Tackling pressure fluctuations in ultra-high performance liquid chromatography to robustly resolve and analyse polar metabolites.

Abstract

The success of modern metabolomics analysis depends on the separation of metabolites in complex samples using methods such as liquid chromatography and mass spectrometry. Herein, we present a protocol for resolving a broad range of polar metabolites, based on hydrophilic interaction liquid chromatography with a zwitterionic bonded phase (HILICz). In optimising this protocol, we encountered pressure fluctuations, a widespread problem that impacts metabolite analysis, restricts batch sizes, and imposes instrument downtime, ultimately incurring substantial time and financial expense. Thus, we use this opportunity as a case study to demonstrate the steps taken to overcome such pressure fluctuations, resulting in a protocol that robustly and consistently resolves polar metabolites in large batches of samples (>100 samples, equating to >40 hours of run-time). This consistency is essential to address the growing demand for repeatable in-depth metabolomics analysis of complex samples.