Development and validation of a method for determination of plasma 25-hydroxyvitamin D3 3-sulfate using liquid chromatography/tandem mass spectrometry.

The quantification of plasma 25-hydroxyvitamin D3 3-sulfate [25(OH)D3S] is expected to be helpful in the assessment of the vitamin D status, especially for infants. In this study, a simple and sensitive method for the quantification of 25(OH)D3S in plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was deproteinized with acetonitrile, purified using an Oasis(®) HLB cartridge, and subjected to LC/ESI-MS/MS operating in the negative-ion mode. Quantification was based on the selected reaction monitoring, and deuterated 25(OH)D3S was used as the internal standard. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 7.9% or lower) and accurate (analytical recovery, 95.8-105.3%) quantification of the plasma 25(OH)D3S using a 20-µL sample, and the limit of quantification was 2.5ng/mL. The developed method was applied to the determination of plasma 25(OH)D3S in infants; the result revealed that preterm infants have lower plasma 25(OH)D3S concentrations.

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

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.