Application Note Separation of Pentose Phosphate Pathway, Glycolysis, and Energy Metabolites Using an ACQUITY Premier System With an Atlantis Premier BEH Z-HILIC Column

Significance of the Topic

Central carbon metabolism, including glycolysis and the pentose phosphate pathway, underpins energy production, redox balance, and biosynthetic precursor supply in cells. Accurate quantification of phosphorylated intermediates from these pathways is critical for understanding disease mechanisms, metabolic fluxes, and biomarker discovery. However, their strong interactions with metal surfaces and high polarity pose analytical challenges.

Objectives and Study Overview

This work aimed to develop and validate a robust UPLC-MS/MS assay for 27 key pentose phosphate, glycolytic, and energy metabolites in biological samples. The study focused on mitigating metal-induced adsorption, optimizing chromatographic conditions at basic pH, and demonstrating applicability in plasma and tissue extracts.

Methodology

The method employs hydrophilic interaction chromatography using a 1.7 μm Atlantis Premier BEH Z-HILIC column maintained at 30 °C and a 0.5 mL/min flow rate. Mobile phases consisted of 15 mM ammonium bicarbonate at pH 9.0 in water (A) and in 90% acetonitrile (B). A gradient elution separated isobaric pairs such as ribose-5-phosphate/ribulose-5-phosphate and 2-phosphoglycerate/3-phosphoglycerate. Calibration curves spanned 0.0025–12.5 μM using stable-isotope internal standards.

Instrumentation

• UPLC system: ACQUITY Premier with MaxPeak High Performance Surfaces (HPS) technology
• Column: Atlantis Premier BEH Z-HILIC, 2.1 × 100 mm, 1.7 μm
• Mass spectrometer: Xevo TQ-S micro tandem quadrupole, positive/negative electrospray
• Data system: MassLynx v4.2

Main Results and Discussion

Optimization revealed that pH 9.0 bicarbonate buffer delivered the sharpest, most symmetric peaks and highest sensitivity. A buffer concentration of 15 mM provided optimal retention and resolution. Method performance characteristics included:

• Correlation coefficients (r²) > 0.99 for all 27 analytes
• Lower limits of quantification ranging from 0.0025 to 1.25 μM
• Effective separation of challenging isobaric compounds
• Reproducible peak areas in rat plasma, liver, and brain extracts

Benefits and Practical Applications

The assay combines base-stable BEH chemistry and metal-resistant HPS hardware to minimize analyte loss and peak distortion. It is suitable for targeted polar metabolomics in research and quality-control laboratories, enabling reliable quantification of central carbon metabolites in biofluids and tissues.

Future Trends and Applications

Advances may include expanding coverage to additional polar metabolites, integrating high-throughput and microflow formats, and coupling with isotopic tracer studies for flux analysis. Ongoing improvements in column chemistries and surface treatments will further reduce adsorption artifacts and enhance sensitivity.

Conclusion

A targeted UPLC-MS/MS method using the ACQUITY Premier system and Atlantis Premier BEH Z-HILIC column achieves high-quality separations and quantification of 27 central carbon metabolites. Its robustness against metal interactions and compatibility with basic mobile phases make it a valuable tool for metabolomics studies.

Reference

1. Murray RK et al. Harper’s Illustrated Biochemistry. 28th ed.; 2009.
2. Ge T et al. The Role of the Pentose Phosphate Pathway in Diabetes and Cancer. Front Endocrinol. 2020;11:365.
3. Walter TH et al. Introducing Atlantis BEH Z-HILIC. Waters App Note; 2021.
4. Lauber M et al. Low Adsorption HPLC Columns Based on MaxPeak HPS. Waters White Paper; 2020.
5. Want EJ et al. Global Metabolomic Profiling of Animal and Human Tissues via UPLC-MS. 2013;8:17–32.