Challenges in method development for analysis of selected phenothizaine drugs in biological samples: oxidation of drugs and metabolites during sample preparation

Abstract

The main goal of this research project was the detection and semi-quantitation of the parent drugs promethazine (PMZ), and chlorpromazine(CPZ) along with selected metabolites promethazine sulfoxide (PMZSO), desmethylpromethazine (DPMZ), chlorpromazine sulfoxide (CPZSO), and desmethylchlorpromazine (DCPZ) in the skeletal remains of rats. We sought to evaluate the relative distribution of structural analogues (promethazine (PMZ) and chlorpromazine (CPZ)), and the relationship of multiple metabolites to the parent drug. The first phase of the project was to consist of method development and validation and the second phase would be examining a large number of rat bone samples exposed to different drug exposure patterns. For the analytical method, drug extraction is completed using microwave assisted extraction (MAE), followed by sample clean-up by microplate solid-phase extraction (MPSPE) and instrumental analysis by ultra-high performance liquid chromatography coupled to photodiode array detection (UHPLC-PDA). During method development and validation, extraneous compounds appeared in the chromatograms. The production of unknown compounds hindered the ability to meet the validation criteria. Thus, the new objective of the research was characterization of the unknown compounds by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS), comparing the extent of degradation for different extraction conditions, and validation of a new method that does not promote degradation.