Researchers have developed a protocol for extracting metabolites and lipids from a single dried blood spot, allowing two omics analyses to be performed without requiring separate samples.
Dried blood spots are commonly used in newborn screening because they require little blood and are relatively easy to collect, transport, and store. Most extraction methods, however, target only one molecular class from each spot. This limits the information available from small or irreplaceable samples.
The method, published in STAR Protocols, uses a modified Folch extraction to separate metabolites and lipids from a 10 µL volumetric dried blood spot. Researchers add a 2:1 mixture of chloroform and methanol, sonicate the sample, add water, and centrifuge it.
This produces two layers: lipids move into the lower organic layer, while metabolites remain in the upper aqueous layer. A second extraction improves recovery before the fractions are separated, dried under nitrogen, and stored or prepared for analysis.
The two extracts are reconstituted separately and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. The published workflow uses a Q Exactive Orbitrap mass spectrometer with heated electrospray ionization.
Metabolites and lipids are analyzed separately in positive- and negative-ion modes. Data-dependent tandem mass spectrometry supports compound identification, particularly lipid annotation. The complete set of four analyses requires approximately 90 minutes of instrument time per sample.
The protocol uses two open-source tools for data processing. Mzmine detects and annotates metabolite features, while LipidMatch processes lipid data. The workflow also includes extraction blanks, pooled samples, injection standards, and other controls to assess contamination, analytical variation, and instrument performance.
Results are semiquantitative. Peak heights or areas can be compared between groups, but absolute concentrations cannot be determined without additional standards and method validation.
Tests of three separate dried blood spot extractions found comparable numbers of metabolite features. The most confidently identified lipid features were generally reproducible, although some differences occurred between extraction trials.
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