Generic multicriteria approach to determine the best precipitation agent for removal of biomacromolecules prior to non-targeted metabolic analysis

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2021-03, Vol.1167, p.122567-122567, Article 122567
• Main Authors: Knudsen, Sofie B., Nielsen, Nikoline J., Qvist, Johan, Andersen, Kim B., Christensen, Jan H.
• Format: Article
• Language: English
• Subjects: Durbin-Watson - component detection algorithm; Metabolomics; Principal component analysis; Protein precipitation; Sample preparation
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2021.122567

•A multicriteria approach is a comprehensive way to examine biomacromolecule removal.•The good quality extracted ion chromatograms reduce the complexity of metabolomic data.•Diluted samples are used to pinpoint the main differences between sample strategies.•The multicriteria approach can be extrapolated to all biofluids. The removal of biomacromolecules from biofluids decreases the sample complexity and lower electrospray suppression effects. Furthermore, it can increase the analysis sensitivity, precision, and selectivity. Often removal approaches evaluate the model based on a single criterion, like protein removed or response of one of few specific metabolites. In this study, we used a multicriteria approach to test the effect of using the solvents methanol and acetonitrile (organic solvent precipitation), trichloroacetic acid (acidic precipitation) and ammonium sulphate (salting out) to remove biomacromolecules from a downstream recovery process from a bacillus fermentation. The downstream recovery process intermediates were analysed using reversed-phase ultra-high-pressure liquid chromatography with electrospray ionisation and high-resolution time-of-flight mass spectrometry detection. To evaluate the pre-treatment agents the following multicriteria was applied i) practical considerations, ii) total amino acid in the precipitated pellet, iii) putative identification of the molecules removed or created by the different treatments, iv) coherence between high quality extracted ion chromatograms (repeatability of DW-CODA) and v) replicate consistency from principal component analysis score values obtained by using the CHEMometric analysis of sections of Selected Ion Chromatograms (CHEMSIC) method. This study presents a generic workflow to find the best pre-treatment for removing bio-macromolecules from biofluids with a multicriteria approach. In our case, the best protein removal strategy for downstream recovery intermediates was acetonitrile precipitation. This method showed high precision, created few artefact peaks compared to simple sample dilution, and mainly removed small peptides.