基于TLC和HPLC-CAD法比较分析天然和市售蜂蜜糖类成分

目的：探讨糖类成分用于鉴别蜂蜜真伪的可行性。方法：通过对14批天然采集蜂蜜和14批市售蜂蜜中糖类成分比较，了解天然蜂蜜糖类特征。首先利用薄层色谱法（TLC法）对蜂蜜中的果糖、葡萄糖、蔗糖和麦芽糖等糖类成分进行快速鉴别，采用薄层硅胶板G60，展开剂为正丁醇-异丙醇-醋酸-水（7：5：1：2），显色剂为苯胺-二苯胺-磷酸溶液；再利用高效液相色谱联用电喷雾检测器法（HPLC-CAD法）对果糖、葡萄糖、蔗糖和麦芽糖进行定量分析，采用氨基分析柱（4.6 mm×250 mm，5 μm），以乙腈（A）-水（B）为流动相梯度洗脱，流速1 mL·min^-1，柱温30℃，进样量5 μL，N₂压力241.325 kPa。结果：天然采集蜂蜜在TLC中具有特征条带且相似，主要为果糖、葡萄糖、蔗糖、麦芽糖；而3批市售蜂蜜样品中含不同于天然蜂蜜的寡糖条带，可能有外源性糖类掺杂。采用HPLC-CAD定量分析，果糖、葡萄糖、蔗糖及麦芽糖线性范围分别为16.4~493.0、17.4~521.0、0.72~62.3和0.68~64.0 μg·mL^-1，线性关系良好（r>0.998 1），平均回收率在87.0%~112.8%之间，RSD为0.7%~6.1%。14批天然蜂蜜和14批市售蜂蜜中果糖含量分别为43.5%~53.5%和45.4%~54.3%，葡萄糖含量分别为41.3%~50.3%和41.0%~50.2%，均无统计学差异（p>0.05）。此外，蜂蜜中还存在有少量蔗糖（0.41%~6.6%）和麦芽糖（0.64%~1.8%）。结论：天然蜂蜜主要含果糖、葡萄糖以及少量蔗糖和麦芽糖，可利用寡糖的存在与否，判断蜂蜜是否有外源性糖类掺入，以评价市售蜂蜜质量。

Objective:To explore the feasibility of carbohydrates in authentication of honey.Methods:Carbohydrates in 14 batches of wild honey samples and 14 batches of commercial honey samples were investigated to reveal the sugar characters of wild honey.The contents of fructose,glucose,sucrose and maltose in honeys were screened by using thin layer chromatography (TLC) on a silica gel 60 plate.The plate was developed with 1-butanol-isopropanol-acedic acid-water (7:5:1:2) and colorized with aniline-diphenylamine-phosphoric acid solution.Quantification was performed by high performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD).An amide column (4.6 mm×250 mm,5 μm) was adopted,the mobile phase consisted of acetonitrile-water with gradient elution at the flow rate of 1 mL·min^-1,and the column temperature was controlled at 30℃.The injection volume was 5 μL and the pressure of N₂ was 241.325 kPa.Results:All wild honeys have a similar TLC profiles,and the main sugars were fructose,glucose,sucrose and maltose.Oligosaccharides were observed in three batches of commercial samples,which might indicate the potential adulterants.Based on HPLC-CAD analysis,good linear relationship was observed for fructose,glucose,sucrose and maltose in the range of 16.4-493.0,17.4-521.0,0.72-62.3 and 0.68-64.0 μg·mL^-1,respectively (r>0.998 1).The average recoveries fell between 87.0%-112.8% with RSD of 0.7%-6.1%.The contents of fructose were 43.5%-53.5% and 45.4%-54.3%,and the contents of glucose were 41.3%-50.3% and 41.0%-50.2%,respectively,in wild (n=14) and commercial (n=14) honey samples,without significant differences in statistics between the two groups (p>0.05).In addition,small amounts of sucrose (0.41%-6.6%) and maltose (0.64%-1.8%) were also detected in both types of honey.Conclusion:The main sugars in wild honey were fructose and glucose,with minor sucrose and maltose.The presence of oligosaccharides could be used as a marker for adulteration,which could be applied to the authentication of commercial honey.