烟草样品溶液中的葡萄糖在葡萄糖氧化酶和过氧化物酶的作用下,可使色原性氧受体4-氨基安替比林与酚定量缩合,生成红色醌类化合物,通过Image软件将溶液颜色转化为灰度值信号,从而确定样品中葡萄糖的含量。样品经干燥、研磨后,分取(0.1±0.0001)g,加入20mL水,于(30±0.5)℃振荡萃取30min。用快速定性滤纸过滤,弃去初始滤液,收集后续的2~3mL滤液备用。移取100μL上述滤液于装有R1和R2试剂各450μL的2mL微量离心管中,混匀后置于(37±0.5)℃的恒温水浴摇床中反应5min。利用智能手机对显色液拍照,通过Image软件将彩色图片转化为灰度模式,得到灰度值。以水为参比,对显色液灰度值进行归一化处理,得到灰度值比值。以葡萄糖浓度的对数为横坐标,灰度值比值为纵坐标绘制标准曲线。结果显示:标准曲线的线性范围为0.100~4.000mmol·L^-1,检出限(3s)为0.0216mmol·L^-1;对相同浓度的葡萄糖、果糖、甘露糖、木糖等9种糖类化合物标准溶液进行测定,除葡萄糖外,其余糖类显色液的灰度值与空白溶液无明显差别;对标准烤烟样品进行精密度和回收试验,测定值的相对标准偏差(n=6)均不大于4.0%,葡萄糖的加标回收率为98.3%~103%;方法用于实际样品分析,并与高效液相色谱法进行比对,结果表明在95%置信度下,两种方法测定结果无显著性差异。

Under the action of glucose oxidase and peroxidase, the glucose in tobacco sample solution could make the quantitative condensation between chromogenic oxygen receptor 4-aminoantipyrine and phenol to generate red quinonoids. The color of the solution was transformed into gray value signal by Image software, so as to determine glucose in the sample. The sample was dried and ground, and an aliquot [(0.1±0.000 1)g] was added into 20 mL of water, then extracted by vibration at (30±0.5)℃ for 30 min. After filtration with rapid qualitative filter paper, the initial filtrate was discard, and 2-3 mL of the subsequent filtrate was collected for use. The above filtrate (100 μL) was transferred into a 2 mL-microcentrifuge tube equipped with R1 and R2 reagents of 450 μL each, mixed well, and the mixture was placed into a constant temperature water bath shaker at (37±0.5)℃ for 5 min. Smart phone was used to take photo of the color solution, then the color was transformed into gray mode by Image software to obtain gray value. Using water as reference, the gray value of the color solution was normalized to obtain the gray value ratio. The standard curve was drawn with the logarithm of concentration of glucose as horizontal coordinate and the gray value ratio as vertical coordinate. It was shown that the linear range of the standard curve was 0.100-4.000 mmol·L^-1, and the detection limit (3s) was 0.021 6 mmol·L^-1. The 9 kinds of carbohydrate standard solutions, including glucose, fructose, mannose, xylose and so on with same concentration were determined. Except for glucose, the gray values of the other carbohydrate color solutions were not significantly different from the blank solution. Tests for precision and recovery on the standard flue-cured tobacco samples showed that RSDs (n=6) of the determined values were not more than 4.0%, and the spiked recoveries of glucose were in the range of 98.3%-103%. This method was applied to the analysis of actual samples, and compared with high performance liquid chromatography. The results showed that there was no significant difference between the two methods at 95% confidence.