pr8b00487_si_001.pdf (1.13 MB)
Trace Phosphate Improves ZIC-pHILIC Peak Shape, Sensitivity, and Coverage for Untargeted Metabolomics
journal contribution
posted on 2018-08-30, 00:00 authored by Jonathan
L. Spalding, Fuad J. Naser, Nathaniel G. Mahieu, Stephen L. Johnson, Gary J. PattiExisting
hydrophilic interaction liquid chromatography (HILIC)
methods, considered individually, each exhibit poor chromatographic
performance for a substantial fraction of polar metabolites. In addition
to limiting metabolome coverage, such deficiencies also complicate
automated data processing. Here we show that some of these analytical
challenges can be addressed for the ZIC-pHILIC, a zwitterionic stationary
phase commonly used in metabolomics, with the addition of trace levels
of phosphate. Specifically, micromolar phosphate extended metabolome
coverage by hundreds of credentialed features, improved
peak shapes, and reduced peak-detection errors during informatic processing.
Although the addition of high levels of phosphate (millimolar) as
a HILIC mobile phase buffer has been explored previously, such concentrations
interfere with mass spectrometric (MS) detection. We show that using
phosphate as a trace additive at micromolar concentrations improves
analysis by electrospray MS, increasing signal for a diverse set of
polar standards. Given the small amount of phosphate needed, comparable
chromatographic improvements were also achieved by direct addition
of phosphate to the sample during reconstitution. Our results suggest
that defects in ZIC-pHILIC performance are predominantly driven by
electrostatic interactions, which can be modulated by phosphate. These
findings constitute both a methodological improvement for untargeted
metabolomics and an advance in our understanding of the mechanisms
limiting HILIC coverage.