, Volume 391, Issue 1, pp 229-238
Date: 18 Dec 2007

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

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Abstract

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 (r 2 = 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.