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.
Key words
4,6-dichloro-2-(propylthio)-5-aminopyrimidine /
5-nitrol-2-(propylthio) pyrimidine-4, 6-diol /
the receptor inhibitors of P2Y12 /
genotoxic impurity /
ticagrelor /
LC-MS/MS
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