BACKGROUND: Hair analysis is the biological matrix of choice for the retrospective assessment of chronic exposure to psychoactive substances over extended time periods. Comprehensive exposure assessment therefore requires the simultaneous determination of biomarkers of licit substance use (e.g., alcohol and tobacco), illicit drugs, novel psychoactive substances (NPS), and prescription medications. However, most existing hair testing protocols are limited to specific drug classes and require multiple extraction procedures and/or relatively large amounts of hair.

OBJECTIVES: This study aimed to develop and validate a sequential multi-platform analytical workflow enabling comprehensive assessment of exposure to licit and illicit psychoactive substances from a single 20-mg hair sample.

METHODS: Following a single liquid extraction, aliquots of the same sample hair extract were used for quantitative determination of ethyl glucuronide (EtG), nicotine (NIC), and cotinine (COT) by LC–MS/MS, and for broad-spectrum screening of drugs of abuse, NPS, and medications by UHPLC–HRMS. The residual hair matrix was then subjected to alkaline digestion and analysed for cannabinoids by GC–MS. The quantitative method was validated over a five-day protocol (linearity, LOD/LOQ, precision, accuracy, carryover, dilution integrity, recovery and matrix effect). The workflow was applied to 40 real hair samples.

RESULTS: Application to real samples showed nicotine detection in 20% of samples, with cotinine never quantifiable; EtG was quantified above the LOQ in 57% of samples (5.2–47.1 pg/mg), reflecting high sensitivity in a range relevant to occasional or moderate alcohol consumption. Drugs of abuse or medications were detected in 45% of samples, and cannabinoids in 15%, with one sample showing a complex polysubstance profile, illustrating the method's ability to capture co-exposure patterns from a single hair aliquot.

CONCLUSIONS: This single-sample, multi-analyte hair workflow provides a sensitive, robust, and resource-efficient tool for comprehensive retrospective exposure assessment to psychoactive substances, suitable for clinical, forensic, and epidemiological monitoring, particularly when hair availability is limited.