Low-abundance plasma and urinary [(15)N]urea enrichments analyzed by gas chromatography/combustion/isotope ratio mass spectrometry

We report a method for determining plasma und urinary [(15)N]urea enrichments in an abundance range between 0.37 and 0.52 (15)N atom% (0-0.15 atom% excess (APE) (15)N) using a dimethylaminomethylene derivative. Compared with conventional off-line preparation and (15)N analysis of urea, this method requires only small sample volumes (0.5 ml of plasma and 25 microl of urine). The (15)N/(14)N ratio of urea derivatives was measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two peaks were separated; one was identified by gas chromatography/mass spectrometry (GC/MS) as the complete derivatized urea. Calibration of the complete urea derivative was performed by linear regression of enrichment values of known standard mixtures. Replicate standard (6-465 per thousand delta(15)N) derivatizations showed a relative standard deviation ranging from 0.1 to 7%. In order to test the feasibility of the method, human subjects and rats ingested a single meal containing either 200 mg of [(15)N]glycine (95 AP (15)N) or 0.4 mg of [(15)N]-alpha-lysine (95 AP (15)N), respectively. Urine and plasma were collected at hourly intervals over 7 h after the meal intake. After (15)N glycine intake, maximum urinary urea (15)N enrichments were 330 and 430 per thousand delta(15)N (0.12 and 0.16 APE (15)N) measured by GC/C/IRMS, whereas plasma [(15)N]glycine enrichments were 2.5 and 3.3 APE (15)N in the two human subjects 2 h after the meal. (15)N enrichments of total urine and urine samples devoid of ammonia were higher enriched than urinary [(15)N]urea measured by GC/C/IRMS, reflecting the presence of other urinary N-containing substances (e.g. creatinine). In rats plasma urea (15)N enrichments were 15-20 times higher than those in urinary urea (10-20 per thousand delta(15)N). The different [(15)N]urea enrichments observed after ingestion of [(15)N]-labeled glycine and lysine confirm known differences in the metabolism of these amino acids.     

Determination of 13C isotopic enrichment of glutathione and glycine by gas chromatography/combustion/isotope ratio mass spectrometry after formation of the N- or N,S-ethoxycarbonyl methyl ester derivatives

The depletion of glutathione (GSH) reported in very-low-birth-weight infants is implicated in several pathologies, especially if deficiency occurs during foetal development. The cause of this depletion is suggested to be modification of GSH turnover. To probe the role of GSH, a reliable non-invasive method adapted to very-low-birth-weight infants is required. In this paper, we report the preparation of the N,S-ethoxycarbonyl methyl ester derivatives of GSH and glycine and their application to the measurement of (13)C/(12)C ratios at natural abundance in erythrocyte samples by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The technique allowed the determination of (13)C/(12)C ratios at natural abundance with a precision <3% and within-day and between-day variabilities both <4%. The method is able to determine accurately low (13)C-enrichments in GSH (0.00241 to 0.00753 Atom Percent Excess) in erythrocyte extracts following incubation with (13)C-glycine at low specific enrichment (approx. 1.5 atom %). Excellent agreement was obtained between the calculated GSH fractional synthesis rate (FSR) in human adult blood (approx. 300% day(-1)) using the low-enrichment (13)C-glycine/GC/C/IRMS protocol and that using highly enriched (13)C-glycine (99 atom %)/GC/MS with the same derivative. The GC/C/IRMS method was shown to be suitable to measure the in vitro GSH FSR (200-660% day(-1)) in human venous and arterial blood from the umbilical cord. This approach provides a good tool for studying the turnover of GSH in vitro in infants, allowing both the use of minimal amounts of tracer and negligible perturbation of endogenous precursor pools.     

Measurement of the 13C/12C ratio of soil-plant individual sugars by gas chromatography/combustion/isotope-ratio mass spectrometry of silylated derivatives

