Distribution of 25-hydroxyvitamin D3 in dried blood spots and implications for its quantitation by tandem mass spectrometry

Dried blood spots (DBS) are a convenient collection and archiving method for blood specimens. The interest in screening certain analytes in neonatal DBS continues to increase for a variety of paediatric disorders. 25-Hydroxyvitamin D3 (25OHD3) is one such analyte. We investigated potential factors that may affect the analysis of 25OHD3 in prospective cohorts of DBS, such as blood spot volume, hole punch position, and paper type. All of these factors were shown to affect 25OHD3 levels measured. When blood volumes of <50 μL were spotted, 25OHD3 concentrations extracted were significantly lower (P < 0.0001). We also observed a chromatographic effect across the surface of blood spots, with 25OHD3 concentrations significantly higher in outer punched spots compared to those punched from the centre (P < 0.0001). This also correlates with a heavier net weight of blood from outer punched spots (P < 0.0001). This effect was reproducible on two types of paper cards (Whatman 903® and FTA®), and paper type was shown to be highly relevant. We also show that the distribution of 25OHD3 in whole blood is essentially extracellular, with over 98% of 25OHD3 residing in the serum component. This may potentially explain why the diffusion properties of blood and type of chromatographic paper may significantly influence the distribution of such analytes in DBS. These factors should be taken into consideration for the prospective collection of DBS and analysis of 25OHD3 in DBS.

► A chromatography effect has been observed leading to assay bias.
► 50 μL spot volume recommended for saturating the paper and maintaining accuracy.
► Significantly higher measured [25OHD3] in spots punched out from the periphery.
► Paper type to be standardised for sample collection in prospective studies using DBS.