Optimization and validation of a SPE-HPLC-PDA-fluorescence method for the simultaneous determination of drugs used in combined cardiovascular therapy in human plasma

This paper reports the chemometrical optimization and the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-Fluo) method for the simultaneous analysis, in human plasma, of drugs usually combined in cardiovascular therapy. Separation of chlorthalidone (CLTD), valsartan (VAL), valsartan-M1 (VAL-M1), fluvastatin (FLUV) and the internal standard (IS) candesartan cilexetil was performed on a dC18 Atlantis column (100 mm × 3.9 mm, 3 μm) using a gradient with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% of formic acid and 10 mM of ammonium formate at pH 4.1. UV and fluorimetric (valsartan, its metabolite and fluvastatin) detectors were used. The sample preparation consisted of protein precipitation using acetonitrile suited to a solid-phase extraction (SPE) on a Strata-X cartridge for sample clean-up. Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) organizations. The method showed good linearity (31–3000 μg/l for chlorthalidone, 20–1000 μg/l for valsartan-M1, 10–5000 μg/l for valsartan and 14–1000 μg/l for fluvastatin), precision and accuracy. Recoveries were in the range of 78–91%. This method allowed the determination of these drugs in human plasma samples obtained from patients under cardiovascular treatment.