Carbohydrate is an important pool in the terrestrial carbon cycle. The potential offered by natural and artificial 13C-labelling techniques should therefore be applied to the investigation of the dynamics of individual sugars in soils. For this reason, we evaluated the method of 13C sugar analysis by gas chromatography/combustion/isotope-ratio mass spectrometry (GC/C/IRMS) after hydrolysis and direct trimethylsilylation. Trimethylsilylation involved the addition of several carbon atoms per sugar. These atoms have to be taken into account in the estimation of the carbon isotope ratio. The analysis of standard and natural pentoses and hexoses of known 13C enrichments revealed that the number of analysed added carbon atoms was less than expected from stoichiometry. This was attributed to incomplete derivatization and/or incomplete oxidation of methylsilyl carbon before IRMS. Using a calibration of the number of analysed added carbon atoms, the isotope excess of enriched samples could be determined with a relative error close to 5%. Concerning the determination of natural abundances by GC/C/IRMS, we could measure the delta 13C of standard C3- and C4-derived sugars with an accuracy of +/-1.5 per thousand using the previous calibration. We were able to apply this technique to plant-soil systems labelled by pulse-chase of 13CO2, revealing the nature and dynamics of sugars in the plant rhizosphere.     

Optimisation of derivatisation procedures for the determination of delta13C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.     

A single glucose derivative suitable for gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry

The incorporation of stable isotopes improves the assessment of glucose metabolism and, with some researchers using two tracers, (2)H-glucose assessed by gas chromatography/mass spectrometry (GC/MS) and (13)C-glucose by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), a common derivative for both is advantageous. The most commonly used derivatives for GC/MS are inappropriate for GC/C/IRMS as additional functional groups dilute the label. We therefore considered the suitability of six derivatives for both GC/MS and GC/C/IRMS. Glucose alkylboronates were prepared by adding the appropriate alkylboronic acid (butyl- or methylboronic acid) in pyridine to desiccated glucose. The derivatisation was completed by reacting this with either (a) acetic anhydride or trifluoroacetic anhydride (acetate derivatives) or (b) bis(trimethylsilyl)trifluoroacetamide BSTFA (TMS derivatives). All six derivatives were assessed using GC/MS and (13)C GC/C/IRMS.Neither TMS derivative exhibited any signal intensity in the molecular ion, although a M-15 ion showed good agreement between experimental and theoretical data and, whilst still low in intensity, could be suitable for isotope work. Similarly, none of the acetate derivatives showed any intensity at the molecular ion although three key fragmentation series were identified. The most attractive sequence, initiated by the loss of 1,2 cyclic boronate, resulted in the main fragment ion of interest, m/z 240, corresponding to the fluorinated methylboronate derivate. Minimal carbon and hydrogen atoms are added to this derivative making it an excellent choice for stable isotope work, while proving suitable for analysis by both GC/MS and GC/C/IRMS.     

Determination of cholesterol in fat and muscle of pig by HPLC and capillary gas chromatography with solvent venting injection

Two methods for determination of cholesterol in fat and muscle of pig were evaluated: extraction with chloroform:methanol (2:1, v/v) followed by saponification (method 1) and direct saponification (method 2). HPLC and GC were used to determine cholesterol concentrations. GC analysis was performed with a capillary column of 100 μm using a PTV injector in the modes of cold split and solvent venting. Cholesterol was analyzed without derivatization. Both methods of extraction did not present significant differences (p > 0.01). Sample analysis by GC with solvent venting injection and HPLC showed the lowest % r.s.d. but GC in the cold split mode allowed to obtain a shorter analysis time. Cholesterol concentrations obtained by HPLC were not statistically different from the results obtained by GC with solvent venting injection and were slightly lower than those previously reported. Cholesterol concentrations in fat and muscle tissues respectively ranged from 52 to 77 mg/100 g and from 55 to 65 mg/100 g.     

Analysis of exogenous dehydroepiandrosterone excretion in urine by gas chromatography/combustion/isotope ratio mass spectrometry.

A detailed procedure for the analysis of exogenous dehydroepiandrosterone (DHEA) in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been established for detecting doping with DHEA. The average delta-value (parts per thousand difference of (13)C/(12)C ratio from the isotope ratio standard) of 26 synthetic steroids commercially available was -30.1 +/- 2.6, and was significantly lower than that of human endogenous DHEA in urine of the world class athletes who had participated in the XVIIth Olympic Winter Games (-20.3 +/- 2.1, n = 446). Although large inter-individual variations of urinary DHEA excretion were observed following a single oral administration of 50 mg of DHEA, no significant inter-individual difference was found when the excretion of exogenous DHEA was monitored in terms of delta-values using GC/C/IRMS; the minimum delta-values were observed around 6-8 h after the administration, and the values returned to the base level at over 72 h after the dosing. Thus, the deviations in delta-values of DHEA and its diol metabolites are considered to be conclusive evidence for detecting doping with DHEA. Some successful cases of detection of doping with DHEA from athletes are also reported.     

