Endogenous Reference Compounds Research Articles

Investigations on changes in 13C/12C ratios of endogenous urinary steroids after pregnenolone administration

For the detection of possible misuse of naturally occurring anabolic androgenic steroids like testosterone (T), anti-doping laboratories use a combination of two techniques. One is molecular steroid profiling to evaluate urinary steroid concentrations and normal diagnostic ratios. The other is isotope ratio mass spectrometry (IRMS), in which the ¹³C/¹²C ratios of target analytes like T are compared to the ¹³C/¹²C ratios of endogenous reference compounds (ERCs). The ¹³C/¹²C of the most commonly used ERC, pregnanediol (5β-pregnane-3α,20α-diol, PD), can be influenced by administration of pregnenolone (3β-hydroxy-pregn-5-en-20-one, PREG). Therefore PREG administration bears the potential to circumvent IRMS testing for doping control samples. In order to investigate the influence of PREG on PD and on other urinary excreted steroids, administration studies with oral and transdermal application of PREG were carried out. The influence of PREG administration on concentrations and ¹³C/¹²C ratios of all investigated target analytes was negligible. Only PD and 5β-pregnan-3α-ol-20-one (3aP) showed significant depletion in both their glucuronidated and sulfated steroids. The results suggest that appropriate alternative ERCs are: 11β-hydroxy-androsterone/etiocholanolone, 5β-pregnane-3α,17,20α-triol, pregn-5-ene-3β,17,20α-triol and cholesterol. Due to its properties to disguise the misuse of anabolic steroids by influencing the ¹³C/¹²C ratio of PD, PREG should be considered to be added to the World Anti-Doping Agency (WADA) list of prohibited substances as a masking agent.

Drug testing data from the 2007 Pan American Games: δ13C 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 13C/ 12C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the δ 13C values (‰) of androsterone, etiocholanolone, 5β-androstane-3α, 17β-diol (5β-diol), 5α-androstane-3α, 17β-diol (5α-diol) and 5β-pregnane-3α, 20α-diol (5β-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 δ values of 5β-diol and 5α-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5β-pdiol, which exceeded the WADA limit of 3‰. 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.

Urine ratio of tetrahydrocortisol to tetrahydrodeoxycortisol to screen for the systemic administration of cortisone and hydrocortisone

We use gas chromatography–mass spectrometry (GC–MS) to determine the urine peak area ratio of tetrahydrocortisol (THF) to tetrahydrodeoxycortisol (THS) in spot urine samples of eight male volunteers after a single intramuscular injection of 100 mg hydrocortisone (HC) and after a single oral administration of 10 mg HC at six different post-treatment times over 24 h with 1 week between the two treatments. Control spot urine samples were also obtained from a group of 100 volunteers of each sex for GC–MS analysis. In addition, one female volunteer was collected for GC–MS and isotope ratio mass spectrometry (IRMS) analysis after a single oral administration of 40 mg HC and 40 mg cortisone (C) at 15 and 10 different post-treatment times over 30 h, respectively. IRMS analysis focused on the acetylated derivative of 11-keto-etiocholanolone (11KE) and 11β-hydroxy-etiocholanolone (11OHE) as target metabolites, and on androsterone (A) as an endogenous reference compound (ERC) for calculating the corresponding δ 13C (‰) depletion values. There was a small but significant sex-related difference for the THF/THS ratio in the control group with mean THF/THS ratio values of 10 and 13.5 for women and men, respectively. A cut-off value of 28 (mean + 2 S.D.) for the THF/THS ratio offered a narrow detection window with 39% of suspicious samples after HC-oral treatment, and a wide detection window with 94% of suspicious samples after HC-intramuscular administration in men. For the woman the same cut-off value offered a wide detection window after HC and C administration with 100% and 90% of suspicious samples, respectively. On the basis of a cut-off value of 3‰ for the δ 13C (‰) depletion, the exogenous origin was widely evidenced for at least one target compound in 93% and 80% of the HC and C samples, respectively. We conclude by discussing the predictive ability of the urine THF/THS ratio and its usefulness in pointing out suspicious samples resulting from the systemic administration of HC and C.

