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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Mata-Granados
J M
JM
SANYRES (GRUPO PRASA), E-14012, Córdoba, Spain. jm.matagranados@gmail.com
Quesada Gómez
J M
JM
Luque de Castro
M D
MD
D001331:Automation; D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D002853:Chromatography, Liquid; D005223:Fats / Q000737:chemistry*; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D014807:Vitamin D / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism*; D014815:Vitamins / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D005223:Fats; D012997:Solvents; D014815:Vitamins; D014807:Vitamin D