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Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry

A rapid determination and LC/MS technique, applied in the field of chromatographic detection, can solve the problems such as the influence of the extract on the sensitivity of the method, the high cost of the enzyme-linked immunosorbent assay, and the interference with the detection results, so as to improve the matrix effect problem, save the concentration time, and reduce the processing time Simple process effect

Inactive Publication Date: 2017-04-26
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the accuracy of the mouse method will vary due to individual differences in mice; the cost of the enzyme-linked immunosorbent method is high, and the gas chromatography-mass spectrometry and liquid chromatography-fluorescence methods need to derivatize TTX with alkaline hydrolysis before detection, and the derivation efficiency and Fluorescent substances originating from the sample can interfere with the detection results, and the steps are complicated and time-consuming
For liquid chromatography-mass spectrometry, the pre-treatment purification needs to meet the requirements of mass spectrometry sample injection. The extraction solution generally uses aqueous solutions such as trichloroacetic acid and acetic acid, and the purified sample uses C 18 Small columns, ion exchange columns, etc., and ultrafiltration, etc., but the obtained extract still has serious matrix effects that affect the sensitivity of the method

Method used

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  • Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry
  • Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry
  • Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry

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Experimental program
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Effect test

Embodiment 1

[0030] The method for the rapid determination of tetrodotoxin content in fish meat by liquid chromatography-mass spectrometry provided by the present embodiment comprises the following steps:

[0031] (1) Sample pretreatment

[0032] The live fish was killed by freezing, peeled, and 50g of the muscle part was taken, diatomaceous earth with 18% of the muscle weight was added, 10mL of 0.1mol / L hydrochloric acid solution was added, and the homogeneous sample maker was thoroughly pulverized, homogeneously pulverized for 23min, and then put into- Freeze at 80°C for 3 hours.

[0033] After freezing, weigh 1.00g of the sample into a 50mL polypropylene centrifuge tube, accurately add 3g of calcium chloride and 8mL of 1.0% acetic acid methanol solution, homogenize for 60s, use a cell disruptor to extract ultrasonically at 60°C for 30min, and centrifuge at 8000r / min For 5 minutes, transfer the supernatant to another clean centrifuge tube; repeat the extraction once with 3 mL of 1% acet...

Embodiment 2

[0047] Embodiment 2 linear range and detection limit

[0048]Weigh 1.00g of the sample in a 50mL polypropylene centrifuge tube, add an appropriate amount of tetrodotoxin standard solution, the spiked levels are respectively 0, 10, 20, 50, 100, 200 μg / kg, and place it for 0.5h according to the method in Example 1 Detect and obtain 0, 10, 20, 50, 100, 200 μg / kg matrix spiked solutions; take the peak area of ​​the TTX standard substance as the ordinate, and the matrix spiked solution concentration (μg / kg) as the abscissa, draw the standard Curve, quantified by external standard method. The results showed that TTX showed good linearity in the range of 10-200μg / kg, and the regression equation was Y=2.34e+003X, r 2 = 0.9994.

[0049] Equally weigh (1.00 ± 0.01) g of red fin puffer fish meat sample, add appropriate amount of standard solution, make the concentration level of TTX be 5, 10, 20, 30 μ g / kg, each level 3 parallel samples, according to the method of embodiment 1 HPLC-MS...

Embodiment 3

[0050] Embodiment 3 recovery rate and precision

[0051] Take by weighing (1.00 ± 0.01) g of cultured negative puffer puffer meat samples that do not contain TTX in 50mL polypropylene centrifuge tubes, divide them into 6 groups, 6 in each group, add an appropriate amount of standard solution, so that the concentrations of TTX in the samples are respectively 10, 15, 30, 50, 100, 200 μg / kg, the recovery rate and precision detection were carried out according to the method of Example 1. The result is as follows:

[0052]

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Abstract

The invention discloses a method for rapidly detecting the content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry. The method comprises (1) sample preprocessing and (2) chromatography-mass spectrometry detection. Diatomite is added in the preprocessing process against the effect problem of a matrix in a fish flesh sample and combining the chemical characteristics of tetrodotoxin, and the tetrodotoxin is separated from muscle tissues in an ultralow temperature freezing manner. A sample undergoes extraction by 1% acetic acid-methanol in a cell crasher at 60 DEG C, obtained extract is purified by a Carbon-GCB SPE small column, and the purified extract is directly introduced and analyzed, so the concentration time is saved. Compared with 0.2% acetic acid-water solution extraction, other SPE purification and ultrafiltration methods in the prior art, the method disclosed in the invention, adopting direct introduction and analysis of the sample, has the advantages of simplicity and low consumption in the processing process, good accuracy, convenience in operation, and good reappearance.

Description

technical field [0001] The invention relates to a method for rapidly determining tetrodotoxin content in fish meat by liquid chromatography-mass spectrometry, and belongs to the technical field of chromatographic detection. Background technique [0002] TTX (tetrodotoxin) is the earliest and extremely toxic small molecule marine toxin, 1000 times more toxic than sodium cyanide. The toxic effect is mainly manifested as selectively blocking sodium ion channels in nerves and muscles, inhibiting sodium ions from passing through nerve cell membranes, paralyzing sensory and motor nerves successively, severe brainstem paralysis leading to respiratory failure; the incubation period of poisoning is short, and the fatality rate is as high as 60%. TTX is a colorless prismatic crystal, soluble in weak acid aqueous solution. The pH value of the solution affects its stability, and it is easy to decompose when the pH value is <3 and >7; when the temperature is higher than 220°C, it ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/16G01N30/18G01N30/72
CPCG01N30/02G01N30/06G01N30/16G01N30/18G01N30/72G01N2030/065G01N2030/167
Inventor 曹文卿吴振兴王曼霞静平田娟
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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