Cocktail Approach for In Vivo Phenotyping of 5 Major CYP450 Isoenzymes: Development of an Effective Sampling, Extraction, and Analytical Procedure and Pilot Study With Comparative Genotyping
Cocktail Approach for In Vivo Phenotyping of 5 Major CYP450 Isoenzymes: Development of an Effective Sampling, Extraction, and Analytical Procedure and Pilot Study With Comparative Genotyping
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DOI：
10.1177/0091270011410570
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发表时间：
2012-08-01
影响因子：
2.9
通讯作者：
Auwaerter, Volker
中科院分区：
文献类型：
--
作者：
Wohlfarth, Ariane;Naue, Jana;Auwaerter, Volker
在这项研究中，作者开发了一种 CYP1A2、2C9、2C19、2D6 和 3A4 的表型分析方法，使用 100 mg 咖啡因、125 mg 甲苯磺丁脲、20 mg 奥美拉唑、30 mg 右美沙芬和 2 mg 咪达唑仑的混合物。建立了一个简单的采样方案，在 0、4 和 24 小时收集 3 个血液样本，然后进行固相萃取和液相色谱/串联质谱分析。添加 8 个氘代内标并萃取后，使用乙酸铵和甲醇梯度洗脱分离分析物。数据采集是在三重四极杆线性离子阱质谱仪上以正电喷雾电离的多反应监测模式进行的。该测定根据国际指南进行了验证：所有分析物的定量限 (LOQ) 均在 0.25 至 1.0 ng/mL 之间，副黄嘌呤和咖啡因 (20 ng/mL) 除外。萃取效率在 77% 到 103% 之间，基质效应在 23% 到 95% 之间；数据的精确度和准确度满足公认的标准。为未稀释和 1:10 稀释血浆建立了从 LOQ 到 1000 ng/mL 的校准曲线 (r > 0.998)。该方法在 14 名志愿者参与的试点研究中进行了测试。先证者的额外基因分型通常证明与测量的表型指数良好一致，但也揭示了某些矛盾的结果。
In this study, the authors developed a phenotyping method for CYP1A2, 2C9, 2C19, 2D6, and 3A4 using a cocktail of 100 mg caffeine, 125 mg tolbutamide, 20 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. A simple sampling scheme was established collecting 3 blood samples at 0, 4, and 24 hours followed by solid-phase extraction and liquid chromatography/tandem mass spectrometry analysis. After addition of 8 deuterated internal standards and extraction, the analytes were separated using gradient elution with ammonium acetate and methanol. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple-reaction monitoring mode with positive electrospray ionization. The assay was validated according to international guidelines: limits of quantification (LOQs) were between 0.25 and 1.0 ng/mL for all analytes, except for paraxanthine and caffeine (20 ng/mL). Extraction efficiencies ranged between 77% and 103% and matrix effects between 23% and 95%; precision and accuracy data fulfilled accepted criteria. Calibration curves from LOQ to 1000 ng/mL were established for undiluted and 1:10 diluted plasma (r > 0.998). The method was tested in a pilot study with 14 volunteers. Additional genotyping of the probands generally demonstrated good accordance with the measured phenotyping indices but also disclosed certain contradictory results.