Comprehensive Analysis of Hydrophilic Metabolites Using Shim-pack™ Mix-HILIC

Significance of the Topic

Biological metabolomics enables comprehensive profiling of small-molecule metabolites to understand physiological states, disease mechanisms, and biotechnological processes.

Objectives and Study Overview

This application demonstrates a unified LC/MS method for simultaneous analysis of 49 hydrophilic metabolites—including amino acids, nucleobases, nucleosides, nucleotides, coenzymes, and organic acids—using the Shim-pack Mix-HILIC column coupled to the LCMS-8060NX.

Methodology and Used Instrumentation

• UHPLC System: Nexera X3
• Mass Spectrometer: LCMS-8060NX triple quadrupole with ESI IonFocus source in MRM mode
• Chromatographic Column: Shim-pack Mix-HILIC (150 mm × 2.1 mm I.D., 5 µm) featuring amine and quaternary ammonium groups on a crosslinked polymethacrylate matrix (pH range 2–13, up to 35 MPa).
• Mobile Phases: A = 40 mM ammonium bicarbonate, pH 9.7 in water; B = acetonitrile. Gradient: 95% B (0–0.5 min) → 40% B (15.5 min) → 0% B (16.5–26.5 min) → 95% B (27.5–35 min), flow rate 0.4 mL/min, column oven 40 °C, injection 5 µL.
• MS Conditions: Nebulizing gas 2.0 L/min, drying gas 10.0 L/min, heating gas 10.0 L/min, DL temp. 250 °C, heat block 400 °C, interface temp. 300 °C, CID gas 270 kPa.
• Sample Preparation: E. coli cells and human plasma extracted by methanol/chloroform/water biphasic partitioning, centrifugation, concentration, and reconstitution in 1:1 water/methanol.

Main Results and Discussion

• All targeted classes are resolved in a single gradient run: uncharged and cationic metabolites elute before 12.8 min, while anionic metabolites elute thereafter.
• Detection performance: 44 of 49 metabolites in E. coli and 25 in plasma, demonstrating broad applicability across sample types.

Benefits and Practical Applications

• Single-run coverage of diverse polar analytes reduces total analysis time, solvent consumption, and sample requirements.
• Suitable for microbial metabolomics, clinical bioanalysis, QA/QC, and industrial analytics where comprehensive polar profiling is required.

Future Trends and Possibilities

• Integration with automated, high-throughput platforms for large-scale studies.
• Method optimization to extend analyte coverage by adjusting buffer pH and gradient profiles.
• Coupling with high-resolution MS to combine targeted quantitation with untargeted discovery workflows.

Conclusion

The Shim-pack Mix-HILIC column paired with LCMS-8060NX offers a robust, high-throughput solution for comprehensive analysis of hydrophilic metabolites under basic conditions, making it a versatile tool for diverse metabolomic investigations.

Used Instrumentation

• Nexera X3 UHPLC
• LCMS-8060NX triple quadrupole mass spectrometer
• Shim-pack Mix-HILIC column (150 mm × 2.1 mm, 5 µm)

References

No explicit references were provided in the source material.