Analysis of N,N-dimethyltryptamine (DMT) and its metabolites using LC–MS/MS for forensic purposes

Ayahuasca contains N,N-dimethyltryptamine (DMT), the primary alkaloid responsible for its psychedelic effects. DMT oxidative deamination yields indole-3-acetic acid (IAA) as the predominant metabolite, while N-oxidation produces N,N-dimethyltryptamine-N-oxide (DMT-NO) as the second most abundant metabolite. An LC–MS/MS method was developed and validated to quantify DMT, IAA, and DMT-NO in human plasma, as well as DMT and DMT-NO in human urine. Protein precipitation using a 75:25 (v/v) acetonitrile:methanol yielded analyte recoveries ≥91% in both plasma and urine. Key parameters including matrix effects, linearity, bias, precision, stability, carryover, and dilution integrity met their respective acceptability criterion outlined by ANSI/ASB 036 recommendations. In plasma, the linear range was 0.5–500 ng/mL (DMT), 0.25–125 ng/mL (DMT-NO), and 240–6000 ng/mL (IAA), while the DMT and DMT-NO range in urine was 2.5–250 ng/mL. Bias was within ±17.5%, and precision was ≤6.4% in both plasma and urine. Analytes were free from exogenous/endogenous interferences, and carryover was negligible. Extracts were also stable in the autosampler compartment (4°C) for 48 hours. A proof-of-concept study was conducted using authentic paired peripheral blood and urine samples. Results showed higher concentrations of DMT and DMT-NO found in urine as compared to plasma, highlighting the rapid metabolism and clearance of DMT and its metabolites. This study proposes the utility of DMT and DMT-NO as direct and distinctive biomarkers for forensic determination of exogenous DMT consumption. While IAA is the predominant metabolite of DMT, IAA should not be relied upon as the sole biomarker due to its substantial endogenous presence in both plasma and urine.