RP-HPLC荧光法测定人血浆中替米沙坦浓度

Determination of Telmisartan Concentration in Human Plasma by Means of RP-HPLC with Fluorescence Detector

目的建立人血浆中替米沙坦浓度RP-HPLC荧光检测法。方法以缬沙坦为内标定量血浆中替米沙坦的浓度,血浆样品在酸性条件下用苯萃取后氮气吹干,残留物用流动相复溶后直接进样。样品分离用ZORBAXEclipseXDB-C18柱(4.6×150mm,5μm)和填充相同材料的预柱(4.6×12.5mm,5μm),流动相为甲醇-乙腈-10mmol/L磷酸二氢钾溶液(pH3.2)(5∶40∶55,v/v/v),流速为1.0mL/min,进样体积为20μL,荧光检测激发波长为250nm,发射波长为375nm,柱温为25℃。结果血浆中内源性物质不干扰测定结果,替米沙坦和内标的保留时间分别为8.8min和10.7min。在浓度1μg/L^180μg/L范围内线性关系良好,线性方程:A=0.2769R+0.03788,相关系数r=0.9995,检测限为1μg/L。浓度为2.0、30.0、70.0μg/L血浆质控样品的平均回收率分别为97.53%、104.6%和105.1%。低、中、高浓度血浆质控样品的日内和日间相对标准偏差(RSD)均小于7%。结论本研究建立的血浆中替米沙坦的浓度测定方法,具有准确、快速、简单,灵敏度高、线性范围宽等特点。该方法更适于替米沙坦血药浓度测定及替米沙坦药物代谢动力学研究。

Objective A RP-HPLC method with fluorescence detector was established to determine telmisartan in human plasma. Methods Internal standard method quantifying telmisartan concentration in plasma was performed and valsartan was used as the internal standard. Plasma samples containing drug were extracted with benzene under acidic condition, followed by drying with nitrogen. The residual products were redissolved with mobile phase prior to analysis. Separation was performed on a reverse phase ZORBAX Eclipse XDB-C18 column（4.6 × 150 mm,5μ m）, equipped with a pre-column of the same material（4.6 ×12.5 mm, 5 μm）. The mobile phase contained methanol-acetonitrile-10 mmol/L potassium dihydrogen phosphate buffer, pH 3.2（5： 40： 55, v/v/v）. The flow rate was set at 1.0 mL/min and the injection volume was 20 μL. The excitation and emission wavelengths were 250 nm and 375 nm, respectively. The column temperature was 25 ℃. Results No intrinsic substances interfered with analysis of telmisartan. The retention times were 8.8 min for telmisartan and 10.7 min for valsartan. The good linear relationship was obtained over the range（ 1 ～ 180） μg/L and the equations were determined by least squares linear regression analysis. The linear equation was A = 0.2769R ＋ 0. 03788. The linearity of the relationship between peak area ratio and amount ratio was demonstrated that the correlation coefficient was 0.9995. The limit quantification（LOQ） was set at 1 μg/L of telmisartan in human plasma. The average recovery rates were 97.53% , 104.6% and 105.1% for plasma quality control（QC） samples at 2.0, 30, 70 μg/L, respectively. The RSDs of inter- and intra-day were all less than 7% for low, medium, high plasma QC samples. The intra and inter-assay precision and accuracy of the quality control and limit of quantification were satisfactory in all cases. Plasma samples were stable in the chromatographic rack for 24 h at room temperature, but we recommended storing processed plasma samples at 4 ℃ until the analysis. Conclusion The developed method was validated with respect to selectivity, linearity, accuracy, LOQ and stability. The experimental datum has proved that the above described method would provide accurate, rapid, simple and sensitive measurements of telmisartan in plasma. It is more suitable to determine telmisartan concentration in plasma and study the pharmacokinetics of telmisartan.