Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1D-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at-80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.
Bibliographical noteFunding Information:
Funding: This research was funded by grants from the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH) to KAS (R01 GM111400) and metabolomics supplements to (R01 GM069438, JGY) and (R01 GM103799, AEJ). The content is solely the responsibility of the authors and does not necessarily present the official views of the NIGMS or the National Institutes of Health.
- 1d-1h nmr
- Preanalytical processing
- Quantitative analysis