Rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in human serum by isocratic adsorption HPLC.

ABSTRACT

The simultaneous determination of alpha-tocopherol and retinol in human serum is reported. The separation is carried out by means of isocratic HPLC on adsorption columns. UV-Detection is possible by using either one wavelength for both compounds (300 nm), or after a lambda change mode with typical wavelengths for alpha-tocopherol (292 nm) and retinol (325 nm). According to short retention times (10 min) and rapid extraction the method is useful for clinical research and allows about 50 analyses per day and operator. Blood from 176 human volunteers was collected and alpha-tocopherol and retinol levels in serum determined with this method. Statistical evaluation of different selected groups shows typical significant differences of alpha-tocopherol and retinol concentrations in smokers and oral contraceptive users.

ABSTRACT

PIP An extraction and separation method for simultaneous vitamin A and E determination was developed to overcome problems of evaporation steps. The method did not involve an evaporation step and separation on adsorption columns by isocratic HPLC. It permits 50 analyses daily and operates with high sensitivity and reproducibility. Blood was collected from 115 healthy male and 63 healthy female volunteers ranging in age from 18-28 years. Retinol (25 mg) was dissolved in isopropanol and taken as stock solution. An aliquot of the stock solution was diluted and its absorbance measured at typical wave length against isopropanol and calculated. Alpha-Tocopherol was dissolved in isopropanol, an aliquot of this stock solution diluted its absorbance measured against isopropanol and calculated. Standard solutions were prepared by appropriate dilution of the measured stock solution. Retinol and a-tocopherol can be extracted in organic solvents after denaturation of the specific binding proteins by adding ethanol. A table shows that small amounts of serum and ethanol are sufficient to extract both vitamins quantitatively. Increasing a-tocopherol concentrations in ethanol can be extracted quantitatively in the hexane phase and, if added to hexane, the vitamin is not lost in the ethanol phase. The recovery study demonstrates different concentrations of the vitamin do not influence the quantitative extraction, and the recovery range indicates that the precision of the method is adequate within the linear range. The stock solution is stable more than 7 weeks when stored at -34 degrees Centigrade in glass vessels; the standard solutions should be prepared for each working day. The serum samples were stable more than 2 weeks when stored at -34 degrees Centigrade. The differences between the retinol values in selected groups are greater than the a-tocopherol differences. Retinol of females using oral contraceptives (OCs) was higher than of nonusers. This elevation was related to the estrogen content of the OCs. Cigarette smoking increased retinol levels slightly; in non-smokers the values were below the normal range. For OC users, the vitamin E levels did not show any significant differences. Cigarette smoking increased plasma vitamin A levels significantly but not vitamin E levels. The rapid simultaneous determination of retinol and a-tocopherol in plasma is useful in several clinical situations and in the nutritional assessment of normal subjects.