Purpose: Tramadol is a centrally acting analgesic indicated for the treatment of mild-severe pain, both acute and chronic. A simple and sensitive high performance liquid chromatography method using tandem mass spectrometry detection (LC-MS/MS) for determination of tramadol and its metabolites, O-desmethyltramadol (ODT) and N-desmethyltramadol (NDT) in human plasma was developed and validated.
Methods: After extraction with methyl t-butyl ether (MTBE), the chromatographic separation of tramadol and its metabolites was carried out using a reversed-phase Luna C18 column (50 mm x 2.0 mm, 5 μm) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5)-methanol (30:70, v/v) and quantified by MS/MS detection in ESI positive ion mode. The mass transition pairs of m/z 264.3→58.1 for tramadol, m/z 250.3→58.2 for ODT, m/z 250.3→44.1 for NDT and m/z 268.2 → 116.1 for internal standard were used, respectively. The flow rate of mobile phase was 0.2 ml/min. The retention time of tramadol, ODT, NDT and internal standard was found to be 0.87, 0.90, 0.83 and 1.26 min, respectively.
Results: The calibration curves were linear over a range of 0.20-400 ng/ml for tramadol and 0.25-200 ng/mL for the metabolites. The mean accuracy and precision for intra- and inter-run validation of tramadol, ODT, and NDT were all within acceptable limits. The intra−run and inter−run accuracies for tramadol, ODT and NDT were 95.5-103.6 % and 91.3−111.4% and 97.4-109.8%, respectively. The intra-run and inter-run precisions for tramadol, ODT and NDT were 2.3-7.9 %, 0.9-8.2 %, and 1.2-13 %, respectively. The pharmacokinetics of tramadol were evaluated after 100 mg single oral administration to healthy volunteers. The mean AUCinf, Cmax, Tmax and T1/2 of tramadol were 2722.6 ± 593.9 ng·hr/mL, 335.9 ± 36.7 ng/mL, 1.3 (1 – 3) hr and 6.2 ± 0.9 hr, respectively.
Conclusion: The present analytical method showed good sensitivity and short running time and successfully applied to a pharmacokinetic study after administration of 100 mg oral dose of tramadol to healthy subjects.