Detection methods and applications of saxitoxin

Abstract

The present invention discloses a method and application for detecting saxitoxin, and relates to the field of compound detection technology. The method comprises detecting a sample using ultra-high performance liquid chromatography-tandem mass spectrometry, treating the sample using proton-assisted ionization technology after liquid chromatography detection, and then performing mass spectrometry detection. By using ultra-high performance liquid chromatography-tandem mass spectrometry detection in combination with proton-assisted ionization technology, the detection sensitivity of saxitoxin is improved, and even if the concentration level of saxitoxin in the sample is extremely low, it can be detected, thereby ensuring the safety of aquatic products.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and in particular to a saxitoxin detection method and application. Background Art

[0002] Shellfish poisoning often occurs in large numbers, is familial, sudden, and regional, and can lead to widespread poisoning and death. The resulting social harm is significant and is attracting increasing public attention. Shellfish toxins include paralytic shellfish toxins, diarrhetic shellfish toxins, neurotoxins, and amnesic shellfish toxins.

[0003] Paralytic shellfish toxin is one of the most toxic toxins, with a toxicity comparable to that of tetrodotoxin. Paralytic shellfish toxin is a highly polar tetrahydropurine tricyclic compound, composed of toxins produced by more than 20 different types of dinoflagellates, which can grow in both tropical and temperate waters. Paralytic shellfish toxin is mainly composed of saxitoxin (STX) and its derivatives. Its toxicity is 80 times that of cobra venom and more than 100,000 times that of general anesthetics. Oral administration of 0.5 to 1.0 mg can be lethal to humans, and the median lethal dose (LD50) of saxitoxin injected intraperitoneally into mice is LD50. 50 The LD50 of 24 nmol / kg was obtained by oral administration. 50 The oral LD50 for humans is 1 237 nmol / kg. 50 The concentration of saxitoxin is 5.7 μg / kg, equivalent to a lethal dose of 0.57 mg upon ingestion. Due to the high toxicity, rapid reaction, and lack of suitable antidotes of saxitoxin, prevention and control present significant challenges. Therefore, there is an urgent need to develop a precise detection method for saxitoxin to provide a technical foundation for ensuring aquatic product safety.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a detection method and application of saxitoxin.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a method for detecting saxitoxin, comprising detecting a sample to be tested using ultrahigh performance liquid chromatography-tandem mass spectrometry, treating the sample to be tested using proton-assisted ionization technology after the liquid chromatography detection, and then performing mass spectrometry detection.

[0008] In an optional embodiment, the proton-assisted ionization technique includes mixing an ionization solvent with a sample to be tested flowing out of a chromatograph and then passing the mixture into a mass spectrometer.

[0009] Preferably, the ionization solvent includes formic acid or acetic acid solution, and the concentration of the ionization solvent is 0.4-0.6%.

[0010] Preferably, the flow rate of the ionization solvent is 3 to 8 μL / min.

[0011] In an alternative embodiment, ultra high performance liquid chromatography-tandem mass spectrometry comprises using a Waters Acquity Ultra Performance LC™-Premier XE MS / MS detector.

[0012] In an optional embodiment, the liquid chromatography column is a Waters ACQUITY UPLC Hilic, the mobile phase A is ammonium acetate-formic acid aqueous solution, the mobile phase B is formic acid methanol solution, the column temperature is 35-45°C, and the sample tray temperature is 2-6°C.

[0013] Preferably, mobile phase A is 5 mM ammonium acetate-0.1% formic acid in water, and mobile phase B is 0.5% formic acid in methanol.

[0014] Preferably, the elution program of the mobile phase includes: 0-2 min, elution with 10% mobile phase A, the balance being mobile phase B; 2-2.1 min, elution with 10-30% mobile phase A, the balance being mobile phase B; 2.1-4 min, elution with 30% mobile phase A, the balance being mobile phase B; 4-4.1 min, elution with 30-60% mobile phase A, the balance being mobile phase B; 4.1-7 min, elution with 60% mobile phase A, the balance being mobile phase B; 7-7.1 min, elution with 60-10% mobile phase A, the balance being mobile phase B; 7.1-12 min, elution with 10% mobile phase A, the balance being mobile phase B.

[0015] Preferably, the injection volume is 1 to 4 μL.

[0016] In an alternative embodiment, mass spectrometry comprises detection using a Waters TQS triple quadrupole mass spectrometer.

