采用超高效液相色谱法(UHPLC)建立28批不同产地狼牙蜜样品的指纹图谱,结合化学模式识别对狼牙蜜进行质量研究,为狼牙蜜的掺伪鉴别和产地来源判断提供依据。称取狼牙蜜样品20 g,加入15 mL水,涡旋溶解,然后加入15 mL乙酸乙酯,涡旋提取,离心后,取上清液,残渣中加入10 mL乙酸乙酯重复提取一次,合并上清液,于30 ℃氮吹至近干,用1 mL甲醇复溶,过0.22 μm有机滤膜。滤液注入超高效液相色谱仪,以ACQUITY UPLC BEH C₁₈色谱柱为固定相,以0.1%(体积分数,下同)甲酸溶液-含0.1%甲酸的甲醇溶液为流动相体系进行梯度洗脱,用二极管阵列检测器在检测波长280 nm下进行测定。利用中药色谱指纹图谱相似度评价系统(2012.130723版),得到28批狼牙蜜样品的指纹图谱和对照指纹图谱,生成22个共有峰。通过与混合对照品溶液色谱图进行比对,初步指认出其中9个共有峰,对应的化合物分别为对羟基苯甲酸、咖啡酸、对香豆酸、阿魏酸、芥子酸、丁香酸甲酯、槲皮素、山奈酚、白杨素。28批狼牙蜜样品的指纹图谱与对照指纹图谱的相似度为0.909~0.993。对掺入不同质量分数(5%~50%)油菜蜜和洋槐蜜的狼牙蜜样品进行模拟掺伪检测分析,得到的掺伪狼牙蜜样品的指纹图谱与对照指纹图谱的相似度为0.251~0.853。采用分层聚类分析、主成分分析、正交偏最小二乘-判别分析对28批狼牙蜜样品进行研究,结果表明,3种方法均将28批狼牙蜜样品分为3类,徽县产区的样品被分为一类,两当县产区的样品被分为两类。对指认出的9种化合物进行定量分析,9种化合物标准曲线的线性范围均为0.5~200.0 mg·L^-1,检出限(3S/N)均为0.01 mg·kg^-1,28批狼牙蜜样品中对羟基苯甲酸、芥子酸、槲皮素、山奈酚含量较高,但各批次样品中每种成分含量差异较大。

The fingerprints of 28 batches of bitter thorn nectar samples from different origins were established by ultra-high performance liquid chromatography (UHPLC), and the quality of samples were studied by UHPLC fringerprints combined with chemical pattern recognition, which provided the basis for the identification of adulteration and discrimination of origin of bitter thorn nectar. Bitter thorn nectar sample (20 g) was taken, and dissolved by 15 mL of water with vortex, and 15 mL of ethyl acetate was added into the mixture for extraction with vortex. After centrifugation, the supernatant was taken, and 10 mL of ethyl acetate was added into the residue for repeat extraction. The supernatants obtained were combined and blown to near dryness by nitrogen at 30 ℃, the residue was redissolved by 1 mL of methanol, and the mixed solution was filtered through a 0.22 μm organic membrane. The filtrate obtained was injected into ultra-high performance liquid chromatograph, with ACQUITY UPLC BEH C₁₈ column as stationary phase, and 0.1% (volume fraction) formic acid solution and methanol solution containing 0.1% formic acid as mobile phase system for gradient elution, and determined by diode array detector at the detection wavelength of 280 nm. The fingerprints of 28 batches of bitter thorn nectar samples and reference fingerprint were obtained by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012.130723 edition), and 22 common peaks were obtained. By comparing with the chromatograms of the mixed reference solutions, 9 common peaks were identified, and the corresponding compounds were p-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, erucinic acid, methyl eugenate, quercetin, kaempferol and albumin. The similarities between fingerprints of 28 batches of bitter thorn nectar samples and reference fingerprint were in the range of 0.909-0.993, and the similarities between fingerprints of pseudo bitter thorn nectar samples and reference fingerprint were in the range of 0.251-0.853 by simulated adulteration detection analysis of rape honey and locust honey mixed with different mass concentrations (5%-50%). The 28 batches of bitter thorn nectar samples were studied by hierarchical cluster analysis, principal component analysis and orthogonal partial least square-discriminant analysis. The results showed that 28 batches of bitter thorn nectar samples were divided into three categories by the three methods, with the samples from Huixian county for one category, and the samples from Liangdang county for two categories. The quantitative analysis of 9 compounds identified showed that the linear ranges of the standard curves of 9 compounds were in the range of 0.5-200.0 mg·L^-1, with the same detection limit (3S/N) of 0.01 mg·kg^-1. The contents of p-hydroxybenzoic acid, erucinic acid, quercetin and kaempferol in 28 batches of bitter thorn nectar samples were higher, but that of each component in each batch of sample was significantly different.