A comprehensive UHPLC–MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti‐doping purposes

Abstract

In the context of steroid analyses, the use of blood could represent a valuable complement to urine. While the blood steroid profile is currently being established to aid unveiling testosterone (T) doping, this matrix is also well suited for detection of exogenous anabolic steroids and steroid esters. In this study, a method to determine a simplified blood steroid profile in combination with the direct detection of exogenous anabolic steroids and steroid esters using just one serum aliquot was developed to obtain a comprehensive analytical workflow. Following the first chromatographic analysis of endogenous and exogenous steroids, samples were derivatised with Girard's reagent T (GT) to improve the ionisation of steroid esters and re‐injected. The quantitative performance for T, androstenedione (A4) and 5α‐dihydrotestosterone (DHT) was evaluated and the method was validated for qualitative analysis of exogenous analogues with estimated limits of detection (LOD) between 50 and 500 pg/ml. To demonstrate the applicability of the method, samples collected from a clinical study with an oral administration of testosterone undecanoate (TU) to 19 male volunteers were then analysed. The individual serum steroid profiles with the endogenous markers T, A4 and DHT were established as well as the concentrations of TU. TU was detected in all 19 volunteers up to 24 h, while DHT represented the most promising biomarker in endogenous steroid profile for the detection of oral TU administration. These results showed that the selected approach to combine exogenous and endogenous steroid analysis has the potential to strengthen T doping detection in the future.

A review of the analytical techniques for the detection of anabolic–androgenic steroids within biological matrices

“…An instance of SLE with DCM extraction solvent recovered 18 free endogenous and exogenous AASs at recovery efficiencies ranging from 42% (methasterone) to 91% (boldenone) from serum when analyzed using an UHPLC-MS/MS (Langer et al, 2022).…”

“…Sixteen estered AASs observed no significant matrix effects and recovery efficiencies of ≥33% from serum when analyzed via UHPLC-MS/MS, with longer chained esters demonstrating reduced recoverability when extracted via ISOLUTE ® SLE+ with DCM. It was suggested heptane be used to improve AAS ester extraction, but to the detriment of free AAS extraction (Langer et al, 2022).…”

Annual banned‐substance review 16th edition—Analytical approaches in human sports drug testing 2022/2023

“…For instance, Langer et al reported on a strategy on how to combine the quantitation of the endogenous steroids T, Adione, and dihydrotestosterone (DHT) with 18 xenobiotic steroids plus 11 intact testosterone esters as well as 5 further AAS esters 45. A volume of 200 μL of serum was prepared by supported liquid extraction (SLE), concentrated, and analyzed by LC-ESI-QqQ MS for the aforementioned serum steroid ABP markers and the non-esterified target analytes.…”

The 40th Manfred Donike workshop on doping analysis

“…These methods facilitated serum steroid profiling 10 and proved applicable also to DBS, 11 where considerable advantages especially regarding steroid ester stabilities were documented. 12 Detecting the manipulation of erythropoiesis with newly approved drugs such as luspatercept has been the subject of in-depth investigations, and two analytical approaches utilizing either an automated capillary immunoassay 13 or gel-based electrophoretic methods 14 were shown to be fit-for-purpose and compatible with serum, DBS, and urine analyses.…”

“…In contrast to urinary steroid (metabolite) analyses, serum and DBS applications employed exclusively liquid chromatographic‐mass spectrometric methods (LC–MS), targeting testosterone, androst‐4‐ene‐3,17‐dione, 5α‐dihydrotestosterone, various testosterone esters, and/or synthetic AAS with and without prior derivatization. These methods facilitated serum steroid profiling 10 and proved applicable also to DBS, 11 where considerable advantages especially regarding steroid ester stabilities were documented 12 …”