Method for synchronously detecting diarrhetic shellfish poisoning through solid-phase extraction and mass spectrometry

Abstract

The invention discloses a method for synchronously detecting diarrhetic shellfish poisoning by solid-phase extraction and mass spectrometry, which relates to the technical field of food detection and comprises the steps of sample pretreatment, chromatographic separation, mass spectrometry detection and qualitative and quantitative analysis. The sample pretreatment comprises methanol extraction and HLB solid-phase extraction column purification; the chromatographic separation adopts a C18 chromatographic column and a specific gradient elution procedure; the mass spectrum detection adopts an ESI < + > / ESI <-> switching scanning mode; according to the method, by optimizing purification conditions of an HLB solid-phase extraction column and a 1% ammonia water methanol elution scheme, toxin loss caused by nitrogen blowing operation is avoided, a pretreatment process is simplified, matrix interference is reduced, and by combining a specific gradient elution program of a C18 chromatographic column and a mass spectrometry detection mode of ESI < + > / ESI <-> switching scanning, the method has the advantages of high efficiency and high reliability. Baseline separation of the isomeride and efficient separation and detection of each toxin are realized, and the method has the advantages of simplicity and convenience in operation, rapidness, high efficiency, high sensitivity, good accuracy and strong specificity.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry. Background Technology

[0002] Diarrhetic shellfish toxins (DSPs) are a class of fat-soluble polyether or macrocyclic lactone compounds, primarily produced by marine algae. After ingestion, they accumulate in shellfish. Humans who consume contaminated shellfish may experience poisoning symptoms such as nausea, vomiting, abdominal pain, and diarrhea, and there is no specific antidote. my country stipulates a limit of 0.2 mg / kg (calculated as OA) for DSPs in shellfish; however, DSP poisoning incidents have continued to occur frequently in coastal areas in recent years, posing a serious threat to food safety.

[0003] Currently, the main detection methods for DSP include mouse bioassay, immunoassay, capillary electrophoresis, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). Among these, LC-MS has become the primary analytical method in poisoning emergency situations due to its advantages such as low sample requirements, rapid detection, and high specificity.

[0004] The existing national standard GB 5009.212-2016 only specifies the detection methods for three toxins: OA, DTX1, and DTX2, which is insufficient to meet the needs of simultaneous detection of multiple components. Some methods are only for single or a few toxins, and the pretreatment is time-consuming and requires a large amount of reagents. The complex shellfish matrix can easily interfere with the mass spectrometry response of the target compound, resulting in problems such as low recovery rate and insufficient sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for simultaneous detection of diarrhetic shellfish toxins using solid-phase extraction mass spectrometry, in order to solve the problems of existing detection methods that only target a single or a few toxins, and that require long pretreatment times and large amounts of reagents; and the fact that complex shellfish matrices can easily interfere with the mass spectrometric response of the target compound, resulting in low recovery rates and insufficient sensitivity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry, comprising sample pretreatment, chromatographic separation, mass spectrometry detection, and qualitative and quantitative analysis steps;

[0007] Diarrhea-causing shellfish toxins include okada spongiform acid (OA), fin algae toxin 1 (DTX1), fin algae toxin 2 (DTX2), scallop toxin 2 (PTX2), scallop toxin (YTX), scallop-like toxin (hYTX), cycloiminotoxin (GYM), spirocyclic lactone toxin 13 (SPX-13), protodinium alginate 1 (AZA1), protodinium alginate 2 (AZA2), and protodinium alginate 3 (AZA3).

[0008] The sample pretreatment includes methanol extraction and HLB solid-phase extraction column purification.

[0009] The chromatographic separation uses C 18 Chromatographic column and specific gradient elution program;

[0010] The mass spectrometry detection uses ESI. + / ESI - Switch scan mode.

[0011] Furthermore, the parameters for the mass spectrometry detection are as follows: electrospray ion source temperature 150℃, capillary voltage 3.0kV, desolvation gas temperature 550℃, desolvation gas flow rate 1000 L / h, and cone gas flow rate 150 L / h.

