Abstract

A more sensitive and accurate high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS) method has been developed and validated simultaneously for analysis of three analytes. The method is sensitive, enough for the study of analyte pharmacokinetics and metabolic pathways of drugs. The current method includes a simple reversed-phase Liquid chromatography–mass spectrometry (LC-MS) assay in determining the plasma concentrations of dextromethorphan, dextrorphan, and midazolam.  Analytes were extracted simply via liquid-liquid extraction with methyl-tert-butyl ether (MTBE). Separation and analysis was done through an Eclipse- XDB® C-8 (150 x 2.1 mm, 3.5 µm particle size, Agilent, Wilmington, DE, USA) analytical column. Analytes were eluted using a mobile phase gradient with good separation and peak shape within 10 min. Using a small sample volume as low as 50 µL of plasma, assay sensitivity was found to be lower limit of quantitation (LLOQ) as low as 0.5 ngmL−1 and the linearity range of 0.5 to 500 ngmL−1 has been achieved for each compound.  The limit of quantitation (LOQ) is 0.5 ngmL−1. The Intra- and inter-day precision was less than 4.0 and 7.0%, respectively. The within and between day accuracies were between 94.3 and 111.4% with a mean of 101.5%. This method is beneficial in the clinical and research evaluation of drug metabolizing enzyme activity for CYP1A2 and CYP3A4.

Key words: Liquid chromatography–mass spectrometry (LC-MS), dextromethorphan, dextrorphan, midazolam, phenotyping.