Enantiomeric determination of tramadol and its main metabolite O-desmethyltramadol in human plasma by liquid chromatography-tandem mass spectrometry

Pharmacokinetic studies require sensitive analytical methods to allow the d etermination of low concentrations of drugs and metabolites. When drugs pre sent an asymmetric center, the enantiomeric determination of the compounds of interest should be performed. The method developed is based on on-line L C-MS-MS using atmospheric pressure chemical ionization as an interface dete rmination of enantiomers of tramadol (T) and its active metabolite O-desmet hyltramadol (ODT) in human plasma. This determination is preceded by an off -line solid-phase extraction (SPE) on disposable extraction cartridges (DEC s), performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was fi rst conditioned with methanol and water. The washing step was performed wit h water and the analytes were finally eluted by dispensing methanol. The co llected eluate was then evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The enantiomeric separati on of tramadol and ODT was achieved on a Chiralpak AD column containing amy lose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phas e was isohexane-ethanol-diethylamine (97:3:0.1, v/v). The LC system was the n coupled to a tandem mass spectrometry system with an APCI interface in th e positive ion mode. The chromatographed analytes were detected in the sele cted reaction monitoring mode. The MS-MS ion transitions monitored were 264 -->58 for tramadol, 250-->58 for ODT, and 278-->58 for ethyltramadol, used as internal standard. The method was validated. The recoveries were around 90% for both T and ODT. The method was found to be linear for each enantiom er of both compounds (r(2)>0.999). The mean RSD values for repeatability an d intermediate precision were 3.5 and 6.4% for T enantiomers and 5.0 and 5. 6% for ODT enantiomers, respectively, Moreover, the method was found to be selective towards other metabolites, N-desmethyltramadol and N,O-desmethylt ramadol (NODT). The method developed was successfully used to investigate p lasma concentration of enantiomers of T and ODT in a pharmacokinetic study. (C) 2000 Elsevier Science B.V. All rights reserved.