[0012] The parent ion, daughter ion, declustering voltage, collision energy, and retention time of diarrhetic shellfish toxins satisfy the following:

[0013] Okada soft sponge acid parent ion m / z 803.46, daughter ion m / z 255.1 / 113;

[0014] Fin-type algal toxin 1: parent ion m / z 817.47, daughter ion m / z 255.2 / 113.1;

[0015] Fin-type algal toxin 2: parent ion m / z 803.46, daughter ion m / z 255.2 / 113;

[0016] Scallop toxin 2: parent ion m / z 876.5, daughter ion m / z 823.5 / 805.4;

[0017] The precursor ion of scallop toxin has a m / z of 1141.47, and the daughter ion has a m / z of 1061.7 / 925.5.

[0018] The precursor ion of the scallop-like toxin has a m / z of 1155.49 and the daughter ions have a m / z of 1075.5 / 869.5.

[0019] The parent ion of the cycloiminotoxin has a m / z of 508.34, and the daughter ions have m / z of 162.1 / 392.3.

[0020] Spirocyclic lactone toxin 13: parent ion m / z 692.5, daughter ion m / z 164 / 444.3;

[0021] The precursor ion of polydinocyanate 1 has a m / z of 842.51 and daughter ions have m / z of 824.5 and 806.5 respectively.

[0022] The precursor ion of polyalginic acid 2 has a m / z of 856.52 and daughter ions have m / z of 838.5 / 820.4.

[0023] The original polyalginate 3 has a parent ion m / z of 828.49 and daughter ions m / z of 362.2 / 658.4.

[0024] Furthermore, the specific steps of the sample pretreatment are as follows:

[0025] After grinding and homogenizing the aquatic product sample, weigh 1g and place it in a 15mL centrifuge tube. Add 3mL of methanol, vortex for 1min, ultrasonically extract at 100-300W and 20-30℃ for 10min, centrifuge at 10000r / min for 5min, take the supernatant and add 6mL of water to shake for 30s.

[0026] The mixture was transferred to an HLB hydrophilic-lipophilic solid-phase extraction column that had been activated by rinsing with 2.5 mL of methanol and 2.5 mL of water. It was then rinsed with 2 mL of 20% methanol-water mixture and eluted with 2 mL of 1% ammonia-methanol mixture. The eluent was collected, and 2 mL of water was added and shaken for 30 s. The solution was then filtered through a 0.22 μm filter membrane to obtain the test solution.

[0027] Furthermore, the method also includes a standard curve plotting step: preparing a mixed standard stock solution containing the diarrhetic shellfish toxin, diluting it with a blank matrix extraction solution to obtain matrix-matched standard working solutions with mass concentrations of 1.00 ng / mL, 2.00 ng / mL, 5.00 ng / mL, 10.0 ng / mL, 20.0 ng / mL, and 50.0 ng / mL (calculated as Okada scleroderma), and plotting a standard curve with mass concentration as the abscissa and peak area as the ordinate after chromatographic separation and mass spectrometry detection.

[0028] Furthermore, the mass concentrations of each toxin in the mixed standard stock solution are as follows: Okada spongiform acid 250 ng / mL, fin algae toxin 1 212.5 ng / mL, fin algae toxin 2 190 ng / mL, scallop toxin 2 220 ng / mL, Yesso scallop toxin 367.5 ng / mL, Yesso scallop-like toxin 431.3 ng / mL, cycloiminotoxin 312.5 ng / mL, spirocyclic lactone toxin 13125 ng / mL, protodinial alginate 1 130 ng / mL, protodinial alginate 2 122 ng / mL, and protodinial alginate 3 147.5 ng / mL, and are stored in a sealed container at -20℃.

[0029] Furthermore, the HLB hydrophilic and lipophilic solid-phase extraction column has specifications of 3cc / 60mg or 6cc / 150mg, a column bed volume of 0.5 to 1.0 mL, and a pore size of 80 to 100 μm.

[0030] Furthermore, the specific conditions for the chromatographic separation are as follows: the chromatographic column is a Waters Acquity UPLC BEH C. 18 (2.1×50mm, 1.7μm), column temperature 40℃, injection volume 10μL, flow rate 0.2mL / min;

[0031] Mobile phase A was 0.2% formic acid-2 mmol / L ammonium formate solution, and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–1.0 min, maintain 20% B; 1.0–3.0 min, linearly increase from 20% B to 55% B; 3.0–6.0 min, linearly increase from 55% B to 95% B; 6.0–7.5 min, maintain 95% B; 7.5–7.6 min, decrease from 95% B to 20% B; 7.6–10.0 min, maintain 20% B.

