目的: 建立LC-MS/MS法检测替格瑞洛中基因毒性杂质4,6-二氯-2-(丙硫基)-5-氨基嘧啶(DCPA)和5-硝基-2-(丙硫基)嘧啶-4,6-二醇(DHPN)。方法: 采用Ultimate® UHPLC XB-Phenyl色谱柱(100 mm×2.1 mm,1.8 μm),以0.1%甲酸溶液为流动相A,0.1%甲酸的甲醇溶液为流动相B,梯度洗脱,流速0.3 mL·min-1,柱温为40 ℃;采用AB SCIEX ExionLC-QTRAP5500三重四极杆串联离子阱液质联用仪进行检测,电喷雾离子源(ESI+)、多反应监测模式(MRM),定量离子对为m/z 240.1→197.8(DCPA)、m/z 232.2→213.9(DHPN)。结果: DCPA质量浓度在0.10~16.1 ng·mL-1范围内与峰面积呈良好线性关系(r=0.999 9);检测限为0.03 ng·mL-1,定量限为0.10 ng·mL-1,平均加样回收率为97.5%(RSD=9.9%,n=9);DHPN质量浓度在0.20~16.1 ng·mL-1范围内与峰面积呈良好线性关系(r=0.999 5),检测限为0.06 ng·mL-1,定量限为0.20 ng·mL-1,平均加样回收率为98.1%(RSD=1.5%,n=9)。重复性和中间精密度实验中,DHPN在100%限度浓度加标供试品溶液(n=12)中的加样平均含量为7.97 μg·g-1,RSD=4.9%;供试品溶液(n=12)中DCPA的平均含量为0.94 μg·g-1,RSD=4.3%,表明精密度良好。对照品溶液、供试品溶液、加标供试品溶液中DCPA和DHPN室温放置24 h内稳定。3批替格瑞洛样品(批号Q19010701G、Q19010702G、Q19010703G)中均未检测到DHPN,DCPA的含量依次为1.2、1.1和1.1 μg·g-1,均符合规定。结论: 该方法简便、准确、可靠,可用于替格瑞洛中DCPA和DHPN的检测。
Objective: To establish an LC-MS/MS analytical method for the detection of 4, 6-dichloro-2- (propylthio)-5-aminopyrimidine (DCPA) and 5-nitrol-2-(propylthio) pyrimidine-4, 6-diol (DHPN) of genotoxic impurities in ticagrelor. Methods: The separation was performed on a Ultimate®UHPLC XB-Phenyl column(100 mm×2.1 mm, 1.8 μm) with mobile phase consisting of 0.1% formic acid solution(mobile phase A) and 0.1% formic acid methanol solution(mobile phase B) by gradient elution at a flow rate of 0.3 mL·min-1. The column temperature was 40 ℃. AB SCIEX ExionLC-QTRAP5500 triple quadrupole tandem ion trap liquid mass spectrometer was used for detection, electrospray ion source (ESI+), multiple reaction monitoring mode (MRM). The quantified ion pairs were m/z 240.1→197.8 (DCPA) and m/z 232.2→213.9 (DHPN). Results: The calibration curve was in a good linearity between the range of the concentration was 0.10-16.1 ng·mL-1 for DCPA and peak area (r=0.999 9). The limit of detection was 0.03 ng·mL-1 and the limit of quantification was 0.10 ng·mL-1; the average recovery was 97.5%(n=9), with RSD of 9.9%. The calibration curve was in a good linearity between the range of the concentration was 0.20-16.1 ng·mL-1 for DHPN and peak area (r=0.999 5). The limit of detection was 0.06 ng·mL-1 and the limit of quantification was 0.20 ng·mL-1. The average recovery was 98.1%(n=9), with RSD of 1.5%. In repeatability and intermediate precision experiments, the average concentration of DHPN was 7.97 μg·g-1 in the 100% limit concentration spiked solutions(n=12), with RSD of 4.9%;the average concentration of DCPA was 0.94 μg·g-1 in the test solutions, with RSD of 4.3%, indicating good precision. At room temperature, DCPA and DHPN were stable within 24 h, in the control solution, test solution and spiked test solution. In three batches of ticagrelor samples (batch number:Q19010701G, Q19010702G and Q19010703G), DHPN wasn’t detected, and the contents of DCPA were 1.2 μg·g-1, 1.1 μg·g-1 and 1.1 μg·g-1, respectively, which all met the requirements. Conclusion: The method is simple, accurate and reliable to detect DCPA and DHPN of ticagrelor.
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