[0017] Preferably, the ion source is an ESI ion source, the ion source temperature is 140-160°C, the capillary voltage is 1.5-2.5 kV, the cone voltage is 50-70 V, the desolvation temperature is 450-550°C, the desolvation gas flow rate is 800-1000 L / h, and the cone gas flow rate is 140-160 L / h.

[0018] In an optional embodiment, the preparation of the sample to be tested includes mixing the shellfish meat with the extracting solution, extracting the mixture, separating the solid and the liquid, repeating the extraction at least twice, and then removing impurities from the extracting solution.

[0019] In an optional embodiment, the extracting comprises vortexing the shellfish flesh with the extraction solution and then ultrasonically extracting it.

[0020] Preferably, the mass volume ratio of shellfish meat to extract is 0.8-1.2 g:4-8 ml.

[0021] Preferably, the extracting solution comprises any one of a formic acid aqueous solution or a formic acid acetonitrile solution.

[0022] Preferably, the content of formic acid in the formic acid aqueous solution is 0.1-0.4%.

[0023] Preferably, the vortex time is 2 to 7 minutes.

[0024] Preferably, the ultrasonic extraction is water bath ultrasonic extraction, the ultrasonic time is 8 to 12 minutes, and the water bath temperature is 40 to 60°C.

[0025] In an optional embodiment, solid-liquid separation includes any one of centrifugation, filtration, and filter press.

[0026] Preferably, the solid-liquid separation is performed by centrifugation, the rotation speed of the centrifuge is 8000-12000 rpm, and the centrifugation time is 8-12 min.

[0027] In an optional embodiment, the impurity removal includes adding an impurity removal solvent to remove impurities and / or solid phase extraction to remove impurities.

[0028] Preferably, the impurity removal using the impurity removal solvent comprises adding the impurity removal solvent to the extract, vortexing to separate the solid and liquid, and retaining the supernatant.

[0029] Preferably, the vortex time is 2 to 5 minutes, the solid-liquid separation is centrifugation, the rotation speed of the centrifuge is 8000 to 12000 rpm, and the centrifugation time is 8 to 12 minutes.

[0030] Preferably, the amount of impurity-removing solvent added is 3 to 12 ml.

[0031] Preferably, the impurity-removing solvent includes at least one of n-hexane and chloroform.

[0032] Preferably, the extraction column for solid phase extraction and impurity removal is at least one of Prime HLB, 6cc, 150mg and MCX, 6cc, 150mg.

[0033] In a second aspect, the present invention provides a method for detecting saxitoxin according to any one of the aforementioned embodiments, and its application in food, drug detection or daily chemical product detection.

[0034] The present invention has the following beneficial effects:

[0035] The present invention provides a method and application for detecting saxitoxin. By adopting ultra-high performance liquid chromatography-tandem mass spectrometry for detection and combining it with proton-assisted ionization technology, the detection sensitivity of saxitoxin is improved. Even if the concentration level of saxitoxin in the sample to be tested is extremely low, it can be detected, thereby ensuring the safety of aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 A schematic diagram of the proton-assisted ionization technology provided by the present invention;

[0038] Figure 2 The chromatogram of the saxitoxin standard solution provided in Example 1 of the present invention;

[0039] Figure 3 The standard curve of saxitoxin provided in Example 1 of the present invention;

[0040] Figure 4 The chromatogram of the saxitoxin standard solution provided in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0042] In a first aspect, the present invention provides a method for detecting saxitoxin, comprising detecting a sample to be tested using ultrahigh performance liquid chromatography-tandem mass spectrometry, treating the sample to be tested using proton-assisted ionization technology after the liquid chromatography detection, and then performing mass spectrometry detection.

[0043] The high toxicity, rapid reaction, and lack of suitable antidotes of saxitoxins pose numerous challenges to their prevention and treatment. Consequently, there is an urgent need to develop a precise detection method for saxitoxins. The inventors have improved the sensitivity of saxitoxin detection by employing ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) combined with proton-assisted ionization (PAI) technology. This allows detection even at extremely low saxitoxin concentrations in test samples, ensuring the safety of aquatic products.

[0044] In an optional embodiment, the proton-assisted ionization technique includes mixing an ionization solvent with a sample to be tested flowing out of a chromatograph and then passing the mixture into a mass spectrometer.

[0045] Preferably, the ionization solvent includes formic acid or acetic acid solution, and the concentration of the ionization solvent is 0.4-0.6%.

[0046] Preferably, the flow rate of the ionization solvent is 3 to 8 μL / min.

