The extract was concentrated in a rotary evaporator at a temperature of 35–37 °C. Next, the carotenoids were dissolved in 25 ml petroleum ether and stored frozen (at about −5 °C) in amber glass flasks until the time for chromatographic analysis. The samples were protected from light throughout the process of chemical analysis using amber
glass ware and aluminum wrapping. The presence of ascorbic acid and carotenoids in fruits was analysed by HPLC using a Shimadzu liquid chromatography system (model SCL 10AT VP) equipped with a high-pressure pump (model LC-10AT VP), automatic loop injector (50 μl; model SIL-10AF), and UV/visible detector (diode array; model SPD-M10A). The system was controlled with the Multi System software, Class VP 6.12. AA was analysed this website using the method optimised by Campos et al. (2009).
The mobile phase consisted of 1 mM monobasic sodium phosphate (NaH2PO4) and 1 mM EDTA, with the pH adjusted to 3.0 with phosphoric acid (H3PO4), and was eluted isocratically on a Lichospher 100 RP18 column Everolimus cost (250 × 4 mm, 5 μm; Merck, Germany) at a flow rate of 1 ml/min. AA was detected at 245 nm. Carotenoids were analysed using the chromatographic conditions described by Pinheiro-Sant’Ana et al. (1998), with some modifications. The mobile phase consisted of methanol:ethyl acetate:acetonitrile (50:40:10) and was eluted isocratically at a flow rate of 2 ml/min on a Phenomenex C18 column (250 × 4.6 mm, 5 μm) coupled to a Phenomenex ODS guard column (C18, 4 × 3 mm). β-Carotene and lycopene were detected at 450
and 469 nm, respectively. AA, lycopene and β-carotene were identified in the samples by comparison of the retention Montelukast Sodium times obtained with those of the respective standards analysed under the same conditions, and by comparison of the absorption spectra of the standards and peaks of interest in the samples using a diode array detector. Recovery of AA, lycopene and β-carotene was analysed, in triplicate, by the addition of the standard to persimmon, acerola and strawberry samples at a proportion of 20–100% of the average original content in the samples. The linear range was determined by injection, in duplicate, of five increasing concentrations of the standard solutions of AA, lycopene and β-carotene under the same chromatographic conditions as those used for sample analysis. The limit of detection was calculated as the minimum concentration able to provide a chromatographic signal three times higher than the background noise (Rodriguez-Amaya, 1999). The limit of quantification was calculated as the minimum concentration able to provide a chromatographic signal five times higher than the background noise (Rodriguez-Amaya, 1999).