Abstract:

Excessive sugar consumption is associated with metabolic health problems. Rare sugars are gradually being used as substitutes for sugar, and their consumption is increasing daily, raising food-safety issues such as false advertising, adulteration, and overdosing. The determination of rare-sugar compounds has attracted considerable attention in recent years. However, no standard method for the simultaneous determination of six rare sugars (allulose, tagatose, trehalose, isomaltulose, erythritol, and mannitol) in solid foods is available. Therefore, establishing a suitable analytical method for these sugars is necessary. In this study, high performance liquid chromatography coupled with evaporative light-scattering detection was used to determine rare sugars in solid foods. The optimum chromatographic and detector conditions were determined by evaluating the instrument parameters. Analysis was carried out on a Zorbax Original NH₂ column (250 mm×4.6 mm, 5 μm) via flow-rate gradient elution (0-15 min, 1.0 mL/min; 15-18 min, 1.0-2.0 mL/min; 18-25 min, 2.0 mL/min) with acetonitrile-water (80∶20, v/v) as the mobile phase. Sharp and symmetric chromatographic peaks were obtained under these conditions. The resolutions for all the six rare sugars were greater than 1.5. Optimization of the evaporative light-scattering detector was extremely important to the responses of the rare-sugar compounds. The two most significant parameters were the nebulizer carrier gas flow rate and drift tube temperature. The detection system was operated under the following conditions: the drift tube temperature was set to 50 ℃, the nebulizer carrier gas was high-purity nitrogen, the carrier gas flow rate was 1.0 mL/min, the nitrogen pressure was regulated to 275.79 kPa, and the gain factor was set to 3. The sample was extracted with 25 mL of water, shaken and vortexed for 10 min, purified with 200 μL of zinc acetate solution and 200 μL of potassium ferricyanide solution, and centrifuged at 4500 r/min for 10 min. Next, 1 mL of the supernatant was passed through a 0.22 μm aqueous-phase filter membrane, and the filtrate obtained was analyzed using the evaporative light-scattering detector. The six rare sugars were quantitatively analyzed using the external standard method and showed good linearity with coefficients of determination (R²) greater than 0.9985. The limits of detection and quantification were 0.020-0.60 and 0.60-1.8 g/100 g, respectively. In addition, when blank solid food samples were spiked with the analytes at three levels, the average recoveries of the six rare sugars were 92.6%-103.2%, with relative standard deviations (RSDs) of 0.7%-4.4%. An RSD of <5% indicated that the method had good precision. Interference experiments were performed to determine whether the sugars and artificial sweeteners commonly found in solid foods affected the targets. The method established in this study was used to analyze the contents of the six rare sugars in actual solid food samples. The experimental results showed various levels of rare glycoconjugates in different solid foods. Moreover, the actual compositions and labeled of rare glycoconjugates in the solid foods were generally consistent. The proposed method features simple operation, rapid results, high sensitivity, and good reproducibility; thus, it meets the requirements for the detection of the six rare sugars in solid foods. It also provides technical support for the development of methodological standards and detection limits for rare sugars in Chinese foods. The results of this study are of great relevance for the daily monitoring of the levels of the six rare sugars in solid foods.

Key words: high performance liquid chromatography (HPLC), evaporative light-scattering detector (ELSD), rare sugar, solid food