimicrobial activities through cross streak method. Enzyme production activity was checked for these actinomycetes for amylase, protease, cellulase and lipase enzymes. Further antimicrobial activity of ethyl acetate extract of the potent strain KS46 was performed. The strain KS46 was identified with 16S rRNA gene sequencing and secondary structure was analysed. Gas chromatography–Mass spectrometry (GC–MS) profiling was conducted to ascertain the presence of bioactive metabolites in the ethyl acetate extract. The collected samples were pre-treated and 70 actinomycetes were isolated. The Streptomyces sp. strain KS46 showed the best antimicrobial activity in primary screening. Ethyl acetate extract of the strain KS46 revealed antimicrobial activity against S. aureus, B. subtilis, B. cereus, E. faecalis, K. pneumoniae, E. coli, S. flexneri, C. albicans and C. glabrata. The 16S rRNA gene sequencing identified the strain KS46 as Streptomyces levis strain KS46. The GC–MS metabolite profiling of the ethyl acetate extract revealed the availability of 42 compounds including fatty acid esters, fatty acid anhydrides, alkanes, steroids, esters, alcohols, carboxylic ester, etc. having antibacterial, antifungal, antiproliferative, antioxidant activities. This study indicated that Devbag and Tilmati beaches being untapped habitats have enormous diversity of promising antimicrobial metabolite producing actinomycetes. Therefore, further exploration should be carried out to characterize the potential actinomycetes, which can be optimistic candidates for generation of novel antimicrobial drugs.

Biogenic copper nanoparticles from Avicennia marina leaves: Impact on seed germination, detoxification enzymes, chlorophyll content and uptake by wheat seedlings.

Biogenic copper nanoparticles (Cu NPs) were synthesized using the aqueous crude extract of mangrove leaves, Avicennia marina (CE). GC-MS metabolite profiling of CE showed that their carbohydrates are mainly composed of D-mannose (29.21%), D-fructose, (18.51%), L-sorbose (12.91%), D-galactose (5.47%) and D-Talose (5.21%). Ultra-fine nanoparticles of 11.60 ±4.65 nm comprising Cu2O and Cu(OH)2 species were obtained with a carbohydrate and phenolic content of 35.6±3.2% and 3.13±0.05 mgGA/g, respectively. The impact of the biogenic Cu NPs on wheat seedling growth was dose-dependent. Upon treatment with 0.06 mg/mL of Cu NPs, the growth was promoted by 172.78 ± 23.11 and 215.94 ± 37.76% for wheat root and shoot, respectively. However, the lowest relative growth % of 81.94 ± 11.70 and 72.46 ± 18.78% were recorded for wheat root and shoot, respectively when applying 0.43 mg/mL of Cu NPs. At this concentration, peroxidase activity (POX) of the germinated wheat seeds also decreased, while ascorbic acid oxidase (AAO) and polyphenol oxidase (PPO) activities increased. Higher uptake of copper was observed in the root relative to the shoot implying the accumulation of the nanoparticles in the former. The uptake was also higher than that of the commercial Cu NPs, which showed an insignificant effect on the seedling growth. By treating the wheat leaves in foliar application with 0.06 mg/mL of Cu NPs, their contents of Chlorophyll a, Chlorophyll b, and total chlorophyll were enhanced after 21 days of application. Meanwhile, the high concentration (0.43 mg/mL) of Cu NPs was the most effective in reducing the leaf content of chlorophyll (a, b, and total) after the same time of application. The findings of this study manifest the potential of utilizing controlled doses of the prepared biogenic Cu NPs for inhibition or stimulation of seedling growth.

