Implementation and Performance of the Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry-Based Method for the Confirmatory Analysis of Endogenous Anabolic Steroids during the Rio de Janeiro Olympic and Paralympic Games 2016.

Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

High-volume steroid isotopic standards developed as working standards for gas chromatography-combustion-isotope ratio mass spectrometry.

High-precision carbon isotope ratio analysis of urinary steroids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is the official test to detect illicit doping of synthetic versions of endogenous steroids, such as testosterone. Our group created the first steroid isotopic standards (SIS) specifically for World Anti-Doping Agency (WADA) accredited laboratories. The standards contain mixtures of steroids as acetates or free steroids at ~400 µg each per ampoule and have been widely distributed to anti-doping laboratories to facilitate comparability of inter-laboratory results. Here we report on the creation and characterization of 3 new high-volume single component SIS suitable for use as working standards. They contain ~50 times more steroid mass per ampoule than previous SIS. The new SIS, coded CU/PCC 40-1, CU/PCC 41-1, & CU/PCC 42-1, contain ~20 mg of androsterone, androsterone-AC, and 5α-cholestane, with determined isotopic values of -27.09 ± 0.07 mUr, -32.82 ± 0.01 mUr, -25.03 ± 0.01 mUr, respectively. We used our previously developed protocol to calibrate the isotopically uniform steroids against the isotopic standard gases methane and ethane in NIST RM 8559 that are traceable to the international standard Vienna PeeDee Belemnite (VPDB). Two sets of data, acquired 7 months apart, of absolute δ13 CVPDB and ∆Δδ13 CVPDB values from 8 randomly selected ampoules of all 3 SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ13 C) < 0.2 mUr Our results show that protocols for SIS extend to creation of high volume working standards that can also be used as internal standards under appropriate GC conditions.

Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5ß-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.

Two-dimensional gas chromatography with heart-cutting for isotope ratio mass spectrometry analysis of steroids in doping control.

The accuracy and precision of gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) measurements are highly dependent on analyte purity. Reliable analysis of urinary steroids for doping control therefore requires extensive and time-consuming sample preparation (i.e. liquid chromatography fraction collection) prior to GC-C-IRMS analysis. The use of two-dimensional GC (GC-GC) with heart-cutting (Deans Switch) as a possible approach to reduce the sample purification required for IRMS analysis is described herein. The system uses a low thermal mass oven (LTM) incorporated into an existing GC-C-IRMS system. GC-GC allowed the use of a cyanopropyl/phenyl column in the first dimension to optimize the separation of underivatized steroids, while a phenyl-methylpolysiloxane column in the second dimension focuses the selectively cut analytes into narrower peaks for more sensitive and reliable MS analysis. In addition, to confirm analyte identity, eluent from the second GC was split, with 20 % entering a scanning MS, and 80 % flowing to the IRMS. As a proof concept, the developed method was then used to analyze a single spot urine (5 ml) from an individual receiving T therapy (2 × 50 mg sachets of Testogel(®)). The T delta value (-27.8 ‰, [T] = 38 ng/ml) was clearly distinct from 11-ketoetiocholanolone (-22.5 ‰) (used as an endogenous reference compound (ERC)), indicating T as being of exogenous origin. The simultaneous analysis by the scanning MS yielded a full scan mass spectrum of the same chromatographic peak, thus confirming the peak to be T.

Examination of the kinetic isotopic effect to the acetylation derivatization for the gas chromatographic-combustion-isotope ratio mass spectrometric doping control analysis of endogenous steroids.

