Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Apr 1;923-924:29-36. doi: 10.1016/j.jchromb.2013.01.031. Epub 2013 Feb 10.
A rapid, simple, and sensitive on-line solid-phase extraction HPLC-DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05-50.0 μg/ml for dapsone and omeprazole, 0.1-50.0 μg/ml for caffeine and 0.2-50.0 μg/ml for metoprolol and chlorzoxazone. The linearity (R(2)) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8-109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms.
已开发并验证了一种快速、简单、灵敏的在线固相萃取 HPLC-DAD 方法，用于同时评估五种 CYP450 同工酶（CYP1A2、CYP2C19、CYP2D6、CYP2E1 和 CYP3A4）的体内活性。五种特定的探针底物包括咖啡因（1A2）、美托洛尔（2D6）、氨苯砜（3A4）、奥美拉唑（2C19）和氯唑沙宗（2E1）。使用 C18 在线固相萃取柱自动预净化血浆并富集分析物。分析物和内标安替比林从柱上洗脱后，在 C18 RP 分析柱上分离，并通过 DAD 进行分析。该方法经过验证，可定量测定氨苯砜和奥美拉唑的浓度范围为 0.05-50.0 μg/ml，咖啡因和美托洛尔以及氯唑沙宗的浓度范围为 0.1-50.0 μg/ml。所有测试分析物的线性（R²）均超过 0.99。日内精密度范围为 0.29-13%，日间精密度范围为 5.0-15%。日内和日间准确度在 86.7%-113.6%之间。所有分析物和内标安替比林的提取回收率在 82.8%-109.9%之间。该方法成功应用于评估 TM208 对大鼠五种 CYP450 同工酶的影响。
