采用液相色谱-同位素质谱联用法(LC-IRMS)建立了测定蜂蜜中外源性γ-淀粉酶残留量的方法.先采用凝胶色谱柱对蜂蜜样品进行预分离,将样品中所含的酶与糖分离开.根据γ-淀粉酶可将底物麦芽糖酶解为葡萄糖的原理,在55℃、pH4.5的0.03 mol/L磷酸盐缓冲液中将γ-淀粉酶与麦芽糖反应48 h后,采用LC分离麦芽糖和葡萄糖,以IRMS测定酶解产物葡萄糖的含量来确定γ-淀粉酶的残留量.本方法的线性范围为5～ 200 U/kg,定量限为5 U/kg,回收率为89.6％～ 108.2％,相对标准偏差为3.3％ ～4.9％.采用本方法对市售蜂蜜和大米糖浆共38个样本进行了考察,γ-淀粉酶的检出率为76.3％.为了进一步验证本方法的检测能力,测定了掺入15％(质量分数)大米糖浆的蜂蜜样品,测得γ-淀粉酶的含量为10.2 U/kg.本方法能够有效地从酶学的角度鉴定蜂蜜中是否含有大米糖浆.%A novel method for the determination of exogenous y-amylase residue in honey using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) was established. After pre-separation by gel column chromatography, the y-amylase in honey samples was separated from the sugars. The y-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 y-amylase in honey can be determined. The linear range of y-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 y-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 y-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.
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