Rapid HPLC assay for the assessment of vitamin K1, A, E and beta-carotene status in children (7-19 years).

The method presented allows the determination of plasma concentrations of the fat soluble vitamins K1, A, E and beta-carotene. After the extraction of the plasma, the only clean-up step required consisted of washing the hexane-layer with a methanol/water mixture. The RP-HPLC system contained a zinc-filled reduction post-column and after chromatographic separation the vitamin K was detected by fluorescence. Only 0.5-1 ml plasma was required for this procedure. 2',3'-Dihydrophylloquinone was added as an internal standard. For phylloquinone the limit of detection was estimated at 0.09 nmol/l (0.04 ng/ml). Investigating a plasma pool with a mean concentration of 0.75 nmol phylloquinone/l plasma, the all-over coefficient of variation was 5.6% for within-day (n = 11) and 7.1% from day to day (n = 8). The rate of recovery was found to be in the range of 92-104% (the same pool as for the precision test was used, and the added amounts were 0.13, 0.5, 2.0 ng phylloquinone). From an aliquot of hexane-extract, the vitamins A, E and beta-carotene were separated by RP-HPLC. The concentrations of vitamins A, E and beta-carotene were measured on the UV and visible detector respectively. Coefficients of variation for all vitamins were < 10%. In a group of 7 to 19 year-old healthy children (n = 298), the concentrations of vitamins A, E, K1 and beta-carotene ranged from 0.34-2.10 mumol/l, 12.0-80.8 mumol/l, 0.09-2.22 nmol/l and 0.23-3.92 mumol/l respectively, and the medians were 1.02 mumol/l, 21.3 mumol/l, 0.49 nmol/l and 0.56 mumol/l respectively.