Volatile metabolites of endophytic Klebsiella aerogenes from Zingiber zerumbet rhizome and its antagonistic effect on soft rot causative Pythium myriotylum

Rhizomes of Zingiber zerumbet collected from their natural habitat and reported earlier to have high zerumbone content were selected for isolation of endophytes. Biochemical and molecular characterization using 16S rRNA sequencing of the endophytes identified the isolates as belonging to genus Klebsiella, Pantoea and Enterobacter. Isolate designated ZzKSD8 identified as K. aerogenes yielded maximal antagonistic activities against P. myriotylum determined as 83.5% ± 0.77. Volatile metabolites produced by ZzKSD8 caused absolute impairment of P. myriotylum hyphal growth compared to control (23.9 ± 0.37 cm). Volatile metabolites were extracted from 48 hour grown stationary phase ZzKSD8 cultures using absolute methanol, ethyl acetate and ethanol (60% v/v). GC-MS metabolite profiling detected alkanes and fatty acid methyl esters as the predominant constituents in the solvent extracts. Major constituents included methyl palmitate (31.37%), methyl stearate (18.57%) and cyclopropaneoctanoic acid, 2-hexyl-, methyl ester (17.05%) in methanol extract; alkanes like tetratetracontane (13.18%) and 2-methyloctacosane (12.10%) in ethyl acetate extracts and 4,22-sigmastadiene-3-one as major metabolite (22.51%) and stigmast-5-en-3-ol, (3.beta) (17.40%) in ethanol extract. Identified metabolites reported to modulate defense strategies in plants against phytopathogens makes ZzKSD8 a potential candidate for development of biological alternatives to control soil-borne soft-rot disease.

Metabolite profiling of whiteleg shrimp Litopenaeus vannamei from super-intensive culture in closed aquaculture systems: a recirculating aquaculture system and a hybrid zero water discharge-recirculating aquaculture system.

The production of the whiteleg shrimp Litopenaeus vannamei now accounts for approximately 75% of the total shrimp production in Indonesia. The techniques used to produce whiteleg shrimp in Indonesia are still dominated by conventional rearing strategies using open-pond systems, which often contribute to unpredictable culture performance and weak sustainability. Alternative production strategies of closed aquaculture systems, including the recirculating aquaculture system (RAS) and hybrid zero water discharge-recirculating aquaculture system (hybrid system), have been developed and implemented for higher productivity, stability and sustainability of whiteleg shrimp grow-out production in Indonesia. Despite the positive aspects of the application of closed aquaculture systems in shrimp aquaculture, the differences in the characteristics of shrimp grown in closed RAS and hybrid systems compared to open-pond systems remain unclear. This study aims to investigate the differences in the metabolite profiles of shrimp grown in intensive closed aquaculture systems, including an RAS and hybrid system, compared to those of shrimp grown in a semi-intensive, open, earthen pond system by means of non-targeted GC-MS metabolite profiling. Shrimp cultured in the closed systems (RAS and hybrid system) and an open system (pond) were harvested and subjected to GC-MS non-targeted metabolomics analysis. A total of 112 metabolites were annotated from shrimp samples and subjected to principal component analysis (PCA). The metabolites annotated from GC-MS mainly included organic compounds, proteinogenic and non-proteinogenic amino acids, sugars, nucleosides and fatty acids. The results of principal component analysis showed several metabolites with high variable importance in projection (VIP) scores, including shikimic acid, β-alanine, uric acid, hypoxanthine, inosine, homocysteine, methionine, phenylalanine, tryptophan and lysine, as the main metabolites differentiating the shrimp grown in the three production systems. Our findings showed that shrimp cultured in different aquaculture systems exhibited distinct metabolite profiles, and the metabolites showing high VIP scores, including shikimic acid, β-alanine, uric acid, hypoxanthine, inosine, homocysteine, methionine, phenylalanine, tryptophan and lysine, may serve as candidate markers to indicate the differences in shrimp from different production systems.

Investigation of α-Glucosidase Inhibitory Metabolites from Tetracera scandens Leaves by GC-MS Metabolite Profiling and Docking Studies.

