HILIC for separation of polar metabolites

April 02, 2015

PTRL West (a division of EAG Inc.)
Metabolism
Team Leader

Overall

Performance

Ease-of-Optimization

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Company:

Phenomenex Inc.

Product Name:

Kinetex HILIC 2.6 um HPLC Column

Catalog Number:

00F-4461-EO

The HILIC kinetex column has been great for separating out those residues that just won't separate by RP HPLC. The biggest challenge has been getting analytes into a solution suitable for HILIC initial conditions. It is currently difficult to judge the robustness as it does not see a ton of action. However, the samples it has seen are "problem" plant and animal extracts and it has held up well so far!

Experimental Design and Results Summary

Application

Metabolism: Exhaustive extract polar unknown metabolites

Starting Material

Hen Liver

Protocol Overview

Liver from hens treated with radiolabeled test substance was initially extracted with acetonitrile water. Additional exhaustive extractions included 0.1 M HCl, 1 M KOH and 24% KOH. Exhaustive extracts were combined, acidified, and partitioned with ethyl acetate. The organic phase and initial ACN/H2O extracts were analyzed by RP HPLC. The aqueous phase when analyzed by RP HPLC resulted in polar residue eluting with the void volume. The aqueous phase was mixed with a small volume of propylene glycol and concentrated to dryness to precipitate salts. The propylene glycol was transferred to a separate vessel and the precipitate rinsed with acetone. Acetone rinse and propylene glycol were pooled and concentrated to near dryness. A portion of the concentrate was diluted with HILIC HPLC initial conditions (10 mM ammonium acetate in ACN/water 9:1). HPLC method used a linear gradient with increasing aqueous conditions.

Tips

It is tricky to get polar analytes into initial conditions when dealing with unknowns. Try to think about solubility and functionality of co-extractants in order to better prepare your sample for analysis. For multiple runs, make sure to allow adequate time for the column to re-equilibrate as HILIC takes a bit longer compared to RP.

Results Summary

Analysis showed multiple residues with one major unknown residue eluting at 26.8 min that accounted for >50% of total peak area. Adequate retention suggests analysis via HILIC LC/MS may produce adequate data for metabolite identification.

Features Summary

Good retention of polar analytes

Additional Notes

None

Image Gallery

Summary

The Good

Good retention of polar analytes

The Bad

Sometimes difficult to prepare samples for injection.

The Bottom Line

It is always a challenge to deal with polar unknown metabolites that will not separate by RP HPLC. Overall, despite the challenges we always face in getting analytes into initial conditions for HILIC analysis; the HILIC column is a great addition to our analytical arsenal and a great tool for dealing with unknown polar metabolites.

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