The challenges of developing and optimising an assay to measure 25-hydroxyvitamin D in saliva

Accurately detecting vitamin D deficiency (defined as concentration in blood of 25-hydroxyvitamin D (25(OH)D), <20 ng/mL) is important for both clinicians and researchers. Drawing blood may be difficult in some populations, such as infants and children. We thus explored the development of a method to measure 25(OH)D concentrations in saliva, using a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay. Using 25(OH)D₃ standards spiked into synthetic saliva, we generated a standard curve with high correlation (r = 0.999, Pearson's); the intra-assay and inter-assay variation were ≤3.2% and ≤13.2% (CV%), respectively. Passive collection of saliva via drooling into glass or polypropylene tubes yielded higher levels of 25(OH)D₃ than chewing on a synthetic swab. Chewing gum for at least 4 min reduced saliva levels of 25(OH)D₃. Differences in the levels of 25(OH)D₃ in saliva between the passive drooling and stimulated swab-chewing methods were normalised by adjusting for measured levels of vitamin D binding protein in saliva. Freezing samples immediately, or after 24 h of refrigeration did not affect 25(OH)D₃ levels. When saliva levels of 25(OH)D₃ were averaged from samples collected daily for three consecutive days, for which an additional centrifugation step was performed after samples were defrosted (to remove mucin), there was a positive (but non-significant) correlation between 25(OH)D₃ levels in saliva and serum (r = 0.57, p = 0.24, Pearson's) with significant correlations (r ≥ 0.88, p < 0.05) observed after further adjusting for saliva flow rate. The time of day of the collection made little difference to 25(OH)D₃ levels measured in saliva. In conclusion, we have developed an LC-MS/MS assay that accurately measures saliva 25(OH)D₃ levels, which correlated with serum levels. However, for a measurement that correlates with serum 25(OH)D it may be necessary to average results from saliva collected on three consecutive days, and adjust for differences in saliva flow rate. This would increase costs, and combined with the processing requirements for samples, could limit the applicability of this assay to large cohort and field studies.