超高效液相质谱联用法测定人血浆中地尔硫及其2个活性代谢产物的浓度
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摘要
目的建立超高效液相质谱联用(UPLC-MS/MS)法测定人血浆中地尔硫(DTZ)及其主要活性代谢产物N-去甲基地尔硫(N-DMeD)和去乙酰基地尔硫(DAc D)的浓度。方法人血浆用乙腈沉淀蛋白法后,用CAPCELL ADME C_(18)色谱柱(2. 1 mm×100. 0 mm,2μm),流动相为乙腈-水(含0. 1%甲酸),梯度洗脱,流速为0. 4 mL·min~(-1)。用电喷雾离子源,正离子模式,多反应监测(MRM)扫描方式进行监测。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、基质效应和稳定性。结果 DTZ,N-DMeD和DAc D分别在0. 2～80. 0,0. 2～40. 0和0. 1～5. 0 ng·mL~(-1)内,线性关系良好,定量下限分别为0. 2,0. 2和0. 1 ng·mL~(-1),提取回收率分别为70. 65%～80. 85%,68. 97%～71. 66%和64. 84%～73. 25%。DTZ,N-DMeD和DAc D都没有明显的基质效应。质控样品日内和日间精密度均小于10. 01%,准确度在94. 55%～110. 83%。结论该方法快速、准确,灵敏度高,专属性强,可用于地尔硫及其2个代谢产物的人体血药浓度检测及盐酸地尔硫片的药代动力学研究。
Objective To develop a rapid and sensitive ultra performance liquid chromatography with tandem mass spectrometry( UPLC-MS/MS) method for the quantification of diltiazem( DTZ) and its active metabolites N-desmethyldiltiazem( N-DMeD) and desacetyldiltiazem( DAcD) simultaneously. Methods Sample preparation was done by protein precipitation. Chromatographic separation was achieved by an CAPCELL ADME C_(18)( 2. 1 mm × 100. 0 mm,2 μm) column with a gradient mobile phase consisting of acetonitrile-water( containing 0. 1% acetic acid) at a flow rate of 0. 4 mL·min~(-1). DTZ,N-DMeD and DAc D were monitored using positive electrospray triple quadrupole mass spectrometer via multiple reaction monitoring( MRM) mode. The specificity,calibration curve,limit of quantification,accuracy,recovery,matrix and stability were validated. Results The validated method had an excellent linearity in the range of 0. 2-80. 0 ng · mL~(-1),0. 2-40. 0 ng·m L~(-1),and 0. 1-5. 0 ng·m L~(-1) for DTZ,N-DMeD and DAcD,with the limits of quantification were 0. 2,0. 2 and 0. 1 ng · mL~(-1).Recovery efficiencies were in the range of 70. 65%-80. 85%,68. 97%-71. 66% and 64. 84%-73. 25% for DTZ,N-DMe D and DAcD. No significant matrix effects were found for the three analytes. The of intra-and inter-day precisions of three quality control concentrations were less than 10. 01%. The intra-and inter-day accuracies were determined to be 94. 55%-110. 83% with all accuracy measurements. Conclusion The validated method was rapid,sensitive,selective and accurate for the quantification of DTZ and its two active metabolites( N-DMe D and DAc D),successfully used to the pharmacokinetics study of DTZ and its active metabolites in human.
Objective To develop a rapid and sensitive ultra performance liquid chromatography with tandem mass spectrometry( UPLC-MS/MS) method for the quantification of diltiazem( DTZ) and its active metabolites N-desmethyldiltiazem( N-DMeD) and desacetyldiltiazem( DAcD) simultaneously. Methods Sample preparation was done by protein precipitation. Chromatographic separation was achieved by an CAPCELL ADME C_(18)( 2. 1 mm × 100. 0 mm,2 μm) column with a gradient mobile phase consisting of acetonitrile-water( containing 0. 1% acetic acid) at a flow rate of 0. 4 mL·min~(-1). DTZ,N-DMeD and DAc D were monitored using positive electrospray triple quadrupole mass spectrometer via multiple reaction monitoring( MRM) mode. The specificity,calibration curve,limit of quantification,accuracy,recovery,matrix and stability were validated. Results The validated method had an excellent linearity in the range of 0. 2-80. 0 ng · mL~(-1),0. 2-40. 0 ng·m L~(-1),and 0. 1-5. 0 ng·m L~(-1) for DTZ,N-DMeD and DAcD,with the limits of quantification were 0. 2,0. 2 and 0. 1 ng · mL~(-1).Recovery efficiencies were in the range of 70. 65%-80. 85%,68. 97%-71. 66% and 64. 84%-73. 25% for DTZ,N-DMe D and DAcD. No significant matrix effects were found for the three analytes. The of intra-and inter-day precisions of three quality control concentrations were less than 10. 01%. The intra-and inter-day accuracies were determined to be 94. 55%-110. 83% with all accuracy measurements. Conclusion The validated method was rapid,sensitive,selective and accurate for the quantification of DTZ and its two active metabolites( N-DMe D and DAc D),successfully used to the pharmacokinetics study of DTZ and its active metabolites in human.
引文
[1]MOLDEN E,JOHANSEN P W,BE G H,et al.Pharmacokinetics of diltiazem and its metabolites in relation to CYP2D6 genotype[J].Clin Pharmacol Ther,2002,72(3):333-342.
[2]ZHOU L Y,ZUO X C,CHEN K,et al.Significant impacts of CYP3A4*1G and CYP3A5*3 genetic polymorphisms on the pharmacokinetics of diltiazem and its main metabolites in Chinese adult kidney transplant patients[J].J Clin Pharm Ther,2016,41(3):341-347.
[3]GEORGITA C,ALBU F,DAVID V,et al.Nonlinear calibrations on the assay of dilitiazem and two of its metabolites from plasma samples by means of liquid chromatography and ESI/MS2 detection:application to a bioequivalence study[J].Biomed Chromatogr,2008,22(3):289-297.
[2]ZHOU L Y,ZUO X C,CHEN K,et al.Significant impacts of CYP3A4*1G and CYP3A5*3 genetic polymorphisms on the pharmacokinetics of diltiazem and its main metabolites in Chinese adult kidney transplant patients[J].J Clin Pharm Ther,2016,41(3):341-347.
[3]GEORGITA C,ALBU F,DAVID V,et al.Nonlinear calibrations on the assay of dilitiazem and two of its metabolites from plasma samples by means of liquid chromatography and ESI/MS2 detection:application to a bioequivalence study[J].Biomed Chromatogr,2008,22(3):289-297.