Sensitive and Robust LC-MS/MS Assay to Quantify 25-Hydroxyvitamin D in Leftover Protein Extract from Dried Blood Spots.

Sanne Grundvad Boelt,Arieh S Cohen,John J Mcgrath,Nadia Sara Jensen Macsween,Marta Jadwiga Thorbek,Lars Melgaard

doi:10.3390/ijns7040082

Abstract

Neonatal dried blood spots (DBS) provide a remarkable resource for biobanks. These microsamples can provide information related to the genetic correlates of disease and can be used to quantify a range of analytes, such as proteins and small molecules. However, after routine neonatal screening, the amount of DBS sample available is limited. To optimize the use of these samples, there is a need for sensitive assays which are integrated across different analytic platforms. For example, after DNA extraction, protein extracts are available for additional analyses. We describe a sensitive and robust LC-MS/MS method for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 optimized for leftover protein extracts from DBS, which has excellent recovery, precision, and accuracy.

Highlights

Neonatal dried blood spots (DBS) have long been used for routine clinical screening for a range of congenital disorders in newborns [1]
A significant challenge using DBS is that only small volumes of clinical matrices are collected, and after routine neonatal screening, even less material is available for subsequent storage and research
The standards, instrumentation, chromatographic condition (Table S1), tandem mass spectrometry detection (Figure S2) and quantification of the vitamin D metabolites were consistent with our previous methods for detecting vitamin D metabolites in DBS [4]

Summary

Introduction

Neonatal dried blood spots (DBS) have long been used for routine clinical screening for a range of congenital disorders in newborns [1]. These samples provide a remarkable resource for risk factor epidemiology as these samples (a) are often part of a large set of unselected samples; (b) can provide DNA for genotyping [2]; (c) allow for the quantification of a wide range of analytes of interest (e.g., proteins and small molecules); and (d) can be linked with electronic health records and health registers [3]. A significant challenge using DBS is that only small volumes of clinical matrices are collected, and after routine neonatal screening, even less material is available for subsequent storage and research. We had access to DBS protein extracts that had previously been used for genotyping [2]. We developed and validated a sensitive and robust LC-MS/MS approach capable of quantifying the two main vitamin D metabolites (25OHD2 and 25OHD3 ) in the leftover of the protein extract from DBS

Methods

Protein

Extraction of Vitamin D from Protein Extract

Results

Discussion