大黄鱼HPLC指纹图谱的建立及其在产地溯源和物种鉴别中的应用
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浙江海洋学院,浙江海洋学院海洋科学与技术学院,浙江海洋学院海洋科学与技术学院,浙江海洋学院海洋科学与技术学院,浙江海洋学院海洋科学与技术学院,浙江海洋学院海洋科学与技术学院

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R 283

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国家科技支撑计划(2011BAD13B08);浙江省水产新品种选育项目(2012C12907-8)


HPLC fingerprint of large yellow croaker (Larimichthys crocea) and its application to geographical origin traceability and species identification
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Zhejiang Ocean University,,Marine Science School of Zhejiang Ocean University,,,

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    摘要:

    通过优化实验条件,选择真空冷冻干燥的方法处理实验原材料,以乙酸乙酯为溶剂采用超声法(功率为250 W、提取时间2 h)制备原材料中的有效成分。运用Agilent 1260DAD-HPLC,采用Thermo ODS-C18色谱柱,利用乙腈-0.1%磷酸水作为流动相,在流速0.5 mL/min、进样量20μL、210 nm条件下,建立了舟山养殖大黄鱼、福建养殖大黄鱼、小黄鱼、黄姑鱼和黑鳃梅童鱼的HPLC指纹图谱,以及具有18个共有峰的指纹图谱共有模式。相似度分析表明,其他几类样品和舟山大黄鱼的相似度排序为福建养殖大黄鱼> 小黄鱼> 黄姑鱼> 黑鳃梅童鱼。应用SPSS18.0数据分析软件,对18个共有峰的相对峰面积进行聚类分析、主成分分析和判别分析。聚类分析结果表明,大黄鱼、小黄鱼、黑鳃梅童鱼和黄姑鱼各自聚为一类,可用于鉴别不同的鱼类。经主成分分析,共选取到3个主成分PC1、PC2和PC3,涵盖了HPLC图谱数据信息的80.36%。根据3个主成分的得分绘制散点图,5种样品各自归于一定的区域,实现了不同产地大黄鱼以及大黄鱼与其他鱼类的鉴别。在此基础上,选择对主成分贡献较大的色谱峰的峰面积建立了5种样品的判别方程式,交互验证的正确率高达 98.4%,可用于鉴别不同产地的大黄鱼及相同产地的相近鱼种。因此,本实验的研究结果为HPLC指纹图谱技术应用于大黄鱼产地溯源提供了理论和数据支持。

    Abstract:

    Large yellow croaker (Larimichthys crocea) is one of the important economic fishes in China. Since 1970s, wild resources of large yellow croaker have been almost extinct due to overfishing. To solve the production problem, artificial breeding and cultured technology for large yellow croaker obtained successful breakthrough and the output of large yellow croaker was improved significantly. However, the quality of the cultured and wild large yellow croaker has great difference. In addition, the quality of cultured large yellow croaker from different environment is uneven. Therefore, the quality control methods for large yellow croaker are important for the development of large yellow croaker breeding industry. At present, fingerprint analysis has been widely applied to quality control systems of traditional Chinese medicines because of its advantages and popularity, and more chromatography and spectral methods including high-performance liquid chromatography (HPLC), gas chromatography, infrared spectrometry and mass spectrometry have been used for the fingerprint analysis, but HPLC fingerprint technology is rarely used in the quality control of aquatic products at present. In this paper, conditions for sample pre-processing, component extraction and HPLC analysis method were optimized, and a validated HPLC method coupled with cluster analysis, principal component analysis and discriminant analysis has been developed for the study of the HPLC fingerprint of large yellow croaker. Under the optimum conditions, muscles from cultured large yellow croaker (L. crocea) from Zhoushan and Fujian, L. polyactis, Nibea albiflora and Collichthys niveatus were freeze-dried and their chemical components were extracted using ethyl acetate. The chemical components from different samples were detected by Agilent 1260 DAD-HPLC on Thermo ODS-C18 (250 mm × 4.6 mm, 5μm) eluted by acetonitrile-water (0.1% H3PO4) under the optimum conditions: Flow rate 0.5 mL/min, inject volume 20μL, and wavelength 210 nm. The HPLC fingerprints and mutual mode of large yellow croaker from Zhoushan and Fujian, L. polyactis, N. albiflora and C. niveatus were established using similarity evaluation system for chromatographic fingerprint of TCM (Version 2004). Similarity analysis results indicated that the similarity order of other samples to large yellow croaker from Zhoushan was large yellow croaker from Fujian > L.polyactis > N. albiflora > C. niveatus. The same species had the similar characteristic peaks of HPLC and could be clustered together, but cluster analysis could not distinguish large yellow croaker from Zhoushan and Fujian. In the principal component analysis, three principal components (PC1, PC2 and PC3) were selected and accounted for 80.36% of the original data. According to the results of principal component scores, the scatter diagram of 62 samples was drawn, and each sample was able to form distinct cluster in the principal component space, then the identification of large yellow croaker from Zhoushan and Fujian, L.polyactis, N. albiflora and C. niveatus was basically achieved. Based on the results of principal component analysis, PC1 was used to develop the discrimination function for distinguishing different type samples. The accuracy of the discrimination function was 98.4%. Therefore, the established method had good stability, precision and reproducibility, and it was also a very reliable and useful method for distinguishing large yellow croaker from different species and/or environment. On the whole, HPLC fingerprint combined with reasonable methods of mathematical statistics provides an effective method for large yellow croaker geographical origin traceability and species identification.

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顾得月,任西杰,迟长凤,王斌,徐梅英,吴常文.大黄鱼HPLC指纹图谱的建立及其在产地溯源和物种鉴别中的应用[J].水产学报,2016,40(2):164~177

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  • 收稿日期:2015-05-02
  • 最后修改日期:2015-08-20
  • 录用日期:2016-01-23
  • 在线发布日期: 2016-03-06
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