Quantitative confirmation of testosterone and epitestosterone in human urine by LC/Q‐ToF mass spectrometry for doping control

Abstract

Testosterone (T) is the primary male sex hormone. In addition to the development of secondary sex characteristics, testosterone has anabolic effects including increases in muscle size and strength and increases in lean body mass, making it an attractive candidate to enhance athletic performance. In the case of exogenous administration of testosterone, the ratio of testosterone to its isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as indicative of possible exogenous testosterone administration. Typically, a sample that screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited number of qualifying ions that can be used for unambiguous identification. We have developed a confirmation method which uses liquid/liquid extraction, followed by room temperature Girard P derivatization, and analysis using LC/MS-ToF. We observe a number of advantages over conventional GC/MS analysis. Analysis time is decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is also increased allowing more confident identification of the analytes. Finally, while this method has been developed on a QToF instrument, it should be easily transferable to any tandem LC/MS/MS system.

Comment on ‘Isolation and detection of steroids from human urine by molecularly imprinted solid-phase extraction and liquid chromatography’ by Gadzala-Kopciuch et al., J. Chromatogr. B 877 (2009), 1177–1184

“…For testosterone, literature data quote the mean amount in urine after deconjugation to be 30.4 ng/mL [6]. Consistent levels of testosterone glucuronides (the major form in urine) of 32 ng/mL [7], 46.8 ng/mL [4] and 34.6 ng/mL [8] have also been published.…”

Automated and sensitive determination of four anabolic androgenic steroids in urine by online turbulent flow solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry: A novel approach for clinical monitoring and doping control

“…Furthermore, AAS are even misused by athletes to improve their sports performance [6,15]. Although the use of AAS has been banned in sports and governed by the World Anti-Doping Agency since 1976, some of the priority controlled substances such as methenolone were still detected in urine at the [19,20] and LC-MS/MS [21][22][23]. Immunoassay is a feasible method in the clinical laboratory but it is susceptible to matrix interference especially for determination of low concentration.…”

Synthesis of magnetic molecularly imprinted polymers with excellent biocompatibility for the selective separation and inhibition of testosterone in prostate cancer cells

“…As can be seen, the present method had a relatively low LOD among the other reported approaches. 9,10,23,24 Meanwhile, Fe 3 O 4 @TSTO-MIPs used as SPE extractant possessed satisfactory selectivity and excellent stability, compared with the standard SPE C18 sorbents 24 and immunoaffinity capillary column, 10 respectively. In conclusion, the present method is reliable, effective, and sensitive.…”