13C/12C Ratios of endogenous urinary steroids investigated for doping control purposes

In order to detect the misuse of endogenous anabolic steroids such as testosterone by athletes a total of n = 1734 suspicious urine samples were investigated by gas chromatography/combustion/isotope ratio mass spectrometry throughout the years 2005, 2006 and 2007. The (13)C/(12)C ratio of a target substance (androsterone, a testosterone metabolite) was compared to the (13)C/(12)C ratio of an endogenous reference compound (11beta-hydroxyandrosterone).N = 1340 samples were investigated due to elevated testosterone/epitestosterone ratios, with n = 87 (6.5%) exceptional findings regarding their isotopic ratios. An additional n = 164 samples were investigated because of elevated dehydroepiandrosterone concentrations, with n = 2 (1.2%) exceptional findings. The remainder were subjected to isotope ratio analysis because of elevated androsterone levels or because this was requested by sports federations.Significant differences between female and male samples were found for the (13)C/(12)C ratios of androsterone and 11beta-hydroxyandrosterone but not for samples taken in or out of competition.A further n = 645 samples originating from other World Anti-Doping Agency accredited laboratories, mainly throughout Europe as well as South America, South Africa and Southeast Asia, were investigated. The (13)C/(12)C ratios of the urinary steroids differ significantly for each geographical region, reflecting the dietary status of the individuals.The system stability over time has been tested by repeated injections of a standard solution and repeated processing of frozen stored blank urine. Despite a drift over time in absolute (13)C/(12)C ratios, no significant change in the difference of (13)C/(12)C (11beta-hydroxyandrosterone) minus (13)C/(12)C (androsterone) could be observed.

Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgens in humans

Carbon isotope ratio of androgens in urine specimens is routinely determined to exclude an abuse of testosterone or testosterone prohormones by athletes. Increasing application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in the last years for target and systematic investigations on samples has resulted in the demand for rapid sample throughput as well as high selectivity in the extraction process particularly in the case of conspicuous samples. For that purpose, we present herein the complimentary use of an SPE-based assay and an HPLC fractionation method as a two-stage strategy for the isolation of testosterone metabolites and endogenous reference compounds prior to GC/C/IRMS analyses. Assays validation demonstrated acceptable performance in terms of intermediate precision (range: 0.1–0.4‰) and Bland–Altman analyses revealed no significant bias (0.2 ‰). For further validation of this two-stage analyses strategy, all the specimens ( n = 124) collected during a major sport event were processed.

Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Metabolites of hydrocortisone (HC) and cortisone (C), namely tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-THF, allo-THE for the main metabolites and 11-hydroxyandrosterone, 11-hydoxyetiocholanolone, 11-ketoandrosterone, and 11-ketoetiocholanolone for the minor metabolites, as well as the two main metabolites of testosterone, androsterone and etiocholanolone, were separated from each other using HPLC fractionation of urine extracts. An isotopic ratio mass spectrometry (IRMS) analysis determined the absolute δ 13C values of 5α-androstanetrione (5α-AT) and 5β-androstanetrione (5β-AT) as the oxidation products (ox-products) of the HC and C metabolites and as target compounds (TCs). We also performed IRMS analysis of 5α-androstanedione (5α-AD) and 5β-androstanedione (5β-AD) as the ox-products of etiocholanolone and androsterone and as endogenous reference compounds (ERCs). Urine samples came from two male volunteers treated with a single 10-mg oral dose and a single 100-mg intramuscular dose of HC hemisuccinate, a male volunteer treated with a single 25-mg oral dose of C acetate, and a control group of 30 drug-free athletes. The mean −3SD of δ 13C depletion values from the controls were −1.46, −1.98, −1.78 and −2.42 for 5β-AT–5β-AD, 5α-AT–5β-AD, 5β-AT–5α-AD and 5α-AT–5α-AD, respectively, indicating −3‰ as a safe cut-off value for differentiating the pharmaceutical from the natural form. In the main metabolite fraction, δ 13C depletion values peaked around −5‰ and −9‰ after oral and intramuscular administration of HC, respectively, and around −6‰ after oral administration of C. In comparison, less impressive results were obtained when IRMS analysis focused on the ox-products of the minor metabolites.

