Bundit Yuangsoi, Orapint Jintasataporn, Nontavit Areechon, and Prathak Tabthipwon

pp. 693 - 700

Abstract

A densitometric Thin-layer Chromatographic (TLC) method of carotenoids such as astaxanthin, lutein, and β-carotene have been established and validated for quantitative determination of carotenoids in fancy carp serum. This study can be used in the evaluation of pharmacokinetic parameters of carotenoids in fancy carp serum. Analyses of carotenoids were performed on TLC glass plates pre-coated with silica gel 60 as the stationary phase. Linear ascending development was carried out in a twin trough glass chamber saturated with mobile phase consisting of petroleum ether-diethyl ether-acetone (75:15:10, v/v/v) at a temperature of 25±2°C. TLC scanner was used for spectrodensitometric scanning and analysis in absorbance mode at 450 nm. The system was found to give compact spots for astaxanthin, lutein, and β-carotene (R_f values of 0.21, 0.17 and 0.97, respectively). The method was validated for linearity, precision, accuracy, LOD, LOQ and HORRAT
value. The linear regression analysis data of astaxanthin, lutein, and β-carotene for the calibration plots showed a good linear relationship with r²
 = 0.999, 0.998 and 0.998, respectively, in a concentration range of 0.01-6.50 ug/spot with respect to the peak area. Precision (% RSD_r) of astaxanthin, lutein, and β-carotene was 2.93, 3.34, and 2.61, respectively. The limit
of detection (LOD) was 0.011, 0.023 and 0.026 µg/spot, respectively. The additionally limit of quantization (LOQ) was 0.036, 0.075 and 0.085 µg/spot, respectively. The percent recoveries of astaxanthin, lutein, and β-carotene spiked to sample blank showed an average of percent recoveries for astaxanthin (0.3-2.0 μg/ml) of 91.70%, for lutein (0.2-3.0 μg/ul) of
90.47%, and for β-carotene (0.1-1.0 μg/ul) of 102.25%. In all carotenoids, the HORRAT values were below the critical value. Therefore, this method enables simple, rapid, economical and precise quantitative determination of carotenoids in fancy carp serum for evaluated pharmacokinetic parameters. It is possible to use the established method for the routine analysis of carotenoids in serum.