Enantiomeric profile of promethazine in metabolic studies in liver microsomes

Promethazine (PMZ) is metabolized in the liver giving rise to chiral metabolites, including promethazine sulfoxide (PMZSO) and desmonomethyl promethazine (DMPMZ). Other metabolites, such as dioxopromethazine (DOPMZ), hydroxy promethazine (PMZOH), N-desmethyl-promethazine sulfoxide, and promethazine N-oxide (PMZNO) can also be formed, but information about them is limited. In this work, the enantiomeric metabolic profile of PMZ in ultra-pool human liver microsomes (HLM) was evaluated. For that, a novel enantioselective analytical method, by liquid chromatography coupled with high-resolution mass spectrometry was established and employed to monitor the enantiomers of PMZ and the formation of its main metabolites PMZSO and DMPMZ in two-hours assay with HLM. The enantioseparation optimized conditions were achieved with two immobilized carbamate amylose-based (3‑chloro-5-methylphenylcarbamate) columns: Lux® 3 µm i-Amylose-3 and Chiralpak® IG-U 1.6 µm. The optimized mobile phase for enantioseparation used buffer and ethanol, a green organic solvent, in a low flow rate. The sample preparation was based only in liquid-liquid extraction. (R)-PMZ consistently exhibited higher concentrations than (S)-PMZ, indicating less extension in metabolization. Regarding the metabolites, PMZSO exhibited higher concentrations compared to DMPMZ. A higher concentration for (S)-PMZSO was found when compared to the (R)-PMZSO and (R)-DMPMZ showing higher concentration compared to (S)-DMPMZ, indicating enantioselectivity in the metabolization process. The metabolites, PMZOH or PMZNO and DOPMZ were also identified. The second enantiomer of DOPMZ also showed a higher proportion than the first eluted enantiomer. These results demonstrated enantioselectivity of liver metabolism of PMZ, as well as confirmed PMZOH, PMZNO, and DOPMZ metabolites formation.