Determination of sugars in foods using HPAE- PAD in dual eluent generation cartridge mode

Significance of the Topic

Determination of individual sugars in food matrices is crucial for nutritional labeling, quality control, and research into carbohydrate metabolism. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) offers direct detection of underivatized saccharides, overcoming limitations of refractive index or derivatization-dependent methods. The adoption of automated eluent generation further enhances reproducibility and simplifies workflows.

Objectives and Overview

This study adapts the AOAC 2018.16 First Action Method for sugar profiling in foods to Thermo Scientific’s Dual Eluent Generation Cartridge (Dual EGC) mode using a Dionex CarboPac PA20-1 mm column. Goals include reducing eluent consumption, eliminating manual reagent preparation, and evaluating method performance—linearity, sensitivity, accuracy, precision, and stability—across model and complex food samples.

Methodology and Instrumentation

• Sample Preparation: Three extraction procedures were applied based on protein/sugar content—low protein, high protein (Carrez cleanup), and high sugar matrices—followed by filtration, centrifugation, vacuum concentration, and dilution.
• Dual EGC Gradient: Series configuration of MSA and KOH eluent generator cartridges produced a reproducible gradient (0–10 mM KOH/0 mM MSA to 200 mM KOH/0 mM MSA, a wash at 100 mM/100 mM, and re-equilibration) at 0.056 mL/min.
• Column and Detection: Dionex CarboPac PA20 guard (1×30 mm) and separation (1×150 mm) columns operated at 28 °C. A pulsed amperometric detector with a disposable gold electrode using a four-potential waveform monitored carbohydrate signals.

Used Instrumentation

• Thermo Scientific Dionex ICS-6000 HPIC system with SP/DP pump, eluent generator, electrochemical detector, and autosampler.
• Eletrochemical detection cell, Ag/AgCl reference electrode, gold on PTFE working electrodes.
• Dual EG eluent degasser, CarboPac PA20 column pack, and Chromeleon CDS software v7.2.9.

Results and Discussion

• Separation: Baseline resolution of galactose, glucose, fructose, sucrose, lactose, maltose, and internal standard arabinose was achieved, with clear separation from potential sugar alcohols and amino sugar interferences.
• Stability: Retention times remained stable over 15 days of continuous operation, confirming robust baseline and gradient performance.
• Calibration: Seven-point curves (0.25–25 mg/L) yielded coefficients of determination ≥0.999 (linear for disaccharides, quadratic for monosaccharides).
• Sensitivity: Limits of detection (S/N=3) ranged from 3.5 to 9.8 µg/L and limits of quantification (S/N=10) from 11.7 to 32.7 µg/L, corresponding to sample LOQs of 7–65 µg/g.
• Accuracy and Precision: Spike recoveries of 91–110% and inter-day peak area RSDs of 0.4–1.6% demonstrated method accuracy and reproducibility.
• Sample Analysis: Infant formula contained ~46 g/100 g lactose and minor glucose/galactose; whey flour showed ~2.7 g/100 g lactose and trace hexoses; chocolate powder required dilution to quantify ~69 g/100 g sucrose.

Benefits and Practical Applications

• Automated eluent generation reduces manual handling, improves reproducibility, and minimizes chemical waste.
• Low flow rates on the 1 mm column lower reagent use while maintaining high resolution.
• Direct detection of underivatized sugars accelerates analysis and reduces potential errors.
• Suitable for routine QA/QC in food, dietary supplements, and complex biological matrices.

Future Trends and Potential Applications

Further developments may include miniaturized systems, integration with on-line sample preparation, coupling with mass spectrometry for expanded carbohydrate profiling, and application to glycan analysis in biomedical and plant research. Advances in eluent generation chemistry could extend dynamic range and accommodate oligosaccharide determination in increasingly complex samples.

Conclusion

The adaptation of AOAC 2018.16 to Dual EGC mode with a CarboPac PA20-1 mm column delivers a robust, accurate, and reproducible HPAE-PAD method for sugar determination in foods. Automated gradient generation simplifies operations, reduces consumable use, and maintains high chromatographic performance.