[0047] See Figure 1 The principle of proton-assisted ionization technology includes: the sample to be tested flowing out of the chromatographic column is first mixed with the ionization solvent before entering the mass spectrometer, and then undergoes electrospray ionization in the mass spectrometer to produce saxitoxin ions, which significantly enhances the ionization efficiency of saxitoxin and thus enhances the detection sensitivity of saxitoxin.

[0048] In an alternative embodiment, ultra high performance liquid chromatography-tandem mass spectrometry comprises using a Waters Acquity Ultra Performance LC™-Premier XE MS / MS detector.

[0049] In an optional embodiment, the liquid chromatography column is a Waters ACQUITY UPLC Hilic, the mobile phase A is ammonium acetate-formic acid aqueous solution, the mobile phase B is formic acid methanol solution, the column temperature is 35-45°C, and the sample tray temperature is 2-6°C.

[0050] Preferably, mobile phase A is 5 mM ammonium acetate-0.1% formic acid in water, and mobile phase B is 0.5% formic acid in methanol.

[0051] Preferably, the elution program of the mobile phase includes: 0-2 min, elution with 10% mobile phase A, the balance being mobile phase B; 2-2.1 min, elution with 10-30% mobile phase A, the balance being mobile phase B; 2.1-4 min, elution with 30% mobile phase A, the balance being mobile phase B; 4-4.1 min, elution with 30-60% mobile phase A, the balance being mobile phase B; 4.1-7 min, elution with 60% mobile phase A, the balance being mobile phase B; 7-7.1 min, elution with 60-10% mobile phase A, the balance being mobile phase B; 7.1-12 min, elution with 10% mobile phase A, the balance being mobile phase B.

[0052] Preferably, the injection volume is 1 to 4 μL.

[0053] In an alternative embodiment, mass spectrometry comprises detection using a Waters TQS triple quadrupole mass spectrometer.

[0054] Preferably, the ion source is an ESI ion source, the ion source temperature is 140-160°C, the capillary voltage is 1.5-2.5 kV, the cone voltage is 50-70 V, the desolvation temperature is 450-550°C, the desolvation gas flow rate is 800-1000 L / h, and the cone gas flow rate is 140-160 L / h.

[0055] By controlling the parameters of ultra-high performance liquid chromatography-tandem mass spectrometry within the above range, the content of saxitoxin in the sample to be tested can be detected more quickly and clearly, providing a reference basis for the detection of saxitoxin in aquatic products.

[0056] In an optional embodiment, ultra high performance liquid chromatography-tandem mass spectrometry is also included to detect a series of saxitoxin standard solutions with concentration gradients, to obtain a saxitoxin standard curve with a concentration-peak area correlation, and to obtain the concentration of the sample to be tested according to the saxitoxin standard curve.

[0057] Preferably, the detection method and parameters of the saxitoxin standard solution and the sample to be tested are the same.

[0058] In an optional embodiment, the preparation of the sample to be tested includes mixing the shellfish meat with the extracting solution, extracting the mixture, separating the solid and the liquid, repeating the extraction at least twice, and then removing impurities from the extracting solution.

[0059] In an optional embodiment, the extracting comprises vortexing the shellfish flesh with the extraction solution and then ultrasonically extracting it.

[0060] Preferably, the mass volume ratio of shellfish meat to extract is 0.8-1.2 g:4-8 ml.

[0061] Preferably, the extracting solution comprises any one of a formic acid aqueous solution or a formic acid acetonitrile solution.

[0062] Preferably, the formic acid aqueous solution can better dissolve saxitoxin. At the same time, in order to ensure the structural stability of saxitoxin, the content of formic acid in the formic acid aqueous solution is 0.1-0.4%.

[0063] Preferably, in order to ensure sufficient dissolution of saxitoxin, the vortex time is 2 to 7 minutes.

[0064] Preferably, the ultrasonic extraction is water bath ultrasonic extraction, the ultrasonic time is 8 to 12 minutes, and the water bath temperature is 40 to 60°C.

[0065] In an optional embodiment, solid-liquid separation includes any one of centrifugation, filtration, and filter press.

[0066] Preferably, the solid-liquid separation is performed by centrifugation, the rotation speed of the centrifuge is 8000-12000 rpm, and the centrifugation time is 8-12 min.

[0067] In an optional embodiment, in order to prevent other components in the sample to be tested from affecting the detection result of saxitoxin, impurity removal includes adding an impurity removal solvent to remove impurities and / or solid phase extraction to remove impurities.