Comparison of gas chromatography and liquid chromatography mass spectrometric measurements for high accuracy analysis of cholesterol in human serum by isotope dilution mass spectrometry

Cholesterol measurements are of vital clinical importance and reliable reference materials are essential for method validation. Gas chromatography with mass spectrometry (GC/MS) is usually used for the high accuracy analysis of cholesterol by isotope dilution. A certified reference material for cholesterol content in human serum was analysed by isotope dilution utilising GC/MS and liquid chromatography mass spectrometry (LC/MS). The use of LC/MS avoided the need for a derivatisation step. Both LC/MS and GC/MS produced results on the measurement of cholesterol that agreed within 0.5% of the certified value. Moreover, the precision obtained for ratio measurement using both techniques are comparable and lead to relative expanded standard uncertainties (with a coverage factor of 2) varying between 0.2 and 0.5%.     

Gas chromatography flow rates for determining deuterium/hydrogen ratios of natural gas by gas chromatography/high-temperature conversion/isotope ratio mass spectrometry

The effects of the gas chromatography flow rate on the determination of the deuterium/hydrogen (D/H) ratios of natural gas utilising gas chromatography/high-temperature conversion/isotope ratio mass spectrometry (GC/TC/IRMS) have been evaluated. In general, the measured deltaD values of methane, ethane and propane decrease with increase in column flow rate. When the column flow rate is 1 mL/min or higher, which is commonly used for the determination of D/H ratios of natural gas, the organic H in gas compounds may not be completely converted into hydrogen gas. Based on the results of experiments conducted on a GC column with an i.d. of 0.32 mm, a GC flow rate of 0.6 mL/min is proposed for determining the D/H ratios of natural gas by GC/TC/IRMS. Although this value may be dependent on the instrument conditions used in this work, we believe that correct deltaD values of organic compounds with a few carbon atoms are obtained only when relatively low GC flow rates are used for D/H analysis by GC/TC/IRMS. Moreover, as the presence of trace water could significantly affect the determination of D/H ratios, a newly designed inlet liner was used to remove trace water contained in some gas samples.     

Determination of total cholesterol in meat and poultry by gas chromatography: single-laboratory validation

The primary objective of this study was to determine the intralaboratory performance of a cholesterol determination method that combines direct saponification of a 1 g meat or poultry sample and GC quantification of liberated cholesterol without derivatization. Cholesterol was detected at 11.96 min using a GC-flame ionization detector (FID) system. With a 0.005 mg/mL 5alpha-cholestane internal standard and 0.008 to 0.020 mg/mL cholesterol standard series, the FID response was linearly correlated to standard concentrations with a coefficient of determination of 0.995 and a response factor of 0.66. The LOD and LOQ were 1.24 and 4.00 mg/100 g, respectively. Cholesterol could be analyzed within 6 days of preparation with high precision (CV of 0.92 to 2.69%) and accuracy (recovery of 93.24 to 100.56%). This simplified procedure allows for decreased errors and increased productivity, and the method proved to be reliable and able to withstand practical variation in procedural application. The method has been applied routinely with excellent precision to update data on the cholesterol content of beef, pork, and chicken in the U.S. Department of Agriculture National Nutrient Database for Standard Reference.     

Quantification of total cholesterol in human milk by gas chromatography

Human milk provides the key nutrients necessary for infant growth and development. The objective of this study was to develop and validate a method to analyze the cholesterol content in liquid human milk samples along lactation. Direct saponification of the sample using ethanolic potassium hydroxide solution under cold conditions was applied and unsaponifiable matter was separated by centrifugation. Cholesterol was converted into its trimethylsilyl ether and the derivative analyzed by gas chromatography coupled with a flame ionization detector. Cholesterol was quantified using epicoprostanol as internal standard. The method is suitable for the determination of cholesterol in only 0.3 g of human milk. It has been validated showing good repeatability (CV(r) < 15%) and intermediate reproducibility (CV(iR) < 15%). The method was used to analyze human milk obtained from five mothers collected at day 30(±3), 60 (±3) and 120 (±3) after delivery. The cholesterol content in human milk slightly decreased from 13.1 mg/100 g at 1 month to 11.3 mg/100 g 120 days after delivery. The method can also be used to determine desmosterol, an intermediate in cholesterol synthesis.     

