Determination of Clopidogrel and Its Three Metabolites Concentrations in Human Plasma by LC-MS/MS
- VernacularTitle:液相色谱-质谱联用法测定人血浆中氯吡格雷及其3种代谢产物的浓度
- Author:
Zhirui LIU
1
;
Pu YAO
1
;
Bo YANG
1
;
Yu WANG
1
;
Wei FENG
1
;
Fengjun SUN
1
Author Information
1. Dept. of Pharmacy，the First Affiliated Hospital of Army Medic al University，Chongqing 400038，China
- Publication Type:Journal Article
- Keywords:
LC-MS/MS;
Clopidogrel;
Metabolites;
Plasma concentration
- From:
China Pharmacy
2019;30(21):2946-2951
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE： To establish the method for simultaneous determination of clopidogrel （CLP）， its intermediate metabolite （2-O-CLP）， inactive metabolite （CLPCA） and active metabolite （CLPTM） in human plasma. METHODS： Totally 90 patients diagnosed as stroke were selected from the First Affiliated Hospital of Army Medical University. They were given one CLP tablet （75 mg/tablet） orally on an empty stomach in the morning. Blood samples were collected 2 h after taking the tablet. CLPTM- D was formed by derivation of CLPTM with 2-bromo-3’-methoxyacetophenone and extracted by precipitation of acetonitrile protein together with the other three substances to be measured. LC-MS/MS method was adopted. The determination was performed on Agilent poroshell 120 EC-C18 column with mobile phase consisted of acetonitrile （0.1％ formic acid） and water （0.1％ formic acid） （90 ∶ 10， V/V）. The quantitation analysis was performed using multiple reaction monitoring at the specific ion transitions of m/z 308.1→198.1 （CLPCA）， 322.3→212.0 （CLP）， 338.3→155.0 （2-O-CLP）， 504.4→354.1 （CLPTM-D） and 264.0→154.1 （ticlopidine， internal standard）， respectively. RESULTS： The retention time of CLPCA， CLP， 2-O-CLP， CLPTM-D and internal standard were 2.01， 3.32， 2.83， 2.68， 1.87 min， respectively. The linear range of CLPCA， CLP， 2-O-CLP and CLPTM-D were 100-10 000， 0.2-20， 0.3-30， 0.5-50 ng/mL （all r≥0.999 5）. The intra-day and inter-day RSD were all less than 9.5％ （n＝5）. Accuracy ranged from 93.5％-98.9％ （n＝5）， and extraction recovery was from 85.4％ to 95.9％ （n＝5）. The matrix effect ranged from 2.7％-6.2％ （n＝5）. In stability tests （storing at －80 ℃ for 3 months， 3 freeze-thaw cycles， storing at 4 ℃ for 8 h）， RE of CLP， CLPCA and CLPTM-D were all lower than 10.0％ （n＝5）. CONCLUSIONS： Established LC-MS/MS method has the advantages of high specificity， accuracy and reliability， and can be used to detect the concentration of CLP and its three metabolites in human plasma.
