Improved carbohydrate analysis of agave syrup using HPAE-PAD in dual eluent generation cartridge mode

Importance of the Topic

Agave syrup has become a popular alternative sweetener owing to its low glycemic index and predominantly fructose composition. Its higher sweetness intensity compared to sucrose or honey allows lower usage rates for the same sweetness, reducing calorie intake. Comprehensive profiling of sugars and oligosaccharides in agave syrup is essential both for nutritional labeling and for detecting economically motivated adulteration by cheaper sweeteners such as high fructose corn syrup or other sugars.

Goals and Study Overview

This study adapts the official Mexican standard method for agave syrup carbohydrate analysis to a system using a high resolution Dionex CarboPac PA200 1 mm column combined with dual eluent generation technology. The objective is to evaluate separation performance, linearity, detection limits, precision, and accuracy for major sugars, polyols and 5-hydroxymethylfurfural. Moreover, enzymatic hydrolysis protocols with amyloglucosidase and fructanase are applied to detect adulteration and quantify total fructan content.

Methodology and Instrumentation

The analysis employs a Thermo Scientific Dionex ICS-6000 HPIC system in dual EGC mode with inline generation of KOH and methanesulfonic acid eluents. Key components include

• Dual EGC cartridges for methanesulfonic acid and potassium hydroxide
• Dionex CarboPac PA200 guard and separation columns (1 × 50 mm and 1 × 250 mm)
• Pulsed amperometric detection on a gold electrode with Ag/AgCl reference
• Autosampler with sample cooling and a 0.4 µL injection loop

Chromatographic conditions feature a low flow rate of 0.063 mL/min, column temperature at 30 C, and a 70 min gradient between KOH and KMSA. Calibration covers seven analytes across relevant concentration ranges. Enzymatic treatments are carried out at 45 C for partial hydrolysis and at 80 C to stop reactions, followed by dilution and filtration prior to analysis.

Main Results and Discussion

The PA200 column yielded baseline separation of inositol, sorbitol, mannitol, HMF, fructose, glucose and sucrose with resolution exceeding 2. Calibration for six analytes was linear with r2 above 0.999, while fructose required quadratic fitting. Limits of detection in syrup matrix ranged from sub-microgram to tens of microgram per gram for each component. Precision for retention time (RSD <0.3 ) and peak area (RSD <1.7 ) met stringent quality criteria. Recovery experiments in real samples delivered 97 to 105 .
Analysis of three commercial agave syrups showed fructose as the dominant carbohydrate (71–75 of total), followed by glucose (1.3–1.5 ), minor polyols inositol and mannitol (0.06–0.3 ) and trace HMF below quantification. Oligosaccharide profiles before and after amyloglucosidase treatment showed minimal glucose increase, indicating absence of high fructose corn syrup adulteration. Fructanase digestion produced characteristic loss of higher oligosaccharides and allowed calculation of total fructan content (1.4–3.3 ).

Benefits and Practical Applications of the Method

This high resolution, dual EGC approach offers

• Elimination of manual eluent preparation
• Reduced reagent consumption and waste
• Excellent separation of carbohydrates and oligosaccharides
• Enhanced precision and system reproducibility
• Direct, derivatization free detection for QA QC and authenticity testing

Future Trends and Potential Applications

Advancements may include automation of enzymatic treatment steps, integration of high throughput sample handling, and application to a broader range of food matrices. Coupling with multivariate data analysis and machine learning for rapid adulteration screening will strengthen quality control. Emerging eluent generation technologies promise further simplification of chromatographic workflows.

Conclusion

The dual EGC HPAE PAD method on a CarboPac PA200 1 mm column meets the official Mexican standard for agave syrup carbohydrate analysis while improving efficiency, precision and ease of operation. It provides reliable quantification of key sugars, polyols and HMF, and enables sensitive detection of adulteration by enzymatic profiling. The approach is well suited for routine QA QC, regulatory compliance and research in food authenticity.

References

1 Wolever T Vuksan V Jenkins A Campbell J Final report on glycemic index of organic blue agave syrup Glycemic Index Laboratories Toronto ON 2011
2 Foster-Powell K Holt SHA Brand-Miller JC International table of glycemic index and glycemic load values Am J Clin Nutr 2002 76 5–56
3 Norma Oficial Mexicana NOM-003-SAGARPA-2016 Official guideline for agave syrup characterization Mexico
4 Thermo Fisher Scientific Application note 73132 Carbohydrate analysis of agave syrup using HPAE PAD
5 Thermo Fisher Scientific Application note 73896 Carbohydrate analysis of agave syrup in dual eluent generation cartridge mode
6 Willems JL Low NH Major carbohydrate polyol and oligosaccharide profiles of agave syrup and application to authenticity analysis J Agric Food Chem 2012 60 8745–54
7 Thermo Scientific Dionex ICS-6000 Operator’s Manual P N 22181-97002
8 Thermo Scientific Electrochemical Detection User’s Compendium P N 065340-02 April 2013
9 Thermo Scientific Dionex CarboPac PA200 Columns Product Manual P N 065734-01 January 2018
10 ICH Guideline Q2B Validation of Analytical Procedures Methodology 1996