Method for detecting TTX and 4, 9-anhydroTTX in shellfish by ion pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting TTX and 4, 9-anhydroTTX in shellfish through ion pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry, and the method comprises the following steps: (1) pretreatment; (2) extracting; (3) enriching and purifying; and (4) loading a sample and detecting. The method for detecting TTX and 4, 9-anhydroTTX in shellfish through ion pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry is established by optimizing extraction, enrichment purification and chromatographic conditions, the method can be used for detecting TTX and 4, 9-anhydroTTX in shellfish samples at the same time, operation is easy and convenient, accuracy, precision and reproducibility are good, and the method can be suitable for quantitative analysis of TTX in shellfish and 4, 9-anhydroTTX homologues of TTX in shellfish.

Description

Technical Field

[0001] The invention relates to the technical field of TTX detection, and in particular to a method for detecting TTX and 4,9-anhydroTTX in shellfish by solid phase extraction column purification-liquid chromatography-tandem mass spectrometry. Background Art

[0002] Tetrodotoxin (TTX) is a highly toxic small molecule alkaloid. It was first isolated from pufferfish. In recent years, it has been found that tetrodotoxin exists not only in fish of the genus Pufferfish, but also in gobies, octopuses, starfish, crabs, shellfish, and even horseshoe crabs and many other marine organisms. Tetrodotoxin is extremely toxic, and consuming 0.5 mg can cause death. TTX binds to sodium ion channel receptors, blocks voltage-dependent sodium channels, and thus blocks action potentials, leading to physiological activity disorders related to it, causing sensory nerve paralysis, quadriplegia, breathing difficulties, and finally death from respiratory depression. There is currently no effective antidote. Due to the serious harm of tetrodotoxin and its stable chemical properties, it cannot be degraded by general cooking methods. It is extremely important to carry out detection and research on TTX in shellfish to ensure the safety of aquatic products and human health.

[0003] At present, the commonly used methods for detecting TTX mainly include mouse biological method, liquid chromatography-fluorescence detection method, etc. The calculation method of mouse biological method is complicated, with poor repeatability, time-consuming and labor-intensive, large individual differences, poor reproducibility, and a certain scale is required to objectively reflect the experimental results; liquid chromatography-fluorescence detection method must first derivatize TTX into a substance with fluorescent absorption, which requires a derivatization device and is relatively cumbersome to operate. Summary of the invention

[0004] The present invention aims to solve the above-mentioned problems existing in the TTX detection method of the prior art, and provides a method for detecting TTX and 4,9-anhydroTTX in shellfish by ion pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry. The method is simple to operate, and has good accuracy, precision and reproducibility, and can be applied to the quantitative analysis of TTX and its homologue 4,9-anhydroTTX in shellfish.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: the method of the present invention for detecting TTX and 4,9-anhydroTTX in shellfish by ion pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry comprises the following steps: (1) Pretreatment: After cleaning the shellfish, peel off the shells, collect the samples and homogenize them to obtain a homogenous slurry. All shellfish samples were prepared in accordance with the national standard GB / T30891-2014 "Aquatic Product Sampling Specifications".

[0006] (2) Extraction: Accurately weigh the homogenized slurry into a centrifuge tube, add 1% by volume acetic acid aqueous solution, vortex and oscillate to perform a first extraction, boil the mixture with hot water after extraction, cool to room temperature, centrifuge, retain the supernatant, add 1% by volume acetic acid aqueous solution to the centrifugal residue, repeat the previous steps to perform a second extraction, combine the supernatants, make up to volume, obtain an extract, mix the extract with an ion pair reagent, and obtain a loading solution.

[0007] (3) Enrichment and purification: Activate the graphitized carbon black solid phase extraction column with acetonitrile, purified water and ion pair reagent in sequence, load the sample solution, rinse with purified water after loading, elute with acetonitrile containing 0.4% by volume of formic acid, blow the eluate to near dryness with nitrogen, reconstitute with the initial mobile phase, filter, and the filtrate is the loading solution.

