Storage stability study of porcine hepatic and intestinal cytochrome P450 isoenzymes by use of a newly developed and fully validated highly sensitive HPLC-MS/MS method.

Microsomes are an ideal medium to investigate cytochrome P450 (CYP450) enzyme-mediated drug metabolism. However, before microsomes are prepared, tissues can be stored for a long time. Studies about the stability of these enzymes in porcine hepatic and intestinal tissues upon storage are lacking. To be able to investigate CYP450 stability in microsomes prepared from these tissues, a highly sensitive and rapid HPLC-MS/MS method for the simultaneous determination of six CYP450 metabolites in incubation medium was developed and validated. The metabolites, paracetamol (CYP1A), 7-hydroxy-coumarin (CYP2A), 1-hydroxy-midazolam (CYP3A), 4-hydroxy-tolbutamide (CYP2C), dextrorphan (CYP2D), and 6-hydroxy-chlorzoxazone (CYP2E) were extracted with ethyl acetate at pH 1.0, followed by evaporation and separation on an Agilent Zorbax Eclipse Plus C18 column. The method was fully validated in a GLP-compliant laboratory according to European guidelines and was highly sensitive (LOQ = 0.25-2.5 ng/mL), selective, had good precision (RSD-within, 1.0-9.1%; RSD-between, 1.0-18.4%) and accuracy (within-run, 83.3-102%; between-run, 78.5-102%), and showed no relative signal suppression and enhancement. Consequently, this method was applied to study the stability of porcine hepatic and intestinal CYP450 isoenzymes when tissues were stored at - 80 °C. The results indicate that porcine CYP450 isoenzymes are stable in tissues at least up to 4 months when snap frozen and stored at - 80 °C. Moreover, the results indicate differences in porcine CYP450 stability compared to rat, rabbit, and fish CYP450, as observed by other research groups, hence stressing the importance to investigate the CYP450 stability of a specific species.