Characterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to Multiplexed Liquid Chromatography-Tandem Mass Spectrometry

BACKGROUND: Immunoassays for 1 alpha,25-dihydroxyvitamin D [1 alpha,25(OH)(2)D] lack analytical specificity. We characterized the cross-reactivity of an anti-1 alpha,25(OH)(2)D antibody with purified vitamin D metabolites and used these data to map the chemical features of 1 alpha,25(OH)(2)D that are important for antibody binding. Additionally, we hypothesized that when combined with isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibody cross-reactivity could be used to semiselectively enrich for structurally similar metabolites of vitamin D in a multiplexed assay. METHODS: Sample preparation consisted of immunoaffinity enrichment with a solid-phase anti-1 alpha,25(OH)(2)D antibody and derivatization. Analytes were quantified with LC-MS/MS. Supplementation and recovery studies were performed for 11 vitamin D metabolites. We developed a method for simultaneously quantifying 25(OH)D-2, 25(OH)D-3, 24,25(OH)(2)D-3, 1 alpha,25(OH)(2)D-2, and 1 alpha,25 (OH)(2)D-3 that included deuterated internal standards for each analyte. RESULTS: The important chemical features of vitamin D metabolites for binding to the antibody were (a) native orientation of the hydroxyl group on carbon C3 in the A ring, (b) the lack of substitution at carbon C4 in the A ring, and (c) the overall polarity of the vitamin D metabolite. The multiplexed method had lower limits of quantification (20% CV) of 0.2 ng/mL, 1.0 ng/mL, 0.06 ng/mL, 3.4 pg/mL, and 2.8 pg/mL for 25(OH)D-2, 25(OH)D-3, 24,25(OH)(2)D-3, 1 alpha,25(OH)(2)D-2, and 1 alpha,25 (OH)(2)D-3, respectively. Method comparisons to 3 other LC-MS/MS methods yielded an r(2) value >0.9, an intercept less than the lower limit of quantification, and a slope statistically indistinguishable from 1.0. CONCLUSIONS: LC-MS/MS can be used to characterize antibody cross-reactivity, a conclusion supported by our multiplexed assay for 5 vitamin D metabolites with immunoenrichment in a targeted metabolomic assay.