Fully automated method for the determination of 24,25(OH)2 and 25(OH) D3 hydroxyvitamins, and vitamins A and E in human serum by HPLC.

A new fully automated method for the determination of metabolites of Vitamin D(3) and Vitamins A and E has been developed. A robotic station for liquid-liquid extraction, connected on line with an automatic system for solid-phase extraction (Prospekt) and a liquid chromatograph were used and the complexity of the overall method was overcome by full automation. The eluate from the chromatograph was monitored by a photodiode-array detector at three wavelengths, namely, 265 nm for Vitamin D(3) metabolites, 291 nm for Vitamin E and 325 nm for Vitamin A-which are the maximum absorption wavelengths for the analytes. The time required per sample analysis was 35 min because of the overlapping development of the steps. The linearity obtained for serum samples (standard addition method) gives correlation coefficients (r(2)) ranging between 0.996 and 0.989, with standard deviation of the slope between 4.0 and 4.9%. The repeatability was between 4.0 and 6.0% and the within-laboratory reproducibility was lower than 10.1% in all cases-both expressed as relative standard deviation-for low concentrations of the analytes, namely, 3 ng/ml for 24,25(OH)(2) dihydroxyvitamin D(3), 10 ng/ml for 25(OH) hydroxyvitamin D(3), 100 ng/ml for Vitamin A and 2 microg/ml for Vitamin E. The method has been validated using a CRM (NIST, SRM968c).