A method for quantitative analysis of vitamin D (both D2 and D3) and its main metabolites - monohydroxylated vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) and dihydroxylated metabolites (1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3) in human serum is here reported. The method is based on direct analysis of serum by an automated platform involving on-line coupling of a solid-phase extraction workstation to a liquid chromatograph-tandem mass spectrometer. Detection of the seven analytes was carried out by the selected reaction monitoring (SRM) mode, and quantitative analysis was supported on the use of stable isotopic labeled internal standards (SIL-ISs). The detection limits were between 0.3-75pg/mL for the target compounds, while precision (expressed as relative standard deviation) was below 13.0% for between-day variability. The method was externally validated according to the vitamin D External Quality Assurance Scheme (DEQAS) through the analysis of ten serum samples provided by this organism. The analytical features of the method support its applicability in nutritional and clinical studies targeted at elucidating the role of vitamin D metabolism.
Keywords: Automation; DEQAS; LC–MS/MS; Metabolites; Serum; Vitamin D.
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