Desenvolvimento de métodos cromatográficos para a determinação simultânea, em plasma humano, de fármacos usados no tratamento de doenças cardiovasculares.

Abstract

Hypertension and dyslipidemias are among the main risk factors for the development of cardiovascular diseases, which kill approximately 17 million people each year. There are several drugs and therapeutic schemes available for the treatment of these diseases. Therefore, methods for simultaneous analysis of several of these drugs can be useful in a wide variety of situations, from pharmacokinetic and therapeutic drug monitoring studies to toxicological analyzes. The objective of this study was to develop methods for the simultaneous determination of drugs used in the treatment of cardiovascular diseases. A method for the determination of olmesartan (OLM), irbesartan (IRB), losartan (LOS) and valsartan (VAL) in human plasma, by high performance liquid chromatography with fluorescence detection (HPLC-FL), was developed. The method development consisted in the optimization of excitation and emission wavelengths, chromatographic conditions and sample preparation. A Luna cyano column (250 x 4.6 mm; 5 µm) and a mobile phase consisting of 0.1% phosphoric acid (v/v) and acetonitrile (ACN) were employed. The analytes were detected using excitation wavelength of 260 nm and emission wavelengths of 370 nm and 410 nm. For sample preparation, two conventional (liquid-liquid extraction - ELL - and protein precipitation - PPT) and one miniaturized (microextraction in packed sorbent - MEPS) techniques were compared. PPT showed similar selectivity to the other techniques and better recoveries for the determination of OLM, IRB, LOS and VAL by HPLC-FL and was used for sample preparation. The method was validated, showing to be selective, linear, precise and accurate. A method for simultaneous determination of 9 antihypertensive and hypocholesterolemic drugs, by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), was also developed. The quantification and confirmation mass transitions for the analytes, the conditions of the ionization source, the chromatographic separation and sample preparation, which was carried out by vortex-assisted liquid-liquid microextraction (VALLME), were optimized. Chromatographic separation was performed using an Onyx Monolithic column and mobile phase consisting of 0.1% (v/v) formic acid and ACN. Suitable recoveries and lower limits of quantification were achieved using VALLME and HPLC-MS/MS with a run time of 8 minutes. The method was validated, showing to be selective, precise, accurate and linear for all analytes, except for verapamil, for which the curves were better fitted employing a quadratic model. After validation, the two developed methods were successfully applied for the analysis of plasma samples obtained from volunteers who are served at Ambulatório de Anticoagulação do Serviço de Hematologia e Oncologia do Hospital das Clínicas da UFMG and are users of antihypertensive and hypocholesterolemic drugs.