Formula omitted]-Values of endogenous urinary steroids

By means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) urinary steroids obtained from a reference population of 56 subjects were analyzed for their C 13 / C 12 -ratios. The analytes encompassed androsterone (A), etiocholanolone (E), 11 β -hydroxyetiocholanolone (OHE), 11 β -hydroxyandrosterone (OHA), and 5 β -pregnane- 3 α , 20 α -diol (PD). A and E represent androgen metabolites (AM). PD, OHE, and OHA have sources independent from androgen metabolism. The δ C 13 -values of the latter compounds may be compared to those of AM in order to detect doping with synthetic androgens and thus may serve as endogenous reference compounds (ERC). In order to allow for classification of conspicuous samples, reference ranges and limits were calculated for δ C 13 -values of selected steroids and differences hereof ( Δ C 13 -values). When A is compared to ERCs, Δ C 13 -values larger than 3 ‰ are very unlikely. A set of additional parameters was surveyed by a questionnaire. Several factors turned out to exert significant influence on the δ C 13 -values of urin ary steroids. These encompass the identity of the steroid itself, sex, oral contraception, travels, and physical activity.

Development and application of stable carbon isotope analysis to the detection of cortisol administration in cattle

The use of anabolic agents in food-producing animals has been prohibited within the EU since 1988. The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as no definitive method and nonambiguous analytical criteria are available. We have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of cortisol to cattle. The method consisted of an efficient combination between OASIS HLB solid-phase extraction (SPE), oxidation, SiOH SPE and semi-preparative high-performance liquid chromatography (HPLC) for glucocorticoid purification. By comparison of the (13)C/(12)C isotopic ratio of the oxidised product of cortisol, i.e. 5 beta-androstane-3,11,17-trione (5 beta AAT), with an endogenous reference compound (ERC), dehydroepiandrosterone (DHEA), the differentiation of cortisol metabolite origin, either endogenous or exogenous, has been achieved. After treatment of an animal, the delta(13)C(VPDB) values of 5 beta AAT reached -30 to -32 per thousand, whereas the delta(13)C(VPDB) values of DHEA remained at -25 per thousand. A significant difference in the delta(13)C(VPDB) values between DHEA and 5 beta AAT was measurable over a period of 3 days after a single administration of cortisol to the animal.

Deuterium/hydrogen ratio analysis of thymol, carvacrol, γ-terpinene and p-cymene in thyme, savory and oregano essential oils by gas chromatography–pyrolysis–isotope ratio mass spectrometry

Isotope ratio mass spectrometry online coupled with capillary gas chromatography (GC–Py–IRMS) on column INNOWAX is used in the origin specific analysis and the authenticity control of the phenolic essential oils (EOs). Isotopic data δ 2H V-SMOW of thymol and carvacrol in natural essential oils were evidently more depleted than synthetic products (from −49 to 7‰ for thymol and −61‰ for carvacrol). δ 2H V-SMOW values of p-cymene, γ-terpinene and thymol in authentic thyme oils ( Thymus vulgaris L. and Thymus zygis L.) were found from −300 to −270‰, from −285 to −248‰ and from −259 to −234‰, respectively. δ 2H V-SMOW values of carvacrol and p-cymene in authentic oregano oils ( Origanum heracleoticum L., Coridothymus capitatus L. and Origanum compactum L.) varied from −223 to −193‰ and from −284 to −259‰, respectively. For authentic Satureja montana subsp. montana essential oils, the mean δ 2H V-SMOW value for aromatic compounds were found to be the following: γ-terpinene −273‰ (SD = 4.6‰) and p-cymene −283‰ (SD = 3.0‰), thymol −245‰ (SD = 1.8‰) and carvacrol −226‰ (SD = 1.7‰). In addition, p-cymene was previously found as a precursor of the biosynthesis of thymol and carvacrol in thyme oil, thus, we considered p-cymene as an endogenous reference compound (ERC) for D/H ratio analysis. The isotopic fractionation factors α thymol/ p-cymene = 1.05 and α carvacrol/ p-cymene = 1.08 were obtained and also used to control the authenticity of the phenolic EOs.

