Development of a liquid-liquid extraction procedure for five 1,4-dihydropyridines calcium channel antagonists from human plasma using experimental design.

A liquid-liquid extraction method using diethyl ether as organic solvent was optimized simultaneously for five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine) belonging to the group of calcium channel blockers. Some experimental tools such as a full factorial design, a central composite design and the Multisimplex program were used to optimise the concentration of NaOH, volume of organic solvent and shaking time as main factors that influence the liquid-liquid extraction procedure. Following the extraction, the quantitation of the 1,4-dihydropyridines concentrations were performed by high-performance liquid chromatography with diode-array detector. Therefore, the studied compounds were separated quantitatively on a Supelcosil ABZ+Plus, 25cmx4.6mm i.d., 5mum column which was set at 30 degrees C, using as mobile phase, a mixture of acetonitrile-water (70:30, v/v) containing 10mM acetate buffer (pH 5) and setting the detector at a wavelength value of 360nm. It was concluded that the main factors that influence in the extraction process were the volume of organic solvent and the shaking time. The Multisimplex program suggested as optimal conditions an average of 6ml of organic solvent and 23min of shaking time. For these values, the optimised liquid-liquid extraction method showed good values of recoveries (80% for amlodipine and higher than 90% for the rest of the compounds) and low values of R.S.D. (<10%) in the reproducibility of the extraction what makes it reliable for the quantification of all the studied compounds in human plasma.