Enantioselective analysis of unbound tramadol, O-desmethyltramadol and N-desmethyltramadol in plasma by ultrafiltration and LC–MS/MS: Application to clinical pharmacokinetics

► Binding of drugs to plasma proteins can be stereoselective. ► Unbound concentrations of tramadol, M1 and M2 enantiomers were analyzed by MS/MS. ► Kinetics of tramadol and M2 was enantioselective in patients with neuropathic pain. ► Fraction unbound of tramadol enantiomers was higher than that of the (+)-M1. This study describes the enantioselective analysis of unbound and total concentrations of tramadol and its main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in human plasma. Sample preparation was preceded by an ultrafiltration step to separate the unbound drug. Both the ultrafiltrate and plasma samples were submitted to liquid/liquid extraction with methyl t-butyl ether. Separation was performed on a Chiralpak® AD column and tandem mass spectrometry consisting of an electrospray ionization source, positive ion mode and multiple reaction monitoring was used as the detection system. Linearity was observed in the following ranges: 0.2–600 and 0.5–250ng/mL for analysis of total and unbound concentrations of the tramadol enantiomers, respectively, and 0.1–300 and 0.25–125ng/mL for total and unbound concentrations of the M1 and M2 enantiomers, respectively. The lower limits of quantitation were 0.2 and 0.5ng/mL for analysis of total and unbound concentration of each tramadol enantiomer, respectively, and 0.1 and 0.25ng/mL for total and unbound concentrations of M1 and M2 enantiomers, respectively. Intra- and interassay reproducibility and inaccuracy did not exceed 15%. Clinical application of the method to patients with neuropathic pain showed plasma accumulation of (+)-tramadol and (+)-M2 after a single oral dose of racemic tramadol. Fractions unbound of tramadol, M1 or M2 were not enantioselective in the patients investigated.