A sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI–MS/MS) method for the determination of 25-hydroxyvitamin D3 [25(OH)D3] 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)D3. 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 D4 was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D3 using a 1.0-ml sample, and the limit of quantitation for 25(OH)D3 was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r2 = 0.830) between the serum 25(OH)D3 level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D3 level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D3 level after the supplementation of vitamin D3.
25-Hydroxyvitamin D3Saliva Liquid chromatography–electrospray ionization–tandem mass spectrometry Derivatization Mobile phase additive Cookson-type reagent