[0032] Furthermore, the qualitative analysis is based on the following criteria: the relative deviation between the retention time of the analyte and the retention time of the standard is ≤2%, and the relative abundance of the characteristic ion is ≤20% compared with that of the standard; the quantitative analysis adopts the external standard method, and the content of each toxin in the sample is calculated based on the standard curve, with a method detection limit of 8-18 μg / kg and a quantitation limit of 24-54 μg / kg.

[0033] Compared with existing technologies, this invention provides a method for simultaneous detection of diarrhetic shellfish toxins using solid-phase extraction mass spectrometry (SPEMS). This method can simultaneously perform qualitative and quantitative analysis of okada spore toxin, fin algae toxin 1, fin algae toxin 2, scallop toxin 2, scallop toxin, scallop-like toxin, cycloiminotoxin, spirocyclic lactone toxin 13, protodinial alginate 1, protodinial alginate 2, and protodinial alginate 3. It effectively solves the problems of existing detection methods, such as limited toxin coverage, long pretreatment time, large reagent volume, and strong matrix interference. By optimizing the HLB solid-phase extraction column purification conditions and the 1% ammonia-methanol elution scheme, it avoids toxin loss caused by nitrogen blowing, simplifies the pretreatment process, and reduces matrix interference. Combined with C... 18 Specific gradient elution programs for chromatographic columns and ESI + / ESI - By switching the mass spectrometry detection mode, baseline separation of isomers and efficient separation and detection of various toxins were achieved. The method detection limit is as low as 8–18 μg / kg, the quantitation limit is 24–54 μg / kg, the spiked recovery rate is 75.2%–121.3%, and the relative standard deviation is only 0.7%–16.5%. It has the advantages of simple operation, rapid efficiency, high sensitivity, good accuracy and strong specificity. It is suitable for daily quality supervision and risk monitoring of diarrhetic shellfish toxins in shellfish and related aquatic products, as well as emergency rapid detection of sudden poisoning events, providing reliable technical support for ensuring the quality and safety of aquatic products. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0035] Figure 1 Chromatograms of OA (RT: 6.11) and DTX2 (RT: 6.31);

[0036] Figure 2 The chromatogram of DTX1;

[0037] Figure 3 The chromatogram of PTX2;

[0038] Figure 4 The chromatogram of YTX;

[0039] Figure 5 The chromatogram of H-YTX is shown below.

[0040] Figure 6 The chromatogram of GYM;

[0041] Figure 7 The chromatogram of SPX-13;

[0042] Figure 8 The chromatogram of AZA1;

[0043] Figure 9 The chromatogram of AZA2;

[0044] Figure 10 The chromatogram of AZA3;

[0045] Figure 11 The figure shows the effect of different solid-phase extraction columns on the recovery rate of 11 toxins in the sample. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0047] As attached Figure 1 To be continued Figure 11 As shown:

[0048] Example:

[0049] A method for simultaneous detection of diarrhetic shellfish toxins using solid-phase extraction mass spectrometry (SPEMS) includes sample pretreatment, chromatographic separation, mass spectrometry detection, and qualitative and quantitative analysis steps, as detailed below:

[0050] S1. Preparation of Instruments and Reagents

[0051] The experimental apparatus is as follows:

[0052] Ultra-high performance liquid chromatography-tandem mass spectrometry system: Waters XEVO TQ-XS (Waters Corporation, USA), equipped with an ESI ion source;

[0053] Electronic balance: MS204TS / 02 (accuracy 0.1mg, Mettler Toledo, Switzerland);

[0054] Vortex mixer: MS3 control (IKA GmbH, Germany);

[0055] High-speed refrigerated centrifuge: Allegra X-30R (Beckman Coulter, USA);

[0056] Ultrasonic cleaner: power 100-300W, temperature adjustable (Shanghai Anpu Scientific Instruments Co., Ltd.);

[0057] Solid phase extraction apparatus: including a vacuum filtration system (Biotage, Sweden);

[0058] Filter membrane: Organic phase needle filter membrane (0.22μm, Shanghai Anpu Experimental Technology Co., Ltd.);

[0059] Centrifuge tubes: 15mL polypropylene centrifuge tubes (enzyme-free and pyrogen-free, Jiangsu Kangda Medical Equipment Co., Ltd.)

