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.

中文翻译：

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猪肝和肠细胞色素P450同工酶的储存稳定性研究，是使用一种新开发并经过充分验证的高灵敏度HPLC-MS / MS方法

微粒体是研究细胞色素P450（CYP450）酶介导的药物代谢的理想培养基。但是，在制备微粒体之前，组织可以保存很长时间。缺乏关于这些酶在猪肝和肠组织中的稳定性的研究。为了能够研究由这些组织制备的微粒体中CYP450的稳定性，开发并验证了一种高灵敏度，快速的HPLC-MS / MS方法，用于同时测定培养培养基中的六种CYP450代谢产物。代谢物是对乙酰氨基酚（CYP1A），7-羟基香豆素（CYP2A），1-羟基咪达唑仑（CYP3A），4-羟基甲苯丁酰胺（CYP2C），右啡烷（CYP2D）和6-羟基氯唑沙宗（CYP2E）。用pH 1.0的乙酸乙酯萃取，然后在Agilent Zorbax Eclipse Plus C18柱上蒸发并分离。该方法已根据欧洲指南在符合GLP的实验室中进行了充分验证，并且具有高灵敏度（LOQ = 0.25–2.5 ng / mL），选择性高，精密度高（RSD-在1.0-9.1％之内； RSD在1.0与1.0％之间）。 –18.4％）和准确度（运行内83.3–102％；运行间78.5–102％），并且没有相对信号抑制和增强。因此，该方法用于研究组织在-80°C下储存时猪肝和肠CYP450同工酶的稳定性。结果表明，猪CYP450同工酶在速冻并保存在-80°C时至少在4个月内是稳定的。此外，结果显示，与其他研究组相比，猪CYP450的稳定性与大鼠，兔子和鱼类的CYP450相比，