Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

Abstract

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.

LC‐MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

“…In the case of metabolite 18-nor-17β-hydroxymethyl-17α-methyl-5β-androsta-1,4,13-triene-3-one 17-glucuronide (MII), GC-MS/MS technology allowed the detection of the aglycon up to 18 and 36 days, in the C and D study, respectively; whereas the direct detection by LC-MS/MS is slightly shorter. [25] So, although the sensitivity for some of the compounds is limited, new improvements on instrumentation and in the sample preparation process would help in the near future. For metabolite MII, which is the metabolite detected for longer time, the sensitivity of the GC-MS/MS method for the aglycon is better than the sensitivity of the LC-MS/MS method for the glucuronoconjugated metabolite as has been described for other AAS metabolites.…”

“…[25] After addition of 20 μL of the IS solution (d3-T-G at 1 μg/mL and d4-And-G at 5 μg/mL), urine sample (2 mL) was passed through a C18 cartridge and treated as described previously. [25] After addition of 20 μL of the IS solution (d3-T-G at 1 μg/mL and d4-And-G at 5 μg/mL), urine sample (2 mL) was passed through a C18 cartridge and treated as described previously.…”

New Potential Biomarker for Methasterone Misuse in Human Urine by Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry

“…Based on our experiences, two biomarkers, parent drug and its C3-reduced metabolite, were employed for monitoring methasterone misuse and an enzymatic hydrolysis step was involved prior to GC-MS analysis in doping control field, which was time-consuming and indirect [15]. Up to now, the application of LC-MS/MS has been increasingly used to identify phase II metabolites [13,16,17]. The objective of this paper is to identify and characterize intact methasterone metabolites (mainly sulfate and glucuronide conjugates) by liquid chromatography coupled with quadrupole time-of-flight mass spectrometric technique (LC-QTOF-MS) and gas chromatography mass spectrometry and find biomarkers to improve our routine analysis in doping control.…”

Androgens

“…Among the adverse analytical findings reported in 2013 from WADA's anti-doping laboratories, 36 different synthetic androgens were detected [46] . Ongoing research is identifying longer-term metabolites of synthetic androgens to further widen the window of detection [74][75][76] . The high risk of detection, as shown by the low incidence of positives for synthetic androgens, provides a strong deterrent to their use for all but illadvised athletes.…”