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首页> 外文期刊>Asian journal of drug metabolism and pharmacokinetics >Simultaneous determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography with UV detection
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Simultaneous determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography with UV detection

机译：紫外检测梯度液相色谱法同时测定多种细胞色素P450探针底物的代谢物

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摘要

Aim Develop a simple practical HPLC method with UV detection for simultaneous determination of several cytochrome P450 (CYP) probe substrate metabolites including 6-hydroxychlorzoxazone (CYP2E1), acetaminophen (CYP1A2), dextrorphan (CYP2D6), 4'-hydroxymephenytoin (CYP2C19), 4-hydroxytolbutamide (CYP2C9) and 6p-hydroxytestosterone (CYP3A4). Methods CYP probe substrate metabolites were prepared from in vitro human and rat microsomal incubations and injected into an HPLC/UV system. The mobile phase consisted of (A) 10 mM potassium phosphate buffer of pH 2.8 and (B) acetonitrile. Separation of the metabolites was achieved using a 5 mum, 3.9 x 150 mm C8 column with gradient elution. The metabolites were quantified by UV absorption at 230 nm. Results The method showed a good linear calibration curve and lower limit of quantitation (LLOQ) of 0.066 uM for acetaminophen, 0.049 uM for dextrorphan, 0.027 muM for 6-hydroxychlorzoxazone, 0.021 uM for 4'-hydroxymephenytoin, 0.035 uM for 4-hydroxytolbutamide and 0.033 uM for 6p-hydroxytestosterone, respectively, Using this method, Michaelis-Menten kinetic parameters K_m and V_(max) for the probe substrates in rat liver microsomes were measured. Conclusion A gradient LC/UV method has been developed for simultaneous and quantitative determination of cytochrome P450 (CYP) probe substrate metabolites. The LC/UV method has been shown to provide sufficient sensitivity and linear concentration range for the analysis of probe substrate metabolites from in vitro microsomal incubations, and can be used to improve throughput and cost-effectiveness in preclinical drug studies.
机译：目的开发一种具有紫外线检测功能的简单实用的HPLC方法，用于同时测定几种细胞色素P450（CYP）探针底物代谢物，包括6-羟基氯唑沙宗（CYP2E1），对乙酰氨基酚（CYP1A2），右旋啡烷（CYP2D6），4'-羟基甲基苯妥英钠（CYP2C19），4 -羟基甲苯丁酰胺（CYP2C9）和6p-羟基睾丸酮（CYP3A4）。方法CYP探针底物代谢物是通过体外人和大鼠微粒体温育制备的，并注入HPLC / UV系统中。流动相由（A）pH 2.8的10 mM磷酸钾缓冲液和（B）乙腈组成。使用5毫米3.9 x 150 mm C8色谱柱通过梯度洗脱实现代谢物的分离。代谢物通过在230 nm处的UV吸收进行定量。结果该方法显示出良好的线性校准曲线，对乙酰氨基酚的定量下限（LLOQ）为0.066 uM，右啡烷为0.049 uM，6-羟基氯唑酮为0.027μM，4'-羟基苯甲酰脲为0.021 uM，4-羟基甲苯基丁酰胺为0.035 uM， 6p-羟基睾丸酮分别为0.033 uM，使用此方法，测量了大鼠肝微粒体中探针底物的Michaelis-Menten动力学参数K_m和V_（max）。结论已经建立了一种梯度LC / UV方法，用于同时和定量测定细胞色素P450（CYP）探针底物代谢产物。 LC / UV方法已显示出足够的灵敏度和线性浓度范围，可用于分析体外微粒体温育中的探针底物代谢物，并可用于提高临床前药物研究的通量和成本效益。

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