A method based on automated on-line solid phase extraction coupled to two-dimensional liquid chromatography with tandem mass spectrometry detection (SPE-2DLC-MS/MS) is here reported for vitamin D metabolite profiling in human serum with absolute quantitation. Two-dimensional LC was configured with two complementary analytical columns, pentafluorophenyl (PFP) and C18 phases, for determination of 25 hydroxyvitamin D3 epimers and the resting bioactive metabolites of vitamin D (D3 and D2)-25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2 and 24,25-dihydroxyvitamin D3. Quantitative determination was supported on the use of a stable isotopic labelled internal standard for each analyte and the resulting method was validated by analysis of a standard reference material certified by the National Institute of Standards & Technology (NIST-972a) and 5 samples provided by the vitamin D External Quality Assurance Scheme (DEQAS). The limits of detection were between 9 and 90pg/mL for the eight analytes, and precision, expressed as relative standard deviation, was lower than 11.6%. Two-dimensional LC has shown to be the key to discriminate between 25 hydroxyvitamin D3 epimers in a quantitative analysis also involving dihydroxyvitamin D metabolites.
Keywords: 3-epi-25(OH)D(3); DEQAS; Mass spectrometry; Mono/dihydroxy metabolites; NIST; Two-dimensional LC; Vitamin D.
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