高效液相色谱串联质谱法测定大鼠血浆中伐地那非浓度的可行性研究

Feasibility of the determination of plasma vardenafil level in rat by performance liquid chromatography-tandem mass spectrometry

目的建立一种基于高效液相色谱串联质谱技术的测定大鼠血浆中伐地那非浓度的方法,并评估其可行性。方法采集正常Sprague-Dawley大鼠血浆样本。采用Phenomenex Synergi Polar-RP 80 A色谱柱(2.0 mm×50 mm,4μm),柱温30℃,流动相A为0.1%甲酸水溶液,流动相B为0.1%甲酸乙腈溶液,流速为0.4 ml/min;质谱采用API 4000Qtrap三重四极杆质谱仪的正离子多反应监测模式监测,伐地那非和内标监测离子质荷比分别为489.3(Q1通道)/151.2(Q3通道)和466.4(Q1通道)/234.2(Q3通道);西沙必利为内标,检测大鼠血浆中添加的伐地那非浓度。然后评估该方法检测伐地那非浓度的专属性、线性及定量范围、精密度和准确度、基质效应、稳定性。结果在选定的色谱和质谱条件下,伐地那非和内标的保留时间分别为2.62和2.80 min,峰形良好。该方法在0.2~200 ng/ml浓度范围内线性良好。该方法检测伐地那非的批内精密度(%CV)和准确度(%DEV)分别在1.5%~9.7%和-6.8%~6.6%之间。批间精密度和准确度分别在3.1%~8.4%和-3.7%~4.6%之间。均在要求的范围内。本样品处理方法,伐地那非的提取回收率在88.2%~104.6%之间,符合提取回收率的考察要求。该样品处理方法,伐地那非各质控浓度经内标归一化的基质效应因子(MF)分别为1.04、0.85、1.04,变异系数(%CV)在1.7%~10.7%之间,符合基质效应的考察要求。伐地那非的短期稳定性、长期稳定性、4次冻融循环稳定性的偏差都在±15%以内,变异系数均在5%以内。结论本研究建立的高效液相色谱串联质谱法特异性好、准确性高,可用于检测大鼠血浆伐地那非浓度。

Objective To develope and validate a reliable and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determination of vardenafil concentration in plasma of rat.Methods Plasma samples of normal Sprague-Dawley rats were collected.A Phenomenex Synergi Polar-RP 80A column(2.0 mm×50 mm,4µm)was used.Column temperature was set at 30℃.Mobile phase A was 0.1%formic acid in water;mobile phase B was 0.1%formic acid in acetonitrile.The flow rate was 0.4 ml/minutes.Quantitative determination was performed by electrospray ionization,operating in positive ion multiple reaction monitoring(MRM)mode.Cisapride was used as the internal standard.The feasibility of the method was evaluated by examining its specificity,linearity and quantitative range,precision and accuracy,matrix effects,and stability.Results Under the selected chromatographic and mass spectrometry conditions,the monitoring ions of vardenafil and internal standard were mass-to-charge ratio(m/z)489.3/151.2 and 466.4/234.2,the retention times of vardenafil and internal standard were 2.62 and 2.80 minutes,respectively,and the peak shape was satisfactory.The method has good linearity in the concentration range of 0.2-200 ng/ml.The intra-batch precision(%CV)and accuracy(%DEV)of vardenafil were 1.5%-9.7%and-6.8%-6.6%,respectively.The inter-batch precision and accuracy of vardenafil were 3.1%-8.4%and-3.7%-4.6%,respectively.In this sample processing method,the extraction recovery rate of vardenafil was obtained at range of 88.2%-104.6%,which met the requirements for the investigation of extraction recovery rate.In this sample processing method,the normalized matrix factor of each quality control concentration of vardenafil was 1.04,0.85,and 1.04,and the coefficient of variation(%CV)was in the range of 1.7%-10.7%,which met the requirements for the investigation of matrix effects.Variations of short-term stability,long-term stability,and stability of 4 freeze-thaw cycles of vardenafil was within±15%,and the coefficient of variation were within 5%.Conclusion The high performance liquid chromatography-tandem mass spectrometry method established in this study is feasible for the measurement of concentration of vardenafil in rat plasma and this method has good specificity and high accuracy,and can be used to detect the concentration of vardenafil in rat plasma.