Borislav Starcevic

Improved method of detection of testosterone abuse by gas chromatography/combustion/isotope ratio mass spectrometry analysis of urinary steroids

The current approach to detection of doping with testosterone is based on measuring the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry. The median T/E for healthy males who have not used T is about 1.0. In a single urine, a T/E lower than six leads to a negative report even though it does not exclude T administration. A value greater than six indicates possible T administration or a naturally elevated ratio. It has been shown previously that the carbon isotope ratio of urinary T changes after T administration. In this study a potential confirmation method for T abuse was optimized. Gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS) was used to analyze two T precursors (cholesterol and 5-androsten-3 beta, 17 beta-diol) and two T metabolites (5 alpha- and 5 beta-androstane-3 alpha, 17 beta-diol) in addition to T itself in each of 25 blind urines collected from eight healthy men before, during or after T administration. The carbon isotope ratios of T and the metabolites were lower after T administration. The relationships among the variables were studied using multivariate analysis and beginning with principal components analysis; cluster analysis revealed that the data are composed of two clusters, and classified the samples obtained after T administration in one cluster and the remainder in the other; discriminant analysis correctly identified T users. The measurement of carbon isotope ratios of urinary androgens is comparable to the T/E > 6 test and continues to show promise for resolving cases where doping with T is suspected.

Analysis of over-the-counter dietary supplements.

ObjectiveTo determine if steroids containing over-the-counter (OTC) dietary supplements conform to the labeling requirements of the 1994 Dietary Supplement Health and Education Act (DSHEA). Design12 brands of OTC supplements containing 8 different steroids were randomly selected for purchase in stores that cater to athletes. There are two androstenediones (4- and 5-androstene-3,17-dione), two androstenediols (4- and 5-androstene-3&bgr;, 17&bgr;-diol), and 4 more are 19-nor cogeners (19-nor-4- and 5-androstene-3,17-dione and 19-nor-4- and 5-androstene-3&bgr;, 17&bgr;-diol). Main Outcome Measures12 brands of OTC anabolic–androgenic supplements were analyzed by high-pressure liquid chromatography. ResultsWe found that 11 of 12 brands tested did not meet the labeling requirements set out in the 1994 Dietary Supplement Health and Education Act. One brand contained 10 mg of testosterone, a controlled steroid, another contained 77% more than the label stated, and 11 of 12 contained less than the amount stated on the label. ConclusionsThese mislabeling problems show that the labels of the dietary steroid supplements studied herein cannot be trusted for content and purity information. In addition, many sport organizations prohibit OTC steroids; thus, athletes who use them are at risk for positive urine test results. In this article we provide the details of the analyses, a summary of the steroids by name and structure, and information on the nature of the positive test results. Athletes and their physicians need this information because of the potential medical consequences and positive urine test results.

Liquid chromatography-tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone.

A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d(3)T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 microl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d(3)T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0-20 ng/ml for T and 0-2.0 ng/ml for d(3)T. The LOQs for T and d(3)T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d(3)T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d(3)T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).

Urinary Testosterone (T) To Epitestosterone (E) Ratios by GC/MS. I. Initial Comparison of Uncorrected T/E in Six International Laboratories

Six laboratories in six countries collaborated to investigate the analytical method for estimating the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry in the context of detecting the application of T as a doping agent in sport. The protocol specified many but not all details of reagents and instrument conditions. The design included the distribution and analysis of four urines with different T/E values, three replicates per value, and one standard. The ranges of mean T/E values for the four urines estimated by peak area (PA) were 0.32-0.42, 0.72-0.94, 0.91-1.14 and 3.19-5.48. The analyses of variance for these data and for the peak height (PH) data were significant for the laboratory factor (p < 0.0001). In addition there was a significant interaction between the urine factor and the laboratory factor which indicates the complexity of the analysis. T/E calculated using PA was not significantly different from that using PH. For within-laboratory precision all values for PH and PA were < 8.3%, and for between-laboratory precision all values were < 11.7% except for one (20.1%). The data represent a baseline for future experiments designed to elucidate the sources of within-and between-laboratory variance, and to harmonize estimates of T/E.

Detection of Prohibited Substances by Liquid Chromatography Tandem Mass Spectrometry for Sports Doping Control

Drug testing for sports doping control programs is extensive and includes numerous classes of banned compounds including anabolic androgenic steroids, β2-agonists, hormone antagonists and modulators, diuretics, various peptide hormones, and growth factors. During competition, additional compounds may also be prohibited such as stimulants, narcotics, cannabinoids, glucocorticosteroids, and beta-blockers depending both on the sport and level of competition. Each of these classes of compounds can contain many prohibited substances that must be identified during the testing procedure. Various methods that have been designed to detect a large number of compounds in different drug classes are highly desirable as initial screening tools. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used by anti-doping testing laboratories for this purpose and several rapid methods have been described to simultaneously detect different classes of compounds. Here, we describe a simple urine sample cleanup procedure that can be used to detect numerous anabolic androgenic steroids, β2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS.

Genetic Variations in UDP-Glucuronosyl Transferase 2B17: Implications for Testosterone Excretion Profiling and Doping Control Programs

The use of drugs and ergogenic substances to augment athletic performance, commonly referred to as doping, has evolved along with sporting events. Ancient Olympic athletes consumed mushrooms, plants, and herbs in an attempt to gain a competitive edge. The modern Olympic Games made their debut in 1896, and mixtures of cocaine, ephedrine, and strychnine were used to enhance performance. Anabolic androgenic steroids (substances similar to the hormone testosterone) were used after World War II by Soviet athletes to increase muscle mass and power in weightlifting and bodybuilding events. Anabolic androgenic steroids rapidly spread to athletes in other sporting events and are still a problem in today’s sports world. To deal with the problem of doping in sports, the International Olympic Committee established a Medical Commission. The first list of prohibited substances was created in 1967; drug testing was implemented at Olympic Games the following year. In 1999, an independent international organization, the World Anti-Doping Agency (WADA), was created to combat doping in sports and provide unified standards for doping control. Anabolic androgenic steroids are the most abused class of prohibited substances, with testosterone accounting for many positive cases. Testosterone abuse is problematic because synthetic testosterone is indistinguishable from endogenous testosterone by routine screening methods such as gas chromatography–mass spectrometry. In the 1980s, it was discovered that testosterone use alters the ratio of testosterone glucuronide to epitestosterone glucuronide (T/E ratio)1 in urine. Epitestosterone is a naturally occurring biologically inactive epimer of testosterone that remains relatively constant in urine. A population-based T/E ratio cutoff of 6.0 was initially used to indicate synthetic testosterone use; the T/E ratio cutoff was lowered to 4.0 in 2005. Based on data from several laboratories, the average T/E ratio ranges from 0.9 to 1.6 for healthy male adolescents and men. At the UCLA Olympic Analytical Laboratory, …