In gas chromatographic-combustion-isotope ratio mass spectrometry (GC-C-IRMS) doping control analysis, endogenous androgenic anabolic steroids and their metabolites are commonly acetylated using acetic anhydride reagent, thus incorporating exogenous carbon that contributes to the measured isotope ratio. Comparison of the endogenous δ(13)C of free, mono-, and di-acetylated steroids requires application of corrections, typically through straightforward use of the mass balance equation. Variability in kinetic isotope effects (KIE) due to steroid structures could cause fractionation of endogenous steroid carbon, resulting in inaccurate results. To test for possible KIE influence on δ(13)C, acetic anhydride of graded isotope ratio within the natural abundance range was used under normal derivatization conditions to test for linearity. In all cases, plots of measured steroid acetate δ(13)C versus acetic anhydride δ(13)C were linear and slopes were not significantly different. Regression analysis of the Δδ(13)C of enriched acetic anhydrides versus Δδ(13)C of derivatized steroids shows that KIE are similar in all cases. We conclude that δ(13)C calculated from the mass balance equation is independent of the δ(13)C of the acetic anhydride reagent, and that net KIE under normal derivatization conditions do not bias the final reported steroid δ(13)C.

Calibration and data processing in gas chromatography combustion isotope ratio mass spectrometry.

Compound-specific isotope analysis (CSIA) by gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) is a powerful technique for the sourcing of substances, such as determination of the geographic or chemical origin of drugs and food adulteration, and it is especially invaluable as a confirmatory tool for detection of the use of synthetic steroids in competitive sport. We review here principles and practices for data processing and calibration of GCC-IRMS data with consideration to anti-doping analyses, with a focus on carbon isotopic analysis ((13)C/(12)C). After a brief review of peak definition, the isotopologue signal reduction methods of summation, curve-fitting, and linear regression are described and reviewed. Principles for isotopic calibration are considered in the context of the Δ(13)C = δ(13)C(M) - δ(13)C(E) difference measurements required for establishing adverse analytical findings for metabolites (M) relative to endogenous (E) reference compounds. Considerations for the anti-doping analyst are reviewed.

Pharmacokinetics, distribution, and metabolism of [14C]sunitinib in rats, monkeys, and humans.

Sunitinib is an oral multitargeted tyrosine kinase inhibitor approved for the treatment of advanced renal cell carcinoma, imatinib-refractory gastrointestinal stromal tumor, and advanced pancreatic neuroendocrine tumors. The current studies were conducted to characterize the pharmacokinetics, distribution, and metabolism of sunitinib after intravenous and/or oral administrations of [(14)C]sunitinib in rats (5 mg/kg i.v., 15 mg/kg p.o.), monkeys (6 mg/kg p.o.), and humans (50 mg p.o.). After oral administration, plasma concentration of sunitinib and total radioactivity peaked from 3 to 8 h. Plasma terminal elimination half-lives of sunitinib were 8 h in rats, 17 h in monkeys, and 51 h in humans. The majority of radioactivity was excreted to the feces with a smaller fraction of radioactivity excreted to urine in all three species. The bioavailability in female rats was close to 100%, suggesting complete absorption of sunitinib. Whole-body autoradioluminography suggested radioactivity was distributed throughout rat tissues, with the majority of radioactivity cleared within 72 h. Radioactivity was eliminated more slowly from pigmented tissues. Sunitinib was extensively metabolized in all species. Many metabolites were detected both in urine and fecal extracts. The main metabolic pathways were N-de-ethylation and hydroxylation of indolylidene/dimethylpyrrole. N-Oxidation/hydroxylation/desaturation/deamination of N,N'-diethylamine and oxidative defluorination were the minor metabolic pathways. Des-ethyl metabolite M1 was the major circulating metabolite in all three species.

Drug testing data from the 2007 Pan American Games: delta13C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS.

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the (13)C/(12)C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the delta(13)C values ( per thousand) of androsterone, etiocholanolone, 5beta-androstane-3alpha, 17beta-diol (5beta-diol), 5alpha-androstane-3alpha, 17beta-diol (5alpha-diol) and 5beta-pregnane-3alpha, 20alpha-diol (5beta-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The delta values of 5beta-diol and 5alpha-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5beta-pdiol, which exceeded the WADA limit of 3 per thousand. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.

