Method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry

A multi-dimensional liquid chromatography, two-dimensional liquid chromatography technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of food matrix interference, false positives, false negatives, etc., to reduce test costs, accurate identification, automation high degree of effect

Pending Publication Date: 2022-02-25
SHANGHAI INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The national standard GB4789.10-2016 "National Food Safety Standard for Food Microbiology Examination of Staphylococcus aureus" uses enzyme linked immunosorbent assay (ELISA) to detect enterotoxins, but this method is susceptible to interference from food matrices. There are cross-reactions between different enterotoxins, which can easily lead to false positive and false negative results

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  • Method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry
  • Method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry
  • Method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry

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Effect test

Embodiment 1

[0051] This embodiment provides a two-dimensional liquid chromatography platform, and the two-dimensional liquid chromatography platform includes a first-dimensional liquid chromatography column, a second-dimensional liquid chromatography column, a trapping column and a six-way valve switching device. The first-dimensional liquid chromatography column, the trapping column and the second-dimensional liquid chromatography column are connected through a six-way valve switching device.

[0052] Two six-way valves are used to build an online two-dimensional liquid chromatography analysis platform, and the connection method is as follows: figure 1 As shown, the first six-port valve port 1 is connected to the syringe, the first six-port valve ports 2 and 5 are connected to the sample loop, the first six-port valve port 3 is connected to the first-dimensional ion-exchange column, and the first six-port The first six-way valve port 4 is connected to the first-dimensional chromatographi...

Embodiment 2

[0055] In this example, the conditions of the first-dimensional ion-exchange liquid chromatography were optimized according to the relevant properties such as the molecular weight of the enterotoxin C protein is 27.6 KDa, and the isoelectric point PI=8.6. Specifically include the following:

[0056] (1) Selection of chromatographic column

[0057] The isoelectric point of enterotoxin C is 8.6, which belongs to basic protein and is suitable for cation exchange chromatography column. Preferably, chromatographic column: strong cation exchange chromatographic column (Shimadzu Shim-pack PA-SP, 5 μm, 8 × 100mm, ), trapping column (Yuexu WelchXtimate C8, 5μm, 2.1×10mm, )

[0058] (2) Optimization of mobile phase pH

[0059] 20mM dipotassium hydrogen phosphate was selected as the buffer salt system, and two pH conditions of pH=6 and pH=5.3 were investigated. When the pH=6, enterotoxin C was not retained in the chromatographic separation process. When the pH was lowered to 5.3, e...

Embodiment 3

[0069] In this example, the conditions of the second-dimensional reversed-phase liquid chromatography were optimized. Specifically include the following:

[0070] (1) Selection of chromatographic column

[0071] The molecular weight of enterotoxin C is 27.6kDa. It is a medium polar compound. It can be separated by C8 column or C4 column. After investigation, the separation effect of C8 column is better than that of C4 column, and it has a good peak shape, such as Figure 6 shown. Preferably, chromatographic column: C8 chromatographic column (Yuexu Welch Xtimate C8, 5 μm, 2.1 × 250mm, ).

[0072] (2) Verification of elution position (combined with SDS-PAGE gel electrophoresis to verify the elution position of enterotoxin in the second dimension)

[0073] SDS-PAGE gel electrophoresis was used to verify the fractions of enterotoxin C second dimension chromatogram with different separation time, Figure 7 It is the gel image after silver staining, the band position shown on ...

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Abstract

The invention discloses a method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry. The method comprises the following steps: S1, selecting a two-dimensional separation mode, and constructing a two-dimensional liquid chromatography platform including first-dimensional ion exchange liquid chromatography and second-dimensional reversed-phase liquid chromatography; and S2, separating milk through the two-dimensional liquid chromatography platform to obtain fractions, and identifying enterotoxin C through mass spectrum identification of complete protein of the fractions and mass spectrum identification of enzymolysis peptide. According to the invention, the method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry has the advantages of accurate identification, rapidness, effectiveness, high sensitivity and the like, overcomes the defect of low accuracy of the existing ELISA method, is high in automation degree, improves the detection flux, reduces the test cost, is convenient and accurate in result judgment, and is more practical.

Description

technical field [0001] The invention relates to the technical field of food safety microorganism detection, in particular to a method for detecting Staphylococcus aureus enterotoxin C in milk by multidimensional liquid chromatography coupled with mass spectrometry. Background technique [0002] Staphylococcus aureus enterotoxin C is a toxin secreted by Staphylococcus aureus, and it is also one of the five classic enterotoxins that most commonly cause Staphylococcus aureus food poisoning. Therefore, the detection of enterotoxin C is beneficial to the protection of food Safe and healthy, prevent food poisoning incidents. The national standard GB4789.10-2016 "National Food Safety Standard for Food Microbiology Examination of Staphylococcus aureus" uses enzyme linked immunosorbent assay (ELISA) to detect enterotoxins, but this method is susceptible to interference from food matrices. There are cross-reactions between different enterotoxins, which can easily lead to false positi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/89G01N30/96
CPCG01N30/89G01N30/96
Inventor 宋明辉林梅英王轩堂秦峰杨美成刘浩
Owner SHANGHAI INST FOR FOOD & DRUG CONTROL
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