Stanozolol‐N‐glucuronide metabolites in human urine samples as suitable targets in terms of routine anti‐doping analysis

• Published in: Drug testing and analysis Vol. 13; no. 9; pp. 1668 - 1677
• Main Authors: Göschl, Lorenz, Gmeiner, Günter, Gärtner, Peter, Stadler, Georg, Enev, Valentin, Thevis, Mario, Schänzer, Wilhelm, Guddat, Sven, Forsdahl, Guro
• Format: Journal Article
• Language: English
• Published: England 01.09.2021
• Subjects: Anabolic Agents - analysis; Anabolic Agents - metabolism; Anabolic Agents - urine; anabolic androgenic steroids; Doping in Sports - prevention & control; Female; glucuronide; Glucuronides - analysis; Glucuronides - urine; high‐resolution mass spectrometry; Humans; Limit of Detection; Male; phase‐II metabolite; Solid Phase Extraction - methods; stanozolol; Stanozolol - analysis; Stanozolol - metabolism; Stanozolol - urine; Substance Abuse Detection - methods; Time Factors; phase-II metabolite; anabolic androgenic steroids; high-resolution mass spectrometry; glucuronide; stanozolol
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3109

The exogenous anabolic‐androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17‐epistanozolol‐1′N‐glucuronide and 17‐epistanozolol‐2′N‐glucuronide in stanozolol‐positive human urine samples due to the access to high‐quality reference standards. Examination of excretion study samples shows large detection windows for the phase‐II metabolites stanozolol‐1′N‐glucuronide and 17‐epistanozolol‐1′N‐glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid‐phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given. The two new stanozolol‐N‐glucuronides, 17‐epistanozolol‐1′N‐glucuronide and 17‐epistanozolol‐2′N‐glucuronide, were unambiguously confirmed and characterized. A comprehensive validation and the analysis of excretion study samples demonstrate the big utility of stanozolol‐N‐glucuronides for anti‐doping purposes. A large detection window of up to 28 days was observed for 17‐epistanozolol‐1′N‐glucuronide.