Development of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry for doping control.

Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry.

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.

Metabolism and disposition of 2,2',4,4',5-pentabromodiphenyl ether (BDE99) following a single or repeated administration to rats or mice.

The metabolism and disposition of 14C-labelled 2,2',4,4',5-pentabromodiphenyl ether (BDE99) were studied in F344 rats and B6C3F1 mice. Approximately 85% of a 1 micromol kg-1 oral dose was absorbed by male rats and mice. Within 24 h following oral doses ranging from 0.1 to 1000 micromol kg-1 to rats, 39-47% of the dose was excreted in the faeces (including 16% unabsorbed), up to 2% was excreted in the urine, and 34-38% remained in the tissues, mostly in adipose tissue. Mice excreted more in the urine and less in the faeces than rats. Tissue accumulation was observed following repeated dosing to rats. Two dihydrohydroxy-S-glutathionyl and two S-glutathionyl conjugates of BDE99, 2,4,5-tribromophenol glucuronide, two mono-hydroxylated BDE99 glucuronides, and three mono-hydroxylated tetrabromodiphenyl ether glucuronides were identified in male rat bile. 2,4,5-Tribromophenol and its glucuronide and sulfate conjugates, were identified in male rat urine. 2,4,5-Tribromophenol, one mono-hydroxylated tetrabromodiphenyl ether, and two mono-hydroxylated BDE99 were characterized in male rat faeces. BDE99 undergoes more extensive metabolism than does BDE47. Half of the absorbed oral dose in male rats was excreted in 10 days mostly as metabolites derived from arene oxide intermediates.

Determination of urinary 13C-caffeine metabolites by liquid chromatography-mass spectrometry: the use of metabolic ratios to assess CYP1A2 activity.

A method using liquid chromatography coupled with mass spectrometry with an atmospheric pressure electrospray source was developed for analysis of labelled caffeine and fourteen of its metabolites in urine. Caffeine metabolic ratios were determined after an oral bolus of labelled caffeine in 20 healthy subjects with different characteristic CYP1A2 activity, relative to smoking habit and oral contraceptive intake. The use of labelled caffeine for the calculation of metabolic ratios avoided taking into account the important background of endogenous caffeine metabolites, very difficult to eliminate even after a specific diet. The selectivity and high sensitivity of mass spectrometry detection allowed urine collections for only a 3h period. Comparison between characteristic groups showed that labelled caffeine metabolic ratios were sensitive markers of changes in CYP1A2 activity.

Noninvasive evaluation of liver metabolism by 2H and 13C NMR isotopomer analysis of human urine.

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.

Identification of the urinary metabolites of 4-bromoaniline and 4-bromo-[carbonyl-13C]-acetanilide in rat.

1. The urinary excretion of 4-bromoaniline and its [carbonyl-(13)C]-labelled N-acetanilide, together with their corresponding metabolites, have been investigated in the rat following i.p. administration at 50 mg kg(-1). 2. Metabolite profiling was performed by reversed-phase HPLC with UV detection, whilst identification was performed using a combination of enzymic hydrolysis and directly coupled HPLC-NMR-MS analysis. The urinary metabolite profile was quantitatively and qualitatively similar for both compounds with little of either excreted unchanged. 3. The major metabolite present in urine was 2-amino-5-bromophenylsulphate, but, in addition, a number of metabolites with modification of the N-acetyl moiety were identified (from both the [(13)C]-acetanilide or produced following acetylation of the free bromoaniline). 4. For 4-bromoacetanilide, N-deacetylation was a major route of metabolism, but despite the detection of the acetanilide following the administration of the free aniline, there was no evidence of reacetylation (futile deacetylation). 5. Metabolites resulting from the oxidation of the acetyl group included a novel glucuronide of an N-glycolanilide, an unusual N-oxanilic acid and a novel N-acetyl cysteine conjugate.