Comparison of gas chromatography–mass spectrometry and gas chromatography–combustion–isotope ratio mass spectrometry analysis for in vivo estimates of metabolic fluxes

Gas chromatography–mass spectrometry (GC–MS) was compared with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for measurements of cholesterol ¹³C enrichment after infusion of labeled precursor ([¹³C_1,2]acetate). Paired results were significantly correlated, although GC–MS was less accurate than GC–C–IRMS for higher enrichments. Nevertheless, only GC–MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC–MS and GC–C–IRMS to analyze in vivo stable isotope studies.     

Screening for inborn errors of metabolism using gas chromatography–mass spectrometry

Screening of newborns for inborn errors of metabolism (IEM) in China is both a challenging and undeveloped area for gynecologists and pediatricians. Since 1999, the Capital Institute of Pediatrics has been studied as regards screening for IEM using advanced gas chromatography–mass spectrometry (GC–MS) method in collaboration with the Matsumoto Institute of Life Science (MILS), Japan, and has successfully diagnosed 51 cases of IEM in a total of 393 patients. Galactosemia, phenylketonuria and methylmalonic acidemia were the most frequent disorders among 51 cases of IEM. Treatment by suitable drugs and/or diet therapy was very effective in the most cases.     

Determination of urinary valproylcarnitine by gas chromatography—mass spectrometry with selected-ion monitoring

A modified method for the determination of valproylcarnitine in urine samples of patients receiving sodium valproate by gas chromatography—mass spectrometry with selected-ion monitoring is described. The chemically analogous internal standard 2-ethylpentanoylcarnitine was added to the urine samples. Valproic acid and its metabolites were removed by extraction with chloroform at pH 5.0. The samples were then applied onto a C₁₈ Sep-Pak column. Inorganic and water soluble compounds were washed out with water. Valproylcarnitine and internal standard were eluted with methanol and were derivatized to the corresponding acyl-containing lactones by heating at 100°C for 60 min in dimethylformamide. Urinary valproylcarnitine levels of epileptic patients receiving valproate were determined according to the present method. The data obtained might be useful for diagnosis of carnitine deficiency.     

Evaluation of urinary dihydrocodeine excretion in human by gas chromatography–mass spectrometry

Urinary metabolic pattern after the therapeutic peroral dose of dihydrocodeine tartrate to six human volunteers has been explored. Using the GC–MS analytical method, we have found that the major part of the dose administered is eliminated via urine within the first 24 h. However, the analytical monitoring of dihydrocodeine and its metabolites in urine was still possible 72 h after the dose was administered. The dihydrocodeine equivalent amounts excreted in urine in 72 h ranged between 32 and 108% of the dose, on average 62% in all individuals. The major metabolite excreted into urine was a 6-conjugate of dihydrocodeine, then in a lesser amount a 6-conjugate of nordihydrocodeine (both conjugated to approximately 65%). The O-demethylated metabolite dihydromorphine was of a minor amount and was 3,6-conjugated in 85%. Traces of nordihydromorphine and hydrocodone were confirmed as other metabolites of dihydrocodeine in our study. This information can be useful in interpretation of toxicological findings in forensic practice.     

Evaluation of plasma enzyme activities using gas chromatography–mass spectrometry based steroid signatures

The simultaneous quantification of 65 plasma steroids, including 22 androgens, 15 estrogens, 15 corticoids and 13 progestins, was developed using gas chromatography-mass spectrometry (GC–MS). The extraction efficiency of the catechol estrogens was improved by the addition of l-ascorbic acid in several steps. All steroids, as their trimethylsilyl derivatives, were well separated with good peak shapes within a 50 min run. The devised method provided good linearity (correlation coefficient, r² > 0.993), while the limit of quantification ranged from 0.2 to 2.0 ng mL^?1. The precision (% CV) and accuracy (% bias) were 2.0–12.4% and 93.5–109.2%, respectively. The metabolic changes were evaluated by applying this method to plasma samples obtained from 26 healthy male subjects grouped according to the pre- and post-administration of dutasteride, which inhibits 5α-reductase isoenzyme types 1 and 2. The levels of three plasma steroids, such as dihydrotestosterone, 5α-androstanedione and allotetrahydrocortisol, were decreased significantly after drug administration, while the levels of testosterone and 5β-androstane-3β,17α-diol were increased. In addition, the ratios of the steroid precursors and their metabolites, which represent the activities of the related enzymes, were z-score transformed for visualization in heat maps generated using supervised hierarchical clustering analysis. These results validated the data transformation because 5α-reductase is an indicator for the biological actions of dutasteride. GC–MS base quantitative visualization might be found in the integration with the mining biomarkers in drug evaluations and hormone-dependent diseases.     

Determination of haloacetic acids in human urine by headspace gas chromatography–mass spectrometry

Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC–MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 μl of dimethylsulphate and 100 μl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na₂SO₄ (6 g per 12 ml of urine), an oven temperature of 70 °C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 μg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water.     

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.     

Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography–mass spectrometry

Introduction

Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers.

Objectives

The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells.

Methods

VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC–MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies.

Results

Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype.

