CN117969726A - Pretreatment method for extracting shellfish toxin in aquatic product - Google Patents
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Landscapes
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a pretreatment method for extracting shellfish toxin in aquatic products, which comprises the following steps: (1) washing and homogenizing the aquatic product; (2) Adding the homogenized sample obtained in the step (1) into a solvent for extraction, and taking supernatant, wherein the solvent is acetonitrile water solution; (3) Freezing the supernatant in the step (2), taking the supernatant, removing the solvent and testing; the shellfish toxin is fat-soluble shellfish toxin field halichondrin, spiral imine compound, fin algae toxin 1, fin algae toxin 2 and/or protopolymethin 1; the method has the advantages of simple steps, less organic reagent consumption and low detection cost, and is suitable for pretreatment of a large number of samples.
Description
Technical Field
The invention relates to a method for extracting harmful substances in aquatic products, in particular to a pretreatment method for extracting shellfish toxins in aquatic products.
Background
Shellfish toxins are produced by algae and enter the shellfish product through the food chain. When people eat the toxic shellfish, poisoning is easy to cause. Shellfish toxins can be classified into water-soluble shellfish toxins and fat-soluble shellfish toxins according to solubility. Common fat-soluble shellfish toxins are okadaic acid toxin (OA), fin algae toxin (DTX), patinopecten Yessoensis Toxoid (YTX), scallop toxoid (PTX), protopolymethine alginic acid toxin (azo), and euglena miltiorrhizae shellfish toxin (GYM), etc. In early studies, toxicity after misfeeding of toxins such as halichondrin (OA) and fin algae toxin (DTX) was mainly diarrhea, and thus it was also called diarrhea shellfish toxin. Research shows that the elimination half-life period of fat-soluble shellfish toxin in shellfish is as long as tens of days or even months, and is not easy to remove and reduce in the processing and eating processes, thus bringing potential health hazard to eaters. The limit of diarrhea shellfish toxin in GB 2733-2015 food safety national standard fresh and frozen animal aquatic products is less than or equal to 0.05MU/g, and the limit of specific various toxins is not specified.
The most common detection method of fat-soluble shellfish toxins is liquid chromatography-tandem mass spectrometry at present, but the method has higher requirements on sample pretreatment. The shellfish product has complex sample matrix, and the sample contains fat, protein and other impurities, which greatly interfere with sample detection. Therefore, when the fat-soluble shellfish toxins are detected by liquid chromatography-tandem mass spectrometry, the pretreatment usually requires sample purification by using a solid-phase extraction column. When the solid phase extraction column is used for sample pretreatment, a certain solvent is needed to be used for pretreatment of the extraction column, sample loading is carried out after activation is finished, and target substances in the sample are adsorbed on the solid phase extraction column filler; then leaching the extraction column by using a solvent to remove interference components; and finally, selecting proper eluent for eluting, and collecting the target object. The solid phase extraction method has complex operation steps and cannot process a large number of samples at the same time. In addition, the method requires extraction consumables, a large amount of organic reagents and corresponding pretreatment equipment, and increases detection cost.
Disclosure of Invention
The invention aims to: the invention aims to provide the pretreatment method for extracting the shellfish toxin in the aquatic products, which has the advantages of simple operation steps, less consumption of organic reagent and low detection cost, and is suitable for pretreatment of batch samples.
The technical scheme is as follows: the pretreatment method for extracting shellfish toxin in aquatic products comprises the following steps:
(1) Washing and homogenizing the aquatic product;
(2) Adding the homogenized sample obtained in the step (1) into a solvent for extraction, and taking supernatant, wherein the solvent is acetonitrile water solution;
(3) Freezing the supernatant in the step (2), taking the supernatant, removing the solvent and testing;
The shellfish toxin is fat-soluble shellfish toxin Okadaic Acid (OA), spiral imine compound (GYM), fin algae toxin 1 (DTX 1), fin algae toxin 2 (DTX 2) and/or protopolymethine alginic acid toxin 1 (AZA 1).
Preferably, in the step (1), the aquatic product is shellfish, and the cleaning includes cutting off the obturator muscle, opening the casing, taking out all soft tissues, cleaning with ultrapure water, removing sediment, and draining.
Preferably, in the step (2), the volume fraction of water in the acetonitrile aqueous solution is 10-30%.
Preferably, in step (2), the ratio of sample to solvent is 1g: 2-10 mL.
Preferably, in the step (2), the extraction is first vortex mixing, then ultrasonic extraction and finally centrifugal treatment.
Preferably, in the step (2), the number of times of extraction is 1 to 3.
Preferably, in the step (3), the freezing temperature is-40 to-4 ℃, and the freezing time is 8 to 24 hours.
Preferably, in step (3), the solvent removal is: and drying with nitrogen at 35-40 ℃.
