Desenvolvimento e validação de métodos analíticos e bioanalíticos para os fármacos anlodipino e olmesartana medoxomila, em dose fixa combinada

Abstract

The treatment of hypertension with a single drug is efficient for only one-third of the patients. In order to maximize the efficacy of the treatment, drugs that act in different pathophysiological mechanisms are associated. The combination of olmesartan medoxomil and amlodipine besylate, an angiotensin II type 1 receptor antagonist and calcium channel blocker, respectively, in fixed dose combination tablets is effective, safe and well tolerated. Because it is a new association that is still protected by a patent, there are few articles in the scientific literature that described analytical and bioanalytical methods for the simultaneous quantification of these drugs. An analytical method by high performance liquid chromatography (HPLC) with ultraviolet detection was developed and validated for simultaneous quantification of amlodipine and olmesartan medoxomil in fixed dose combination. The chromatographic analysis was performed in a C18 column, mobile phase composed of acetonitrile:methanol:0.3% triethylamine (30:30:40) pH 2.75 and a flow rate of 1.0 mL/min. Injection volume was 10 µL, detection was at ë 238 nm and run time was 5.5 minutes. Then, the analytical method was transferred to ultra performance liquid chromatography (UPLC) with ultraviolet detection using mathematical formulas to calculate the new injection volume and new flow rate of mobile phase, which were 0.7 µL and 0.613 mL/min., respectively. Adjustments in the solvent composition of mobile phase were performed to improve the efficiency of the UPLC method. The optimized mobile phase was composed of acetonitrile:methanol:0.3% triethylamine (26:26:52) pH 2.75. At this condition, the run time was 1.25 minutes, which provided a reduction of approximately 77% in the analysis time and 86% in solvent consumption. After the method optimization by UPLC, the method was validated. The HPLC method showed to be statistically equivalent to the UPLC method after analysis of three batches of the product BenicarAnlo®. Thus, both methods may be used to perform quality control of olmesartan medoxomil and amlodipine in fixed dose combination tablets. A bioanalytical method was also developed and validated by HPLC with tandem mass spectrometry detection and electrospray ionization in the positive mode for quantifying amlodipine and olmesartan in human plasma. Felodipine and valsartan were used as internal standards of amlodipine and olmesartan, respectively. The sample preparation was carried out using the technique of liquid-liquid extraction. The chromatographic analysis was performed in a C18 column, mobile phase composed of methanol:0.05% formic acid (75:25) and a flow rate of 1.0 mL/min. The transitions employed for the quantification of amlodipine, olmesartan, felodipine and valsartan were m/z 409.4 237.7, m/z 447.6 206.8, m/z 384.7 338.2, m/z 436.5 291.0, respectively. The bioanalytical method showed to be selective, precise, accurate and linear over the range of 1 to 100 ng/mL for amlodipine and 3 to 1800 ng/mL for olmesartan. Therefore, it may be applied in future studies of therapeutic drug monitoring and bioequivalence.