Analysis of unbound plasma concentration of oxcarbazepine and the 10-hydroxycarbazepine enantiomers by liquid chromatography with tandem mass spectrometry in healthy volunteers

This study describes the development and validation of a method for the analysis of unbound plasma concentrations of oxcarbazepine (OXC) and of the enantiomers of its active metabolite 10-hydroxycarbazepine (MHD) [S-(+)- and R-(−)-MHD] using liquid chromatography with tandem mass spectrometry (LC–MS/MS). Additionally, the free fraction of the drug is described in healthy volunteers (n = 12) after the oral administration of 300 mg OXC/12 h for 5 days. Plasma aliquots of 200 μL were submitted to ultrafiltration procedure and 50 μL of the ultrafiltrate were extracted with a mixture of tert-butyl methyl ether:dichloromethane (2:1, v/v). OXC and the MHD enantiomers were separated on a OD-H chiral phase column. The method was linear in the range of 4.0–2.0 μg/mL for OXC and of 20.0–6.0 μg/mL plasma for the MHD enantiomers. The limit of quantification was 4 ng for OXC and 20 ng for each MHD enantiomer/mL plasma. The intra- and inter-day precision and inaccuracy were less than 15%. The free fraction at the time of peak plasma concentration of OXC was 0.27 for OXC, 0.37 for S-(+)-MHD and 0.42 for R-(−)-MHD. Enantioselectivity in the free fraction of MHD was observed, with a higher proportion of R-(−)-MHD compared to S-(+)-MHD