Stock solutions of α-, β-, δ- and γ-tocopherol were prepared by d

Stock solutions of α-, β-, δ- and γ-tocopherol were prepared by dissolving about 50 mg of each tocopherol fraction in 25 mL of hexane. Note that these stock solutions have the four tocopherol fractions in the same concentration. Serial dilution (37.50, 25.00, 17.50, 10.00, 5.00 and 2.50 mg L−1) of a 2 mg mL−1 tocopherol solution was carried out. Tocotrienols were quantified based on the area of tocopherol homologues. In the same way, stock solutions of β-carotene were prepared

by dissolving 5 mg in 25 mL of hexane. Serial dilution (10.00, 5.00, 2.50, 1.00, 0.50, 0.25, 0.10 and 0.05 mg L−1) of the 0.2 mg mL−1 β-carotene solution was then performed. Total carotenes were quantified based on the area of β-carotene. These calibration standards were freshly prepared in triplicate for each analytical run. Triplicates of quality control samples were prepared in hexane using the concentrations of 5.00 (LOQ), 15.00 and 35.00 mg L−1 for the tocopherol system and in concentrations of 0.10, 0.35 and 9.00 mg L−1 for β-carotene, as described above for the calibration standards. These quality control samples were used to investigate intra- and inter-run variations. A chromatographic validation run included

a set of calibration samples Cilengitide in vivo assayed in triplicate and quality control samples at three levels in triplicate, which was carried out on six separate occasions. The method validation was performed in accordance with the previously reported procedures (Marin et al., 2007, Shah et al., 2000 and USDHHS, 2001). Calibration curves

in the range of 2.5–37.5 mg L−1 for each tocopherol in hexane and in the range of 0.05–10.00 mg L−1 for β-carotene were plotted based on the peak-areas of each compound (axis y) against the respective nominal concentrations (axis x). All calibration curves were required to have a correlation coefficient of at least 0.9800. The intra- and inter-run accuracy and precision of the assays were assessed by the average relative percentage deviation (DEV%) from the nominal concentrations and the coefficient of variance (C.V.%) values, respectively, based on reported guidelines (Marin et al., 2007, Shah et al., 2000 and USDHHS, 2001). Precision (C.V.) and accuracy much (DEV%) were calculated from Eqs. (1 and 2): equation(1) CV(%)=SDAverage calculated concentration×100 equation(2) DEV(%)=1-Average calculated concentrationNominal concentration×100where SD stands for standard deviation. Intra-run precision and accuracy measurements were performed on the same day using tocopherol concentrations (n = 3) of 5.00, 15.00 and 35.00 mg L−1 in hexane and β-carotene concentrations (n = 3) of 0.10, 0.350 and 9.000 mg L−1. Inter-run precision and accuracy of the analytical method were determined simultaneously from the results of the calibration curve and quality control samples run on six days. Each set of quality control samples containing tocopherols or β-carotene was evaluated from recently obtained calibration curves.

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