Urinary 19-norandrosterone purification by immunoaffinity chromatography: application to gas chromatography/combustion/isotope ratio mass spectrometric analysis

The detection of exogenous 19-norandrosterone (19-NA) in urines was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). 19-NA is, for the first time to our knowledge, isolated from urinary matrix by specific immunoaffinity chromatography (IAC) before analysis. The sample preparation consisted of a preliminary purification of urine by solid-phase extraction after hydrolysis by beta-glucuronidase. Unconjugated 19-NA was thus isolated by IAC and directly analysed by GC/C/IRMS. Optimisation of IAC purification was achieved and the reliability of the technique for anti-doping control is discussed.     

A comprehensive procedure based on gas chromatography–isotope ratio mass spectrometry following high performance liquid chromatography purification for the analysis of underivatized testosterone and its analogues in human urine

The confirmation by GC/C/IRMS of the exogenous origin of pseudo-endogenous steroids from human urine samples requires extracts of adequate purity. A strategy based on HPLC sample purification prior to the GC/C/IRMS analysis of human urinary endogenous androgens (i.e. testosterone, androsterone and/or androstenediols), is presented. A method without any additional derivatization step is proposed, allowing to simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis, without the need of correcting the measured delta values for the contribution of the derivatizing agent. The HPLC extracts were adequately combined to both reduce the number of GC/C/IRMS runs and to have appropriate endogenous reference compounds (ERC; i.e. pregnanediol, 11-keto-etiocholanolone) on each GC–IRMS run. The purity of the extracts was assessed by their parallel analysis by gas chromatography coupled to mass spectrometry, with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision below 0.3 ‰ ¹³C delta units and between assay precision below 0.6 ‰ ¹³C delta units for most of the compounds investigated) fulfilling the World Anti-Doping Agency requirements.     

Validation of pentaacetylaldononitrile derivative for dual 2H gas chromatography/mass spectrometry and 13C gas chromatography/combustion/isotope ratio mass spectrometry analysis of glucose

A reference method to accurately define kinetics in response to the ingestion of glucose in terms of total, exogenous and endogenous glucose is to use stable‐isotope‐labelled compounds such as ²H and ¹³C glucose followed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. The use of the usual pentaacetyl (5Ac) derivative generates difficulties in obtaining accurate and reproducible results due to the two chromatographic peaks for the syn and anti isomers, and to the isotopic effect occurring during acetylation. Therefore, the pentaacetylaldononitrile derivative (Aldo) was validated for both isotopes, and compared with the 5Ac derivative. A correction factor including carbon atom dilution (stoichiometric equation) and the kinetic isotopic effect (KIE) was determined. Analytical validation results for the ²H GC/MS and ¹³C GC/C/IRMS measurements produced acceptable results with both derivatives. When ²H enrichments of plasma samples were ≤1 mol % excess (MPE), the repeatability (RSD_{Aldo Intra assay and Intra day} <0.94%, RSD_{5Ac Intra assay and Intra day} <3.29%), accuracy (Aldo <3.4%, 5Ac <29.0%), and stability of the derivatized samples were significantly better when the Aldo derivatives of the plasma samples were used (p < 0.05). When the glucose kinetics were assessed in nine human subjects, after glucose ingestion, the plasma glucose ²H enrichments were identical with both derivatives, whereas the ¹³C enrichments needed a correction factor to fit together. Due to KIE variation, this correction factor was not constant and had to be calculated for each batch of analyses, to obtain satisfactory results. Mean quantities of exogenous glucose exhibit marked difference (20.9 ± 1.3g (5Ac) vs. 26.7 ± 2.5g (Aldo)) when calculated with stoichiometric correction, but fit perfectly when calculated after application of the correction factor (22.1 ± 1.3g (5Ac) vs. 22.9 ± 1.9g (Aldo)). Finally, the pentaacetylaldononitrile derivative, used here in GC/C/IRMS for the first time, enables measurement of ²H and ¹³C enrichments in plasma glucose with a single sample preparation. Copyright © 2009 John Wiley & Sons, Ltd.     

Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta(13)C analysis of small polyfunctional compounds

Carbohydrates and proteins are among the most abundant naturally occurring biomolecules and so suitable methods for their reliable stable isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) are required. Due to the non-volatile nature of these compounds they require hydrolytic cleavage to their lower molecular weight subunits and derivatisation prior to GC/C/IRMS analysis. The addition of carbon to the molecules and any kinetic isotopic fractionation associated with derivatisation must be accounted for in order to provide meaningful stable isotope values and estimates of propagated errors. To illustrate these points amino acid trifluoroacetate/isopropyl esters and alditol acetates were prepared from authentic amino acids and monosaccharides, respectively. As predicted from the derivatisation reaction mechanisms, a kinetic isotope effect was observed which precludes direct calculation of delta(13)C values of the amino acids and monosaccharides by simple mass balance equations. This study shows that the kinetic isotope effect associated with derivatisation is both reproducible and robust, thereby allowing the use of correction factors. We show how correction factors can be determined and accurately account for the addition of derivative carbon. As a consequence of the addition of a molar excess of carbon and the existence of a kinetic isotope effect during derivatisation, errors associated with determined delta(13)C values must be assessed. We illustrate how such errors can be quantified (for monosaccharides +/-1.3 per thousand and for amino acids between +/-0.8 per thousand and +/-1.4 per thousand). With the magnitude of the errors for a given delta(13)C value of a monosaccharide or amino acid quantified, it is possible to make reliable interpretations of delta(13)C values, thereby validating the determination of delta(13)C values of amino acids as TFA/IP esters and monosaccharides as alditol acetates.     

Measurement of insulin sensitivity indices using 13C-glucose and gas chromatography/combustion/isotope ratio mass spectrometry

Important aspects of glucose metabolism can be quantified by using the minimal model of glucose kinetics to interpret the results of intravenous glucose tolerance tests. The power of this methodology can be greatly increased by the addition of stable isotopically labelled tracer to the glucose bolus dose. This allows the separation of glucose disposal from endogenous glucose production and also increases the precision of the estimates of the physiological parameters measured. Until now the tracer of choice has been deuteriated glucose and the analytical technique has been gas chromatography/mass spectrometry (GC/MS). The consequence of this choice is that nearly 2 g of labelled material are needed and this makes the test expensive. We have investigated the use of (13)C-labelled glucose as the tracer in combination with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) as the analytical technique. This methodology offers superior analytical precision when compared with the conventional method and so the amount of tracer used, and hence the cost, can be reduced considerably. Healthy non-obese male volunteers were recruited for a standard intravenous glucose tolerance test (IVGTT) protocol but 6,6-(2)H-glucose and 1-(13)C-glucose were administered simultaneously. Tracer/tracee ratios were derived from isotope ratio measurements of plasma glucose using both GC/MS and GC/C/IRMS. The results of these determinations indicated that the two tracers behaved identically under the test protocol. The combination of these results with plasma glucose and insulin concentration data allowed determination of the minimal model parameters S*g and S*i. The parameter relating to insulin-assisted glucose disposal, S*i, was found to be the same in the two techniques, but this was not the case for the non-insulin-dependent parameter S*g.     

Determination of denzimol, a new anticonvulsant agent, and its main metabolite in biological material by gas chromatography and high-performance liquid chromatography

Two methods, using gas chromatography (GC) and high-performance liquid chromatography (HPLC), were developed in order to investigate the pharmacokinetics of denzimol hydrochloride, N-[beta-[4-(beta-phenylethyl)phenyl]-beta-hydroxyethyl] imidazole hydrochloride, which is a new anticonvulsant drug, and of its main metabolite, N-[beta-[4-(beta-phenyl-beta(alpha)-hydroxyethyl)phenyl] -beta-hydroxyethyl]-imidazole (referred to as M2), in humans. Both methods involve the use of a homologue of denzimol as an internal standard. The GC method is more sensitive (sensitivity limit 2.5 ng/ml for denzimol and 15 ng/ml for M2) and was utilized for the determination of denzimol and M2 in plasma. The GC method is specific, precise (relative standard deviations are 3.26, 2.12 and 1.72% at 10, 100 and 1000 ng/ml for denzimol and 6.45, 4.17 and 3.38% at 50, 500 and 1000 ng/ml for M2) and accurate (mean recovery +/- S.D. is 102.58 +/- 4.10% for denzimol and 99.72 +/- 7.75% for M2). The HPLC method is very simple and quick to perform. This method has a sensitivity limit of 0.5 micrograms/ml for denzimol and 1 microgram/ml for M2, and allows the determination of both compounds in urine with high selectivity, reproducibility (relative standard deviations are 2.05, 3.50 and 1.02% for denzimol and 2.78, 2.80 and 1.73% for M2, at concentrations of 15, 35 and 70 micrograms/ml) and accuracy (mean recovery +/- S.D. is 103.57 +/- 2.97% for denzimol and 95.91 +/- 1.59% for M2). The common anticonvulsants, when present in plasma, do not interfere with the monitoring of denzimol levels.     

