Quantification of 1α,25-Dihydroxy Vitamin D by Immunoextraction and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

BACKGROUND: 1α,25-dihydroxy vitamin D [1,25(OH)2D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution/ultra-performance liquid chromatography (UPLC)–tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)2D. We developed a method for simultaneous quantification of 1,25(OH)2D2 and 1,25(OH)2D3 with a 4.6-min instrument cycle time. Results are available 36 h after sample preparation begins. METHODS: Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)2D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization–tandem mass spectrometry. We used hexadeuterated 1,25(OH)2D3 and 1,25(OH)2D2 as internal standards and performed method comparisons against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory. RESULTS: 1,25(OH)2D3 intraassay and interassay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)2D2 intraassay and interassay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were both 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of −4.1, and Pearson correlation (r2) of 0.31. Comparison with a reference LC-MS/MS assay had a proportional bias of 0.89, constant bias of 3.7, and r2 of 0.88. CONCLUSIONS: Protein precipitation with antibody-based extraction is effective for sample preparation before LC-MS/MS analysis of derivatized 1,25(OH)2D. This method appears to have improved specificity over a clinically used RIA with low imprecision and limits of detection.