DEVELOPMENT AND VALIDATION OF THE UPLC-ESIMS/ MS METHOD FOR METABOLITE ANALYSIS IN ARTEMISININ BIOSYNTHESIS

• Main Author: Adiwijaya, Royyan
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/70489
• Institution: Institut Teknologi Bandung

Background and purpose: Malaria is a disease with a very high severity rate, especially in tropical areas where P. falciparum predominates. Artemisinin is the best and safest curative option after the previously used chloroquine derivatives experienced cases of high resistance. Artemisinin is obtained from conventional or semisynthetic methods involving artemisinin biosynthesis to convert substrates into metabolites. This study aims to develop and validate the UPLC-ESI-MS/MS method to analyze the biosynthetic metabolites of artemisinin. Methods: The method of extracting dried Artemisia annua leaf samples is ultrasonication while the standard analysis method for artemisinic acid (AA), dihydroartemisinic acid (DHAA), artemisinin (ART), and extracts is ultra-high performance liquid chromatographyelectrospray ionization tandem mass spectrometry (UPLC-ESI- MS/MS). Results: The specificity test showed the absence of solvent peaks on all standard chromatograms of metabolites. The linearity of the standard curve is above 0,995, respectively. The sensitivity value of the instrument to artemisinic acid was LOD: 0,35 mg/L and LOQ: 1,049 mg/L. The sensitivity values for dihydroartemisinic acid are LOD: 0,17 mg/L and LOQ: 0,53 mg/L. Sensitivity values for artemisinin were LOD:0,018 mg/L and LOQ: 0,054 mg/L. In intra-day testing, the percent recovery of all standards was in the range of 97-103% (100,48% AA, 99,43% DHAA, and 101,26% ART) and %RSD of all standards was below the 2% requirement for AUC response and retention time. Extract test results showed a content of 0,048% w/w AA, 0,21% w/w DHAA, and 0,80% w/w ART relative to dry leaves weight. Conclusion: From the experimental results, it can be concluded that the UPLCESI- MS/MS method has met the validation requirements by testing the parameters of specificity, linearity, sensitivity (LOD and LOQ), accuracy, and precision. The validated UPLC-ESI-MS/MS method successfully analyzed samples of the methanol extract of Artemisia annua cultivated locally in Lembang, Bandung City, Indonesia.