[0008] (4) Sample loading detection: The sample solution is extracted with an injection needle, loaded according to the set chromatography-mass spectrometry conditions, and detected by ultra-high performance liquid chromatography tandem mass spectrometry. The ion spectrum peaks of TTX and 4,9-anhydroTTX are obtained, and the mass concentrations of TTX and 4,9-anhydroTTX are calculated based on the ion spectrum peak areas and the standard curve of TTX and 4,9-anhydroTTX. Obtaining the standard curve of TTX and 4,9-anhydroTTX is a conventional method in the art.

[0009] Preferably, in step (2), 5±0.01 g of homogenized slurry is weighed into a 50 mL centrifuge tube, the mass ratio of the homogenized slurry to 1% by volume acetic acid aqueous solution is 1:1; vortex oscillation is performed for 1 to 3 min, and hot water is boiled for 10 to 12 min; centrifugation is performed at 3 to 5° C. and 10,000 rpm for 5 to 6 min; the mass ratio of the centrifugal residue to 1% by volume acetic acid aqueous solution is 1:0.4; the supernatant is diluted to 10 mL with purified water; the ion pair reagent is perfluoroheptanoic acid, and 0.5 mL of the extract is mixed with 0.1 mL of 25 mmol / L ion pair reagent. Shellfish contain a large amount of interfering compounds such as proteins, amino acids and minerals. The above substances will produce matrix effects when they elute with the target, affecting the accuracy of liquid-to-mass quantitative results. The inventors chose to extract the spiked samples with 0.1%, 0.5%, 1%, 2% and 2.5% acetic acid aqueous solutions, and investigated the effect of acetic acid ratio on the extraction rate of TTX and 4,9-anhydroTTX. The results showed that when the acetic acid ratio was 0.1%-1%, the recovery rates of TTX and 4,9-anhydroTTX increased with the increase of acidity. When the acidity increased further, the extraction effect decreased. The 1% acetic acid aqueous solution was able to completely extract the target, so it was selected as the optimal extraction solution. Based on this, the inventors used 1% acetic acid aqueous solution as the extraction solution and chose to perform a single extraction on the shellfish spiked samples at a solid-liquid ratio of 1:1, 1:2 and 1:3. The results of the recovery rate showed that the solid-liquid ratio had no significant effect on the recovery rate of TTX and 4,9-anhydroTTX during the first extraction. Under different solid-liquid ratio conditions, the recovery rate of TTX was lower than 88%, and the recovery rate of 4,9-anhydroTTX was lower than 70%. The inventors took the solid-liquid ratio of 1:1 as the optimal condition for the first extraction, and selected 1:0.2, 1:0.4, 1:0.8 and 1:1.2 for the secondary extraction of the residue after extraction, and further investigated whether the secondary extraction could increase the recovery rate of TTX and 4,9-anhydroTTX. The results showed that after the secondary extraction with a solid-liquid ratio of 1:0.4, the recovery rates of TTX and 4,9-anhydroTTX increased by 9.8% and 8.4%, respectively. Therefore, 1:1 and 1:0.4 were finally selected as the optimal solid-liquid ratio conditions for the first extraction and the second extraction. Ion pair reagents are formed by strongly hydrophilic ions, which react to the neutral ion pairs of sample molecules, and can enhance the retention of highly polar compounds on reversed-phase analytical columns.Under acidic pH conditions, the guanidine group of tetrodotoxin carries a positive