Tackling pressure fluctuations in ultra-HPLC to robustly resolve and analyze polar metabolites

The success of modern metabolomics analysis depends on the separation of metabolites in complex samples using methods such as LC and mass spectrometry. Herein, we present a protocol for resolving a broad range of polar metabolites, based on hydrophilic interaction LC with a zwitterionic-bonded phase (HILICz). In optimizing 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 h of run time). This consistency is essential to address the growing demand for repeatable in-depth metabolomics analysis of complex samples.