[0060] The experimental reagents are as follows:

[0061] Acetonitrile: chromatographic grade (Thermo Fisher Scientific, USA);

[0062] Formic acid: LC / MS grade (Thermo Fisher Scientific, USA);

[0063] Ammonium formate: analytical grade (purity ≥98.0%, Sinopharm Chemical Reagent Company);

[0064] Methanol: chromatographic grade (Thermo Fisher Scientific, USA);

[0065] Ammonia solution: analytical grade (concentration 25%–28%, Sinopharm Chemical Reagent Company);

[0066] Experimental water: ultrapure water (resistivity ≥18.2MΩ・cm, prepared by Milli-Q ultrapure water system);

[0067] Standards: Okada spongiform acid, fin algae toxin 1, fin algae toxin 2, scallop toxin 2, scallop toxin, scallop-like toxin, cycloiminotoxin, spirocyclic lactone toxin 13, protodinolic acid 1, protodinolic acid 2, protodinolic acid 3 (all with a purity ≥90%, NRCC, Canada).

[0068] Solid phase extraction column: HLB hydrophilic and lipophilic type (specifications 3cc / 60mg or 6cc / 150mg, column bed volume 0.5-1.0mL, pore size 80-100μm, Waters Corporation, USA).

[0069] S2, Sample Pretreatment

[0070] Sample preparation: Commonly available aquatic products (oysters, scallops, mussels, clams, abalone, snails, cockles, etc.) were selected as test samples. After removing the shells, the edible parts were taken, ground and homogenized using a high-speed tissue homogenizer, and placed in a clean, sealed container. The samples were then refrigerated at 4°C for later use (testing was completed within 24 hours).

[0071] Extraction: Accurately weigh 1.000g of homogenized sample into a 15mL centrifuge tube, add 3mL of chromatographically pure methanol, and vortex for 1min (oscillation frequency 3000r / min). Then place it in an ultrasonic cleaner and extract ultrasonically for 10min at 20-30℃ and 200W power. After extraction, place it in a high-speed centrifuge and centrifuge at 10000r / min for 5min. Carefully aspirate the supernatant into another clean centrifuge tube.

[0072] Purification: Add 6 mL of ultrapure water to the supernatant and vortex for 30 s to mix thoroughly to obtain the solution to be purified; Activate the HLB solid-phase extraction column in advance: Elute the column sequentially with 2.5 mL of methanol and 2.5 mL of ultrapure water, controlling the flow rate at 1-2 drops / s, and discard the eluent; Slowly inject the solution to be purified into the activated HLB column, and after the liquid has completely passed through the column, rinse the column with 2 mL of 20% methanol-water (volume ratio, methanol:water = 20:80) to remove impurities such as fat and protein, and discard the eluent; Finally, elute with 2 mL of 1% ammonia-methanol (volume ratio, ammonia:methanol = 1:99), and collect the eluent into a 10 mL centrifuge tube.

[0073] Pretreatment before chromatographic analysis: Add 2 mL of ultrapure water to the collected eluent, vortex for 30 s to mix, reduce the proportion of methanol in the eluent to improve the peak shape, then filter the mixture through a 0.22 μm organic phase needle filter membrane, collect the filtrate into a sample vial, and wait for chromatography-mass spectrometry detection.

[0074] S3, Standard Curve Plotting

[0075] Preparation of mixed standard stock solution: Accurately transfer the solutions of each toxin standard into a 2.0 mL volumetric flask, dissolve them in chromatographic grade methanol, and dilute to a final volume. Prepare a mixed standard stock solution with the following concentrations of toxins: Okada hygroscopic acid 250 ng / mL, fin algae toxin 1 212.5 ng / mL, fin algae toxin 2 190 ng / mL, scallop toxin 2 220 ng / mL, Yesso scallop toxin 367.5 ng / mL, Yesso scallop-like toxin 431.3 ng / mL, cycloiminotoxin 312.5 ng / mL, spirocyclic lactone toxin 13 125 ng / mL, protodinial alginate 1 130 ng / mL, protodinial alginate 2 122 ng / mL, and protodinial alginate 3 147.5 ng / mL. After preparation, seal and store at -20°C. Shelf life is 3 months.

