Development and application of an enantioselective liquid chromatography – tandem mass spectrometry method for the quantification of enantiomers to evaluate in vivo chiral inversion in beagle dog

Abstract

To assess whether a suspected metabolic chiral inversion of a new chemical entity (NCE) with a single chiral centre occurred in an in vivo pharmacokinetic (PK) study of UCB-1, a quantitative enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. Chromatographic screening of authentic standards of the two enantiomers was carried out using a range of Phenomenex Lux chiral columns under various isocratic conditions to achieve optimum chromatographic resolution. Protein precipitation was used for analyte extraction from 40 µL of dog plasma. The subjects were given a po dose of either 1, 3 or 10 mg/kg followed by blood sample collection at timepoints over a 72-hour period. For the analysis, a Shimadzu liquid chromatography system was coupled to a Sciex QTRAP 5500 mass spectrometer operated using multiple reaction monitoring (MRM) in positive ion mode. The regression for the two enantiomers was (R2 ≥ 0.999) over the range of 10 – 20,000 ng/mL while employing a quadratic fit, with a lower limit of quantification (LLOQ) of 10 ng/ml. The method was validated for intra- and inter-day precision and accuracy, which were within +/- 20%. The enantiomers were stable in dog plasma at room temperature for 4 h and after three freeze-thaw cycles. The method was successfully applied to a pharmacokinetic study in dog, after po administration of UCB-1 where a significant level of chiral inversion was observed in plasma.