Liquid chromatography-tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D3:: a noninvasive tool for the assessment of vitamin D status

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of 25-hydroxyvitamin D-3 [25(OH)D-3] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC-MS/MS. The PTAD derivative was much more easily ionized in positive-ESI-MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D-3. Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D-4 was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D-3 using a 1.0-ml sample, and the limit of quantitation for 25(OH)D-3 was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r(2) = 0.830) between the serum 25(OH)D-3 level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D-3 level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D-3 level after the supplementation of vitamin D-3.