Conclusions

We demonstrate that a GC–MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

     

Microscale analysis of amino acids using gas chromatography–mass spectrometry after methyl chloroformate derivatization

To conduct studies of stable isotope incorporation and dilution in growing plants, a rapid microscale method for determination of amino acid profiles from minute amounts of plant samples was developed. The method involves solid-phase ion exchange followed by derivatization and analysis by gas chromatography–mass spectrometry (GC–MS). The procedure allowed the eluent to be derivatized directly with methyl chloroformate without sample lyophilization or other evaporation procedures. Sample extraction and derivatization required only ca. 30 min and quantification of the 19 amino acids eluted from the cation exchange solid-phase extraction step from a single cotyledon (0.4 mg fresh weight) or three etiolated 7-day-old Arabidopsis seedlings (0.1 mg fresh weight) was easily accomplished in the selected ion monitoring mode. This method was especially useful for monitoring mass isotopic distribution of amino acids as illustrated by Arabidopsis seedlings that had been labeled with deuterium oxide and ¹⁵N salts. Sample preparation was facile, rapid, economical, and the method is easily modified for integration into robotic systems for analysis with large numbers of samples.     

Extractive alkylation of 6-mercaptopurine and determination in plasma by gas chromatography—mass spectrometry

An analytical procedure was developed for the determination of 6-mercaptopurine in plasma. Owing to the polar character and low plasma concentrations of the compound, extraction and derivatization was carried out directly from the plasma sample by extractive alkylation. Determination was made using gas chromatography—mass spectrometry with multiple-ion detection.Conditions with respect to the rate of formation and the stability of the derivative formed in the extractive alkylation step were evaluated. The selectivity of the method to azathioprine and to metabolites was thoroughly investigated. No 6-mercaptopurine was formed from azathioprine added to water or plasma and run through the method. The method enables the detection of 2 ng of 6-mercaptopurine in a 1.0-ml plasma sample. Quantitative determinations were done down to 10 ng/ml 6-mercaptopurine in plasma.     

Detection of β-blockers in urine by solid-phase extraction-supercritical fluid extraction and gas chromatography-mass spectrometry

The most convenient way to perform supercritical fluid extraction (SPE) of liquid sample matrices is to combine it with solid-phase extraction (SPE). β-Blockers from urine were collected on an Empore disc, which was then placed into an extraction cell for derivatization and SPE. SPE recovery was best at pH 10. Effects of temperature, pressure and volume of pyridine on the acetylation and SFE processes were studied. Without acetylation the β-blockers were not significantly soluble in CO₂. SFE temperatures of 70°C and 150°C together with 200 μl of acetic anhydride and 400 μl pyridine gave the best results. With the SPE-SFE-GC-MS method developed here, β-blockers like oxprenolol, metoprolol and propranolol could easily be detected in urine samples, and the limit of detection (LOD) for these compounds was found to be 20 ng/ml, 30 ng/ml and 40 ng/ml, respectively.     

Simplified determination of lorazepam and oxazepam in biological fluids by gas chromatography—mass spectrometry

Lorazepam and oxazepam in plasma and urine were measued by gas chromatography—mass spectrometry. Oxazepam was used as an internal standard in the assay of lorazepam and vice versa. After removal of interfering substances with n-hexane, the drugs were extracted with benzene and converted to N₁,O₃-bistrimethylsilyl derivatives. Glucuronide forms of the drugs were extracted after hydrolysis with β-glucuronidase. A common fragment ion at m/e 429 was used to monitor the two drugs. The sensitivity was 2 ng/ml for both drugs, which was sufficient to determine plasma and urine concentrations after therapeutic doses to humans.     

Revised method for routine determination of urinary dialkyl phosphates using gas chromatography–mass spectrometry

Among urinary organophosphorus pesticide (OP) metabolites, dialkyl phosphates (DAPs) have been most often measured as a sensitive biomarker in non-occupational and occupational OP exposure risk assessment. In our conventional method, we have employed a procedure including simple liquid–liquid extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide, PFBBr) and clean-up (multi-layer column) for gas chromatography–mass spectrometry (GC–MS) analysis starting from 5-mL urine samples. In this study, we introduce a revised analytical method for urinary DAPs; its main modification was aimed at improving the pre-derivatization dehydration procedure. The limits of detection were approximately 0.15 μg/L for dimethylphosphate (DMP), 0.07 μg/L for diethylphosphate (DEP), and 0.05 μg/L for both dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) in 2.5-mL human urine samples. Within-run precision (percent of relative standard deviation, %RSD) at the DAP levels varying in the range of 0.5–50 μg/L was 6.0–19.1% for DMP, 3.6–18.3% for DEP, 8.0–25.6% for DMTP and 9.6–27.8% for DETP. Between-run precision at 5 μg/L was below 15.7% for all DAPs. The revised method proved to be feasible to routine biological monitoring not only for occupational OP exposure but also for environmental background levels in the general population. Compared to our previous method, the revised method underscores the importance of adding pre-derivatization anhydration for higher sensitivity and precision.     

