Bioanalysis of amphetamines in alternative matrices using a sensitive and validated liquid chromatography-tandem mass spectrometry method and its application to real samples

Amphetamines abuse remains a major concern in both clinical and forensic toxicology. While conventional matrices like blood and urine are commonly used, their detection window is limited. This study aimed to validate a UHPLC-ESI-MS/MS method for amphetamines detection across both conventional and alternative biological specimens and apply it to real patient samples. A validated UHPLC-ESI-MS/MS method was used to quantify amphetamines in whole blood, urine, oral fluid, scalp hair, and fingernail specimens collected from fifty amphetamine abusers. Method validation followed international guidelines, assessing parameters including linearity, sensitivity, precision, and carry-over. Decontamination protocols and wash analyses were performed to exclude external contamination for keratinized matrices. The method demonstrated excellent linearity (r² = 0.995–0.998) and sensitivity (LOD = 2 ng/mL or ng/mg) across all matrices. Oral fluid showed strong correlation with recent drug use and was a reliable non-invasive alternative to blood and urine. Hair yielded the highest amphetamine concentrations due to melanin binding, whereas nail showed lower levels but provided a complementary window for retrospective exposure. Simultaneous quantification of amphetamines in five matrices provides a comprehensive assessment of both recent and chronic drug use. Hair remains the superior matrix for long-term detection, but nail offers valuable supplementary information. Oral fluid emerged as a practical and reliable tool for recent exposure monitoring. This is the first study to report amphetamine concentrations in all five matrices from the same individuals, offering critical insights for toxicological interpretations.