Human seminal fluid as a novel Forensic matrix for flualprazolam and α-hydroxy flualprazolam: development and validation of a sensitive SPE–UHPLC–MS/MS method and application to real samples

Flualprazolam is a novel designer benzodiazepine frequently reported in drug-facilitated sexual assaults, yet validated methods for its quantification in human seminal fluid remain limited. A sensitive method was therefore developed and validated for quantification of flualprazolam and α-hydroxy flualprazolam in human seminal fluid. An ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed using solid-phase extraction, a phenyl-hexyl reversed-phase column, positive electrospray ionization and multiple reaction monitoring. Results: The method showed linearity over 0.5–200 ng/mL (R2 ≥ 0.99). Limits of detection (LOD) and lower limits of quantification (LLOQ) were 0.250 and 0.500 ng/mL, respectively. Accuracy and precision were within ±20%, with extraction recovery >81%. The method was applied to 100 seminal-fluid samples. The validated method enables sensitive quantification of flualprazolam and α-hydroxy flualprazolam in human seminal fluid for forensic toxicological applications.