LC-MS/MS法测定血浆中达比加群浓度及在房颤消融围术期抗凝治疗中的应用

Determination of dabigatran concentration in plasma by LC-MS/MS and its application in anticoagulant therapy for atrial fibrillation ablation

目的建立一种快速、灵敏的液相色谱-串联质谱(LC-MS/MS)法,用于测定房颤患者服用达比加群酯抗凝治疗后血浆中达比加群浓度。方法以达比加群-d3为内标,血浆样品经甲醇蛋白沉淀,使用色谱柱为Agilent Eclipse XDB-C18柱(100 mm×2. 1 mm,3. 5μm),流动相为0. 5%甲酸水溶液和0. 5%甲酸甲醇溶液,梯度洗脱。流速0. 3 mL·min-1,柱温50℃。在电喷雾电离源正离子条件下,采用多反应监测模式,分别监测反应离子对达比加群和达比加群-d3,m/z 472. 3→289. 1和m/z 475. 3→292. 3。结果本法测定的达比加群线性范围为5. 0~400μg·L-1(r=0. 999 9),定量下限为5. 0μg·L-1。低、中、高3种浓度下方法的日内和日间相对标准差(RSD)均<10%,准确度(RE)均在±5%以内,提取回收率为97. 72%~101. 22%。结论该法快速、准确、灵敏和专属性强,适用于房颤患者消融围术期抗凝治疗的监测和安全性研究。

AIM To establish a rapid and sensitive LC-MS/MS method for determination of dabigatran con- centration in plasma of atrial fibrillation patients. METHODS Dabigatran-d3 was used as the internal standard. A single-step precipitation was used for plasma sample preparation by methanol. The separation was performed on an Agilent Eclipse XDB-C18 analytical column （100mm×2.1mm,3.5μm） using a gradient elution mode with a column temperature of 50 ℃. The mobile phase was a mix of distilled water containing 0.5% formic acid and methanol containing 0.5% formic acid at a flow rate of 0.3 mL · min -1 Detection and quantification were per- formed by eleetrospray ionization （ESI） in the positive ionization mode by multiple reaction monitoring （MRM） of the transitions at m/z 472.3→289, 1 for dabigatran and m/z 475, 3→292, 3 for dabigatran-d3. RESULTS The linear range of dabigatran was 5.0 - 400 μg ·L - 1 （ r = 0. 999 9） with lower limit of quantitation of 5.0 μg · L - 1 The intra- and inter-day RSDs were all below 10% and accuracy were within ± 5% at low, moderate and high con- centrations. The mean extract recoveries were 97.72% - 101.22% for dabigatran. CONCLUSION The method established is rapid, accurate, sensitive and reliable for the determination of dabigatrau in human plasma. Themethod is suitable for the therapeutic drug monitoring of dabigatran and can be applied in safety study of anticoagu- lant therapy for atrial fibrillation ablation.