[0068] Preferably, the impurity removal using the impurity removal solvent comprises adding the impurity removal solvent to the extract, vortexing to separate the solid and liquid, and retaining the supernatant.

[0069] Preferably, the vortex time is 2 to 5 minutes, the solid-liquid separation is centrifugation, the rotation speed of the centrifuge is 8000 to 12000 rpm, and the centrifugation time is 8 to 12 minutes.

[0070] Preferably, the amount of impurity-removing solvent added is 3 to 12 ml.

[0071] Preferably, the impurity-removing solvent includes at least one of n-hexane and chloroform.

[0072] Preferably, the extraction column for solid phase extraction and impurity removal is at least one of Prime HLB, 6cc, 150mg and MCX, 6cc, 150mg.

[0073] In a second aspect, the present invention provides a method for detecting saxitoxin according to any one of the aforementioned embodiments, and its application in food, drug detection or daily chemical product detection.

[0074] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0075] Example 1

[0076] S01. Draw the standard curve of saxitoxin

[0077] Saxitoxin was dissolved in methanol solution to prepare a saxitoxin stock solution with a concentration of 100 μg / mL. The saxitoxin stock solution was diluted to concentrations of 5 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL and 1000 ng / mL in sequence to obtain a series of saxitoxin standard solutions. The saxitoxin standard solutions were sequentially detected by ultra high performance liquid chromatography-tandem mass spectrometry to obtain chromatograms of the standard solutions. The chromatogram of the 25 ng / L standard solution was as follows: Figure 2 As shown, by calculating the peak area of ​​saxitoxin in the chromatogram of each standard solution, the peak area is corresponded to the concentration of the standard solution to draw a standard curve, as shown in FIG. Figure 3 The correlation coefficient (R) of the target compound of saxitoxin standard curve is shown in Figure 2. 2 ) is 0.9992, indicating that there is a good quantitative relationship between the chromatographic peak area and the compound concentration, which can meet the requirements of target analysis.

[0078] During the detection process, the standard solution is first passed into the chromatographic column for ultra-high performance liquid chromatography separation, and then the response intensity of the standard solution is enhanced using proton-assisted ionization technology before being passed into the mass spectrometer.

[0079] The chromatographic conditions of ultrahigh performance liquid chromatography included: a Waters ACQUITY UPLC Hilic liquid chromatography column, mobile phase A consisting of 5 mM ammonium acetate-0.1% formic acid aqueous solution, mobile phase B consisting of 0.5% formic acid methanol solution, a column temperature of 40°C, a sample tray temperature of 4°C, and an injection volume of 2 μL.

[0080] The elution program of the mobile phase includes: 0-2 minutes, elution with 10% mobile phase A, and the balance is mobile phase B; 2-2.1 minutes, elution with 10-30% mobile phase A, and the balance is mobile phase B; 2.1-4 minutes, elution with 30% mobile phase A, and the balance is mobile phase B; 4-4.1 minutes, elution with 30-60% mobile phase A, and the balance is mobile phase B; 4.1-7 minutes, elution with 60% mobile phase A, and the balance is mobile phase B; 7-7.1 minutes, elution with 60-10% mobile phase A, and the balance is mobile phase B; 7.1-12 minutes, elution with 10% mobile phase A, and the balance is mobile phase B.

[0081] Proton-assisted ionization includes: mixing an ionization solvent with a sample flowing out of a chromatograph and then passing the mixture into a mass spectrometer. The ionization solvent is a 0.5% formic acid solution, and the flow rate of the ionization solvent is 3 to 8 μL / min.

[0082] The mass spectrometry conditions included: the mass spectrometer was a Waters TQS triple quadrupole mass spectrometer, the ion source was an ESI ion source, the ion source temperature was 150°C, the capillary voltage was 2 kV, the cone voltage was 60 V, the desolvation temperature was 500°C, the desolvation gas flow rate was 900 L / h, and the cone gas flow rate was 150 L / h.

[0083] S02. Preparation of saxitoxin test samples

[0084] Take shellfish sample 1 after washing with clean water, wipe off the water, remove the shell and take out the shellfish meat, then put it into a homogenizer for homogenization, take 1 g of the homogenized shellfish meat into a 15 mL centrifuge tube, add 6 mL of 0.2% formic acid aqueous solution, vortex for 5 minutes, and then ultrasonically extract in a 50°C water bath for 10 minutes. After the extraction is completed, centrifuge at 10,000 rpm for 10 minutes, transfer the supernatant to a 50 mL centrifuge tube, repeat the above extraction operation once, combine the two supernatants to obtain the extract.

