目的:建立HPLC-MS/MS法测定人EDTA抗凝血浆中维格列汀的浓度,并将其应用于药代动力学研究。方法:以稳定同位素标记13C-15N-维格列汀为内标,血浆样品用乙腈进行蛋白沉淀处理,采用Hypurity C18(150 mm×2.1 mm,5 μm)色谱柱,以甲醇-5 mmol·L-1甲酸铵水溶液为流动相,梯度洗脱,流速0.5 mL·min-1,柱温40 ℃,进样量2 μL,采用电喷雾离子源(ESI源),多反应监测正离子模式进行检测。用于定量分析的维格列汀监测离子对m/z 304.3→154.2,内标监测离子对m/z 310.3→160.3。考察其专属性、标准曲线、定量限、精密度、回收率、基质效应、稳定性,并使用该方法对健康受试者的血浆维格列汀浓度进行测定。结果:血浆中维格列汀质量浓度在1.11~534.0 ng·mL-1范围内线性关系良好。4个浓度水平的批内、批间精密度(RSD)均在0.9%~8.5%,准确度在99.8%~109.3%。低浓度血浆样品室温放置0.5、1、2 h的准确度分别为92.0%、87.6%、71.2%,冰上放置0.5、1、2 h的准确度分别为102.0%、94.5%、86.6%,该结果提示维格列汀在血浆中可能存在不稳定现象。提取回收率、基质效应以及其他稳定性结果等均符合生物样品分析的相关要求。8例健康受试者药代动力学研究结果:t1/2为(1.49±0.37) h,tmax为(2.06±1.11) h,Cmax为(290.94±100.36) ng·mL-1,AUC0-24 h为(1 343.46±186.89) ng·h·mL-1,AUC0-∞为(1 351.31±188.79) ng·h·mL-1。结论:该方法操作简便,特异性好,灵敏度高,成功应用于8名健康受试者空腹口服给药50 mg维格列汀片后的药代动力学研究,可作为一种可靠的检测方法用于人体药动学研究和治疗药物监测。
Objective: To develop a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method for the determination of vildagliptin in human anticoagulant plasma with ethylenediamine tetra acetic acid and apply it to the study of pharmacokinetics. Methods: 13C-15N-vildagliptin was used as internal standard (IS). After extraction from human plasma by protein precipitation with acetonitrile, all components were separated by a Hypurity C18 column (150 mm×2.1 mm,5 μm), using a gradient elution procedure consisting of methanol and 5 mmol·L-1 ammonium formate at a flow rate of 0.5 mL·min-1,and the column temperature was 40 ℃. Injection volume was just 2 μL. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 304.3→154.2 for vildagliptin and m/z 310.3→160.3 for internal standard. Specificity,standard curve, lower limit of quantification,precision,recovery,matrix effect and stability were examined. Then this method was used to determine the plasma concentration of veragliptin in healthy subjects. Results: The calibration curve of vildagliptin in human plasma was linear over the concentration range of 1.11 to 534.0 ng·mL-1. The lower limit of quantitation was 1.11 ng·mL-1. The intra-and inter-day precisions at four quality control levels were within 0.9%-8.5%,and the accuracy was within 99.8%-109.3%. The data of short-term stability at room temperature displayed that the accuracy percentage of LQC samples was 92.0% for 0.5 h exposure, 87.6% for 1 h exposure, 71.2% for 2 h exposure. These of LQC samples chilled on ice was 102.0% for 0.5 h exposure, 94.5% for 1 h exposure, 86.6% for 2 h exposure. These results showed a phenomenon that there was a possible degradation of vildagliptin in plasma. The results of extraction recovery and matrix effect and other stability met the requirements of biological sample analysis. The pharmacokinetic study results of 8 healthy subjects showed that t1/2 was (1.49±0.37) h, tmax was (2.06±1.11) h, Cmax was (290.94±100.36) ng·mL-1, AUC0-24 h was (1 343.46±186.89) ng·h·mL-1, AUC0-∞ was (1 351.31±188.79) ng·h·mL-1. Conclusion: This method is easy to operate, has high specificity, and sensitivity. It has been successfully applied to the pharmacokinetic study of 8 healthy subjects after oral administration of 50 mg vigagliptin tablets on an empty stomach. Therefore, it can be used as a reliable detection method for human pharmacokinetic research and therapeutic drug monitoring.
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