Detection of exogenous intake of natural corticosteroids by gas chromatography/combustion/isotope ratio mass spectrometry: application to misuse in sport

A detailed procedure for the analysis of exogenous hydrocortisone and cortisone in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is proposed. As urinary levels of hydrocortisone are rather low for GC/C/IRMS analysis, the focus is on the main corticosteroid metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Following different solid phase extraction purifications, THE and THF are oxidized to 5beta-androstanetrione before analysis by GC/C/IRMS. Significant differences in delta(13)C per thousand values of synthetic natural corticosteroids and endogenous human corticosteroids have been observed. Therefore, a positive criterion, to detect exogenous administration of synthetic corticosteroids in anti-doping control, is proposed.     

2H/H] Isotope ratio analyses of [2H5]cholesterol using high-temperature conversion elemental analyser isotope-ratio mass spectrometry: determination of cholesterol absorption in normocholesterolemic volunteers

This paper validates the use of high-temperature conversion elemental analyser isotope-ratio mass spectrometry (TC-EA/IRMS) for measuring the [(2)H/H] enrichment of plasma [(2)H(5)]cholesterol. From a molecular point of view, the free cholesterol is initially separated from plasma by thin-layer chromatography (TLC) and then injected onto the TC-EA reactor which converts cholesterol molecules into CO and H(2) gases. The slope of the curve of the experimental mole percent excess (MPE((exp.))) versus MPE((theor.)) was very close to 1, demonstrating that no significant isotopic fractionation was observed during all processing of the samples (i.e., isolation of plasma free cholesterol by TLC and pyrolysis in the TC-EA reactor). Excellent linearity (r(2) = 0.9994, n = 4) of delta ( per thousand ) of [(2)H/H] isotopic measurements versus mole percent (MP) was assessed over the range 0 to 0.1 MP. The precision of the [(2)H/H] measurement, evaluated with two calibration points processed with TLC, was delta(2)H(V-SMOW) = -192.5 +/- 3.4 per thousand and delta(2)H(V-SMOW) = -136.9 +/- 2.9 per thousand. The standard deviations of the within-assay and between-assay repeatabilities of the analytical process, evaluated using the quality control (QC) of plasma samples, were 4.6 and 6.1 per thousand, respectively. Plant sterols are known to reduce cholesterol absorption and therefore were used as a positive control in a clinical study performed with normocholesterolemic volunteers. This present method produces biological results consistent with those already reported in the literature.     

A streamlined approach to the analysis of volatile fatty acids and its application to the measurement of whole-body flux

Volatile fatty acids (VFAs) are produced in the human colon by the bacterial breakdown of carbohydrates that escape digestion and absorption in the small intestine. They have important local and systemic effects on gastrointestinal and nutritional functions. Measuring their production is difficult because of inaccessibility of sampling sites and low circulating concentrations. Stable isotope tracer techniques are a way to measure VFA production but require measurement of isotope dilution in blood and other biological fluids. We have developed a streamlined and robust method to measure the concentration and enrichment of [(2)H]-labelled VFAs by gas chromatography/mass spectrometry (GC/MS) and [(13)C]-labelled VFAs by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Both types of analysis were carried out on the same samples allowing multiple tracer studies to be conducted. Good accuracy and repeatability were found for GC/MS analysis of [(2)H]-labelled VFAs. Careful handling of the background contribution, especially acetate, allowed quantitation of concentration and enrichment within the analysis. GC/C/IRMS analysis of [(13)C] VFAs was also achieved with good accuracy and repeatability. This methodology was used to determine whole-body acetate production in two subjects using multiple tracers ([(2)H(3)]- and [1-(13)C]acetate) and blood and urine sampling. Whole-body acetate flux was similar when measured either with [(2)H(3)]- or [1-(13)C]acetate, and when flux was determined from plasma or urine tracer enrichment. This new method will permit rapid and accurate measurement of VFA flux using [(2)H]- and/or [(13)C]-labelled VFAs as tracers. Measurements of the contribution of colonic VFA production to whole-body VFA flux are now possible.