Application of Stable Carbon Isotope Analysis to the Detection of Testosterone Administration to Cattle

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.

Use of isotope ratio mass spectrometry to detect doping with oral testosterone undecanoate: Inter-individual variability of 13C/ 12C ratio

The metabolic effect of multiple oral testosterone undecanoate (TU) doses over 4 weeks was assessed in seven voluntary men. The protocol was designed to detect accumulation of the substance by choosing the appropriate spot urines collections time and to study the urinary clearance of the substance after weeks of treatment. Urines were analysed by a new GC/C/isotope ratio mass spectrometry (IRMS) method to establish the δ 13C-values of testosterone metabolites (androsterone and etiocholanolone) together with an endogenous reference compound (16(5α)-androsten-3α-ol). The significant differences in inter-individual metabolism following TU intake was illustrated by large variations in δ 13C-values of both T metabolites (maximum Δ δ 13C-values = 5.5‰), as well as by very stable longitudinal T/E profiles and carbon isotopic ratios in the first hours following administration. According to T/E ratios and δ 13C-values, the washout period after 80 mg TU intake was less than 48 h for all subjects and no accumulation phenomenon was observed upon chronic oral 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.

Urea as an endogenous surrogate in human microdialysis to determine relative recovery of drugs: Analytics and applications

During in vivo microdialysis studies time-consuming and laborious bedside calibration methods, e.g. retrodialysis, have to be performed. To reduce the burden on the patient it would be desirable to establish a reliable, time-saving calibration technique to obtain the in vivo recovery describing the relative drug transfer across the membrane of the microdialysis probe. The performed study aimed to evaluate and validate the use of urea as an endogenous reference compound to determine relative in vivo recovery of anti-infectives, e.g. linezolid used herein as model drug. In order to meet the special requirements imposed by microdialysis to measure urea concentrations in very small sample volumes (∼10 μL) a photometric assay in 96-well microtiter plates was established based on the method of Berthelot. Subsequently, concentration- and flow rate-dependence were evaluated in vitro to determine the relative recovery (RR) of urea. Finally, urea and linezolid concentrations in human microdialysis samples were measured. The developed assay was validated according to international guidelines and met all requirements. Relative in vitro recovery was found to be independent from concentration and dependent on flow rate. Subsequently, relative in vivo recovery of urea was correlated with relative in vivo recovery of linezolid obtained by the traditional retrodialysis method. In healthy volunteers, the mean ratio of the relative recovery of linezolid to the relative recovery of urea was 0.6 for the subcutaneous (s.c.: CV 33.4%, n = 48) and 0.7 for the intramuscular probe (i.m.: CV 18.8%, n = 40), respectively. In critically ill patients this ratio was 0.7 for both tissues (s.c.: CV 32.8%, n = 18; i.m.: CV 22.1%, n = 17). Successful calibration of the urea reference technique without the need to use in vitro data will further promote the application of microdialysis in clinical studies especially in critically ill patients, as it reduces the imposed burden to a minimum.

Application of stable carbon isotope analysis to the detection of 17β-estradiol administration to cattle

The use of anabolic agents in food producing animals is prohibited within the EU since 1988 (96/22/EC directive). The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as far as no definitive method and non-ambiguous analytical criteria are available. The ability of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of 17beta-estradiol to bovine has been investigated in this paper. By comparison of 13C/12C isotopic ratio of main urinary estradiol metabolite, i.e. 17alpha-estradiol, with two endogenous reference compounds (ERCs), i.e. dehydroepiandrosterone (DHEA) and 5-androstene-3beta,17alpha-diol, the differentiation of estradiol metabolite origin, either endogenous or exogenous, has been proved to be achievable. After treatment, the delta(13)C(VPDB)-values of 17alpha-estradiol reached -27 per thousand to -29 per thousand, whereas delta13CVPDB-values of DHEA remained between -13 per thousand and -20 per thousand depending on the diet, maize and grass, respectively. A significant difference of delta13CVPDB between ERCs and 17alpha-estradiol was measurable over a period of 2 weeks after estradiol ester administration to the animal.