charge and can form ion pairs with anions of hydrophobic alkyl chain ion pair reagents, thereby enhancing the retention capacity on the extraction column. Therefore, the inventors selected three ion pair reagents, perfluoropentanoic acid (NDFHA), perfluorohexanoic acid (UDFHA) and perfluoroheptanoic acid (TDFHA) (n=3) according to the length of the alkyl chain, and investigated the retention effect of CNW bond carbon GCB on TTX and 4,9-anhydroTTX, in order to further improve the enrichment and purification effect of the target. First, 60ngTTX and 4,9-anhydroTTX were added to 0.5mL of extract, and 5mmol / L ion pair reagent was added as the loading solution at a volume ratio of 5:1. After loading, 2ml of purified water was used for elution, and the eluent was collected in batches of 0.5ml and diluted with the initial mobile phase (5% by volume of 5mmol / L ammonium acetate solution containing 0.1% formic acid + 95% by volume of acetonitrile). After filtration with a 0.22μm filter membrane, the liquid was subjected to mass analysis. According to the loss of the target during the elution process, when perfluorohexanoic acid was used as the ion pair reagent, as the elution volume increased, the target combined with perfluorohexanoic acid became worse. Under the 2mL water elution condition, TTX and 4 The loss rates of 4,9-anhydroTTX and 4,9-anhy-droTTX were 75.1% and 62.6% respectively; when perfluoropentanoic acid was used as an ion pair reagent, TTX loss mainly occurred in the first 2mL of eluent, with a loss rate of 40.1%; 4,9-anhydroTTX loss mainly occurred in the first 1.5mL of eluent, with a loss rate of 36.5%; as the elution volume increased further, the loss of the two was not obvious; when perfluoroheptanoic acid was used as an ion pair reagent, under the same elution conditions mentioned above, TTX and 4,9-anhy-droTTX did not lose anything until the eluent volume increased to 2.5mL, and TTX and 4,9-anhy-droTTX lost 4.5% and 3.6% respectively. It can be seen that perfluoroheptanoic acid has the strongest retention capacity for TTX and 4,9-anhydroTTX among the three ion pair reagents, so perfluoroheptanoic acid and 2mL of purified water elution were selected as the purification conditions.Based on this, the inventors continued to add 60ngTTX and 4,9-anhydroTTX to 0.5mL of the extract, and added 5mmol / L perfluoroheptanoic acid as the loading solution at a volume ratio of 5:1 to investigate the effect of the matrix on the enrichment and purification of TTX and 4,9-anhydroTTX. The results showed that the recovery rates of TTX and 4,9-anhydroTTX dropped to 45.1-49.3%, indicating that the shellfish matrix affected the binding ability of the ion pair reagent and the target. Therefore, the inventors chose to further increase the concentration of perfluoroheptanoic acid (5, 10, 15, 20, 25 and 30mmol / L) to investigate the effect of TTX and 4,9-anhydroTTX on CNW bond carbon. The retention of GCB showed that with the increase of perfluoroheptanoic acid concentration, the loss rate of TTX and 4,9-anhydroTTX decreased. When the concentration of perfluoroheptanoic acid increased to 25 and 30 mmol / L, no loss of the target was found. Considering that too high a concentration of perfluoroheptanoic acid would lead to an increase in the volume of the eluent, 25 mmol / L perfluoroheptanoic acid was finally selected as the optimal loading condition. The ion pair reagent and the target will form a hydrophobic ion pair complex in the filler

