Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC₅₀=78.38±0.15) which suggests its antioxidant potential.     

Gas chromatography/mass spectrometry in metabolic profiling of biological fluids

One of the objectives of metabonomics is to identify subtle changes in metabolite profiles between biological systems of different physiological or pathological states. Gas chromatography mass spectrometry (GC/MS) is a widely used analytical tool for metabolic profiling in various biofluids, such as urine and blood due to its high sensitivity, peak resolution and reproducibility. The availability of the GC/MS electron impact (EI) spectral library further facilitates the identification of diagnostic biomarkers and aids the subsequent mechanistic elucidation of the biological or pathological variations. With the advent of new comprehensive two dimensional GC (GCxGC) coupled to time-of-flight mass spectrometry (TOFMS), it is possible to detect more than 1200 compounds in a single analytical run. In this review, we discuss the applications of GC/MS in the metabolic profiling of urine and blood, and discuss its advances in methodologies and technologies.     

Detection of metabolites of lysergic acid diethylamide (LSD) in human urine specimens: 2-oxo-3-hydroxy-LSD,a prevalent metabolite of LSD

Seventy-four urine specimens previously found to contain lysergic acid diethylamide (LSD) by gas chromatography–mass spectrometry (GC–MS) were analyzed by a new procedure for the LSD metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD) using a Finnigan LC–MS–MS system. This procedure proved to be less complex, shorter to perform and provides cleaner chromatographic characteristics than the method currently utilized by the Navy Drug Screening Laboratories for the extraction of LSD from urine by GC–MS. All of the specimens used in the study screened positive for LSD by radioimmunoassay (Roche Abuscreen^®). Analysis by GC–MS revealed detectable amounts of LSD in all of the specimens. In addition, isolysergic diethylamide (iso-LSD), a byproduct of LSD synthesis, was quantitated in 64 of the specimens. Utilizing the new LC–MS–MS method, low levels of N-desmethyl-LSD (nor-LSD), another identified LSD metabolite, were detected in some of the specimens. However, all 74 specimens contained O-H-LSD at significantly higher concentrations than LSD, iso-LSD, or nor-LSD alone. The O-H-LSD concentration ranged from 732 to 112 831 pg/ml (mean, 16 340 pg/ml) by quantification with an internal standard. The ratio of O-H-LSD to LSD ranged from 1.1 to 778.1 (mean, 42.9). The presence of O-H-LSD at substantially higher concentrations than LSD suggests that the analysis for O-H-LSD as the target analyte by employing LC–MS–MS will provide a much longer window of detection for the use of LSD than the analysis of the parent compound, LSD.     

A high-throughput method for microbial metabolome analysis using gas chromatography/mass spectrometry

An analytical high-throughput method based on gas chromatography/mass spectrometry (GC/MS) was developed for fast metabolome investigation. By parallelization and partial automation the time needed for the preanalytical steps could be reduced. In addition a strong decrease of the relative standard deviation of metabolite concentrations from independent samples on the same microtiter plate from 25 to 13% was achieved. Between different plates the relative standard deviation is comparable to the one observed in standard experiments with shaking flasks. Using a fast GC the time need for the full GC/MS-based metabolome analysis could be decreased from 60 to 18 min per run, allowing the measurement of 72 single samples per day and GC/MS machine. In samples of the model organism Corynebacterium glutamicum more than 1000 peaks in the total ion current could be detected in a single fast GC/MS run of which 650 were strong enough to be quantified. Approximately 150 compounds of these were identified using our metabolite MS-library. Correlation analysis of the concentration vectors of independent wild-type samples raised under the same conditions show very high correlations of 0.99+/-0.01 (logs). In conclusion this method allows screenings of large mutant libraries for genetically induced metabolic perturbations.     

Metabolite profiling of maize grain: differentiation due to genetics and environment

A comparative metabolite profiling approach based on gas chromatography-mass spectrometry (GC/MS) was applied to investigate the impact of genetic background, growing location and season on the chemical composition of maize grain. The metabolite profiling protocol involved sub-fractionation of the metabolites and allowed the assessment of about 300 distinct analytes from different chemical classes (polar to lipophilic), of which 167 could be identified. A comparison, over three consecutive growing seasons, of the metabolite profiles of four maize cultivars which differed in their maturity classification, was carried out using principal component analysis (PCA). This revealed a strong separation of one cultivar in the first growing season, which could be explained by the immaturity of the kernels of this cultivar compared with others in the field trial. Further evaluations by pair-wise comparison using Student’s t-test and analysis of variance (ANOVA) showed that the growing season was the most prominent impact factor driving variation of the metabolite pool. An increased understanding of metabolic variation was achieved by analysis of a second sample set comprising one cultivar grown for 3 years at four locations. The applied GC/MS-based metabolite profiling demonstrated the natural variation in maize grain metabolite pools resulting from the interplay of environment, season, and genotype.     