Urinary analysis of 16(5α)-androsten-3α-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5α)-androsten-3α-ol together with 5β-pregnane-3α,20α-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5αA), 5β-androstane-3α,17β-diol (5βA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6‰, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual δ13C-values for 16(5α)-androsten-3α-ol and 5β-pregnane-3α,20α-diol are less than 0.9‰. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50 mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5α)-androsten-3α-ol and 5β-pregnane-3α,20α-diol, whereas the isotopic ratio values of 5β-pregnane-3α,20α-diol vary by more 5‰ upon ingestion of pregnenolone. We have observed δ13C-value changes lower than 1‰ for 16(5α)-androsten-3α-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5β-pregnane-3α,20α-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.

Urinary analysis of 16(5α)-androsten-3α-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5α)-androsten-3α-ol together with 5β-pregnane-3α,20α-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5αA), 5β-androstane-3α,17β-diol (5βA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6‰, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual δ 13C-values for 16(5α)-androsten-3α-ol and 5β-pregnane-3α,20α-diol are less than 0.9‰. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50 mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5α)-androsten-3α-ol and 5β-pregnane-3α,20α-diol, whereas the isotopic ratio values of 5β-pregnane-3α,20α-diol vary by more 5‰ upon ingestion of pregnenolone. We have observed δ 13C-value changes lower than 1‰ for 16(5α)-androsten-3α-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5β-pregnane-3α,20α-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.

Validation of urea as an endogenous reference compound for the in vivo calibration of microdialysis probes

Microdialysis is a widely used experimental technique, which offers the opportunity to measure drug and metabolite concentrations in the interstitial space fluid in animals and humans. However, microdialysis probes need to be calibrated in vivo to obtain a recovery factor, which describes the relative drug transfer across the membrane. Recently, novel time-saving calibration techniques, based on the use of reference compounds, have been developed. In particular, the use of endogenous urea levels has been advocated. In the present study we set out to validate the use of the urea reference technique for microdialysis probe calibration in healthy volunteers, employing glucose and paracetamol as model analytes. Urea calibration was compared with the results of two standard calibration techniques, i.e. the no net flux technique and the retrodialysis technique. For glucose, recovery values, calculated by the urea reference technique differed significantly from those values, which were assessed by the no net flux technique (p < 0.05), whereas for paracetamol recovery values did not differ significantly, albeit a high intramethod variability was observed (CV=66%). As a conclusion, we could not confirm the hypothesis that recovery values calculated by the urea reference technique provide equivalent results compared with standard calibration techniques.

The use of stable carbon isotope analysis to detect the abuse of testosterone in cattle.

The use of stable carbon isotope analysis to detect the administration of anabolic steroids to cattle was investigated. Samples were extracted by solid-phase extraction on C18 cartridges. Stable isotope ratios (13C:12C) were measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) of the underivatised extracts. A programmed temperature vaporiser (PTV) injector was installed in the GC-IRMS system, which conferred a number of advantages. First, it allowed large volumes of sample to be injected whilst the injector liner was cool. The solvent was subsequently vented to the atmosphere prior to transfer of the sample to the GC column. Thus a significantly greater amount of sample could be presented for analysis, thereby increasing the sensitivity. Second, by this means virtually all the solvent could be removed prior to analysis. This eliminates solvent peak tailing, which can be a major problem in GC-IRMS. Finally, the PTV allowed the use of higher initial GC oven temperatures, which in turn facilitated the analysis of underivatised steroids by reducing the GC run time and improving the chromatographic peak shape. The carbon isotope composition of 5 beta-androstane-3 alpha,17 alpha-diol, the major metabolite of testosterone found in bovine bile, was measured in bile samples from untreated cattle and from cattle injected intramuscularly with testosterone or a mixture of testosterone esters. There was considerable inter-animal variation in the values obtained and there was no significant difference between samples from treated and untreated animals. However, when the isotopic composition of the metabolite was normalised with respect to that of an endogenous reference compound (cholesterol) in the same sample, the difference between treated and untreated animals become statistically significant.