HPLC-PDA METHOD FOR THE QUANTIFICATION OF PARACETAMOL IN PLASMA: APPLICATION TO PK/PD STUDIES WITH ARTHRITIC RATS

摘要

Objective: To develop and validate an easy, rapid, sensitive and selective high-performance liquid chromatography with photodiode diode-array (HPLC-PDA) detection method for quantification of paracetamol and to demonstrate its application in a pharmacokinetic–pharmacodynamic study with arthritic rats. Methods: Paracetamol was separated from plasma samples (50-100 μl) by a single protein precipitation step, prior to HPLC-PDA detection. The separation was performed on a Knauer Eurospher II, C 18 column 5 μm, 150 × 4.6 mm. The mobile phase comprised a mixture of water: methanol (75:25) and the flow rate was 1.1 ml/min. The detection wavelength was set at 245 nm. All analyses were carried out at room temperature (25 °C). Pharmacodynamics data were obtained with a gout-type pain model in rats. Results: The method was linear within a range of 0.2-200 μg/ml (R 2 ≥0.99). The intra-day and inter-day precision and accuracy expressed as coefficient of variation and relative error, respectively were below 10%. The lower limit of quantification was 0.2 μg/ml. Plasma samples were stable at least for 5 w at ?20° C. Conclusion: The validated method is sensitive, precise, accurate and specific as other more complex high-performance liquid chromatographic methods coupled to mass spectrometry (HPLC-MS), using small plasma samples (50-100 μl) and with a short time analysis (<5 min). The method was successfully applied to a pharmacokinetic-pharmacodynamic study of paracetamol in arthritic rats.