Abstract
There is a need for comprehensive analysis of the dried blood spot (DBS) metabolome to study both new and known inborn errors of metabolism (IEM). The purpose of this study was to complete and evaluate an untargeted metabolomics method using liquid chromatography – electrospray ionization – Q Exactive Orbitrap mass spectrometry (MS), for analysis of one punch (3.2 mm diameter corresponding to about 3 µL whole blood) of a DBS. The criteria for evaluation and measurements were inspired by stringency applied by the World Anti-Doping Agency. In this regard, the instrument repeatability and assay reproducibility were satisfactory with relative standard deviation (% RSD) of peak areas below 10 % and retention times below1 %, using a pooled control sample, injected three times each day during sample analysis for 11 days. Compared to perimeter punches, analyzing center punches improved the repeatability. An increased organic solvent amount in the reconstitution solution, and reduced m/z range of the MS (focusing more on each m/z increases sensitivity) increased the number of hydrophobic and low-abundant compounds detected, respectively. The quantification of most of the method evaluation compounds (acylcarnitines and amino acids) was satisfactory, with linear correlation of R2 above 0.99 in signal vs. concentration plots, using 1-4 punches (about 3, 6, 9 and 12 µL whole blood) of the DBS, but some polar compounds were affected by matrix effects. Tandem MS data was acquired to secure better identification. Combining the method with bioinformatics, one could identify changes between samples taken after free diet, overnight fasting (12 hours) and prolonged fasting (36 hours), using only 3 µL blood on paper. The method will be used to detect differences in the metabolome of patient and healthy samples in research and in future diagnostics.