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A novel Cookson-type reagent for enhancing sensitivity and specificity in assessment of infant vitamin D status using liquid chromatography/tandem mass spectrometry


Correspondence to: T. Higashi, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278–8510, Japan.




25-Hydroxyvitamin D3 [25(OH)D3] is the best-established indicator of vitamin D status. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD), a representative Cookson-type reagent, has often been employed for enhancing the sensitivity in the trace determination of 25(OH)D3 in a neonatal dried blood spot (DBS), which contains only 2.65 μL of whole blood, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The objective of this study was the development of a novel Cookson-type reagent surpassing PTAD in terms of sensitivity and specificity in the LC/ESI-MS/MS assay of 25(OH)D3.


A novel Cookson-type reagent, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), was synthesized from 4-dimethylaminobenzoyl chloride. The DAPTAD-derivative of 25(OH)D3 was prepared and its LC/ESI-MS/MS behavior was examined. The applicability of the DAPTAD-derivatization in the determination of 25(OH)D3 in neonatal DBSs was also examined.


The derivatization was completed at room temperature within 1 h. The DAPTAD-derivative of 25(OH)D3 provided a characteristic product ion derived from the cleavage of the vitamin D skeleton during MS/MS. The limit of detection of the DAPTAD-derivative during selected reaction monitoring was 0.25 fmol on the column, which was 30 and 2 times lower than those of the intact 25(OH)D3 and the PTAD-derivative, respectively. The DAPTAD-derivatization followed by LC/ESI-MS/MS enabled the detection of a trace amount (in the low-ng/mL range) of 25(OH)D3 in DBSs with a simple pretreatment (only methanol extraction) and short chromatographic run time (10 min). The DAPTAD-derivatization was also useful for the separation of 25(OH)D3 from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]. On the contrary, the assay using the PTAD-derivatization might lead to overestimation of the true 25(OH)D3 levels due to the co-elution of 25(OH)D3 and 3-epi-25(OH)D3.


We developed DAPTAD for enhancing the sensitivity and specificity of the LC/ESI-MS/MS assay of 25(OH)D3. Our new method using DAPTAD can reduce the overestimation of the 25(OH)D3 levels, and will prove helpful in the diagnosis of vitamin D deficiency in infants. Copyright © 2013 John Wiley & Sons, Ltd.

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