Automated method for the determination of fat-soluble vitamins in serum

Abstract

A new fully automated high-performance liquid chromatography (HPLC) method using 1 ml of serum has been developed for the determination of retinol (Vitamin A), α-tocopherol (Vitamin E), 25-hydroxyvitamin D 3 and 24 R,25-hydroxyvitamin D 3. The eluate was monitored with a photodiode-array detector at three wavelengths—namely: 265 nm for Vitamin D 3, 291 nm for Vitamin E and 325 nm for Vitamin A. The detection limits were equal to or lower than 1 ng ml −1 for all vitamins. The linearity obtained with serum samples (standard addition method) gives correlation coefficients ( r 2) ranging between 0.999 and 0.996 in all cases, with standard deviation of the slope between 3.2 and 1.6%. The repeatability was between 4.0 and 6.0% and the within-laboratory reproducibility was lower than 10% in all cases. The most outstanding features of the present method are its ease of use, its rapidity and fully automation, which enables its use for routine analysis. The time required per sample was 30 min, because the overlapped development of the steps. This method was used for the determination of normality range of these vitamins in healthy people in the 18–80-year-old interval.