Simultaneous Determination of Reducing Sugars in Honey by Capillary Zone Electrophoresis with LIF Detection Using Low-Toxicity 2-Picoline Borane and APTS for Pre-Capillary Derivatization

TL;DR

This paper presents a new method to accurately measure reducing sugars in honey using a safer and more sensitive technique.

Contribution

A safer, low-toxicity pre-capillary derivatization method for CZE-LIF analysis of reducing sugars in honey is developed.

Findings

Optimal derivatization conditions for glucose, mannose, and maltose were identified at 50 °C in 0.5 M citric acid.

Fructose showed low reactivity due to its ketose structure.

Liquid–liquid extraction with ethyl acetate was the most effective cleanup strategy for reducing background signals.

Abstract

This study aimed to develop a reliable method for profiling reducing sugars in honey using capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). Reducing sugars were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in the presence of 2-picoline borane, a safer alternative to sodium cyanoborohydride. Key parameters influencing the derivatization efficiency—temperature, pH, incubation time, and reagent concentrations—were systematically optimized. The highest labeling efficiency for glucose, mannose, and maltose was achieved at 50 °C in 0.5 M citric acid with 0.1 M APTS, while fructose showed low reactivity due to its ketose structure. To reduce the background signal from excess reagents, three cleanup strategies were evaluated. Liquid–liquid extraction with ethyl acetate effectively removed unreacted APTS without significant analyte loss, whereas solid-phase extraction and microextraction caused substantial losses of hydrophilic sugars. The method showed good linearity (0.5–10 mM, R2 > 0.994), precision (RSD 0.81–13.73%), and accuracy (recoveries 93.47–119.75%). Stability studies indicated that sugar standards should be stored at –20 °C. The method was successfully applied to the analysis of four nectar honeys—rapeseed, acacia, phacelia, and dandelion—revealing differences in glucose and fructose content related to botanical origin. The results confirm the suitability of CZE-LIF for sensitive and selective carbohydrate analyses in complex food matrices.

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