Purpose: Venlafaxine (VEN) is a widely clinically used antidepressant, databases reveal a significant increase in the non-medical use of VEN. This study aimed to develop and validate a method to detect VEN and its major metabolite O-desmethylvenlafaxine (ODV) in human hair using a gas chromatography-tandem mass spectrometry (GC-MS/MS) and apply it to real cases.

Methods: Drug-free hair samples were decontaminated, cryogenically ground, and extracted with methanol. The supernatant was dried under nitrogen, reconstituted, and analyzed by GC–MS/MS. Characteristic fragment ion pairs of VEN and ODV were identified in multiple reaction monitoring (MRM) mode. After validation of selectivity, linearity, limits of detection (LOD), low limits of quantification (LLOQ), recovery, and matrix effects, the method was applied to authentic hair samples.

Result: The LOD for VEN and ODV in hair were 0.5 ng/mg, and the LLOQ for both analytes were 1.0 ng/mg. Calibration curves exhibited good linearity. Matrix effects ranged from 90.1% to 110%, extraction recoveries exceeded 93.0%, and analyte stability under the tested conditions was greater than 86.0%. Accuracy and precision met the predefined acceptance criteria for the assay. Application of the method to hair samples that were compatible with non-medical use of VEN successfully detected both VEN and ODV.

Conclusion: The developed GC–MS/MS method is simple, selective, and reliable for the qualitative and quantitative determination of VEN and ODV in human hair. Cryogenic grinding provided the highest extraction recovery. In addition, both were found to accumulate at higher levels in black hair compared to white hair.