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

Abstract

BACKGROUND 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. METHODS 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. RESULTS 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%. CONCLUSIONS 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.

Newborn vitamin D levels in relation to autism spectrum disorders and intellectual disability: A case–control study in california

“…The major circulating form, 25‐hydroxyvitamin D (25(OH)D), also known as 25‐hydroxycholecalciferol, is a reliable indicator of cumulative production and is therefore used clinically to measure vitamin D sufficiency. Total 25‐Hydroxyvitamin D (sum of 25(OH)D2 and 25(OH)D3, hereinafter referred to as 25(OH)D) was measured by a sensitive isotope dilution liquid chromatography–tandem mass spectrometry method for dried blood spots as previously described [Eyles et al, ; Kvaskoff et al, ]. One sample was mislabeled so could not be linked; of the remaining 1189, 25(OH)D3 was detectable in all newborn blood spots, of which 82 samples also had detectable 25(OH)D2, which is obtained from dietary sources or supplements.…”

Neonatal vitamin D status in relation to autism spectrum disorder and developmental delay in the CHARGE case–control study

“…We report total 25‐Hydroxyvitamin D (25[OH]D) concentrations as the sum of 25(OH)D2 and 25(OH)D3. 25(OH)D was measured by an isotope dilution liquid chromatography–tandem mass spectrometry assay validated for dried blood spots (DBSs) [Eyles, Anderson, et al, ; Eyles, Feron, et al, ; Kvaskoff et al, ] by a participatory laboratory of the Vitamin D External Quality Assessment Scheme. Assay accuracy was assessed using NIST sera calibrants (SRM 972a Levels 1–4) in replicates of 3 for each run.…”

Vitamin D treatment during pregnancy prevents autism-related phenotypes in a mouse model of maternal immune activation

“…Two vitamin D metabolites 24,25OHD3 and 25OHD3 were examined in the same maternal blood in which cytokines were analyzed using a liquid chromatography/tandem mass spectrometry method [46]. Therefore, this represents samples taken 4 h post 1,25OHD administration.…”