[0076] Preparation of matrix-matched standard working solutions: Take a blank aquatic product sample (confirmed by testing to be free of the target toxin), and prepare a blank matrix extraction solution according to the pretreatment steps in S2; accurately pipette an appropriate amount of mixed standard stock solution, and dilute it stepwise with the blank matrix extraction solution to prepare a series of matrix-matched standard working solutions with mass concentrations (calculated as Okada scleroderma) of 1.00 ng / mL, 2.00 ng / mL, 5.00 ng / mL, 10.0 ng / mL, 20.0 ng / mL, and 50.0 ng / mL, respectively. Prepare fresh each time you need to use the solution.

[0077] Standard curve plotting: Inject a series of matrix-matched standard working solutions sequentially under the chromatographic-mass spectrometry conditions of S4, and record the peak area of ​​each toxin in each standard working solution; plot the standard curve using the mass concentration of each toxin as the abscissa (x) and the corresponding peak area as the ordinate (y), and obtain the linear equation and correlation coefficient (r) for each toxin. The correlation coefficient r is required to be greater than 0.99 to meet the requirements of quantitative analysis.

[0078] S4, Chromatography-Mass Spectrometry Detection

[0079] The chromatographic conditions are as follows:

[0080] Chromatographic column: Waters Acquity UPLC BEH C 18 (2.1×50mm, 1.7μm);

[0081] Column temperature: 40℃;

[0082] Injection volume: 10 μL;

[0083] Flow rate: 0.2 mL / min;

[0084] Mobile phase: Phase A is 0.2% formic acid-2mmol / L ammonium formate solution (volume ratio, formic acid:water = 0.2:99.8, ammonium formate is added to make the concentration 2mmol / L), and Phase B is acetonitrile;

[0085] Gradient elution program: 0–1.0 min, maintain 20% B; 1.0–3.0 min, linearly increase from 20% B to 55% B; 3.0–6.0 min, linearly increase from 55% B to 95% B; 6.0–7.5 min, maintain 95% B; 7.5–7.6 min, decrease from 95% B to 20% B; 7.6–10.0 min, maintain 20% B (equilibrate the column).

[0086] The mass spectrometry conditions are as follows:

[0087] Ionization mode: ESI + / ESI - Switch scan;

[0088] Electrospray ion source temperature: 150℃;

[0089] Capillary voltage: 3.0kV;

[0090] Desolvation gas temperature: 550℃;

[0091] Desolvation gas flow rate: 1000 L / h;

[0092] Conical orifice air flow rate: 150L / h;

[0093] Mass spectrometry parameters of each toxin:

[0094] Toxin name Molecular formula Parent ion (m / z) Daughter ions (m / z) Declustering voltage (V) Collision energy (V) polarity Retention time (min) OA <![CDATA[C 44 H 68 O 13 ]]> 803.46 255.1*113 30,65 47,50 ESI- 6.11 DTX1 <![CDATA[C 45 H 70 O 13 ]]> 817.47 255.2*113.1 80 45,60 <![CDATA[ESI - ]]> 6.82 DTX2 <![CDATA[C 44 H 68 O 13 ]]> 803.46 255.2*113 70 50,65 <![CDATA[ESI - ]]> 6.31 PTX2 <![CDATA[C 47 H 70 O 14 ]]> 876.5 823.5*805.4 40 30,30 <![CDATA[ESI + ]]> 6.37 YTX <![CDATA[C 55 H 82 O 21 S2]]> 1,141.47 1,061.7*925.5 30,40 36,55 <![CDATA[ESI - ]]> 6.23 H-YTX <![CDATA[C 56 H 84 O 21 S2]]> 1,155.49 1,075.5*869.5 37 36,55 <![CDATA[ESI - ]]> 6.24 GYM <![CDATA[C 32 H 45 NO4]]> 508.34 162.1*392.3 60 55,35 <![CDATA[ESI + ]]> 3.78 SPX-13 <![CDATA[C 42 H 63 NO7]]> 692.5 164*444.3 47 60,55 <![CDATA[ESI + ]]> 4.46 AZA1 <![CDATA[C 47 H 71 NO 12 ]]> 842.51 824.5*806.5 35 30,23 <![CDATA[ESI + ]]> 6.72 AZA2 <![CDATA[C 48 H 73 NO 12 ]]> 856.52 838.5*820.4 35 30,44 <![CDATA[ESI + ]]> 6.89 AZA3 <![CDATA[C 46 H 69 NO 12 ]]> 828.49 362.2*658.4 47,35 55,40 <![CDATA[ESI + ]]> 6.42

