Carbohydrate analysis of agave syrup using HPAE-PAD

Importance of the Topic

Agave syrup has emerged as a popular low-glycemic sweetener alternative to honey and sucrose, owing to its fructose-rich composition and sweeter taste. Accurate profiling of its carbohydrate content is critical both for nutritional labeling—impacting dietary recommendations—and for authentication, since agave syrup is prone to economically motivated adulteration with cheaper sweeteners like high-fructose corn syrup.

Study Aims and Overview

This work evaluates the Official Mexican Standard NOM-003-SAGARPA-2016 method for analyzing agave syrup carbohydrates using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Key objectives include adapting the method to a Thermo Scientific Dionex CarboPac PA1-2 mm column, assessing chromatographic performance (separation, linearity, sensitivity, precision, accuracy), and applying enzymatic hydrolysis (amyloglucosidase, fructanase) to detect adulteration and quantify fructans.

Methodology and Instrumentation

Chromatographic separation employed a Dionex CarboPac PA1 guard (2×50 mm) and separation (2×250 mm) columns at 30 °C, with a three-eluent gradient of NaOH and sodium acetate at 0.25 mL/min. Detection used a Dionex ICS-5000+ HPIC system with pulsed amperometric gold electrode. Sample preparation encompassed water dilution, 0.2 µm filtration, and enzyme treatments to hydrolyze starch-like and fructan polymers. Calibration covered seven analytes (inositol, sorbitol, mannitol, HMF, glucose, fructose, sucrose) over relevant concentration ranges.

Results and Discussion

• Separation and Calibration: All seven analytes were baseline-resolved (resolution >2.0) with linear correlations (r²>0.999) except fructose, which followed a quadratic fit (r²=0.9991).
• Sensitivity: Limits of detection ranged from 1.4 to 15 µg/L and quantification limits from 4.6 to 51 µg/L, corresponding to 0.92–10.2 µg/g and 3.06–34.1 µg/g in sample.
• Precision and Accuracy: Retention time RSDs were <1.3%, peak area RSDs ≤2.8%. Recoveries for spiked sorbitol, glucose, and sucrose fell between 85% and 115%.
• Sample Composition: Three commercial agave syrups contained 70–73% fructose, 1.3–1.6% glucose, <0.1% sucrose, 0.06–0.11% inositol, 0.10–0.30% mannitol; HMF was below detection. Enzymatic profiles confirmed absence of starch-derived sugars and indicated 2.3–3.2% total fructan content.
• Adulteration Assessment: Minimal change in oligosaccharide profiles after amyloglucosidase treatment ruled out high-fructose corn syrup adulteration.

Benefits and Practical Applications

The optimized HPAE-PAD method delivers rapid, sensitive, and accurate carbohydrate profiling for quality control and regulatory compliance. Reduced eluent consumption with the 2 mm column lowers operating costs. The approach enables simultaneous quantification of mono-, di-, and oligosaccharides along with HMF, facilitating comprehensive compositional and authenticity assessments.

Future Trends and Applications

Emerging directions include high-throughput and automated workflows for routine quality control, further miniaturization of columns to reduce solvent use, integration with mass spectrometry for structural elucidation of novel oligosaccharides, and application to other complex natural sweetener matrices such as maple or palm syrups.

Conclusion

The Dionex CarboPac PA1-2 mm HPAE-PAD method effectively implements the Mexican NOM guideline for agave syrup carbohydrate analysis, demonstrating excellent chromatographic performance, robust quantification, and reliability for detecting adulteration. This protocol supports both nutritional monitoring and authenticity verification in food quality laboratories.

Reference

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3. NORMA Official Mexicana NOM-003-SAGARPA-2016.
4. Willems JL, Low NH. Carbohydrate, polyol, and oligosaccharide profiles of agave syrup and implications for authenticity. J Agric Food Chem. 2012;60(35):8745–54.
5. Thermo Scientific Dionex ICS-5000+ Ion Chromatography System Operator’s Manual P/N 065446, 2014.
6. Thermo Scientific Electrochemical Detection User’s Compendium P/N 065340-02, 2013.