Enantiospecific determination of DL-methylphenidate and DL-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: application to human ethanol interactions

Abstract

In humans, concomitant DL-methylphenidate (DL-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite DL-ethylphenidate (DL-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between D- and L-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic-pharmacodynamic correlations regarding the MPH-ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of D- and L-MPH as well as D- and L-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r(2)>0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed.

Fig 1

Representative LC-MS/MS chromatograms of blank human plasma (A) and of plasma spiked with 0.25 ng/ml of dl-MPH (B), dl-EPH (C), and d3-dl-MPH (D), and human plasma sample collected at 2 h after administration of 0.3 mg/kg dl-MPH, then 0.6 g/kg ethanol (E). The inserts in Fig 1E show enlarged images of l-MPH and d-EPH chromatographic peaks. The m/z transitions of dl-MPH, dl-EPH, and d3-dl-MPH were m/z 234>84, 248>84, and 237>84, respectively.

Fig 2

Plasma concentration-time profile of dl-MPH and dl-EPH isomers from a healthy male volunteer administered 0.3 mg/kg dl-MPH with or without ethanol (0.6 g/kg).