Stone leaf (Tetracera scandens) is a Southeast Asian medicinal plant that has been traditionally used for the management of diabetes mellitus. The underlying mechanisms of the antidiabetic activity have not been fully explored yet. Hence, this study aimed to evaluate the α-glucosidase inhibitory potential of the hydromethanolic extracts of T. scandens leaves and to characterize the metabolites responsible for such activity through gas chromatography–mass spectrometry (GC–MS) metabolomics. Crude hydromethanolic extracts of different strengths were prepared and in vitro assayed for α-glucosidase inhibition. GC–MS analysis was further carried out and the mass spectral data were correlated to the corresponding α-glucosidase inhibitory IC50 values via an orthogonal partial least squares (OPLS) model. The 100%, 80%, 60% and 40% methanol extracts displayed potent α-glucosidase inhibitory potentials. Moreover, the established model identified 16 metabolites to be responsible for the α-glucosidase inhibitory activity of T. scandens. The putative α-glucosidase inhibitory metabolites showed moderate to high affinities (binding energies of −5.9 to −9.8 kcal/mol) upon docking into the active site of Saccharomyces cerevisiae isomaltase. To sum up, an OPLS model was developed as a rapid method to characterize the α-glucosidase inhibitory metabolites existing in the hydromethanolic extracts of T. scandens leaves based on GC–MS metabolite profiling.

BBX31 promotes hypocotyl growth, primary root elongation and UV-B tolerance in Arabidopsis

ABSTRACTPhotomorphogenesis is an important developmental process that helps the seedlings adapt to external light conditions. B-Box proteins are a family of transcription factors that regulate photomorphogenic responses. BBX31 negatively regulates photomorphogenesis under visible light. In contrast, it promotes photomorphogenesis under UV-B and enhances tolerance to high doses of UV-B radiation. BBX31 and HY5 independently and oppositely regulate the ability of seedlings to adapt to varying light intensities. BBX31 also regulates primary root elongation under low intensities of white light. GC-MS and HPLC-based metabolite profiling identified differential accumulation of multiple primary and secondary metabolites in 35S:BBX31 that might enhance tolerance to UV-B.

Metabolomic Investigation of Tenderness and Aging Response in Beef Longissimus Steaks

A study was conducted to identify molecular changes reflective of beef tenderness variation and tenderization during postmortem aging. Carcasses (U.S. Select) were selected to represent extremes in tenderness (n = 20; 10 per class). Two pairs of adjacent longissimus lumborum steaks from each strip loin were blocked by location and assigned to each aging time (2, 7, 14, or 28 d postmortem). One steak from each pair was designated for slice shear force determination and the other was used for sarcomere length, western blotting for desmin, and non-targeted LC- and GC–MS metabolite profiling. Tough steaks had higher (P < 0.001) slice shear force values than tender steaks, and increasing aging time decreased (P < 0.001) slice shear force values. Tender steaks had a greater (P < 10–4) proportion of desmin degraded than tough steaks, and increasing aging time increased (P < 10–22) desmin degradation in steaks from both classes. From 2,562 profiled metabolites, 102 metabolites were included in the final analysis after statistical screening. Twenty-eight metabolites could be annotated and loosely categorized into amino acids/peptides (n = 16), metabolism intermediates (n = 7), glycosides (n = 4), and fatty acids and phospholipids (n = 3). Amino acids were primarily associated with desmin degradation. Increased glucose levels were strongly associated to the tender classification and moderately associated to increased proteolysis, while increased glucose-6-phosphate was strongly related to the tender class but was related to decreased proteolysis. Increased malic acid was strongly associated to the tough classification, increased slice shear force, and decreased proteolysis. Increased levels of 3-phosphoglyceric acid and glycerol-3-phosphate was moderately associated with increased slice shear force and decreased proteolysis. These data indicate that accumulation of amino acids during aging is strongly related to postmortem proteolysis and may provide evidence of the fate of proteins degraded postmortem. Measures of glucose, glucose-6-phosphate, and malic acid concentrations may provide a metabolic fingerprint indicative of tenderness differences in beef longissimus.

GC-MS metabolite profiling for specific detection of dwarf somaclonal variation in banana plants.

