Talanta 67 (2005) 933–941 Development of a liquid–liquid extraction procedure for five 1,4-dihydropyridines calcium channel antagonists from human plasma using experimental design A.B. Baranda, N. Etxebarria, R.M. Jim´ enez, R.M. Alonso ∗ Department of Analytical Chemistry, Science and Technology Faculty, University of the Basque Country/EHU, Apdo. 644, 48080 Bilbao, Spain Received 15 November 2004; received in revised form 7 March 2005; accepted 7 April 2005 Available online 31 May 2005 Abstract 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, 25 cm × 4.6 mm i.d., 5 m column which was set at 30 ◦ C, using as mobile phase, a mixture of acetonitrile–water (70:30, v/v) containing 10 mM acetate buffer (pH 5) and setting the detector at a wavelength value of 360 nm. 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 6 ml 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. © 2005 Elsevier B.V. All rights reserved. Keywords: 1,4-Dihydropyiridines; Calcium channel antagonists; LLE; Plasma and method optimization 1. Introduction Calcium channel antagonists (CCA) and angiotensin en- zyme inhibitors (ACE) are used today as first-line therapy for hypertension. The 1,4-dihydropyridines (1,4-DHPs) are the largest class of CCAs and are typified by the drug nifedipine. The characteristic skeleton of this important group among the CCAs is the 1,4-dihydropyridinic structure exhibiting phenyl substitution in position 4. Structural variations of this proto- type concern primarily the ester functions and variations in phenyl substitution as well as changes in position 2 (Fig. 1). ∗ Corresponding author. Tel.: +34 94 601 2686; fax: +34 94 601 8500. E-mail address: qapalror@lg.ehu.es (R.M. Alonso). The 1,4-dihydropyridines studied in this work are: am- lodipine, nitrendipine, felodipine, lacidipine and lercanidip- ine. They act upon the L-type channel, which has a specific dihydropyridines site on its extracellular surface and bind more selectively to vascular calcium channel to those in the myocardium. Thus, the principal effect of the 1,4-DHPs is to reduce blood pressure, with minimal change in heart rate or cardiac output [1–6]. The determination of some of the above-mentioned com- pounds in plasma, serum or urine has been mainly carried out by liquid chromatographic methods with photometric, electrochemical or mass spectrometric detection [7–15]. Gas chromatography with different kinds of detection has also been widely used for this purpose [16–26]. 0039-9140/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2005.04.028