Multiplex LC–MS/MS for simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D₃, albumin, and vitamin D-binding protein with its isoforms: One-step estimation of bioavailable vitamin D and vitamin D metabolite ratio

Bioavailable vitamin D and vitamin D metabolite ratio (VMR) have emerged as potential novel vitamin D markers. We developed a multiplex liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to determine all elements necessary for the calculation of bioavailable vitamin D and VMR, including 25-hydroxyvitamin D [25-(OH)D] and 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂D₃], VDBP and its isoforms, and albumin. Following separate reactions of hexane extraction and trypsin digestion, serum samples were analyzed using LC–MS/MS to measure 25-(OH)D₃, 25-(OH)D₂, 24,25-(OH)₂D₃, VDBP and its isoforms, and albumin. Analytical performances were assessed. Korean (n = 229), Arab (n = 98), White (n = 99) and Black American (n = 99) samples were analyzed. Bioavailable vitamin D and VMR were calculated. All target molecules were clearly separated and accurately quantified by LC–MS/MS. Analytical performances, including imprecision, accuracy, ion suppression, limit of quantification, linearity, and comparison with existing methods were within acceptable levels. The allele frequencies of VDBP isoforms in various races resulted similar to previously known values. The levels of bioavailable vitamin D were highest in White Americans and lowest in Black Americans. We have successfully developed a multiplex LC–MS/MS-based assay method that can simultaneously perform the measurement of all parameters needed to calculate bioavailable vitamin D and VMR. Our devised method was robust and reliable in terms of analytical performances and could be applied to routine clinical samples in the future to more accurately assess vitamin D status.