Abstract: A novel method for the determination of exogenous γ-amylase residue in honey using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) was established. After pre-separation by gel column chromatography, the γ-amylase in honey samples was separated from the sugars. The γ-amylase was then used to catalyze maltose into glucose. This enzymatic reaction was under the conditions of 55 ℃ and 0.03 mol/L phosphate buffer solution (pH 4.5) for 48 h. The maltose and glucose in the above enzymatic reaction solution were separated using liquid chromatography. By measuring the content of glucose with isotope ratio mass spectrometry, the γ-amylase in honey can be determined. The linear range of γ-amylase was 5~200 U/kg with the quantification limit of 5 U/kg. The recoveries were between 89.6% and 108.2% with the relative standard deviations from 3.3% to 4.9%. This method was used to analyze 38 honey and rice syrup samples, and the detection rate of γ-amylase was 76.3%. To further verify the detection capability of this method, an authentic honey was adulterated with 15% (mass fraction) rice syrup. The γ-amylase content in this sample was 10.2 U/kg. This method can effectively identify honey adulteration with rice syrups from the perspective of enzymology.

Key words: γ-amylase, adulteration, enzymatic reaction, gel column chromatography, honey, rice syrup, liquid chromatography-isotope ratio mass spectrometry (LC-IRMS)