22 μm PTFE syringe filter. For the stationary phase, a monomeric C18 ODS2, 5 μm, 4.6 × 150 mm (Waters Spherisorb®, Wilmington, USA) was used. The mobile phase consisted of acetonitrile (containing 0.05% of triethylamine), methanol and ethyl acetate. A concave see more gradient was used for C. moschata ‘Menina Brasileira’ from 95:5:0 to 60:20:20 for 20 min, maintaining this proportion until the end of the run. For the C. maxima ‘Exposição’, a gradient from 98:2:0 to 60:20:20 for 20 min was used. Reequilibration took 15 min in both cases. The flow rate was 0.5 ml/min and column temperature was kept at 35 °C. The identification of the carotenoids was performed

considering (a) the combined information from chromatographic parameters (retention time and elution order), (b) UV–visible spectrum parameters (λmax and spectral fine structure % III/II) compared to standards and to data available in literature, (c) co-chromatography with standards and (d) chemical reactions to verify the type Selleck ZD1839 and position of the

substituents in the xanthophylls ( Pfander et al., 1994 and Schiedt and Liaaen-Jense, 1995). The chemical reactions were acetylation of secondary hydroxyl groups with acetic anhydride, methylation of hydroxyl groups in allylic position with acidified methanol, iodine catalysed isomerisation and epoxide-furanoxide rearrangement (5,6-epoxide–5,8-epoxide) with dilute HCl ( Rodriguez-Amaya, 1999). The major carotenoids in each sample were quantified by using calibration curves prepared from standards,

and the results were expressed as μg/g of sample. Standard curves were constructed with five different concentrations for each carotenoid, each point in duplicate, with lines passing by origin and coefficients of co-relation greater than or similar to 0.95. Violaxanthin was quantified with the standard curve of lutein, and the cis-isomers of β-carotene with the standard curve of all-trans isomer ( Assunção & Mercadante, 2003). Due to the difficulty of isolating the ζ-carotene standard, its quantification was performed Benzatropine through the standard curve of the all-trans-β-carotene. The standards used in this work were isolated from other plant species, such as carrots and green vegetables, by using open column chromatography (OCC), according to Kimura and Rodriguez-Amaya (2002), with a glass column of 2.5 × 25 cm packed with MgO:Hyflosupercel (1:1), activated for 2 h at 110 °C and developed with petroleum ether containing varying quantities of acetone and ethyl ether. Concentrations of the standard solutions were determined through a spectrophotometer (Hitachi, U-1800, Tokyo, Japan) and corrected according to their purity through HPLC, considering 90% as minimum purity to be used as standard. Many studies that propose to investigate retention of carotenoids in processed foods do not take into account the gain or loss of weight during processing through incorporation or through loss of water or water soluble solids.