Baitmet,a computational approach for GC–MS library-driven metabolite profiling

Introduction

Current computational tools for gas chromatography—mass spectrometry (GC–MS) metabolomics profiling do not focus on metabolite identification, that still remains as the entire workflow bottleneck and it relies on manual data reviewing. Metabolomics advent has fostered the development of public metabolite repositories containing mass spectra and retention indices, two orthogonal properties needed for metabolite identification. Such libraries can be used for library-driven compound profiling of large datasets produced in metabolomics, a complementary approach to current GC–MS non-targeted data analysis solutions that can eventually help to assess metabolite identities more efficiently.

Results

This paper introduces Baitmet, an integrated open-source computational tool written in R enclosing a complete workflow to perform high-throughput library-driven GC–MS profiling in complex samples. Baitmet capabilities were assayed in a metabolomics study involving 182 human serum samples where a set of 61 metabolites were profiled given a reference library.

Conclusions

Baitmet allows high-throughput and wide scope interrogation on the metabolic composition of complex samples analyzed using GC–MS via freely available spectral data. Baitmet is freely available at http://CRAN.R-project.org/package=baitmet.

     

Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice

We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC x GC) gas chromatography-time-of-flight (TOF)-mass spectrometry. This method was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining 1D- and GC x GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.     

Trapping and identification of the dichloroacetate radical from the reductive dehalogenation of trichloroacetate by mouse and rat liver microsomes

A key question in the risk assessment of trichloroethylene (TRI) is the extent to which its carcinogenic effects might depend on the formation of dichloroacetate (DCA) as a metabolite. One of the metabolic pathways proposed for the formation of DCA from TRI is by the reductive dehalogenation of trichloroacetate (TCA), via a free radical intermediate. Although proof of this radical has been elusive, the detection of fully dechlorinated metabolites in the urine and the formation of lipid peroxidation by-products in microsomal incubations with TCA argue for its existence. We report here the trapping of the dichloroacetate radical with the spin-trapping agent PBN, and its identification by GC/MS. The PBN/dichloroacetate radical adduct was found to undergo an intramolecular rearrangement during its extraction into organic solvent. An internal condensation reaction between the acetate and the nitroxide radical moieties is hypothesized to form a cyclic adduct with the elimination of an OH radical. The PBN/dichloroacetate radical adduct has been identified by GC/MS in both a chemical Fenton system and in rodent microsomal incubations with TCA as substrate.     

Assay of ezlopitant, a substance P receptor antagonist, and metabolites in biological matrices by gas chromatography with mass spectrometric detection: simultaneous analysis of a benzyl alcohol and alkene

A method for the analysis of the substance P antagonist ezlopitant and two active metabolites in serum using solid-phase extraction followed by GC–MS analysis is described. The linear dynamic range was 1.0 to 100 ng/ml and precision and accuracy over this range were within 15%. Upon injection of reconstituted sample extracts into the hot injector port of the gas chromatograph, the benzyl alcohol metabolite undergoes a small amount of spontaneous dehydration to the alkene metabolite. We have incorporated an additional hexadeuterated internal standard of the benzyl alcohol into the assay to permit measurement of the extent of dehydration in each sample. This novel approach should be generally applicable to the simultaneous determination of benzyl alcohols and corresponding alkenes by GC–MS when the possibility exists that the alcohol can undergo spontaneous dehydration to the alkene in the injector port of GC instrumentation.     

Temporal characterization of serum metabolite signatures in lung cancer patients undergoing treatment

Lung cancer causes more deaths in men and women than any other cancer related disease. Currently, few effective strategies exist to predict how patients will respond to treatment. We evaluated the serum metabolomic profiles of 25 lung cancer patients undergoing chemotherapy ± radiation to evaluate the feasibility of metabolites as temporal biomarkers of clinical outcomes. Serial serum specimens collected prospectively from lung cancer patients were analyzed using both nuclear magnetic resonance (¹H-NMR) spectroscopy and gas chromatography mass spectrometry (GC–MS). Multivariate statistical analysis consisted of unsupervised principal component analysis or orthogonal partial least squares discriminant analysis with significance assessed using a cross-validated ANOVA. The metabolite profiles were reflective of the temporal distinction between patient samples before during and after receiving therapy (¹H-NMR, p < 0.001: and GC–MS p < 0.01). Disease progression and survival were strongly correlative with the GC–MS metabolite data whereas stage and cancer type were associated with ¹H-NMR data. Metabolites such as hydroxylamine, tridecan-1-ol, octadecan-1-ol, were indicative of survival (GC–MS p < 0.05) and metabolites such as tagatose, hydroxylamine, glucopyranose, and threonine that were reflective of progression (GC–MS p < 0.05). Metabolite profiles have the potential to act as prognostic markers of clinical outcomes for lung cancer patients. Serial ¹H-NMR measurements appear to detect metabolites diagnostic of tumor pathology, while GC–MS provided data better related to prognostic clinical outcomes, possibility due to physiochemical bias related to specific biochemical pathways. These results warrant further study in a larger cohort and with various treatment options.     

