A rapid non‐target screening method for determining prohibited substances in human urine using liquid chromatography/high‐resolution tandem mass spectrometry

Target analysis using liquid chromatography-tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non-target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid-phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high-resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%-131%), matrix effects (35%-237%), limites of detection (0.0002-100 ng/mL), and intra- and inter-day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non-target screening method is efficient for conducting anti-doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.