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, ß2-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, ß2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS.

Low-fat high-fiber diet decreased serum and urine androgens in men.

To validate our hypothesis that reduction in dietary fat may result in changes in androgen metabolism, 39 middle-aged, white, healthy men (50-60 yr of age) were studied while they were consuming their usual high-fat, low-fiber diet and after 8 wk modulation to an isocaloric low-fat, high-fiber diet. Mean body weight decreased by 1 kg, whereas total caloric intake, energy expenditure, and activity index were not changed. After diet modulation, mean serum testosterone (T) concentration fell (P < 0.0001), accompanied by small but significant decreases in serum free T (P = 0.0045), 5 alpha-dihydrotestosterone (P = 0.0053), and adrenal androgens (androstendione, P = 0.0135; dehydroepiandrosterone sulfate, P = 0.0011). Serum estradiol and SHBG showed smaller decreases. Parallel decreases in urinary excretion of some testicular and adrenal androgens were demonstrated. Metabolic clearance rates of T were not changed, and production rates for T showed a downward trend while on low-fat diet modulation. We conclude that reduction in dietary fat intake (and increase in fiber) results in 12% consistent lowering of circulating androgen levels without changing the clearance.

Another designer steroid: discovery, synthesis, and detection of 'madol' in urine.

Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.

Testosterone metabolic clearance and production rates determined by stable isotope dilution/tandem mass spectrometry in normal men: influence of ethnicity and age.

The metabolic clearance rate (MCRT) and production rate (PRT) of testosterone (T) were measured using constant infusion of trideuterated (d3) T and quantitating serum d3T by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Serum unlabeled T (d0T) was measured by LC-MS-MS, and serum total T (d3T + d0T) was measured by RIA. Mean MCRR (measured by LC-MS-MS) in young white men (1272 +/- 168 liters/d) was not significantly different from young Asian men (1070 +/- 166 liters/d). Mean PRT was also not significantly different between the two ethnic groups (whites, 9.11 +/- 1.11 mg/d; Asians, 7.22 +/- 1.15 mg/d; P = 0.19 using d0T data). Both the mean MCRR (812 +/- 64 liters/d; P < 0.01) and the PRT (3.88 +/- 0.27 mg/d; P < 0.001) were significantly lower in middle-aged white men when compared with their younger counterparts. The mean MCRR and PRR calculated using serum total T or d0T data showed a diurnal variation, with levels at midday significantly higher than those measured in the evening in the young (MCRT, P < 0.01; PRT, P < 0.001) and to a lesser extent in the older men (MCRT, P < 0.05; PRT, P < 0.05 using total T and P < 0.001 using d0T data). We conclude that using LC-MS-MS to detect d3T in serum after constant infusion of stable isotope-labeled T allows the measurements of MCRT and PRT, which can be used to study androgen metabolism repeatedly after physiological or pharmacological interventions.

Tetrahydrogestrinone: discovery, synthesis, and detection in urine.

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.

Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry.

The diagnosis of male hypogonadism requires the demonstration of a low serum testosterone (T) level. We examined serum T levels in pedigreed samples taken from 62 eugonadal and 60 hypogonadal males by four commonly used automated immunoassay instruments (Roche Elecsys, Bayer Centaur, Ortho Vitros ECi and DPC Immulite 2000) and two manual immunoassay methods (DPC-RIA, a coated tube commercial kit, and HUMC-RIA, a research laboratory assay) and compared results with measurements performed by liquid chromatography-tandem mass spectrometry (LC-MSMS). Deming's regression analyses comparing each of the test results with LC-MSMS showed slopes that were between 0.881 and 1.217. The interclass correlation coefficients were between 0.92 and 0.97 for all methods. Compared with the serum T concentrations measured by LC-MSMS, the DPC Immulite results were biased toward lower values (mean difference, -90 +/- 9 ng/dl) whereas the Bayer Centaur data were biased toward higher values (mean difference, +99 +/- 11 ng/dl) over a wide range of serum T levels. At low serum T concentrations (<100 ng/dl or 3.47 nmol/liter), HUMC-RIA overestimated serum T, Ortho Vitros ECi underestimated the serum T concentration, whereas the other two methods (DPC-RIA and Roche Elecsys) showed differences in both directions compared with LC-MSMS. Over 60% of the samples (with T levels within the adult male range) measured by most automated and manual methods were within +/- 20% of those reported by LC-MSMS. These immunoassays are capable of distinguishing eugonadal from hypogonadal males if adult male reference ranges have been established in each individual laboratory. The lack of precision and accuracy, together with bias of the immunoassay methods at low serum T concentrations, suggests that the current methods cannot be used to accurately measure T in females or serum from prepubertal subjects.