Determination of low- and non-caloric sweeteners in food and beverages by HPAE-PAD

Importance of the topic

Consumption of low- and non-caloric sweeteners has grown as consumers seek to reduce sugar intake, address obesity concerns, and manage blood glucose. Accurate quantification of sugar substitutes like erythritol, allulose and sucralose in foods and beverages is critical for nutritional labeling, quality control, and regulatory compliance. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) offers direct, sensitive analysis of these compounds without chemical derivatization.

Objectives and overview

This study aimed to develop and validate a robust HPAE-PAD method for simultaneous determination of three sugar substitutes—erythritol, allulose, and sucralose—in a variety of food and beverage matrices. The work included analytical method design, comparison of two reference electrodes, and application to commercial samples.

Methodology

• Eluent gradient: 100 mM NaOH initial condition, followed by a sodium acetate gradient (up to 300 mM NaOAc in 100 mM NaOH) to achieve baseline separation of early-eluting sugars (glucose, fructose, allulose, sucrose) and later-eluting sucralose.
• Sample preparation: Pulverization of solid products, dissolution in deionized water, centrifugation, 0.2 µm filtration, and dilution to appropriate levels.
• Calibration: Stock standards (1000 mg/L) diluted to nine mixed standards (0.156–40 mg/L). Linear regression applied to all analytes except erythritol, which used a quadratic fit.
• Validation parameters: Linearity (r2 > 0.999), precision (RSD <0.5% for retention times and <5% for peak areas), accuracy (spike recovery range 83–116%), and electrode comparison.

Instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC system with dual pump, dual temperature chromatography compartment, and electrochemical detector cell.
• Thermo Scientific Dionex AS-AP autosampler with tray cooling.
• Dionex CarboPac PA20 Guard (3 × 30 mm) and Analytical (3 × 150 mm) columns at 30 °C.
• Gold working electrode with either PdH or Ag/AgCl reference electrodes in a pulsed amperometric detection cell.
• Degassed 100 mM NaOH and 1 M sodium acetate/100 mM NaOH eluents maintained under helium or nitrogen at 5–8 psi.

Main results and discussion

The optimized gradient separated six sugars within a 25 min cycle. Sucralose elution was influenced by its electronegative chlorine atoms, necessitating sufficient hydroxide concentration for ionization. Comparison of PdH and Ag/AgCl reference electrodes revealed similar background (~25 nC) and noise (~35 pC/min) levels, with 0–9% higher peak areas using the PdH electrode.

Five commercial products (two protein bars, a keto cookie mix, flavored water, and a sports drink) were analyzed. Erythritol, allulose, and sucrose were quantified in bars and mix; flavored water contained erythritol only; sports drink contained sucralose only. Results from both electrodes agreed within ±5%.

Calibration curves exhibited excellent linearity for allulose, sucrose, and sucralose over their respective ranges; erythritol required a quadratic model to accommodate detector response at higher levels.

Spike recovery experiments at two levels returned 83–116% for all analytes when matrix effects were controlled by appropriate dilution. Method precision across nine injections showed RSD <0.5% for retention times and <5% for peak areas.

Benefits and practical applications

• No sample derivatization is required, simplifying workflow and reducing analysis time.
• The method provides high sensitivity and selectivity for a wide range of sweeteners in complex matrices.
• Gradient control of acetate concentration allows tunable retention and resolution within a single run.
• Applicability to diverse products supports routine quality control and label verification in food and beverage industries.

Future trends and possibilities

As interest in rare and low-calorie sugars expands, coupling HPAE-PAD with mass spectrometry may offer enhanced specificity and lower detection limits. Automation of sample prep and online filtration systems could further increase throughput in high-volume testing laboratories. The method may also be extended to additional sugar alcohols and novel sweeteners emerging in the market.

Conclusion

An HPAE-PAD method on the Dionex CarboPac PA20 column was successfully developed and validated for simultaneous determination of erythritol, allulose, and sucralose in various food and beverage matrices. The approach demonstrated excellent linearity, precision, and accuracy, with interchangeable use of PdH and Ag/AgCl reference electrodes. Its simplicity and robustness make it well suited for regulatory compliance, product development, and quality assurance.

References

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