[0085] Add 10 mL of n-hexane to the extract, vortex for 2 minutes, and centrifuge at 10,000 rpm for 10 minutes. Discard the supernatant. Add 5 mL of chloroform to the lower layer, vortex for 5 minutes, and centrifuge at 10,000 rpm for 10 minutes. Transfer the supernatant to a 15 mL centrifuge tube for later use.

[0086] Remove 5 mL of the reserved supernatant and pass it through a Prime HLB extraction column (6 cc, 150 mg). Collect the filtrate in a 15 mL centrifuge tube. Add 1 mL of 0.2% formic acid solution to the filtrate and pass it through a solid-phase extraction column. Collect the effluent again in a centrifuge tube. Add methanol to the centrifuge tube to make the volume 10 mL. Vortex to mix thoroughly, then remove 1 mL of the solution and filter it for ultra-high performance liquid chromatography-tandem mass spectrometry analysis.

[0087] The parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry method are the same as those in step S01. The peak area of ​​the sample to be tested is substituted into the saxitoxin standard curve to obtain a saxitoxin concentration of 25.36 ng / kg in the sample to be tested.

[0088] The method of Example 1 was used to test another four different shellfish samples, and the concentrations of saxitoxin in the samples were shown in Table 1:

[0089] Table 1 Saxitoxin content in different shellfish samples

[0090] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Concentration ng / kg 25.36 71.34 76.32 59.12 66.61

[0091] As can be seen from Table 1, the detection method provided in the embodiment of the present invention can detect the saxitoxin content in different shellfish samples.

[0092] Comparative Example 1

[0093] This comparative example provides a method for detecting saxitoxin, and its specific steps are similar to those of Example 1, except that proton-assisted ionization technology is not used. The chromatogram of the obtained 25 ng / L standard solution is as follows: Figure 4 As shown by Figure 4 and Figure 2 It can be seen that the embodiment of the present invention uses proton-assisted ionization technology to significantly reduce the response concentration of saxitoxin, so that lower concentrations of saxitoxin in the test sample can be detected, thereby improving the detection sensitivity of saxitoxin.

[0094] Comparative Example 2

[0095] This comparative example provides a method for detecting saxitoxin. Samples 1 to 5, identical to those in Example 1, were used as raw materials. The specific detection method was similar to that in Example 1, except that 6 mL of a 0.5% methanolic formic acid solution was added during extraction and vortexed for 5 minutes. The peak concentration obtained was applied to the saxitoxin standard curve to obtain the saxitoxin concentrations in the test samples, as shown in Table 2.

[0096] Table 2 Saxitoxin content in different shellfish samples

[0097] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Concentration ng / kg 20.1 30.57 43.92 21.66 30.18

[0098] As shown in Table 2, an increase in the formic acid content in the formic acid methanol solution added during extraction will lead to a decrease in the detected amount of saxitoxin in shellfish samples.

[0099] Comparative Example 3

[0100] This comparative example provides a method for detecting saxitoxin. Samples 1 to 5, the same as in Example 1, were used as raw materials. The detection method was similar to that in Example 1, except that the mass spectrometry conditions were changed to a capillary voltage of 0.5 kV. The peak concentration obtained by the detection was substituting into the saxitoxin standard curve to obtain the saxitoxin concentration in the test sample, as shown in Table 3.

[0101] Table 3 Saxitoxin content in different shellfish samples

[0102] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Concentration ng / kg 10.58 22.73 18.72 9.52 19.92

[0103] As shown in Table 3, a decrease in the capillary voltage in the mass spectrometry conditions will lead to a decrease in the detection amount of saxitoxin in shellfish samples.

[0104] Test Example 1

[0105] A repeatability experiment was carried out using a 50 ng / L saxitoxin standard solution as a QC sample. The saxitoxin in the QC sample was detected using the methods of Example 1 and Comparative Examples 1 to 3, respectively. The spiked recovery and standard relative deviation (RSD) value were calculated to obtain the results shown in Table 4.

[0106] Table 4 Accuracy of the detection method for saxitoxin

[0107] Detection concentration (ng / L) Spiked recovery (%) RSD (%) Example 1 44.9 89.8 6.7 Comparative Example 1 36.55 73.1 8.8 Comparative Example 2 32.47 64.9 7.2 Comparative Example 3 29.81 59.6 9.3

[0108] As can be seen from Table 4, the detection method provided by the embodiment of the present invention has a higher spike recovery rate and a lower standard relative deviation RSD value, which can accurately and reliably detect the content of saxitoxin in the sample, providing a basis for the detection of saxitoxin.