[0095] S5. Qualitative and Quantitative Analysis

[0096] Compare the chromatographic-mass spectrometry results of the sample solution with the results of the standard working solution:

[0097] Retention time matching: The relative deviation between the retention time of the analyte in the sample and the retention time of the standard is ≤2%;

[0098] Characteristic ion matching: The relative abundance of the characteristic ions of the analyte in the sample (two characteristic ions are selected for each toxin, one of which is a quantitative ion and the other is a qualitative ion) deviates from the relative abundance of the corresponding ion in the standard by ≤20%; if both of the above conditions are met, the sample is determined to contain the diarrhetic shellfish toxin.

[0099] Quantification was performed using the external standard method: the peak area of ​​each toxin in the sample was substituted into the corresponding linear equation of the standard curve to calculate the mass concentration (c) of each toxin in the sample. Then, the actual content (X) of each toxin in the sample was calculated using the following formula: X = (c × V × f) / m where:

[0100] X: Content of the target toxin in the sample (unit: μg / kg);

[0101] c: Concentration of the target toxin in the test solution calculated from the standard curve (unit: ng / mL).

[0102] V: Final volume of the test solution (unit: mL). In this example, V = 4 mL (2 mL of eluent + 2 mL of water).

[0103] f: Dilution factor. In this embodiment, no additional dilution was performed during sample pretreatment, so f=1.

[0104] m: Sample mass (unit: g), in this example m=1.000g.

[0105] S6, Methodological Validation

[0106] A series of standard working solutions were prepared according to S3, and tests were performed to plot standard curves. The results showed that the 11 diarrhetic shellfish toxins exhibited good linearity within their respective linear ranges, with correlation coefficients r > 0.99 for all. The limit of detection (LOD) was calculated with a signal-to-noise ratio (S / N) of 3, and the limit of quantitation (LOQ) was calculated with an S / N of 10. The results are shown in the table below.

[0107] Toxin name Linear range (ng / mL) Linear equations Correlation coefficient r Limit of detection (μg / kg) Limit of quantitation (μg / kg) OA 1.0～50.0 <![CDATA[y= 4.46e 5 x+2.56e 5 ]]> 0.9983 15.0 45.0 DTX1 0.85～42.5 <![CDATA[y= 1.14e 5 x+2.46e 5 ]]> 0.9948 13.0 39.0 DTX2 0.76～38.0 <![CDATA[y= 5.27e 4 x+1.08e 5 ]]> 0.9932 13.0 39.0 PTX2 0.88～44.0 <![CDATA[y= 1.72e 6 x+1.08e 6 ]]> 0.9988 13.0 39.0 YTX 1.47～73.5 <![CDATA[y= 9.97e 4 x+1.39e 4 ]]> 0.9933 18.0 54.0 H-YTX 1.73～86.3 <![CDATA[y= 3.27e 5 x-4.7e 4 ]]> 0.9990 18.0 54.0 GYM 1.25～62.5 <![CDATA[y= 1.81e 6 x+9.26e 5 ]]> 0.9994 18.0 54.0 SPX-13 0.50～25.0 <![CDATA[y= 1.01e 7 x+5.96e 5 ]]> 0.9997 8.0 24.0 AZA1 0.52～26.0 <![CDATA[y= 2.12e 7 x+6.41e 6 ]]> 0.9991 8.0 24.0 AZA2 0.49～24.4 <![CDATA[y= 2.27e 7 x+5.36e 6 ]]> 0.9993 8.0 24.0 AZA3 0.59～29.5 <![CDATA[y= 2.27e 6 x+2.47e 5 ]]> 0.9996 8.0 24.0

