Minimizing Matrix Effects for the Accurate Quantification of 25-Hydroxyvitamin D Metabolites in Dried Blood Spots by LC-MS/MS.

David Kvaskoff, Alicia K Heath, Henry A Simila, Pauline Ko, Dallas R English, Darryl W Eyles

The noncalcemic actions of vitamin D in multiple organs are now widely recognized. Vitamin D status has been linked with a wide variety of conditions, which has led to an increasing demand for vitamin D screening. In particular, there is intense interest in the impact of vitamin D on a variety of developmental conditions. The most readily accessible pediatric samples are dried blood spots, and health organizations are increasingly archiving such samples for later assessment of the antecedents of disease. In 2009, we developed a method to quantify the major circulatory form of vitamin D, 25-hydroxyvitamin D, in archived dried blood spots. Over the last 6 years, we have made substantial alterations to the published method to enhance throughput, sensitivity, and assay robustness. With the alterations, the assay was 3 times faster than the previously published assay and had a >10-fold increase in signal strength. Intraassay imprecision decreased from 13.4% to 6.9%, and there was a 5-fold reduction in interfering phospholipids. In actual use over 2 years, the assay showed an interassay imprecision of 11.6%. This assay has performed reliably over the past 6 years. The practical changes we have made should allow clinical chemists to successfully adapt this method.