Desenvolvimento de métodos analíticos para quantificação de artemeter e lumefantrina em comprimidos de dose fixa combinada e em plasma humano.

Abstract

Nowadays, malaria is the worlds most incident parasitic infection. The artemisinin based combination therapy (ACT) has been advocated as a promising treatment for malaria and artemetherlumefantrine (20+120 mg) is the main recommended association in endemic areas. In spite of the wide use of this association, the scientific literature is still scarce regarding analytical methods for the quantitation of artemether and lumefantrine in pharmaceutical products and biological matrices. The aim of the present work was developing and validating analytical methods for the quantitation of artemether and lumefantrineisolated in pharmaceutical raw materials and simultaneously in fixed dose combination tablets and human plasma. The analytical method described in the International Pharmacopeia 4th edition by HPLC-UV wasadapted and validated for the quantitation of artemether in pharmaceutical raw material. Three analytical methods were developed and statistically compared for lumefantrine determination: HPLC, UV and non aqueous titration. The chromatographic analysis was performed in a C18 column and mobile phase composed of methanol and 0.05% trifluoroacetic acid (80:20). The robustness of the HPLC method wasevaluated by means of Youdens test, which allowed analysing the influence of seven analytical parameters in the final result. In the spectrophotometric method, the detection was performed at 335 nm. The titulant employed in the volumetric method was 0.1 M perchloric acid in glacial acetic acid. All methods showed to be adequate for the quantitation of the drug in raw material, while for the tablet assay, HPLC and UV provided the most reliable results. The simultaneous quantitation of artemether and lumefantrine in fixed dose combination tablets was carried out by means of artemether standard addition method, by HPLC with cyano column, mobile phase composed of acetonitrile and 0.05% trifluoroacetic acid (60:40) and UV detection at 210 nm. The developed method complied with all required validation parameters e showed to beadequate for routine analysis. A bioanalytical method by HPLC with detection by mass spectrometry and eletrospray ionization in the positive mode was developed for the quantitation of artemether and lumefantrinein human plasma. For the extraction of the drugs from plasma, protein precipitation and artesunate as internal standard were employed. The chromatographic analysis was performed using a cyano column andthe employed transitions for the quantitation of artemether, lumefantrine and artesunate were m/z 316 m/z 267, m/z 530 m/z 348 e m/z 402 m/z 267, respectivelly. The method showed to be selective, precise,acurate, besides providing recovery rates higher than 80% for all drugs. Linearity of the method for artemether was proved in the range from 10 to 1000 ng/ml, while for lumefantrine, a quadratic curve was obtained in the range from 10 to 18000 ng/ml. The clinical study with volunteers allowed obtaining plasmatic absorption curves, as well as the pharmacokinetic parameters. The maximum plasmatic concentration of artemether, 57.4 ng/ml, was reached 1.9 h after the drug administration, whereas for lumefantrina, a maximum concentration of 1979.9 ng/ml was reached after 5.8 h. The study of the fragment structures and fragmentation routes of artemether and lumefantrine was also performed. The development of simple and rapid analytical methods is extremely important to evaluate the quality of the antimalarials distributed nowadays and to contribute to assuring the treatment efficacy, while the developed bioanalytical methodrepresents a useful tool for bioavailability and bioequivalence studies.Key wo