Premise of the StudyThe production of banana (Musa spp.; Musaceae) plants is affected by various types of somaclonal variations (SV), including dwarfism. However, methods for specific detection of SV are still scarce. To overcome this, a metabolite‐based method for detection of dwarf variants was evaluated.MethodsThe gas chromatography–mass spectrometry (GC‐MS) metabolite profile of dwarf banana variants was investigated and compared to that of normal‐healthy (N) and cucumber mosaic virus (CMV)–infected plants using principal components analysis and partial least squares discriminant analysis (PLS‐DA).ResultsSignificant differences among the sample groups were observed in 82 metabolites. Rhamnose was exclusively present in dwarf plants but allothreonine and trehalose were present in all but SV samples. Cellobiose was only detected in N plants, while 45 other metabolites, including methyl‐glucopyranoside, allopyranose, lactose, phenylalanine, and l‐lysine were detected in all but CMV‐infected samples. PLS‐DA models were able to detect SV, CMV, and N plants with 100% accuracy and specificity.DiscussionThe GC‐MS metabolite profile can be used for the rapid, specific detection of SV at early plant production stages. This is the first metabolite‐based characterization and detection of somaclonal variation in plants.

Identification of physiological changes and key metabolites coincident with postharvest internal browning of pineapple (Ananas comosus L.) fruit

Chilling causes biochemical and physiological dysfunction in pineapple fruit, evidently as internal browning (IB). Here, putative biomarkers of the early responses to chilling stress, prior to the appearance of IB, were investigated using a combination of GC–MS profiling and physiological and biochemical assay. Two cultivars – one tolerant and one susceptible – were used, and attention was made to tissue sampling from the early reversible to late irreversible phase of chilling injury. At these stages, electrolyte leakage and the levels of phenolic compounds were not altered even after prolonged chilling stress; however, changes in ascorbic acid levels and antioxidant activity coincided with IB occurrence. These data suggest that antioxidant capacity and ascorbic acid are the measurable and ‘early’ responses to IB. The GC–MS metabolite profiling data of chilled pineapple fruit presented here is the first reported. The tolerant pineapple showed greater shifts in metabolism and accumulated higher levels of amino acids and organic acids, especially, valine, cysteine, aspartate and galacturonate. In contrast, the lactic acid content of susceptible cultivar was higher upon chilled storage.

Metabolite profiling of tartary buckwheat-an underutilized neutraceutical crop of Kashmir Himalaya

<p>The aim of the present study was to explore the possible metabolites in the methanolic extract of root, stem, groat and hull of the neutraceutical crop, <em>Fagopyrum tataricum</em> using GC-MS technique. From GC-MS metabolite profiling, over 90 different metabolites were identified among root, stem, groat and hull extract. The most prevailing compounds were 3, 3’, 4’, 5, 7-pentahydroflavone-3-rhamnoglucoside (71.94%) in groat, 9, 12-octadecadienoic acid (49.38%) in root, 6-octadecanoic acid, a steric acid (70.46%) in hull and Cis-9-hexadecanal (13.38%) in stem. Present investigation reveals that <em>F. tataricum</em> is an excellent source of many metabolites such as, fatty acids, hydrocarbons, steroids, terpenoids, esters, organic acids and aldehydes with excellent pharmaceutical properties. These results suggest that tartary buckwheat could be a promising alternative in the functional food sector and neutraceutical to improve social well-being and diminish malnutrition.</p>

Quantitative profiling of polar primary metabolites of two chickpea cultivars with contrasting responses to salinity.

This study reports a GC-QqQ-MS method for the quantification of forty-eight primary metabolites from four major classes (sugars, sugar acids, sugar phosphates, and organic acids) which can be applied to a number of biological systems. The method was validated in terms of linearity, reproducibility and recovery, using both calibration standards and real samples. Additionally, twenty-eight biogenic amines and amino acids were quantified using an established LC-QqQ-MS method. Both GC-QqQ-MS and LC-QqQ-MS quantitative methods were applied to plant extracts from flower and pod tissue of two chickpea (Cicer arietinum L.) cultivars differing in their ability to tolerate salinity, which were grown under control and salt-treated conditions. Statistical analysis was applied to the data sets using the absolute concentrations of metabolites to investigate the differences in metabolite profiles between the different cultivars, plant tissues, and treatments. The method is a significant improvement of present methodology for quantitative GC-MS metabolite profiling of organic acids and sugars, and provides new insights of chickpea metabolic responses to salinity stress. It is applicable to the analysis of dynamic changes in endogenous concentrations of polar primary metabolites to study metabolic responses to environmental stresses in complex biological tissues.