Improved sensitivity of serum/plasma 1α,25-dihydroxyvitamin D quantification by DAPTAD derivatization

Background: Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)(2)D quantification. An accurate method is required for the determination of the 1,25(OH)(2)D status. Methods: We designed a serum/plasma 1,25(OH)(2)D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)(2)D-3 concentrations were determined in 232 healthy Japanese individuals. Results: The intra- and inter-assay CVs for 1,25(OH)(2)D-3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)(2)D-3 was 7.1 pg/ml. Rheumatoid factor (RF) at concentrations below 517 IU/ml did not affect serum 1,25(OH)(2)D analysis. No significant differences were observed for various blood collection tubes, repeated freeze thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r = 0.786), but serum 1,25(OH)(2)D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)(2)D-3 concentrations by LC-MS/MS were 18.7-53.9 pg/ml. Conclusion: A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)(2)D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)(2)D concentrations in the clinical setting.