目的：建立人血浆中主要CYP450 酶探针药及其特异代谢物的快速、准确定量分析方法,以开展药物-药物相互作用（DDI）的临床评价。方法：针对5种主要CYP450 亚型,选择咖啡因（CYP1A2）、氯沙坦（CYP2C19）、奥美拉唑（CYP2C9）、右美沙芬（CYP2D6,CYP3A4）和咪哒唑仑（CYP3A4）组成鸡尾酒探针药组合。建立一种online SPE-LC-MS/MS 方法对给予探针药后的人血浆样本进行分析。血浆样品通过葡萄糖醛酸酶水解进行前处理,采用Spark HySphere-C18HD（10 mm×2 mm,7 μm）为固相萃取柱,Shiseido MGⅢ（2.0 mm×100 mm,5 μm）为分析柱,柱温40 ℃。以含0.01% 甲酸的甲醇-乙腈（1∶1）-水溶液为流动相,梯度洗脱,流速为0.35 mL·min-1。在质谱ESI 源、正离子扫描方式下检测5种探针药及相应的6种代谢产物的母离子-子离子对。结果：所建立的分析方法可在5 min 完成11种待测成分的分析。对方法学的验证表明所建立的方法对血浆样品分析具有专属性,定量范围内线性良好,无基质效应影响。中和高浓度下待测成分的批内和批间样品的RSD 为2.1%~14.2%,LLOQ 浓度下为2.1%~15.4%,准确度在86.4%~107.2% 范围内。样品在室温、长期冻存、反复冻融及样品架放置条件下稳定性良好。采用该方法对服用鸡尾酒探针药后的人血浆样本进行了分析,检测出的5种探针药及6种代谢物浓度均在定量区间内,其中右美沙芬代谢物3-甲氧基吗啡喃浓度最低,均低于2 ng·mL-1。结论：所建立的Online SPE-LCMS/MS 分析方法快速、灵敏、准确,可应用于采用鸡尾酒探针的临床DDI 研究和代谢酶活性评估。
张颖, 李军梅, 苗兰, 孙明谦, 林力, 刘建勋, 徐立
. Online SPE-LC-MS/MS 法快速同时分析人血浆中5种CYP450 酶探针药及6种代谢物*[J]. 药物分析杂志, 2021
, 41(4)
: 603
-612
.
DOI: 10.16155/j.0254-1793.2021.04.06
Objective: To establish a rapid,accurate and simultaneous quantitative analysis method for probe drugs of major cytochrome P450（CYP450） enzymes and their specific metabolites,in order to be used in clinical evaluation of drug-drug interaction（DDI）. Methods: A“ probe cocktail” approach was employed to evaluate the activities of CYP450s composed of caffeine（CYP1A2）,losartan（CYP2C19）,omeprazole（CYP2C9）, dextromethorphan（CYP2D6,CYP3A4） and midazolam（CYP3A4）. An online-SPE-LC-MS/MS method was developed to analyze the human plasma after“ probe cocktail” was administrated. Plasma samples were hydrolyzed by glucuronidase and subjected to online solid-phase extraction with Spark HySphere-C18HD（10 mm×2 mm, 7 μm） thereafter. Separation of 11 analytes was accomplished by a gradient elution on a Shiseido MGⅢ（2.0 mm×100 mm,5 μm） analytical column using methanol-acetonitrile（1∶1）-water with 0.01% formic acid as the mobile phase at a flow rate of 0.35 mL·min-1. The column temperature was maintained at 40 ℃ . Mass spectrometry was performed using a triple-quadrupole mass spectrometer operated in positive ion ESI mode, with MS/MS transitions of 5 probe drugs and the corresponding 6 metabolites monitored. Results: The established analysis method could complete the detection of 11 analytes in 5 minutes. The validation of methodology showed that the method had specificity for the analysis of plasma samples,exhibited good linearity in the quantitative ranges and little matrix effect. The RSD values of the intra-and inter-batch quality control samples of the analytes were 2.1%-14.2% at medium and high concentrations and 2.1%-15.4% at LLOQ level along with the accuracy in the range of 86.4% to 107.2%. The samples had good stability at room temperature,long-term freezing,repeated freeze-thaw and placement at autosampler. The method was applied to analyze plasma samples from subjects taken the“ probe cocktail”. The results showed that the concentrations of 5 probe drugs and 6 metabolites were all within the quantitative range. Among them,the concentration of dextromethorphan's metabolite 3-methoxymorphan was the lowest,all of which were lower than 2 ng·mL-1. Conclusion: The established online SPE-LC-MS/MS method provides a rapid and sensitive analytical tool to serve the investigation of multiple DDI and the CYP450 enzymes phenotyping in the clinic.
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