Abstract
Species of Copaifera genus (Fabaceae – Caesalpinoiodidaeae) produces an important commercial oleoresin that displays many medicinal properties. Copaifera oleoresins (COR) are composed mainly of a mixture of diterpenes and sequiterpenes, and the main reported acid diterpenes for this genus are kaurenoic, copalic, hardwickiic and polyaltic acids. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for identification and quantification of nine acid diterpenes. The developed method was applied in the analyses of 10 authentic COR samples collected in the North and Southeast of Brazil and six commercial COR samples. Samples preparation consisted of simple dilution of oleoresins in methanol followed by filtration. Validation parameters of the method for nine acid diterpenes were satisfactory: selectivity/specificity was defined by retention time and MS/MS analyses for each analyte; generally all analytical curves presented r2>0.99, Lack-of-fit test not significant and RSD<20% for all concentration levels; limit of detection and limit of quantification were on the scale of nanogram per milliliter; inter- and intra-day precision and accuracy were adequate. Regarding the robustness, the method was sensible to small deliberate variations of temperature and additives to the mobile phase, such as formic acid and ammonium hydroxide. Results of 16 analyzed samples of COR showed qualitative and quantitative differences of acid diterpenes among all samples. The diterpenes ent-kaurenoic acid 1, ent-polyalthic acid 3, ent-copalic acid 5 and, ent-3-β-acetoxy copalic acid 9 were found with more frequency in COR analyzed samples. Additionally, the content of the acid diterpenes found in 16 Copaifera oleoresin samples was analyzed by Principal Component Analysis (PCA), suggesting a botanical origin for the commercial samples. The developed UPLC method was shown to be reliable for the analysis of acid diterpenes in commercial Copaifera oleoresins.
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