Fully automatic method for the determination of fat soluble vitamins and vitamin D metabolites in serum

Fat soluble vitamins and vitamin D metabolites are key compounds in bone metabolism. Unfortunately, variability among 25(OH)D assays limits clinician ability to monitor vitamin D status, supplementation, and toxicity.

0.5 ml serum was mixed with 0.5 ml 60% acetonitrile 150 mM sodium dodecyl sulfate, vortexed for 30 s and injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration, then on-line transferred to a reversed-phase analytical column by a 15% methanol-acetonitrile mobile phase at 1.0 ml/min for individual separation of the target analytes. Ultraviolet detection was performed at 265 nm, 325 nm and 292 for vitamin D metabolites, vitamin A and alpha- and delta-tocopherols, respectively.

Detection limits were between 0.0015 and 0.26 microg/ml for the target compounds, the precision (expressed as relative standard deviation) between 0.83 and 3.6% for repeatability and between 1.8 and 4.62% for within laboratory reproducibility. Recoveries between 97-100.2% and 95-99% were obtained for low and high concentrations of the target analytes in serum. The total analysis time was 20 min.

The on-line coupling of SPE-HPLC endows the proposed method with reliability, robustness, and user unattendance, making it a useful tool for high-throughput analysis in clinical and research laboratories.