Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS over the time course of 10 years, using an untargeted LC-MS/MS based metabolomics protocol. We found that a majority (79%) of the DBS metabolome was stable during 10 years of storage at -20 C. However, we found trends of degradation for lipid-related metabolites, such as phosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1-0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage.

The authors have declared no competing interest.

This research has been conducted using the Danish National Biobank resource supported by the Novo Nordisk Foundation.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study was conducted in accordance with the Declaration of Helsinki and the protocol complies with the Danish Ethical Committee law by not being a health research project (Section 2,1), but a method development study not requiring an ethical approval. The Committees on Health Research Ethics for the Capital Region of Denmark waived ethical approval for this work.

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