Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC–MS/MS

• Simultaneous enantioselective measurement of tramadol and its three main metabolites.
• A novel method based on reversed phase LC–MS/MS on a chiral stationary phase.
• Quality controls prepared from racemic and pure enantiomeric standards were used.
• Validation showed high sensitivity, good accuracy and excellent precision.
• The method was successfully applied to authentic whole blood samples.

The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (−)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC–MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5 g were fortified with internal standards (tramadol-13C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid–liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20 mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X2, in the range of 0.25–250 ng/g), limit of quantitation (0.125–0.50 ng/g), repeatability (2–6%) and intermediate precision (2–7%), accuracy (83–114%), dilution integrity (98–115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100 mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations.

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