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Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma. Use of incurred samples to test back-conversion.

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2010-10-20)
Luigi Silvestro, Mihaela Cristina Gheorghe, Isabela Tarcomnicu, Silviu Savu, Simona Rizea Savu, Adriana Iordachescu, Constanta Dulea
ABSTRACT

Quantitative methods using LC-MS/MS allow achievement of adequate sensitivity for pharmacokinetic studies with clopidogrel; three such methods, with LLOQs as low as 5 pg/mL, were developed and fully validated according to the well established FDA 2001 guidelines. The chromatographic separations were performed on reversed phase columns Ascentis RP-Amide (15 cm x 2.1 mm, 5 μm), Ascentis Express C8 (10 cm x 2.1 mm, 2.7 μm) and Ascentis Express RP Amide (10 cm x 2.1 mm, 2.7 μm), respectively. Positive electrospray ionization in MRM mode was employed for the detection and a deuterated analogue (d3-clopidogrel) was used as internal standard. Linearity, precision, extraction recovery, matrix effects and stability tests on blank plasma spiked with clopidogrel and stored in different conditions met the acceptance criteria. During the analysis of the real samples from the first pharmacokinetic study, a significant increase (>100%) of the measured clopidogrel concentrations in the extracts kept in the autosampler at 10 °C was observed. Investigations led to the conclusion that most probably a back-conversion of one or more of the clopidogrel metabolites is occurring. The next methods were optimized in order to minimize this back-conversion. After a series of experiments, the adjustment of the sample preparation (e.g. processing at low temperature and introducing a clean-up step on Supelco HybridSPE-Precipitation cartridges) has proven to be the most effective in order to improve the stability of the extracts. Incurred samples of real subjects were successfully used in the validation of the last two analytical methods to evaluate the back-conversion, while tests using only the known metabolites could not detect this important problem.

MATERIALS
Product Number
Brand
Product Description

Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 15 cm × 75 μm
Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 500 μm
Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 75 μm
Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 200 μm
Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 300 μm
Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 100 μm
Supelco
Ascentis® C8 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 10 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 10 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 3 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 21.2 mm
Supelco
Ascentis® Express C8 Capillary, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 15 cm × 500 μm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 2 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 5 cm × 2.1 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 3 cm × 3 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 10 cm × 2.1 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 15 cm × 3 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 10 cm × 3 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 3 cm × 4.6 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 3 cm × 2.1 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 2.1 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 3 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 7.5 cm × 3 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 5 cm × 4.6 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 7.5 cm × 4.6 mm
Supelco
Ascentis® Express RP-Amide, 2.7 μm HPLC Column, 2.7 μm particle size, L × I.D. 10 cm × 4.6 mm