[100] . It is necessary to determine a suitable eluent to effectively recover the target and it should be compatible with the subsequent liquid phase analysis conditions. Tetrodotoxin and its homologues are unstable under alkaline conditions. After purification of the extraction column, it is not suitable to use alkaline solutions such as ammonia water for elution. Some scholars used 1-2 mL of methanol solution containing hydrochloric acid to elute TTX, but the matrix inhibition effect was obvious. Considering that strong acids such as hydrochloric acid are corrosive, formic acid was used instead of hydrochloric acid in this experiment. It was found that a formic acid content of 0.4% could ensure the complete elution of TTX and 4,9-anhydroTTX. Further investigation of the effect of the ratio of acetonitrile to aqueous solution (100%, 80%, 60%, 40%, 20%) and elution volume on the elution effect of TTX and 4,9-anhydroTTX showed that different ratios of acetonitrile to aqueous solution had no significant effect on the recovery rate, and 2 mL volume was sufficient to completely elute the target. Given that pure acetonitrile can achieve a concentrated effect after nitrogen blowing, the optimal elution condition was determined to be 2 mL of acetonitrile containing 0.4% formic acid.

[0010] Preferably, in step (3), 2.5 mL of acetonitrile, 2.5 mL of purified water, and 2.5 mL of 5 mmol / L ion pair reagent are used to activate the graphitized carbon black solid phase extraction column in sequence; the sample loading volume is 0.5 ml; after the sample loading is completed, it is rinsed with 2 mL of purified water and eluted with 2 mL of acetonitrile containing 0.4% by volume of formic acid; the eluent is blown to near dryness with nitrogen at 40° C.; the initial mobile phase is 5% by volume of 5 mmol / L ammonium acetate solution containing 0.1% formic acid and 95% by volume of acetonitrile; and filtered with a 0.22 μm filter membrane. The extract not only contains the target TTX and 4,9-anhydroTTX, but also contains a large amount of matrix co-extracts. The carrying capacity of the graphitized carbon black SPE column is limited. Excessive loading not only affects the binding ability of the target with the column filler, but also affects the subsequent elution. The inventor added 50ngTTX and 4,9-anhydroTTX to the blank matrix, and systematically investigated the effect of 0.2mL, 0.5mL, 1mL, 1.5mL and 2mL loading volumes on the recovery rate. The results showed that the recovery rates of TTX and 4,9-anhydroTTX were the highest under the loading volume of 0.5mL, and then the recovery rates of the two decreased with the increase of the loading volume. The presence of shellfish matrix can interfere with the binding of the target with the filler to a certain extent, thereby causing TTX and 4,9-anhydroTTX to lose more during the loading process. Therefore, 0.5mL is selected as the optimal loading volume in the present invention. The ion pair reagent and the target will form a hydrophobic ion pair complex in the filler, so it is necessary to determine a suitable eluent to effectively recover the target, and it should be compatible with the subsequent liquid phase analysis conditions. Tetrodotoxin and its homologues are unstable under alkaline conditions. After purification of the extraction column, it is not suitable to use alkaline solutions such as ammonia water for elution. Some scholars use 1-2mL methanol solution containing hydrochloric acid to elute TTX, but the matrix inhibition effect is obvious. Considering that strong acids such as hydrochloric acid are corrosive, the inventors replaced hydrochloric acid with formic acid and found that when the formic acid content is 0.4%, TTX and 4,9-anhydroTTX can be completely eluted. The effects of the proportion of acetonitrile aqueous solution (100%, 80%, 60%, 40%, 20%) and the elution volume on the elution effect of TTX and 4,9-anhydroTTX were further investigated. The results showed that different proportions of acetonitrile aqueous solution had no significant effect on the recovery rate, and a volume of 2mL was sufficient to completely elute the target. In view of the fact that pure acetonitrile can achieve a concentration effect after nitrogen blowing, the best eluent was determined to be 2mL acetonitrile containing 0.4% formic acid.

[0011] Preferably, the graphitized carbon black solid phase extraction column is a CNW bond carbon GCB graphitized carbon black solid phase extraction column. The inventor selected four graphitized carbon black solid phase extraction columns, namely Supelclean ENVI Carb (250 mg / 3 mL, Supelco, USA), Agilent BoodElut carbon (500 mg / 6 mL, Agilent, Germany), CNW bond carbon GCB (250 mg / 3 mL, Shanghai Anpu Scientific Instrument Co., Ltd.) and Aega Cleanert pesti carb (250 mg / 3 mL, Aegla Technologies, USA), and investigated their purification effects on blank matrix spiked samples (TTX and 4,9-anhydroTTX 50 ng were added respectively). Under the optimal purification conditions of the four solid phase extraction columns, the recovery rates of TTX were 86.7%, 81.2%, 85.7% and 75.25%, respectively, and the recovery rates of 4,9-anhydroTTX were 75.5%, 83.4%, 88.7% and 89.1%, respectively. According to the results, CNWbondcarbonGCB has a more ideal purification and enrichment effect, with recoveries of 85.7% and 88.7% respectively, and was finally selected as the optimal solid phase extraction column for purifying the target.