Preferably, in step (3), the sample to be tested further includes: the sample was added to methanol, then filtered, and finally subjected to instrumental analysis.
Preferably, the filtration uses a 0.22um microporous filter membrane.
The mechanism of the invention is as follows: according to the pretreatment method of shellfish toxins in aquatic products, disclosed by the invention, the fat-soluble shellfish toxins in the aquatic products can be extracted from acetonitrile, and the content of interfering substances such as protein, amino acid and the like in crude extract can be reduced by adding water. Because the target compound and the interfering substances such as protein have differences in melting points, the interfering substances can be condensed into ice in the water phase under the low-temperature condition, and the target substance is remained in the liquid acetonitrile, so that the effect of purifying the sample is achieved.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the method has simple operation steps, does not need extraction materials and additional organic reagents, has less organic reagent consumption, reduces detection cost, and is suitable for detecting mass samples.
Detailed Description
The technical scheme of the invention is further described below by referring to examples.
Example 1
The pretreatment method for extracting shellfish toxin in aquatic products comprises the following steps:
(1) Washing the appearance of shellfish sample with clear water, cutting off the muscle of the shell closure, and opening the shell. Taking out all soft tissues, cleaning with ultrapure water, removing sediment, draining, and homogenizing.
(2) Accurately weighing 1g of the sample prepared in the step (1), adding 5mL of 80% acetonitrile water solution (volume fraction) into a centrifuge tube, vortex mixing for 1min, ultrasonic extracting for 10min, centrifuging for 5min at 7000r/min, and removing supernatant.
(3) Repeating the extraction of the precipitate in the step (2) once, combining the two supernatants as in the step 2.
(4) Freezing the supernatant obtained in the step (3) at-20 ℃ for 16 hours, taking 4mL of supernatant, blowing nitrogen at 40 ℃ to near dryness, adding 800 mu L of methanol for dissolution, carrying out vortex oscillation for 60 seconds, passing through a 0.22 mu m microporous filter membrane, and carrying out instrument analysis and determination.
Reagent: five fat-soluble shellfish toxin standards ：OA(10.08±0.24μg/mL)、GYM(2.50±0.10μg/mL)、DTX1(10.01±0.48μg/mL)、DTX2(3.83±0.18μg/mL)、AZA1(1.22±0.06μg/mL), acetonitrile (chromatographic grade), ultrapure water.
Instrument: homogenizer, vortex mixer, ultrasonic cleaner, centrifuge, freezer, nitrogen blower, and 0.22 μm microporous filter membrane.
Example 2
The organic phase obtained by the pretreatment method for extracting fat-soluble shellfish toxin in shellfish by using the frozen liquid-liquid extraction method is detected by adopting a liquid chromatography tandem mass spectrometry. The experiment is as follows:
Instrument: 1290-6470 LC-TQ liquid chromatography triple quadrupole mass spectrometer (Agilent technologies Co., USA); vortex-Genie 2 Vortex oscillator (company SCIENTIFIC INDUSTRIES, usa); QQ10-250A ultrasonic cleaner (Shanghai Qian electronic technology Co., ltd.); centrifuge 5430R low temperature high speed Centrifuge (Eppendorf company, germany); milli-Q,18.2 M.OMEGA.ultra-pure water preparation instrument (Miibos, france), nitrogen blower (America Organomation), ice chest (Heer Intelligence Co., ltd.).
Reagent and material: five fat-soluble shellfish toxin standards ：OA(10.08±0.24μg/mL)、GYM(2.50±0.10μg/mL)、DTX1(10.01±0.48μg/mL)、DTX2(3.83±0.18μg/mL)、AZA1(1.22±0.06μg/mL)( Qingdao Pribon bioengineering Co., ltd.), acetonitrile (chromatographic grade), ultrapure water, ammonia water, ammonium formate (analytical grade), 0.22 μm microporous filter membrane (Tianjin Teng Co.).
Test conditions:
The chromatographic conditions were as follows:
Chromatographic column: ACQUITY UPLC BEH C18 (2.1 mm. Times.100 mm,1.7 μm); mobile phase: a is (0.01% ammonia water, 2mmol/L ammonium formate) aqueous solution, B is (0.01% ammonia water, 2mmol/L ammonium formate) 95% acetonitrile water; gradient procedure: 0-2 min,30% of B; 2-10 min, 30-90% of B; 10-12 min,90% B; 12-14 min, 90-30% B; 14-16 min,30% of B; sample injection amount: 2. Mu.L; flow rate: 0.3mL/min; column temperature: 40 ℃.
