LC-MS with hydrophilic interaction liquid chromatography (HILIC) separations facilitates identification and quantification of hydrophilic species due to the orthogonality and enhanced sensitivity compared to reversed phase. Zwitterionic columns, having 1:1 ratio of positive and negative charges, provides separation by HILIC partitioning plus weak attraction and repulsion interactions with their abundant balanced charges.

We performed LC-MS of charged and neutral compounds on zwitterionic HILIC columns with complementary charge orientation. One column type had phosphorylcholine functional groups (SeQuant ZIC-cHILIC), comprising inner negatively charged phosphate and distal positively charged quaternary ammonium whereas the second had functional groups of sulfobetaine(SeQuant ZIC-HILIC), i.e., inner positively charged quaternary ammonium and distal negatively charged sulfonate.

Compounds analyzed comprised ranges of charged and neutral metabolites including 1) organic acids; malate & fumarate, 2) nucleotides; AMP, ADP & ATP, 3) quaternary ammonium compounds; carnitine & acetylcarnitine, 4) amino acids; leucine & iso-leucine, plus 5) aminoglycosides; streptomycin, gentamycin & neomycin. Typical gradients ranged from 90-50% acetonitrile, and contained volatile buffers of formic acid or ammonium acetate for control of pH and ionic interactions.

The LC-MS studies showed that the retention of nucleotides, organic acids and amino acids increased 10-30% when separated on the phosphorylcholine-based HILIC column, whereas the retention of carnitines and aminoglycosides decreased with similar amounts, relative to the sulfobetaine-based HILIC column. Separation selectivity (alpha) was less influenced and changed differently from compound to compound. Peak resolution typically increased with retention (weak electrostatic attraction), although for the multiply charged and very hydrophilic aminoglycosides it was noted that a weaker interaction (weak electrostatic repulsion) resulted in improved separation and peak shape.

For separation of critical peak pairs such as leucine & iso-leucine on the column that showed higher peak resolution for this pair (ZIC-cHILIC), the retention time precision was determined to 0.5% (n=8) for control samples injected during analysis of 80 samples with methanol-water leukemia cell extracts.

We could thus conclude that the spatial arrangement of zwitterionic charges in HILIC columns influences the separation selectivity, especially for charged polar hydrophilic molecules, and that this can be a useful feature for separation tuning. The distal charge dominated over the inner charge, although the close proximity of the two charges in the zwitterions resulted in an overall weak ionic interactions that can be optimized at low buffer concentrations to meet precision requirements of LC-MS analysis.