LC-MS/MS法评价CYP3A酶活性

Evaluation of CYP3A Activity by LC-MS/MS

目的用高效液相色谱-质谱联用(LC-MS/MS)法测定尿液中氢化可的松和6β-羟基氢化可的松的含量,计算6β-羟基氢化可的松和氢化可的松的比值以反映人体CYP3A酶的活性,为其研究提供有效的分析手段。方法采用色谱柱:Phenomenex Kinetex C18(1.7μm,50×2.10mm),柱温40℃,以0.1%甲酸水溶液-乙腈为流动相梯度洗脱,流速为0.5m L·min^(-1),以醋酸氟氢化可的松为内标。质谱采用大气压离子源,以多反应离子监测模式进行定量分析,尿液样品用pH=5缓冲液和乙酸乙酯进行液-液萃取。结果氢化可的松在0.5~200ng·mL^(-1)浓度范围线性关系良好;6β-羟基氢化可的松在10~1250ng·mL^(-1)浓度范围线性关系良好。日内、日间RSD均小于11%(n=6),提取回收率>65%。结论本文建立的方法操作简单、灵敏度高、分析时间短,适合于人体尿样中氢化可的松和6β-羟基氢化可的松的含量测定及人体CYP3A酶的活性评价。

Objective To evaluate the activity of CYP3A enzyme and to establish an effective method of LC-MS/MS for determining the concentration of 6β-hydroxycortisol and cortisol in urine.Methods The analysis was carried out on a Phenomenex Kinetex C_(18) column(2.1×50mm,1.7μm).The column temperature was 40℃,with linear gradient elution using 0.1% formic acid solution and acetonitrile,and the flow rate was 0.5mL·min^(-1).The quantitation was done by multiple reaction monitoring(MRM)mode of Heated Nebulizer,with fludrocortisone acetate as internal standard(IS).Urine samples were adjusted to(pH=5.0)and performed by liquid-liquid extraction.Results The linear range was(0.5~200)ng·mL^(-1) for cortisol,10~1250 ng·mL^(-1) for 6β-hydroxycortisol.Both intra-and inter-day RSD were less than 11%(n=6),the recoveries were more than65%.Conclusion The method is accurate,sensitive,simple and suitable for the evaluation of the activity of CYP3A.