Tramadol and O-desmethyltramadol quantification in hair samples by GC–MS

Purpose: Abuse of tramadol and related fatal intoxications has been increasing. Hair analysis is the ideal matrix to evidence cumulative long-term exposure. Besides tramadol, it is important to quantify its main metabolite, O-desmethyltramadol (M1), since when present in hair represents internal exposure and it is much more active than tramadol itself. Until now, there is no validated technique to simultaneously quantify tramadol and M1 in hair. Methods: A gas-chromatography/mass spectrometry (GC–MS) method with solid-phase-extraction (SPE) for simultaneous determination of tramadol andM1 in human hair samples was developed and validated. Hair samples (60 mg), were subjected to decontamination with dichloromethane, water and acetone, and then extracted with methanol in ultrasonic bath. Samples were then cleaned-up with solid-phase extraction using mixed-mode MCX cartridges. Derivatization was performed using ethyl acetate and bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1%TMCS) and then analytes were analyzed by GC–MS. Results of the study: Validation of the method was performed working with spiked hair samples. The method proved to be selective since there were no matrix interferences. The regression analysis for tramadol and M1 was linear in the range of 0.1–20 ng/mg with detection and quantification limits of 0.0003 and 0.0013 ng/mg for tramadol and 0.0001 and 0.0006 ng/mg for M1 respectively. The present method was further applied to six clinical cases of longterm tramadol administration.