New LC–MS Method Developed for Anti-Doping Testing of Endogenous Steroids in Blood

Researchers at the University of Turin in Turin, Italy have developed a single-run ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for the simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes. The study, published in the journal Talanta, presents an alternative to the urinary steroidal module of Athlete Biological Passport (ABP) that is currently used to detect anti-doping rule violations related to the abuse of endogenous anabolic androgenic steroids (EAAS) (1).

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EAAS are naturally occurring hormones in the human body that promote the growth and development of muscle tissue, bone density, and red blood cell production. These hormones play a crucial role in the development and maintenance of male sexual characteristics. When athletes abuse EAAS, they can enhance athletic performance by increasing muscle mass, strength, and endurance. EAAS also aid in the recovery process, allowing athletes to train harder and more frequently, which can ultimately lead to improved athletic performance. However, the use of EAAS is prohibited in sports due to their potential health risks and unfair advantage.

The ABP’s steroidal module monitors the ratios of five androgens in urine, which can be altered by synthetic forms of EAAS, and determines individual limits by a Bayesian adaptive model. However, this approach has limitations due to confounding factors that can alter the steroid profile, and the lack of sensitivity in measuring the T/E ratio in individuals with certain genotypes.

To overcome these limitations, the researchers developed a LC–MS method for the quantification of major circulating steroid hormones, as well as an extended panel of glucuro- and sulpho-conjugated phase II metabolites of androgens, in serum. The method was optimized by comparing the performance of three different C18 stationary phases and selecting mobile phases that could separate all the target steroids, including numerous isomeric/isobaric compounds.

The researchers fine-tuned MS parameters to achieve the sensitivity required to measure the target analytes, which have specific serum concentrations ranging from low pg/mL to μg/mL. They also developed a sample preparation protocol for the extraction of steroid hormones from 200 μL of serum and evaluated its performance in terms of extraction recovery and matrix effect.

The final method was applied to authentic serum samples collected from healthy volunteers, and the results showed serum concentrations of the targeted steroids, including previously undiscovered phase II metabolites. The researchers were also able to efficiently separate dihydrotestosterone sulphate from its isomer, epiandrosterone sulphate, which circulates in blood at high concentrations.

The study’s lead author, Federico Ponzetto, commented, “The promising results of this study are encouraging for the measurement of blood steroid profile markers in serum and plasma samples for Athlete Biological Passport purposes.”

This new LC–MS method could be a valuable tool for detecting testosterone abuse in females and individuals with deletion of UGT2B17 enzyme, and may help improve the detection capabilities of doping practices with endogenous anabolic androgenic steroids.

Reference

(1) Ponzetto, F.; Parasiliti-Caprino, M.; Gesmundo, I.; Marinelli, L.; Nonnato, A.; Nicoli, R.; Kuuranne, T.; Nicoli, R.; Kuuranne, T.; Mengozzi, G.; Ghigo, E.; Settanni, F. Single-run UHPLC–MS/MS method for simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes. Talanta 2023, 255. DOI: https://doi.org/10.1016/j.talanta.2022.124218