Metabolite profiling of small cerebrospinal fluid sample volumes with gas chromatography–mass spectrometry: application to a rat model of multiple sclerosis

Analysis of metabolites in biofluids by gas chromatography–mass spectrometry (GC–MS) after oximation and silylation is a key method in metabolomics. The GC–MS method was modified by a modified vial design and sample work-up procedure in order to make the method applicable to small volumes of cerebrospinal fluid (CSF), i.e. 10 μL, with similar coverage compared to the standard procedure using ≥100 μL of CSF. The data quality of the modified GC–MS method was assessed by analyzing a study sample set in an animal model for multiple sclerosis, including repetitively analysed quality control rat CSF samples. Automated normalization and intra- and inter-batch correction significantly improved the data quality with the majority of metabolites showing a relative standard deviation <20 %. The modified GC–MS method was successfully applied in rat model of multiple sclerosis where statistical analysis of 93 metabolites, of which 73 were (tentatively) identified, in 10 μL of rat CSF showed statistically significant differences in metabolite profiles of rats at the onset and peak of experimental autoimmune encephalomyelitis compared to rats in the control group. The modified GC–MS method presented proved to be a valid and valuable metabolomics method when only limited sample volumes are available.     

Separation methods used in the determination of choline and acetylcholine

Cholinergic neurotransmission has been the subject of intensive investigations in recent years due to increasing recognition of the importance of its roles in physiology, pathology and pharmacology. The fact that the disposition of a neurotransmitter may reflect its functional status has made the measurement of acetylcholine and/or its precursors and metabolites in biological fluids an integral part of cholinergic research. With evolving complexity in experimental approaches and designs, and correspondingly increasing demand on sensitivity, specificity and accuracy matching advancements in sophistication in analytical methods have been made. The present review attempts to survey the array of analytical techniques that have been adopted for the measurement of acetylcholine or its main precursor/metabolite choline ranging from simple bioassays, radioenzymatic assays, gas chromatography (GC) with flame ionization detection, GC with mass spectrometry (GC–MS) detection, high-performance liquid chromatography (HPLC) with electrochemical detection (ED), HPLC with MS (HPLC–MS) to the sophisticated combination of micro-immobilized enzymatic reactor, microbore HPLC and modified electrode technology for the detection of ultra-low levels with particular emphasis on the state of the art techniques.     

Sequential GC/MS analysis of sialic acids,monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.     

A metabolite profiling approach to follow the sprouting process of mung beans (Vigna radiata)

A metabolite profiling methodology based on capillary gas chromatography/mass spectrometry (GC/MS) was employed to investigate time-dependent metabolic changes in the course of the sprouting of mung beans (Vigna radiata). Intact mung beans and sprout samples taken during the germination process were subjected to an extraction and fractionation procedure covering a broad spectrum of lipophilic (e.g. fatty acid methyl esters, hydrocarbons, fatty alcohols, sterols) and hydrophilic (e.g. sugars, acids, amino acids, amines) low molecular weight constituents. Investigation of the obtained fractions by GC resulted in the detection of more than 450 distinct peaks of which 146 were identified by means of MS. Statistical assessment of the metabolite profiling data via principal component analysis demonstrated that the metabolic changes during the sprouting of mung beans are reflected by time-dependent shifts of the scores which were comparable for two spouting processes independently conducted under the same conditions. Analysis of the loadings showed that polar metabolites were major contributors to the separation along the first principal component. The dynamic changes of single metabolites revealed significantly increased levels of monosaccharides, organic acids and amino acids and a decrease in fatty acid methyl esters in mung bean sprouts.     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Formation of 13,14-dihydro-prostaglandin E1 during intravenous infusions of prostaglandin E1 in patients with peripheral arterial occlusive disease.

Formation of 13,14-dihydro-prostaglandin (PG) E1 during intravenous infusions of PGE1 in patients with peripheral arterial occlusive disease was investigated. Using both high performance liquid chromatography (h.p.l.c.) combined with radioimmunoassay and gas chromatography/triple stage quadrupole mass spectrometry (GC/MS/MS) basal levels of 13,14-dihydro-PGE1 were found to be close to or below the detection limits of the assay methods. Levels of the PGE1 metabolite increased significantly during the infusion periods and decreased after their end. Since 13,14-dihydro-PGE1, in contrast to its precursors 15-keto-PGE1 and 15-keto-13,14-dihydro-PGE1, is biologically active, its formation could contribute to the beneficial effects of PGE1 administered intravenously in patients with peripheral arterial occlusive disease.     

7-Hydroxyphthalide: a new natural salicylaldehyde analog from Oulenzia sp. (Astigmata: Winterschmitiidae)

A new salicyl lactone was detected from the unidentified Oulenzia sp. and its chemical structure was elucidated as 7-hydroxyphthalide (7-hydroxy-3H-isobenzofuran-1-one), based on its GC/MS and GC/FT-IR data and SiO2 column behavior. The compound was synthesized by NaBH4 reduction of 3-hydroxyphthalic anhydride. The GC/MS and GC/FT-IR spectra of the natural compound were consistent with those of the synthetic product. Although the compound is known as a medical material, this is the first example of its presence in nature.