[0108] Blank oyster samples were selected, and mixed standard working solutions with low, medium, and high spiking levels were added respectively. Six samples were prepared in parallel for each spiking level. Pretreatment and detection were performed according to steps S2 to S4. Spike recoveries and relative standard deviations (RSD) were calculated. The results are shown in the table below:

[0109] Toxin name Low spiking level (mg / kg) Recovery rate (%) RSD (%) Sino-Canadian standard level (mg / kg) Recovery rate (%) RSD (%) High spiking level (mg / kg) Recovery rate (%) RSD (%) OA 0.06 96.1 7.7 0.12 99.1 5.3 0.30 97.9 5.3 DTX1 0.05 89.5 16.5 0.10 95.6 6.3 0.25 78.7 3.8 DTX2 0.05 75.2 11.6 0.10 78.6 11.7 0.25 81.2 14.3 PTX2 0.05 111.4 2.4 0.10 102.8 0.7 0.25 121.3 1.0 YTX 0.09 93.1 15.7 0.18 114.3 10.5 0.45 79.8 13.1 H-YTX 0.09 109.4 11.0 0.18 95.6 7.3 0.45 115.3 1.7 GYM 0.08 104.0 3.3 0.16 95.2 2.2 0.40 101.9 3.7 SPX13 0.03 104.6 2.5 0.06 96.4 2.1 0.15 101.5 2.4 AZA1 0.03 102.6 2.6 0.06 102.9 1.6 0.15 116.9 1.6 AZA2 0.03 97.3 4.7 0.06 98.8 3.1 0.15 111.4 1.8

[0110] The results showed that the average recovery rate of the 11 toxins was 75.2%–121.3%, and the relative standard deviation (RSD) was 0.7%–16.5% (n=6), which met the requirements of food safety testing methods for recovery rate and precision (RSD<20%).

[0111] S7 Actual Sample Testing

[0112] This method was used to test 116 batches of aquatic product samples, including 56 batches purchased online (scallops, mussels, oysters, clams, etc.) and 60 batches purchased offline (oysters, abalone, snails, clams, fish, etc.). The results showed that one batch of scallop sample tested positive for cycloiminotoxin at a content of 167.4 μg / kg; one batch of clams tested positive for cycloiminotoxin at a content of 50.4 μg / kg; the remaining 114 batches did not test positive for the target toxin. All results were within the limits for diarrhetic shellfish toxins stipulated in Chinese national standards (0.2 mg / kg, calculated as okadaic acid), indicating that this method is suitable for routine monitoring, risk surveillance, and emergency detection of diarrhetic shellfish toxins in aquatic products.

[0113] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry, characterized in that, This includes sample pretreatment, chromatographic separation, mass spectrometry detection, and qualitative and quantitative analysis steps; Diarrhea-causing shellfish toxins include okada spongiform acid, fin algae toxin 1, fin algae toxin 2, scallop toxin 2, scallop toxin, scallop-like toxin, cycloiminotoxin, spirocyclic lactone toxin 13, protodinolic acid 1, protodinolic acid 2, and protodinolic acid 3. The sample pretreatment includes methanol extraction and HLB solid-phase extraction column purification. The chromatographic separation uses C 18 Chromatographic column and specific gradient elution program; The mass spectrometry detection uses ESI. + / ESI - Switch scan mode.

2. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 1, characterized in that, The parameters for mass spectrometry detection are as follows: electrospray ion source temperature 150℃, capillary voltage 3.0kV, desolvation gas temperature 550℃, desolvation gas flow rate 1000 L / h, and cone gas flow rate 150 L / h. The parent ion, daughter ion, declustering voltage, collision energy, and retention time of diarrhetic shellfish toxins satisfy the following: Okada soft sponge acid parent ion m / z 803.46, daughter ion m / z 255.1 / 113; Fin-type algal toxin 1: parent ion m / z 817.47, daughter ion m / z 255.2 / 113.1; Fin-type algal toxin 2: parent ion m / z 803.46, daughter ion m / z 255.2 / 113; Scallop toxin 2: parent ion m / z 876.5, daughter ion m / z 823.5 / 805.4; The parent ion of scallop toxin has a m / z of 1141.47, and the daughter ion has a m / z of 1061.7 / 925.

