Sharpen Your Peaks: Novel Column Hardware for Improved HILIC Polar Metabolite Chromatography

Significance of the Topic

Hydrophilic interaction liquid chromatography (HILIC) is a cornerstone technique for profiling polar metabolites in biological samples. However, traditional stainless-steel (SS) hardware can interact with metal-sensitive analytes, requiring lengthy conditioning steps that delay sample analysis. The introduction of deactivated SS columns and a quick-change inline filter promises to streamline workflows, improve peak shapes, and maintain reproducible retention times, enabling broader adoption of HILIC metabolomics by novice and expert users alike.

Objectives and Study Overview

This work evaluates novel deactivated SS column hardware against standard phosphonated SS columns through three complementary studies:

• Direct comparison of peak shape, resolution, and retention time between deactivated SS (with abbreviated preparation) and conventional phosphonated SS columns.
• Longevity assessment of the deactivated SS column over 14 days and 700+ plasma injections, monitoring retention time stability for 173 metabolites.
• Inter-laboratory reproducibility involving four independent labs running 40 plasma injections each to test transferability of retention times and method robustness.

Methodology and Instrumentation

The standard HILIC metabolomics protocol utilized an Agilent 1290 Infinity III Bio LC coupled to a 6495D LC/TQ mass spectrometer. Key conditions included:

• Column: HILIC-Z, 2.1 × 150 mm, 2.7 µm in both SS and deactivated SS formats with guard column and InfinityLab Quick Change Bio Inline Filter.
• Mobile phases: A) 20 mM ammonium acetate, pH 9.3 with 5 µM medronic acid in water; B) acetonitrile.
• Gradient: 90% B (0 min) to 10% B (15 min) at 0.400 mL/min; column temperature 15 °C; injection volume 4 µL.

Main Results and Discussion

Direct comparison revealed that the deactivated SS column with abbreviated preparation (~10 h) matched the phosphonated SS in peak shape, resolution, and retention time precision, supporting use of existing dMRM databases. Longevity testing demonstrated stable retention times across 700+ injections, with over half of the 173 metabolites exhibiting <1% RSD and all within the predefined acquisition window. The inter-lab study confirmed method transferability: more than 150 analytes showed retention time RSDs below 5% across four sites and different operators.

Benefits and Practical Applications

• Significantly reduced column conditioning time (from >30 h to ~10 h) accelerates time to data.
• Lower risk of metal-analyte interactions enhances sensitivity for phosphate-rich compounds (e.g., ATP, creatine phosphate).
• Inline filter integration prolongs column life and maintains reproducibility during large sample batches.
• Proven inter-laboratory robustness supports adoption in multi-site studies and clinical workflows.

Future Trends and Opportunities

Continued development of inert column materials and inline components will likely expand HILIC coverage to even more polar metabolites and complex matrices. Automation of sample preparation, integration with high-throughput platforms, and coupling to advanced data analysis pipelines will drive large-scale metabolomics in clinical diagnostics, drug development, and systems biology.

Conclusion

The newly developed deactivated SS HILIC column and quick-change Bio Inline Filter deliver comparable chromatographic performance to traditional phosphonated SS hardware while dramatically reducing preparation time and maintaining retention time stability over extensive use. Their ease of transfer across laboratories underscores their potential to simplify and standardize polar metabolite analysis in diverse research and QA/QC environments.

References

1. Yannell KE, Simmermaker C, Durham SD, Hsiao JJ. An End-to-End Targeted Metabolomics Workflow. Agilent Application Note 5994-5628EN. 2023.
2. Yannell KE, Simmermaker C, Durham SD, Hsiao JJ. Mastering HILIC-Z Separation for Polar Analytes. Agilent Application Note 5994-5949EN. 2023.
3. Sartain M, Durham SD. Enabling Automated, Low-Volume Plasma Metabolite Extraction with the Agilent Bravo Platform. Agilent Application Note 5994-2156EN. 2020.