Method for quickly detecting tetrodotoxin content in fish liver employing liquid-mass chromatography

A rapid determination, tetrodotoxin technology, applied in the field of chromatographic detection, to achieve the effect of good reproducibility, reliable results, and convenient operation

Inactive Publication Date: 2017-05-10
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the problem of matrix effect in the prior art, the technical problem to be solved by the present invention is to provide a method for quickly determining the content of tetrodotoxin in fish liver by liquid chromatography-mass spectrometry

Method used

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  • Method for quickly detecting tetrodotoxin content in fish liver employing liquid-mass chromatography
  • Method for quickly detecting tetrodotoxin content in fish liver employing liquid-mass chromatography
  • Method for quickly detecting tetrodotoxin content in fish liver employing liquid-mass chromatography

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Experimental program
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Effect test

Embodiment 1

[0027] The method for the rapid determination of tetrodotoxin content in fish liver by liquid chromatography-mass spectrometry provided by this embodiment comprises the following steps:

[0028] (1) Sample pretreatment

[0029] (1) The live puffer fish was killed by freezing, peeled, and the liver part was taken to be fully pulverized by a homogeneous sample maker, and 50 g of the liver sample was taken after crushing, and 15-20 g of kaolin was added, and continued to be pulverized by a homogeneous sample maker, and homogenized Crush for 10-15 minutes, then freeze in a -40°C refrigerator for 1-2 hours;

[0030] After freezing, weigh (1.00±0.01) g of the sample into a 50 mL polypropylene centrifuge tube, accurately add 5 mL of 30% ethanol aqueous solution and 5 mL of 1.0% acetic acid methanol solution, homogenize for 30-50 s, and extract by ultrasonic at 60 °C with a cell disruptor 25min, centrifuge at 8000r / min for 5min, transfer the supernatant to another clean centrifuge tu...

Embodiment 2

[0042] Embodiment 2 linear range and detection limit

[0043]Weigh the sample (1.00±0.01)g into a 50mL polypropylene centrifuge tube, add an appropriate amount of tetrodotoxin standard solution, the spiked levels are 0, 10, 20, 50, 100, 200μg / kg respectively, after standing for 0.5h, according to Embodiment 1 method, obtain the matrix spiked solution of 0, 10, 20, 50, 100, 200 μ g / kg; Take the peak area of ​​TTX standard substance as the ordinate, and the matrix spiked solution concentration (μg / kg) as the abscissa, A standard curve was drawn and quantified by the 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.

[0044] Equally weigh puffer fish liver samples (1.00 ± 0.01) g, add an appropriate amount of standard solution, so that the concentration level of TTX is 5.0, 10, 20, 30 μg / kg, 3 parallel samples at each level, carry out according to the method of Exa...

Embodiment 3

[0047] Recovery and precision

[0048] Take by weighing (1.00 ± 0.01) g of cultured negative puffer fish liver samples that do not contain TTX in 50mL polypropylene centrifuge tubes, divide them into 3 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, analyzed by HPLC-MS / MS according to the method of Example 1. The matrix spiked calibration curve was quantified by the external standard method, and the recovery and precision data are shown in Table 3. A blank matrix was used for the recovery experiment, and the results were as follows:

[0049] Table 3 recovery rate and precision data

[0050] Added level / (μg.kg -1 )

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Abstract

The invention discloses a method for quickly detecting the tetrodotoxin content in a fish liver employing a liquid-mass chromatography. The method comprises the steps of (1) sample pretreatment; and (2) chromatography-mass spectrometry detection. For the problem of a matrix effect in a fish liver sample, by combining the chemical characteristics of tetrodotoxin, kaolin is added during pretreatment, and the tetrodotoxin is separated to the maximum extent. Meanwhile, acidolysis is carried out at 60 DEG C, ultrasonic extraction is carried out by adopting a cell disruptor, and after Carbon-GCB SPE purification, a sample directly injected for analysis, so that the treatment process is simple, fast, low in consumption. The method is accurate, convenient to operate and good in reproducibility and can meet the technical requirements of fast detection of the tetrodotoxin in the fish liver.

Description

technical field [0001] The invention relates to a method for quickly determining tetrodotoxin content in fish liver 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): G01N30/02
CPCG01N30/02
Inventor 曹文卿吴振兴静平王曼霞许艳丽
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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