A liquid chromatography/electrospray ionization–tandem mass spectrometry‐based method was developed for the identification of the conjugation positions of the monoglucuronides of 25‐hydroxyvitamin D3 [25(OH)D3] and 24,25‐dihydroxyvitamin D3 [24,25(OH)2D3] in human urine. The method employed derivatization with 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D3‐3‐ and ‐25‐glucuronides, or 24,25(OH)2D3‐3‐, ‐24‐ and ‐25‐glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D3 and 24,25(OH)2D3, respectively. The 3‐glucuronide was not present for 24,25(OH)2D3, unlike 25(OH)D3, thus we found that selective glucuronidation occurs at the C24‐hydroxy group for 24,25(OH)2D3.

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. Copyright © 2015 John Wiley & Sons, Ltd.

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