Detection and quantification of selected cannabinoids in hair samples by liquid-liquid extraction and LC-MS/MS

Cannabis remains the most used illicit drug worldwide, with rising use linked to medical and recreational decriminalization. This has driven the development of analytical techniques to detect cannabinoids in biological matrices. Hair offers key advantages due to its non-invasive collection, extended detection window, stability, and easy storage. This study presents the development and validation of a robust method for extracting natural cannabinoids from hair, following ANSI/ASB 2019, FDA, and Society of Hair Testing guidelines. Hair samples were washed with methanol and cut into small pieces. Approximately 20 mg of hair was incubated with 1 M NaOH and methanol (30 min, 50 °C). The mixture was acidified with acetic acid and underwent liquid-liquid extraction using hexane/ethyl acetate (90/10, v/v). The organic phase was evaporated and reconstituted in 1-pentanol/methanol (50/50, v/v). Analysis was conducted by LC-MS/MS using multiple reaction monitoring (MRM) and triple-stage mass spectrometry (MS³). The method was selective, specific, precise, and linear, with working ranges of 5-2000 pg/mg for THC, CBN, and CBD; 50-2000 pg/mg for THC-OH; and 0.2-20 pg/mg for THC-COOH. Ion suppression was observed but did not affect sensitivity, with LLOQs and LODs from 0.2 to 50 pg/mg. Over 25 hair samples from university students tested positive for cannabis. THC ranged from 5.9 to 2430.7 pg/mg; one sample had THC-OH above LLOQ (61.4 pg/mg); THC-COOH ranged from 0.3 and 36.4 pg/mg; CBN from 5.7 to 461.0 pg/mg; and CBD from 5.7 to 850.2 pg/mg. Results aligned with self-reported use, confirming the method's forensic suitability.