Determination of Carbohydrates in Urine by Capillary HPAE-PAD

Significance of the Topic

Carbohydrate markers such as mannitol, rhamnose, xylose, 3-O-methylglucose, and lactulose are widely used to assess intestinal permeability and disease states in clinical and veterinary research. Accurate, sensitive analysis of these sugars in urine is essential for diagnostics and monitoring.

Study Objectives and Overview

This work demonstrates direct determination of monosaccharides and disaccharides in synthetic urine using capillary high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) on a Thermo Scientific Dionex ICS-4000 HPIC capillary system. Building on previous analytical-scale methods (AN1006), the method aims to achieve low-picomole sensitivity, reduced eluent consumption, and continuous uptime.

Methodology and Instrumentation

• Separation on a Dionex CarboPac PA20 capillary guard and analytical columns (0.4 × 35 mm and 0.4 × 150 mm).
• Eluent generation via RFIC™ with EGC-KOH and CR-ATC cartridges, delivering a gradient of 10–35 mM KOH at 0.008 mL/min.
• Temperature control: column at 30 °C, compartment at 27 °C; injection volume 0.4 µL.
• Detection by PAD on a gold/PTFE disposable electrode with a four-potential carbohydrate waveform; reference electrode Ag/AgCl.
• System components include Dionex ICS-4000 HPIC, electrochemical detector, AS-AP autosampler, and precision capillary fittings.

Main Results and Discussion

• Baseline separation of ten carbohydrates in 16 minutes, with partial resolution of critical pairs (3-O-methylglucose/rhamnose and lactose/lactulose).
• Comparable performance to analytical-scale methods, while reducing eluent and waste to ~15 mL/day and extending cartridge life to ~18 months.
• Overlay of water and synthetic urine matrices showed consistent retention times and minimal matrix interference, aside from a salt elution void peak.
• Detection limits reach the low picomole range, with stable baselines and reproducible peak areas.

Benefits and Practical Applications

• No derivatization required, lowering cost and preparation time.
• Capillary format enables high-throughput, 24/7 operation with minimal reagent use.
• Eluent automation avoids manual errors and carbonate contamination.
• Applicable to clinical intestinal permeability tests, pharmacokinetic studies, and non-invasive diagnostics.

Future Trends and Applications

• Coupling HPAE-PAD with mass spectrometry for structural confirmation and increased sensitivity.
• Fully automated workflows for routine clinical and veterinary laboratories.
• Extension to novel glycan biomarkers such as sialic acids and glycopeptides.
• Development of microfluidic or point-of-care platforms for rapid carbohydrate analysis.

Conclusion

The capillary HPAE-PAD method on the Dionex ICS-4000 HPIC system provides a robust, sensitive, and sustainable approach for urinary carbohydrate analysis, supporting diagnostic and research applications with reduced solvent usage and continuous operation.

Reference

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