Comparative evaluation of new Cookson-type reagents for LC/ESI-MS/MS assay of 25-hydroxyvitamin D3 in neonatal blood samples.

The screening of vitamin D deficiency in neonatal infants, which is based on the blood 25-hydroxyvitamin D3 [25(OH)D3 ] quantification, is important for the early detection, diagnosis and health risk assessment of several diseases. In this study, two new Cookson-type reagents, 4-(4-diethylaminophenyl)-1,2,4-triazoline-3,5-dione (DEAPTAD) and 4-(6-quinolyl)-1,2,4-triazoline-3,5-dione, were designed and synthesized, then compared with the previous reagents, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), in terms of sensitivity and specificity in the assay of 25(OH)D3 in neonatal blood samples by liquid chromatography/electrospray ionization-tandem mass spectrometry. Among the reagents, DEAPTAD was found to be the most promising. The limit of detection (0.38 fmol on the column) of the DEAPTAD-derivatized 25(OH)D3 was 60 and 2 times lower than those of the intact 25(OH)D3 and the PTAD derivative, respectively. 25(OH)D3 was more clearly detected in the plasma sample as the DEAPTAD derivative than the DAPTAD derivative owing to the lower background noise. DEAPTAD derivatization was also useful for the separation of 25(OH)D3 from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 . By using DEAPTAD, a trace amount of 25(OH)D3 in dried blood spots was reproducibly determined without interference from coexisting compounds. Thus, DEAPTAD was proved useful in the measurement of 25(OH)D3 in neonatal blood samples.