Desenvolvimento de metodologias cromatográficas para o aprimoramento do controle de qualidade e autenticidade de óleos de copaíba (Copaifera sp. – Fabaceae)

Abstract

Copaifera multijuga oleoresins are predominantly found in the northern region of Brazil, occurring in the Amazon biome. They are used in natura consumption and in pharmaceutical formulations, due to their anti-inflammatory potential; being used as a raw material in the cosmetics and perfume industries as well, to obtain their essential oil and β-caryophyllene. In this study, two methodologies were developed using high performance thin layer chromatography (HPTLC), with the purpose of contributing to the improvement of the quality control of copaiba oils in a simple, fast and accessible way. The esterification of these oleoresins faces some obstacles, which ones were attenuated with an in situ esterification methodology proposed, using the reagent trimethylsilyldiazomethane (TMSD) and comparing it with the current and usual methodologies. The authenticity of copaiba oils was confirmed by gas chromatography – mass spectrometry (GC-MS), which is the most classic technique used for analysis of copaiba oils. The GC-MS analysis led up to the identification of 44 constituents, with a predominance of sesquiterpenes (86.21% - 96.26%). β-caryophyllene was the major constituent in 9 of the 10 oils analyzed (24.87% - 70.67%) and trans-α-bergamotene was the major constituent in only 1 oil (37.64%). The quantification of β-caryophyllene and its oxide showed a great variation among the specimens, and the concentration of β-caryophyllene ranged from 31.7 µg.mL-1 to 886 µg.mL-1 and of caryophyllene oxide varied from 4.0 µg.mL-1 to 27.2 µg.mL-1. The Relative Response Factor (RRF) was essential to quickly identify the main chemical markers of copaiba oils, without the need to isolate the constituents or esterify the oils, which is a necessary condition for the identification of diterpenes by GC. The developed method was applied to three C. multijuga oils and their main chemical markers were satisfactorily identified. The HPTLC also proved to be adequate in the detection of adulterants in the oils of this species. Adulterations with vegetable oils showed greater sensitivity in HPTLC and had a lower limit of visual detection (2.3%), unlike adulterations with diesel oil, which were detected only above 23%. The adulterations were confirmed by HPTLC-MS and chemometric analysis of HCA, PCA and PLS-DA. Regarding to the esterification methodologies, all of them caused some sort of damage to copaiba oils, some promoted the degradation and oxidation of the constituents and others, the formation of artifacts; however, all methodologies proved to be inefficient in the esterification of diterpenes; the esterification with sulfuric acid was the closest to the expected profile for an authentic copaiba oil, except by the number of diterpenes observed that was lower than expected. The esterification with TMSD proved to be adequate for copaiba oils, promoting the complete esterification of the diterpenes in a short reaction time, with a reduced number of steps and minimum sample amounts. All the proposed methodologies proved to be suitable for copaiba oils and can contribute satisfactorily to the improvement of the quality control of these oleoresins, in a great, speedy and accessible way.