Background: With the increasing focus of metabolomic methods on obesityrelated
diseases, it is important to consider how sample handling may need to be adapted for
the high compositions of lipids that can occur in such subjects.
Introduction: High-lipid (cloudy, milky appearances; a.k.a. lipemic) biofluids are common
in very high BMI subjects. Organic extractions of biofluids are useful for removing protein
backgrounds, inactivating capsid viruses, and yielding relatively stable samples with excellent
spectroscopic characteristics. This work considered how acetonitrile extractions, which
are widely used, perform on lipemic sera.
Results: In this technical note, we report the observation and remediation of a liquid-liquid
phase separation in acetonitrile extractions of many lipemic sera. This unexpected behavior
can be challenging to identify, especially if working with small volumes. The liquid-liquid
separation shows a high miscibility of proteins in both liquid phases that impairs NMR data
quality. We also report a simple temperature-based adaption of the acetonitrile extraction
procedure that consistently results in a single aqueous phase and eliminates unwanted constituents.
Conclusion: A robust approach to achieving reproducible, high quality samples of aqueous
metabolites from lipemic sera from very high BMI subjects should be of utility in expanding
metabolomics applications to lipemic biofluids.