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

Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)₂D quantification. An accurate method is required for the determination of the 1,25(OH)₂D status.

We designed a serum/plasma 1,25(OH)₂D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)₂D₃ concentrations were determined in 232 healthy Japanese individuals.

The intra- and inter-assay CVs for 1,25(OH)₂D₃ were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)₂D₃ was 7.1pg/ml. Rheumatoid factor (RF) at concentrations below 517IU/ml did not affect serum 1,25(OH)₂D 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)₂D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)₂D₃ concentrations by LC-MS/MS were 18.7-53.9pg/ml.

A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)₂D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)₂D concentrations in the clinical setting.