A sample extraction and chromatographic strategy for increasing LC/MS detection coverage of the erythrocyte metabolome

Reproducible and comprehensive sample extraction and detection of metabolites with a broad range of physico-chemical properties from biological matrices can be a highly challenging process. A single LC/MS separation method was developed for a 2.1 mm × 100 mm, 1.8 μm ZORBAX SB-Aq column that was used to separate human erythrocyte metabolites extracted under sample extraction solvent conditions where the pH was neutral or had been adjusted to either, pH 2, 6 or 9. Internal standards were included and evaluated for tracking sample extraction efficiency. Through the combination of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques in both positive (+) and negative (−) ion modes, a total of 2370 features (compounds and associated compound related components: isotopes, adducts and dimers) were detected across all pHs. Broader coverage of the detected metabolome was achieved by observing that (1) performing extractions at pH 2 and 9, leads to a combined 92% increase in detected features over pH 7 alone; and (2) including APCI in the analysis results in a 34% increase in detected features, across all pHs, than the total number detected by ESI. A significant dependency of extraction solvent pH on the recovery of heme and other compounds was observed in erythrocytes and underscores the need for a comprehensive sample extraction strategy and LC/MS analysis in metabolomics profiling experiments.