title: Two-dimensional liquid chromatography coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3-epimer-25-monohydroxyvitamin D3.
abstract: 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.
authors.affiliation: University of Córdoba, Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, Córdoba, Spain; University of Córdoba, Agroalimentary Excellence Campus, ceiA3, Córdoba, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Mena-Bravo
authors.affiliation: University of Córdoba, Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, Córdoba, Spain; University of Córdoba, Agroalimentary Excellence Campus, ceiA3, Córdoba, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain. Electronic address: q72prcaf@uco.es.
authors.forename: F
authors.initials: F
authors.lastname: Priego-Capote
authors.affiliation: University of Córdoba, Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, Córdoba, Spain; University of Córdoba, Agroalimentary Excellence Campus, ceiA3, Córdoba, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain.
authors.forename: M D
authors.initials: MD
authors.lastname: Luque de Castro
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood; D002112:Calcifediol / Q000097:blood* / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling; D057230:Limit of Detection; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry / Q000378:metabolism*; D014815:Vitamins / Q000097:blood / Q000378:metabolism
chemical_list: D014815:Vitamins; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol
keywords: 3-epi-25(OH)D(3); DEQAS; Mass spectrometry; Mono/dihydroxy metabolites; NIST; Two-dimensional LC; Vitamin D