Simultaneous determination of retinol, carotenoids and tocopherols in human serum by high pressure liquid chromatography

The determination of serum vitamins having antioxidant properties has gained in importance in recent years. This is mainly due to the observation that an inverse correlation exists between blood levels of these vitamins, including retinol, carotenoids and tocopherol, and diet-related chronic diseases such as coronary heart disease and cancers. This laboratory has been carrying out a series of studies into the nutritional and analytical aspects of retinol and carotenoids. A simple reversed-phase HPLC method has been developed in an effort to improve methodologies for the separation and quantitation of carotenoids and retinol in foods and biological specimens, especially blood serum. As an extension to these studies, trials were carried out to determine the feasibility of analysing tocopherols using the same chromatographic procedure. With the addition of another detector wavelength, the same procedure detected and quantitated 3 major tocopherols simultaneously with retinol and five carotenoids. Within-day and between-day precision of the procedure was satisfactory. Trials carried out were able to improve recovery of the vitamins. Experiments conducted also showed that the addition of ascorbic acid to the extracting ethanol was beneficial for the analytical procedure. The presence of peroxide in ethyl acetate used in the chromatography mobile phase caused drastic destruction to the vitamins analysed. The addition of ascorbic acid during sample preparation was able to inhibit this destruction. The method was used for the analysis of sera from 65 apparently healthy Malaysians with a mean age of 52.8 years (range 24-76 years). Mean retinol concentration of the group was 69.8 ± 18.8 mg/dl. The mean β-carotene concentration of the subjects studied was 33.8 ± 24.3 mg/dl, while the mean total carotenoid concentration was 180.2 ± 3.0 mg/dl. The most abundant carotenoid in the serum samples studied was lutein, comprising about one-third of all carotenoids quantitated. The concentrations of δ- and γ-tocopherols in the serum samples studied were too low to be identified with certainty and quantitated accurately. The mean α-tocopherol level was 1840 ± 528 μg/dl. For retinol, α-tocopherol and most of the carotenoids determined, there was no statistically significant difference in the mean levels between male and female subjects as well as among the three different ethnic groups. Results obtained in this study were very similar to those previously reported by this laboratory. It is hoped that more data on the serum concentrations of these vitamins can become available for various population groups, including during various disease conditions.