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography–mass spectrometry

A GC–MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography–electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-βDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1–20 μg/mL (R² ≥ 0.998). Intra-day accuracies ranged between 97.2–104.9%, 96.1–103.2%, and 97.3–102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2–105.7%, 99.1–105.2%, and 96.5–101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.     

Neonatal ketosis is not rare: experience of neonatal screening using gas chromatography–mass spectrometry

The causes and effects of transient neonatal ketosis, discovered during a pilot study of screening for abnormalities in neonatal metabolism using gas chromatography–mass spectrometry, were investigated. Of the 21 342 neonates that were screened, 47 had significant ketosis. The organic acid profile accompanying ketosis in the urine of neonates followed the pattern of ketotic dicarboxylic aciduria in approximately half of the cases. Ketosis was more often found in neonates nourished by breast feeding (33 out of 47). Over half of the neonates showing ketosis (28 out of 47) were asymptomatic. When normal neonates and neonates testing positive for ketosis were compared, no statistically significant correlations were found with regard to birth mass, gestational period, or gender. However, neonates with ketosis tended to have low mass gain rates in the 5 days from birth and a statistically significant difference was found in this regard in comparison to normal neonates (P<0.0001). From the above results, development of ketosis in neonates was found to be possible even in normal subjects. Most ketosis in neonates was also found to depend largely on nourishment after birth. Existence of an asymptomatic ketosis category was also suggested.     

Identification of flavanone metabolites in rat urine by combined gas–liquid chromatography and mass spectrometry

1. The metabolism of flavanone in the rat was studied after oral or intraperitoneal administration of the compound. Flavone and flav-3-ene together with five other unidentified minor metabolites were excreted in the urine. 2. The formation of flavanone metabolites was not suppressed by the administration of high doses of the antibacterial compounds aureomycin and phthaloylsulphathiazole. 3. No aromatic acids that could be attributed to ring cleavage of flavanone were detected. 4. Administration of 100 or 200mg of flavanone daily per rat caused some deaths during the 7-14-day period. 5. The application of combined gas-liquid chromatography/mass spectrometry and proton nuclear-magnetic-resonance spectroscopy to the separation and identification of the flavanone metabolites is described. 6. Measurement of the two major flavanone metabolites was carried out by gas-liquid chromatography.     

Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.     

Comparative metabolomics of Japanese Black cattle beef and other meats using gas chromatography–mass spectrometry

Progress in metabolomic analysis now allows the evaluation of food quality. This study aims to identify the metabolites in meat from livestock using a metabolomic approach. Using gas chromatography–mass spectrometry (GC/MS), many metabolites were reproducibly detected in meats, and distinct differences between livestock species (cattle, pigs, and chickens) were indicated. A comparison of metabolites between tissues types (muscle, intramuscular fat, and intermuscular fat) in marbled beef of Japanese Black cattle revealed that most metabolites are abundant in the muscle tissue. Several metabolites (medium-chain fatty acids, etc.) involved in triacylglycerol synthesis were uniquely detected in fat tissue. Additionally, the results of multivariate analysis suggest that GC/MS analysis of metabolites can distinguish between cattle breeds. These results provide useful information for the analysis of meat quality using GC/MS-based metabolomic analysis.

ABBREVIATIONS: GC/MS: gas chromatography-mass spectrometry; NMR: nuclear magnetic resonance; MS: mass spectrometry; IS: 2-isopropylmalic acid; MSTFA: N-Methyl-N-trimethylsilyltrifluoroacetamide; CV: coefficient of variation; TBS: Tris-buffered saline; MHC: myosin fast type; PCA: principal component analysis; OPLS-DA: orthogonal partial least-squares discriminant analysis; O2PLS: two-way orthogonal partial least-squares     

Determination of styrene and styrene-7,8-oxide in human blood by gas chromatography–mass spectrometry

Methods of isotope-dilution gas chromatography–mass spectrometry (GC–MS) are described for the determination of styrene and styrene-7,8-oxide (SO) in blood. Styrene and SO were directly measured in pentane extracts of blood from 35 reinforced plastics workers exposed to 4.7–97 ppm styrene. Using positive ion chemical ionization, styrene could be detected at levels greater than 2.5 μg/l blood and SO at levels greater than 0.05 μg/l blood. An alternative method for measurement of SO employed reaction with valine followed by derivatization with pentafluorophenyl isothiocyanate and analysis via negative ion chemical ionization GC–MS–MS (SO detection limit=0.025 μg/l blood). The detection limits for SO by these two methods were 10–20-fold lower than gas chromatographic assays reported earlier, based upon either electron impact MS or flame ionization detection. Excellent agreement between the two SO methods was observed for standard calibration curves while moderate to good agreement was observed among selected reinforced plastics workers (n=10). Levels of styrene in blood were found to be proportional to the corresponding air exposures to styrene, in line with other published relationships. Although levels of SO in blood, measured by the direct method, were significantly correlated with air levels of either styrene or SO among the reinforced plastics workers, blood concentrations were much lower than previously reported at a given exposure to styrene. The two assays for SO in blood appear to be unbiased and to have sufficient sensitivity and specificity for applications involving workers exposed to styrene and SO during the manufacture of reinforced plastics.