OBJECTIVE

2

To establish a method for determining the contents of clopidogrel （CLP）， clopidogrel carboxylate （CLP-C）， clopidogrel acyl-

β

-D-glucuronide （CLP-G） and contents of clopidogrel active metabolite （CAM） in rat plasma， and to investigate their

in vivo

pharmacokinetic characteristics.

METHODS

2

The Shisedo CAPCELL ADME column was used with a mobile phase consisting of water and acetonitrile （both containing 0.1% formic acid） in a gradient elution. The flow rate was 0.4 mL/min， and the column temperature was maintained at 20 ℃. The injection volume was 2 μL. The analysis was performed in positive ion mode using electrospray ionization with multiple reaction monitoring. The ion pairs for quantitative analysis were

m

/

z

322.1→211.9 （for CLP），

m

/

z

308.1→197.9 （for CLP-C），

m

/

z

322.1→154.8 （for CLP-G），

m

/

z

504.1→154.9 [for racemic CAM derivative （CAMD）

]

. Six rats were administered a single intragastric dose of CLP （10 mg/kg）. Blood samples were collected before medication and at 0.08， 0.33， 0.66， 1， 2， 4， 6， 10， 23 and 35 hours after medication. The established method was used to detect the serum contents of various components in rats. Pharmacokinetic parameters were then calculated using WinNonlin 6.1 software.

RESULTS

2

The linear ranges for CLP， CLP-C and CAMD were 0.08-20.00， 205.00-8 000.00， and 0.04-25.00 ng/mL， respectively （

r

≥0.990）. The relative standard deviations for both intra-day and inter-day precision tests were all less than 15%， and the relative errors for accuracy ranged from －11.68% to 14.40%. The coefficients of variation for the matrix factors were all less than 15%， meeting the requirements for bioanalytical method validation. The results of the pharmacokinetic study revealed that， following a single intagastric administration of CLP in rats， the exposure to the parent CLP in plasma was extremely low. Both the area under the drug concentration-time curve （AUC

0-35 h

） and the peak concentration of the parent CLP were lower than those of its metabolites.The AUC

0-35 h

of the active metabolite CAM was approximately 43 times that of CLP， though it had a shorter half-life （2.53 h）. The inactive metabolite CLP-C exhibited the highest exposure level， but it reached its peak concentration the latest and was eliminated slowly. The AUC

0-35 h

of CLP-G was about four times that of CAM， and its half-life was similar to that of CLP-C.

CONCLUSIONS

2

This study successfully established an liquid chromatography-tandem mass spectrometry method for the determination of CLP and its three metabolites， and revealed their pharmacokinetic characteristics in rats. Specifically， the parent drug CLP was rapidly eliminated， while the inactive metabolites CLP-C and CLP-G exhibited long half-lives， and active metabolite CAM displayed a transient exposure pattern.