[0109] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for detecting saxitoxin, characterized in that: The method comprises the following steps: using ultra-high performance liquid chromatography-tandem mass spectrometry to detect the sample to be tested, wherein after the liquid chromatography detection is completed, the sample to be tested is treated with proton-assisted ionization technology and then subjected to mass spectrometry detection; The proton-assisted ionization technique includes mixing an ionization solvent with a sample to be tested flowing out of a chromatograph and then passing the mixture into a mass spectrometer; The ionization solvent includes formic acid or acetic acid solution, and the concentration of the ionization solvent is 0.4-0.6%; The ultra-high performance liquid chromatography-tandem mass spectrometry method includes using Waters Acquity Ultra Performance LCTM-PremierXE MS / MS detection; Mass spectrometry included detection using a Waters TQS triple quadrupole mass spectrometer; The ion source was ESI, with a source temperature of 140–160°C, a capillary voltage of 1.5–2.5 kV, a cone voltage of 50–70 V, a desolvation temperature of 450–550°C, a desolvation gas flow rate of 800–1000 L / h, and a cone gas flow rate of 140–160 L / h. The preparation of the sample to be tested comprises mixing shellfish meat with an extracting solution, extracting the mixture, separating the solid and the liquid, repeating the extraction at least twice, and then removing impurities from the extracting solution; The extraction comprises vortexing the shellfish meat and the extracting solution and then ultrasonically extracting; the extracting solution comprises any one of a formic acid aqueous solution or a formic acid acetonitrile solution; the formic acid content in the formic acid aqueous solution is 0.1-0.4%, the vortexing time is 2-7 minutes; the ultrasonic extraction is a water bath ultrasonic extraction, the ultrasonication time is 8-12 minutes, and the water bath temperature is 40-60°C; The impurity removal includes adding an impurity removal solvent to remove impurities or solid phase extraction to remove impurities; the impurity removal solvent includes at least one of n-hexane and chloroform; the extraction column for solid phase extraction and impurity removal is at least one of Prime HLB, column volume 6cc, filler mass 150mg or MCX, column volume 6cc, filler mass 150mg; The liquid chromatography column was a Waters ACQUITY UPLC Hilic, mobile phase A was ammonium acetate-formic acid in water, mobile phase B was formic acid in methanol, column temperature was 35–45°C, and sample tray temperature was 2–6°C; Mobile phase A was 5 mM ammonium acetate-0.1% formic acid in water, and mobile phase B was 0.5% formic acid in methanol; The elution program of the mobile phase included: 0-2 min, elution with 10% mobile phase A, the balance being mobile phase B; 2-2.1 min, elution with 10-30% mobile phase A, the balance being mobile phase B; 2.1-4 min, elution with 30% mobile phase A, the balance being mobile phase B; 4-4.1 min, elution with 30-60% mobile phase A, the balance being mobile phase B; 4.1-7 min, elution with 60% mobile phase A, the balance being mobile phase B; 7-7.1 min, elution with 60-10% mobile phase A, the balance being mobile phase B; 7.1-12 min, elution with 10% mobile phase A, the balance being mobile phase B; The injection volume is 1~4μL.

2. The detection method according to claim 1, wherein The flow rate of the ionization solvent is 3-8 μL / min.

3. The detection method according to claim 1, wherein The mass volume ratio of the shellfish meat to the extract is 0.8-1.2 g: 4-8 ml.

4. The detection method according to claim 1, wherein The solid-liquid separation includes any one of centrifugation, filtration and filter pressing.

5. The detection method according to claim 4, characterized in that Solid-liquid separation is performed by centrifugation, the speed of the centrifuge is 8000~12000rpm, and the centrifugation time is 8~12min.

6. The detection method according to claim 1, characterized in that The impurity removal process using the impurity removal solvent includes adding the impurity removal solvent to the extract, vortexing the extract to separate the solid and the liquid, and retaining the supernatant.

7. The detection method according to claim 6, characterized in that The vortex time is 2 to 5 minutes, the solid-liquid separation is centrifugation, the centrifuge speed is 8000 to 12000 rpm, and the centrifugation time is 8 to 12 minutes; The amount of impurity removal solvent added is 3~12ml.

8. Use of the saxitoxin detection method according to any one of claims 1 to 7 in food, drug detection or daily chemical products.

Images