5. The precursor ion of the scallop-like toxin has a m / z of 1155.49 and the daughter ions have a m / z of 1075.5 / 869.

5. The parent ion of the cycloiminotoxin has a m / z of 508.34, and the daughter ions have m / z of 162.1 / 392.

3. Spirocyclic lactone toxin 13: parent ion m / z 692.5, daughter ion m / z 164 / 444.3; The precursor ion of polydinocyanate 1 has a m / z of 842.51 and daughter ions have m / z of 824.5 and 806.5 respectively. The precursor ion of polyalginic acid 2 has a m / z of 856.52 and daughter ions have m / z of 838.5 / 820.

4. The original polyalginate 3 has a parent ion m / z of 828.49 and daughter ions m / z of 362.2 / 658.

4.

3. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 1, characterized in that, The specific steps of the sample pretreatment are as follows: After grinding and homogenizing the aquatic product sample, weigh 1g and place it in a 15mL centrifuge tube. Add 3mL of methanol, vortex for 1min, ultrasonically extract at 100-300W and 20-30℃ for 10min, centrifuge at 10000r / min for 5min, take the supernatant and add 6mL of water to shake for 30s. The mixture was transferred to an HLB hydrophilic-lipophilic solid-phase extraction column that had been activated by rinsing with 2.5 mL of methanol and 2.5 mL of water. It was then rinsed with 2 mL of 20% methanol-water mixture and eluted with 2 mL of 1% ammonia-methanol mixture. The eluent was collected, and 2 mL of water was added and shaken for 30 s. The solution was then filtered through a 0.22 μm filter membrane to obtain the test solution.

4. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 1, characterized in that, The standard curve plotting step also includes: preparing a mixed standard stock solution containing the diarrhetic shellfish poison, diluting it with a blank matrix extraction solution to obtain matrix-matched standard working solutions with mass concentrations of 1.00 ng / mL, 2.00 ng / mL, 5.00 ng / mL, 10.0 ng / mL, 20.0 ng / mL, and 50.0 ng / mL, and plotting a standard curve with mass concentration as the abscissa and peak area as the ordinate after chromatographic separation and mass spectrometry detection.

5. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 4, characterized in that, The mass concentrations of each toxin in the mixed standard stock solution are as follows: Okada spongiform acid 250 ng / mL, fin algae toxin 1212.5 ng / mL, fin algae toxin 2190 ng / mL, scallop toxin 2220 ng / mL, Yesso scallop toxin 367.5 ng / mL, Yesso scallop-like toxin 431.3 ng / mL, cycloiminotoxin 312.5 ng / mL, spirocyclic lactone toxin 13125 ng / mL, protodinial alginate 1130 ng / mL, protodinial alginate 2122 ng / mL, and protodinial alginate 3147.5 ng / mL. The solution is stored in a sealed container at -20℃.

6. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 1, characterized in that, The HLB hydrophilic and lipophilic solid-phase extraction column has specifications of 3cc / 60mg or 6cc / 150mg, a column bed volume of 0.5 to 1.0 mL, and a pore size of 80 to 100 μm.

7. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 1, characterized in that, The specific conditions for chromatographic separation are: column temperature 40℃, injection volume 10μL, and flow rate 0.2mL / min; Mobile phase A was 0.2% formic acid-2 mmol / L ammonium formate solution, and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–1.0 min, maintain 20% B; 1.0–3.0 min, linearly increase from 20% B to 55% B; 3.0–6.0 min, linearly increase from 55% B to 95% B; 6.0–7.5 min, maintain 95% B; 7.5–7.6 min, decrease from 95% B to 20% B; 7.6–10.0 min, maintain 20% B.

8. The method for simultaneous detection of diarrhetic shellfish poisoning using solid-phase extraction mass spectrometry according to claim 1, characterized in that, The qualitative analysis is based on the following criteria: the relative deviation between the retention time of the analyte and the retention time of the standard is ≤2%, and the relative abundance of the characteristic ion is ≤20% compared with that of the standard. The quantitative analysis adopts the external standard method, and the content of each toxin in the sample is calculated based on the standard curve. The method detection limit is 8-18 μg / kg, and the quantitation limit is 24-54 μg / kg.

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