A green and non-invasive approach for quantitative analysis of psychoactive substances in exhaled breath

Exhaled breath has emerged as a promising alternative biological matrix in forensic toxicology due to its non-invasive collection and its potential to reflect recent substance use. In this study, a custom-made breath collection device was developed using 3D printing technology, and its analytical performance for detecting psychoactive substances was evaluated. A quantitative analytical method for target analytes in exhaled breath was validated in accordance with ANSI/ASB guidelines and subsequently applied to samples collected from individuals undergoing substance use treatment. The method demonstrated excellent linearity (r² > 0.99) within the range of 0.5-500 pg/filter, with LOQs ranging from 0.4 to 48.1 pg/filter for 24 analytes. Accuracy and precision were within ±20%, and recovery values exceeded 80% for all compounds. Stability studies indicated that analytes were stable for up to seven days at -20°C, while ambient conditions caused partial degradation. The environmental sustainability was assessed using the AGREE metric, resulting in a score of 0.55. Application to real samples (n = 20), revealed that 95% of participants were positive for at least one psychoactive substance. Methamphetamine (METH) was the most frequently detected compound (90%), followed by amphetamine (AMP), pregabalin (PRG), and gamma-aminobutyric acid (GABA). Notably, several substances were detected in exhaled breath despite negative blood or urine results, underscoring the high sensitivity of this matrix for identifying recent drug intake. Overall, this study demonstrates that exhaled breath is a practical, sensitive, and environmentally conscious matrix suitable for forensic toxicology, with potential applications in roadside screening, workplace drug testing, and rehabilitation monitoring.