e. trueness and precision (Dias, Camões, & Oliveira, 2008). This was, in fact, one of the goals of the present article: to validate the HPLC method previously developed
by our INCB024360 research group to quantify simultaneously total carotenes, tocopherols and tocotrienols. Furthermore, the method was used to quantify the presence of compounds in some Amazon oils. All solvents and reagents used in this study were of HPLC grade. The mobile phase used in the HPLC system was vacuum-filtered through a 0.45 μm filter (USA). Hexane was purchased from Mallinckrodt (USA) and isopropanol from Tedia (Brazil). α-, β-, δ- and γ-Tocopherol standards were purchased from Calbiochem (USA) and
the β-carotene standard from Fluka (Germany). Chromatographic analyses were carried out using a Shimadzu HPLC, series LC-20AT (Japan), equipped with a quaternary pump, an autosampler (SIL-20A), a degasser, and a SPD-M20A spectrophotometric detector (Photo Diode Array detector – PDA), which was set at 292 and 455 nm, and a RF-10AXL fluorescence detector, which was set at 290 nm for excitation and 330 nm for emission. Chromatographic separation of the compounds was achieved at 30 °C, using a normal-phase Lichrospher column (Merck, 250 × 4.6 mm id; 5 μm particle size) with a guard column (10 × 4.6 mm) purchased from Merck (Germany). The concentration gradient used was as follows: 0–7 min 99.5% hexane and 0.5% isopropanol; Etofibrate 7–9 min linear gradient of 0.5–1% isopropanol; Metformin 9–20 min 99.0% hexane and 1.0% isopropanol; 20–25 min reconditioning of the column with 0.5% isopropanol isocratic for 10 min. The flow gradient was: 0–4 min 1.0 mL min−1, 4–7 min linear gradient of 1–1.5 mL min−1, 7–9 min 1.0 mL min−1, 9–15 min linear gradient of 1.5–2.0 mL min−1, 15–17 min linear gradient of
2.0–1.0 mL min−1, 17–35 min 1.0 mL min−1. The total chromatographic run time was 35 min, being the time required for analysis of tocopherols. Although the analyses of carotenes and tocopherols were carried out simultaneously, calibration curves were performed separately due to the ease of preparing the standards separately. For the calibration curve of β-carotene, a 5 min run was used with a mobile phase composed of 99.5% hexane and 0.5% isopropanol, and a flow rate of 1.0 mL min−1. System control, data acquisition and processing were performed with an Intel-Celeron D PC, operated with Microsoft Windows XP Professional version 2002 and LC Solutions® version 2002 chromatography software with the system suitability option installed. Calibration curves were calculated by linear regression analysis of the peak area versus the concentration of the nominal standard for each compound.