A Validated Method for the Quantification of Clopidogrel in Human Plasma at the 2.5 pg/mL Level Using Xevo TQD, ACQUITY UPLC H-Class System, and UNIFI Scientific Information System

Importance of the Topic

Clopidogrel is a widely prescribed antiplatelet prodrug crucial for preventing atherosclerotic events. Accurate quantification of its low circulating concentrations is essential for pharmacokinetic studies, therapeutic monitoring, and drug development.

Objectives and Study Overview

This study aimed to develop and validate a highly sensitive LC-MS/MS method for the quantification of clopidogrel in human plasma at a lower limit of quantification of 2.5 pg/mL, employing solid phase extraction and rapid UPLC separation.

Methodology

Sample preparation involved Oasis MCX µElution SPE of 350 µL plasma spiked with deuterated internal standard and buffered. After conditioning and washing, analytes were eluted with ammonium hydroxide in IPA/ACN, diluted, and a 10 µL aliquot injected. Chromatographic separation was achieved on a C18 column (2.1×50 mm, 1.7 µm) under a 3-minute gradient at 600 µL/min. Detection used positive ion electrospray MRM transitions 322→212 for clopidogrel and 326→216 for the internal standard.

Instrumentation Used

• ACQUITY UPLC H-Class System
• Xevo TQD Mass Spectrometer
• Oasis MCX µElution SPE Plate
• UNIFI Scientific Information System

Results and Discussion

Clopidogrel eluted at 2.10 minutes with sharp, symmetrical peaks and negligible carryover. The assay demonstrated linearity from 2.5 to 500 pg/mL (r ≥ 0.9988) with a 3-minute runtime enabling analysis of a 96-well plate in 5 hours. Intra-day precision at the LLOQ ranged from 5.8% to 12.2% (bias -6.9% to -13.6%), and for high QC (300 pg/mL) precision was 1.2% to 2.9% (bias 1.4% to 1.6%). Inter-day precision at the LLOQ was 8.5% (bias -9.4%) and at high QC 2.3% (bias 1.5%).

Benefits and Practical Applications

The validated method offers exceptional sensitivity and throughput for bioanalytical studies, enabling reliable pharmacokinetic profiling and therapeutic monitoring of clopidogrel at trace levels.

Future Trends and Opportunities

Advancements may include automation of sample processing, integration with high-resolution MS for metabolite profiling, and extension to personalized medicine approaches and multi-analyte panels.

Conclusion

A robust LC-MS/MS assay with an LLOQ of 2.5 pg/mL for clopidogrel in human plasma was validated, showing excellent precision, accuracy, and throughput. This method supports low-level quantification needs in pharmacokinetic and clinical research.

Reference

1. Pereillo JM, Maftouh M, Andrieu A, Uzabiaga MF, Fedeli O, Savi P, Pascal M, Herbert JM, Maffrand JP, Picard C. Structure and stereochemistry of the active metabolite of clopidogrel. Drug Metabolism and Disposition. 2002;30:1288-1295.