A novel analytical strategy based on gas chromatography-Orbitrap high-resolution mass spectrometry combined with solid-phase extraction for the monitoring of stanozolol misuse in human urine.

Abstract

Stanozolol, an anabolic androgenic steroid listed in Part S1 of the World Anti-Doping Agency Prohibited List, exhibits a low response and significant matrix interference in urine samples when using liquid-liquid extraction-gas chromatography-mass spectrometry (GC-MS). Enhancing sample preparation techniques remains essential for the effective detection of stanozolol and its metabolites. A method for determining stanozolol and its metabolites (3'-OH-stanozolol, 4β-OH-stanozolol, and 16β-OH-stanozolol) in human urine was developed and validated using GC-Orbitrap high-resolution MS combined with optimized mixed-mode solid-phase extraction (SPE). This method was applied to urine samples from two volunteers who orally administered a single dose of stanozolol, with samples collected over a 30-day period post-administration. The optimized mixed-mode SPE method reduced matrix interference and achieved satisfactory extraction efficiency and high sensitivity, enabling confident identification of all targets in human urine. Validation showed extraction recovery of 74% to 81% and limits of detection from 0.1 to 0.25 ng mL-1. The method was successfully applied to detect urinary excretion profiles of stanozolol and its metabolites in positive volunteer samples. This study presents a novel detection protocol for stanozolol and its metabolites, enhancing the monitoring of stanozolol abuse and contributing to the integrity of sports competitions. This protocol offers a robust tool for anti-doping laboratories, aiding in the accurate detection of stanozolol misuse and supporting the enforcement of fair play in athletics.