The Limit of detection
(LOD) was defined as the lowest concentration to be detected, taking into consideration a signal-to-baseline noise ratio larger than 3 (Marin et al., 2007, Shah et al., 2000 and USDHHS, 2001). According to the FDA Guidance, solvent evaporation stability during storage in the autosampler for a 24 h period was established at five concentrations of 37.50, 25.00, 17.50, 10.00 and 5.00 mg L−1 in triplicate and was tested only for tocopherols. Considering that solvent evaporation would affect the concentrations of tocopherols and carotenes in the same proportion, INCB018424 nmr no specific stability test was required for carotenes. Three Amazon oils were selected: Buriti (Mauritia Flexuosa), Patawa (Oenocarpus bataua) and Tucuma (Astrocaryum aculeatum). Samples were dissolved in hexane and aliquots of 20 μL were injected in the HPLC system. The following fruits pulps were purchased at local markets in the Amazon Region:
Buriti pulp was acquired in Abaetuba (Pará, Brazil), and Patawa and Tucuma pulps in Belém (Pará, Brazil), during harvest time. Thirty fruits of each specie were gathered in three different places which were separated by a distance of at least two kilometers from each other, adding up to 90 fruits from each specie. The Bligh and Dyer (1959) method was used to extract oils from the dried pulps. The total lipid fraction was learn more extracted by exhaustive maceration with
chloroform and methanol, followed by filtration of solids and separation of the solvent/fat layer. Dried samples (10% moisture) were used to facilitate extraction with organic solvents. All data are presented as mean values ±SD and the mean values were analysed by one-way ANOVA and Tukey-HSD triclocarban at p < 0.05 with SAS. Reproducible separation of β-carotene was obtained in the same silica normal-phase column used for tocopherol analysis. Retention time of β-carotene is 1.9 min, showing that this compound has lower affinity with the column. Peaks are sharp, symmetrical and all homologues were efficiently separated (Fig. 1). Tocopherols were analysed using both PDA and fluorescence detectors. Retention times for tocopherols using the fluorescence detector were, respectively, 7.6, 16.6, 19.9 and 29.1 min for the α-, β-, γ- and δ-tocopherol homologues. For the PDA detector, retention times were 7.2, 16.4, 19.3 and 28.5 min, respectively, for the α-, β-, γ- and δ-tocopherol homologues. Note that retention times for PDA were lower than for fluorescence. This difference is due the system configuration: the samples pass through the PDA detector and then the fluorescence detector. It is also important to highlight that retention times can vary slightly on different days and analysis.