中国临床药学杂志

目的建立和验证人肝微粒体中CYP3A4探药代谢物6β-羟基睾酮和1-羟基咪达唑仑的测定方法 ,以准确体外评价CYP3A4的活性。方法在人肝微粒体温孵系统中加入睾酮37℃温孵生成6β-羟基睾酮,经过碱化后用乙酸乙酯提取微粒体样本,采用Phenomenex Gemina C₁₈分离,流动相由甲醇和水组成,梯度洗脱,流速1.0 mL·min^-1,检测波长245 hm,测定微粒体中6β-羟基睾酮浓度;或人肝微粒体温孵系统中加入咪达唑仑,37℃温孵生成1-羟基咪达唑仑,经过乙腈处理人肝微粒体样本,采用Diamosil C₁₈分离,20 mmoL·L^-1醋酸铵-乙腈(60:40,V/V)流动相等度洗脱,流速1.0mL·min^-1,检测波长254 nm,测定1-羟基咪达唑仑浓度。结果 6β-羟基睾酮线性范围为0.111～8.90 mg·L^-1,1-羟基咪达唑仑线性范围为20～1 000 mg·L^-1,两代谢物的批内、批间差异<15%,准确度为85%～115%。结论建立的测定微粒体中6β-羟基睾酮和1-羟基咪达唑仑浓度的方法符合生物样品测定的要求,能准确测定微粒体中目标物浓度,可用于评价CYP3A4的活性。

AIM To establish and validate the HPLC methods of determining the concentrations of 6β-hydroxytestosterone and 1-hydroxymidazolam in human liver microsomes to accurately evaluate the activity of CYP3A4 in vitro. METHODS For 6β-hydroxytestosterone,after incubation with testosterone in the microsome system at 37℃,the samples containing 6β-hydroxytestosterone were extracted with ethyl acetate after being basified.Separation was achieved using a Phenomenex Gemina C₁₈ column and a gradient mobile phase of methanol and water at a flow rate of 1 mL·min^-1.The concentrations of 6β-hydroxytestosterone were determined using a UV detector set at a wavelength of 245 nm.For 1-hydroxymidazolam, after incubation with midazolam in the microsome system at 37℃,the samples containing 1-hydroxymidazolam were extracted using a protein precipitation with acetonitrile.Separation was achieved on a Diamosil C₁₈ column with isocratic mobile phase of 20 mmol·L^-1 ammonium acetate-acetonitrile(60:40,V/V) at a flow rate of 1.0 mL·min^-1,The concentrations of 1-hydroxymidazolam were determined using a UV detector set at a wavelength of 254 nm. RESULTS The linearity ranges of 6β-hydroxytestosterone and 1-hydroxymidazolam were 0.111-8.90 mg·L^-1 and 20-1 000μg·L^-1,respectively.RSD of intra- and inter- batches for both metabolites were below 15%and accuracies were within 85%-115%.CONCLUSION The methods for the quantification of 6β-hydroxytestosterone and 1-hydroxymidazolam in human liver microsomes meet the requirements for biological sample determination,and are able to be applied in the evaluation of the activity of CYP3A4 in vitro.