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

Rationale 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. Methods 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. Results 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. Conclusion 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.