Optimization of Chromatographic Conditions for the Analysis of Polar Metabolites in Aqueous Solutions by UPC2-MS

Importance of the Topic

Analysis of polar, low-molecular-weight metabolites in aqueous biological fluids underpins metabolic phenotyping and precision medicine. Reliable, high-throughput methods are essential to measure endogenous compounds in urine, plasma, and cell extracts for diagnostics, biomarker discovery, and pharmacokinetic studies.

Objectives and Overview of the Study

This work aimed to optimize ultraperformance convergence chromatography with mass spectrometry detection (UPC2-MS) for broad-scope analysis of polar metabolites in aqueous solutions. Key variables including column chemistry, mobile phase gradient, organic co-solvent composition, water content, buffer strength, and acidification were systematically evaluated to maximize resolution, peak shape, and throughput.

Methodology and Instrumentation

A test mixture of 33 representative polar analytes (nucleobases, nucleosides, organic acids, alkaloids, pharmaceuticals, and phthalates) at 10 µg/mL was prepared in water. Chromatography was performed on an ACQUITY UPC2 system with Binary Solvent Manager, Sample Manager, Automatic Back-Pressure Regulator, and photodiode array detector. Separation used an ACQUITY UPC2 BEH column (1.7 µm, 3.0 × 100 mm) at 35 °C with a CO2/MeOH gradient (initial 98:2 ramped to 40–60% co-solvent). Methanol co-solvent was modified with water, ammonium formate buffer (5–40 mM), and formic acid (0.2–1%). The effluent was monitored by PDA at 273 nm or a Waters Quattro XE MS in positive ESI mode (capillary 3.4 kV, cone 30 V, m/z 100–800). Post-column makeup flow was added via splitter into the MS. Data were processed with MassLynx v4.1.

Main Results and Discussion

The optimized method—linear gradient to 60% MeOH containing 40 mM ammonium formate, 2% water, and 0.2% formic acid—achieved baseline separation of 31 out of 33 analytes in a 12 min gradient (total cycle 16 min). Peak capacity exceeded 200, with asymmetry factors averaging 1.1. Critical isomeric pairs (cytidine/2′-deoxycytidine) displayed resolution >4.5. Addition of water improved peak symmetry; buffer enhanced selectivity and retention; low-level formic acid corrected tailing and promoted early elution of acidic analytes. Use of acetonitrile in the co-solvent increased retention but reduced peak capacity.

Advantages and Practical Applications

• High throughput: rapid re-equilibration and 16 min sample cycle
• Broad polarity range: effective for acids, bases, nucleosides, and small drugs
• Compatibility with aqueous extracts: direct injection of urine/plasma samples
• High resolution: peak capacity >200 enables complex mixture profiling
• MS detection: sensitive and selective identification of endogenous metabolites

Future Trends and Opportunities

UPC2-MS is poised for integration into large-scale metabolomics and clinical workflows. Future directions include coupling with high-resolution MS, expanding stationary phase chemistries for tailored selectivity, automated online sample preparation, and multimodal omics platforms. Advances in supercritical fluid modifiers may further enhance peak shape and polarity coverage.

Conclusion

The study demonstrates that UPC2-MS with optimized co-solvent composition, buffer strength, and acidification provides a robust, generic screening method for polar metabolites in aqueous samples. The approach offers an attractive alternative to HILIC-UPLC, delivering high resolution, throughput, and compatibility with biofluid extracts.

Reference

1. Pinkston JD, Stanton DT, Wen D. Elution and preliminary structure-retention modelling of polar and ionic substances in supercritical fluid chromatography using volatile ammonium salts. J Sep Sci. 2004;27:115–123.
2. Ashraf-Khorassani M, Taylor LT. Subcritical fluid chromatography of water-soluble nucleobases on various polar stationary phases facilitated with alcohol-modified CO2 and water as the polar additive. J Sep Sci. 2010;33:1682–1691.
3. Taylor LT. Packed column supercritical fluid chromatography of hydrophilic analytes via water-rich modifiers. J Pharm Biomed Anal. 2012;43:464–470.
4. Liu Y, Li X, Yang C, Tai S, Zhang X, Liu G. UPLC-MS/MS method for simultaneous determination of caffeine, tolbutamide, metoprolol, and dapsone in rat plasma and its application to cytochrome P450 activity study. J Chrom Sci. 2013;51:26–32.
5. Cazenave-Gassiot A, Boughtflower B, Caldwell J, et al. Effect of increasing concentration of ammonium acetate as an additive in supercritical fluid chromatography using CO2–methanol mobile phase. J Chrom A. 2009;1216:6441–6450.