The mass spectrometry conditions were as follows:
Ion source: dual jet electrospray ion source (Dual AJF ESI), scan mode: positive and negative ion mode, detection mode: multiple Reaction Monitoring (MRM); atomizer pressure: 45psi; gas temperature: 300 ℃, gas flow rate: 5L/min; sheath gas (N 2) temperature: sheath gas (N 2) flow rate at 250 ℃): 11L/min; capillary voltage: 3500V; nozzle voltage: 500V. The detailed mass spectrum parameters are shown in table 1.
Mass spectrum parameters of 15 fat-soluble shellfish toxins
Note that the quantitative ion is shown.
Standard solution preparation
Experimental details
Linear range and detection limit: the shellfish toxin standard is diluted to 1.00, 5.00, 10.00, 25.00, 50.00, 100.00, 150.00 mug/L with blank matrix (clam) liquid to obtain serial concentration standard solutions. And 5 kinds of detection targets are taken as an ordinate, and the concentration of toxin added into the blank matrix liquid is taken as an abscissa, so that a linear regression equation of a standard curve is obtained. The linear range, correlation coefficient (R 2), detection limit of the method (3-fold signal-to-noise calculation), and quantification limit (10-fold signal-to-noise calculation) are shown in table 2.
Table 25 Linear relation, detection limit and quantitative limit of fat-soluble shellfish toxins
The results in Table 2 show that the 5 detection targets have good linearity in the corresponding concentration range, and the correlation coefficient (R 2) is 0.9975-0.9997, so that the requirement of instrument analysis is met. It is possible to explain the detection method of the present embodiment.
Example 3
Recovery rate and precision experiment: a sample of shellfish (clam) was taken, and shellfish toxin was added at three concentration levels of 15. Mu.g/kg, 40. Mu.g/kg, 60. Mu.g/kg, and the labeling recovery test was performed, with each level repeated 6 times, and the results are shown in Table 3.
Table 3 results of measurement of recovery rate of 5 fat-soluble shellfish toxins in blank shellfish (n=6)
As can be seen from Table 3, the average recovery rate of 5 fat-soluble shellfish toxins after pretreatment of the labeled sample by this method is 87.60% -96.91% and the Relative Standard Deviation (RSD) is 2.01% -9.16%. The method has good recovery rate and precision, and can meet detection requirements.
Conclusion: the method utilizes the frozen liquid-liquid extraction method to extract the fat-soluble shellfish toxin in the shellfish, has simple operation steps, requires less experimental consumables and reagents, reduces the detection cost, is suitable for pretreatment of a large number of samples, and provides technical support for detection of the shellfish toxin in the aquatic products.
Claims (10)
1. A pretreatment method for extracting shellfish toxin in aquatic products is characterized by comprising the following steps:
(1) Washing and homogenizing the aquatic product;
(2) Adding the homogenized sample obtained in the step (1) into a solvent for extraction, and taking supernatant, wherein the solvent is acetonitrile water solution;
(3) Freezing the supernatant in the step (2), taking the supernatant, removing the solvent and testing;
the shellfish toxin is fat-soluble shellfish toxin okadaic acid, spiral imine compound, fin algae toxin 1, fin algae toxin 2 and/or protopolymethin 1.
2. The pretreatment method for extracting shellfish toxins in aquatic products according to claim 1, wherein in the step (2), the volume fraction of water in the acetonitrile aqueous solution is 10-30%.
3. The pretreatment method for extracting shellfish toxin in aquatic products according to claim 1, wherein in the step (2), the ratio of the sample to the solvent is 1g: 2-10 mL.
4. The pretreatment method for extracting shellfish toxin in aquatic products according to claim 1, wherein in the step (3), the freezing temperature is-40 to-4 ℃ and the freezing time is 8 to 24 hours.
5. The pretreatment method for extracting shellfish toxin in aquatic products according to claim 1, wherein in the step (3), the solvent removal is: and drying with nitrogen at 35-40 ℃.
6. The pretreatment method for extracting shellfish toxins in aquatic products according to claim 1, wherein in the step (2), the extraction is performed by first vortex mixing, then ultrasonic extraction, and finally centrifugation.
7. The pretreatment method for extracting shellfish toxin in an aquatic product according to claim 1, wherein in the step (2), the number of extraction times is 1 to 3.
8. The pretreatment method for extracting shellfish toxin in aquatic products according to claim 1, wherein in step (3), the sample to be measured further comprises: the sample was added to methanol, then filtered, and finally subjected to instrumental analysis.
9. The pretreatment method for extracting shellfish toxins in aquatic products of claim 8, wherein the filtering is performed with a 0.22um microporous filter membrane.
10. The pretreatment method for shellfish toxin extraction in an aquatic product according to claim 1, wherein in the step (1), the aquatic product is shellfish, and the washing comprises cutting off the obturator muscle, opening the obturator muscle, taking out all soft tissues, washing with ultrapure water, removing sediment, and draining.
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