Methods for Untargeted Analysis of Milk Metabolites: Influence of Extraction Method and Optimization of Separation

Cow's milk is a complex food, and research into its metabolome can provide information useful in the study of animal health, farming practices, food safety and the adulteration of milk. Comparative interlaboratory metabolic analysis is hampered by the lack of standardized methods-a requirement addressed in this study.

We studied the influence of the chromatography column and extraction solvent on the metabolites isolated during untargeted metabolomics.

After studying fifteen columns and four extraction solvents, it was determined that an HILIC column offered the best compromise between retention time and separation of metabolites. Each extraction solvent covered a different area of the metabolome, only overlapping with previously annotated compounds. Extraction mixtures containing methanol tend to give better recovery.

The choice of extraction solvent was crucial when looking at the difference between samples, but if interest lies only in previously annotated compounds, then there is little difference between the solvents.