Quantitative Determination of Clopidogrel and Clopidogrel Carboxylic Acid in Human Plasma by LC-MS-MS
Authors
Michael P. Sullivan, Orlando E. Espinosa, Christopher J.L. Buggé, Sherilyn Adcock, Jeffrey G. Stark
Introduction
Clopidogrel is an inhibitor of ADP-induced platelet aggregation. After administration clopidogrel is rapidly hydrolyzed to the inactive carboxylic acid metabolite, which routinely has been studied for PK response due to its longer residence time. Little information is available on the pharmacokinetics of the active clopidogrel itself. We developed a single method to determine concentrations of clopidogrel and its acid metabolite. We then looked at plasma concentrations of clopidogrel and its metabolite after administration of 75 mg of the reference drug in a single volunteer to determine the PK profile of both analytes.
Experimental
Sample Extraction- 0.200 mL of K 3 -EDTA human plasma
- Add internal standard solution
- Acidify sample
- Extract with organic solvent
- Evaporate organic extract and reconstitute
- Inject 3 µL onto the LC-MS-MS system
- HPLC Column: Varian Monochrom 3u, 100 X 4.6 mm
- Mobile Phase: acetonitrile/ammonium acetate (2mM) (600:140)
- Flow Rate: 1.0 mL/min
- Detector: Sciex 4000 mass spectrometer in positive ion mode (TurboIonspray)
- Ions Monitored:
Clopidogrel m/z 322 --> 155 Clopidogrel-D3 m/z 325 --> 158 Clopidogrel Carboxylic Acid m/z 308 --> 198 Clopidogrel Carboxylic Acid-D4 m/z 312 --> 202
Results
Analytical Methodology
Ranges:
Clopidogrel = 0.02 to 4.0 ng/mL
Clopidogrel Carboxylic Acid = 20.0 to 4000 ng/mL
Recovery:
Clopidogrel = 80%
Clopidogrel Carboxylic Acid = 60%
Stability
Extract Stability: 18 hours
Room Temperature Stability: 24 hours
Freeze/Thaw Stabitlity: 4 Cycles
Pharmacokinetic Data
Clopidogrel concentrations rose rapidly to a maximum of 2.1 ng/mL at 1h and clopidogrel carboxylic acid reached a maximum of 3020 ng/mL within 30 min of administration of a single 75 mg dose of clopidogrel. Quantifiable clopidogrel data were obtained over approximately 30 hours. Clopidogrel carboxylic acid concentrations were obtained over approximately 20 hours (time estimate from extrapolated concentration time-profile).
Conclusions
Clopidogrel may be the more important indicator for pharmacokinetics since it is the active moiety. However, the inactive metabolite has been extensively monitored in the past since it has been the easier to measure of the two. We have a rugged and sensitive method designed to monitor both the parent compound and the acid metabolite during investigations of oral dosages, and have tested its performance with clinical samples. The validated assay range is appropriate for pharmacokinetic studies of clopidogrel.