[0012] Preferably, in step (4), the chromatographic conditions are: Waters ACQUITY UPLC BEE H Amide chromatographic column, column size 50 mm×2.1 mm, 1.7 μm; sample chamber temperature: 4° C.; chromatographic column temperature: 40° C.; injection volume: 5 μL; mobile phase A: 5 mmol / L ammonium acetate solution containing 0.1% formic acid; mobile phase B: acetonitrile; flow rate: 0.3 mL / min, and the gradient elution conditions are: 0 min, 5% mobile phase A, 95% mobile phase B→0.5 min, 5% mobile phase A, 95% mobile phase B→6.0 min, 55% mobile phase A, 45% mobile phase B→8.0 min, 55% mobile phase A, 45% mobile phase B→8.1 min, 5% mobile phase A, 95% mobile phase B→10.0 min, 5% mobile phase A, 95% mobile phase B. TTX and 4,9-anhydroTTX are highly polar compounds, so the present invention uses ACQUITY UPLC BEE H Amide chromatographic column, which can provide better retention and separation ability; ammonium acetate as a buffer solvent can improve the separation effect and obtain a good peak shape. In addition, adding a certain concentration of organic acid to the mobile phase can not only improve the separation of the components to be measured, but also enhance its ionization effect. The inventors added 0.05%, 0.1% and 0.2% formic acid to solutions containing 2mmol / L and 5mmol / L ammonium acetate, and compared their effects on the response value and separation effect of the target. The results showed that when chromatographic separation was performed using 5mmol / L ammonium acetate solution containing 0.1% formic acid as the mobile phase, the peak shape of TTX and 4,9-anhydroTTX was sharper and the response value was the best. Therefore, 5mmol / L ammonium acetate solution containing 0.1% formic acid was determined to be the most suitable mobile phase; and in order to reduce the effect of impurity peaks in the matrix on TTX In order to avoid the interference of 4,9-anhydroTTX, the inventors further adjusted the gradient elution conditions and finally determined the gradient elution conditions as follows: 0 min, 5% mobile phase A, 95% mobile phase B→0.5 min, 5% mobile phase A, 95% mobile phase B→6.0 min, 55% mobile phase A, 45% mobile phase B→8.0 min, 55% mobile phase A, 45% mobile phase B→8.1 min, 5% mobile phase A, 95% mobile phase B→10.0 min, 5% mobile phase A, 95% mobile phase B, with a total separation time of 10 min. The peaks of TTX and 4,9-anhydroTTX appeared at 6.67 and 6.10 min, respectively.

[0013] Preferably, in step (4), the mass spectrometry conditions are: electrospray ion source positive ion scanning; monitoring mode: multiple reaction monitoring mode; ion source temperature: 120°C; capillary voltage: 1.5 kV; desolvation gas temperature: 600°C; cone gas: 3.5 kV; desolvation: high-purity nitrogen, flow rate 800 L / h.

[0014] Therefore, the present invention has the following beneficial effects: a method for detecting TTX and 4,9-anhydroTTX in shellfish by ion pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry is established by optimizing extraction, enrichment purification and chromatographic conditions, which can simultaneously determine TTX and 4,9-anhydroTTX in shellfish samples. The method is simple to operate, and has good accuracy, precision and reproducibility, and is suitable for the quantitative analysis of TTX and its homologue 4,9-anhydroTTX in shellfish. DETAILED DESCRIPTION

[0015] The present invention is further described below through specific implementation modes.

[0016] The materials and equipment in the following examples are as follows: standard TTX and 4,9-anhydroTTX, from Dr. Ehrenstorfer, Germany; nonafluoropentanoic acid (NDFHA), perfluorohexanoic acid (UDFHA), tridecafluoroheptanoic acid (TDFHA), from Sigma, USA; formic acid and sodium acetate (both chromatographically pure), from Sigma, USA; methanol and acetonitrile (both chromatographically pure), from Meck, Germany; acetic acid (analytical pure), from Shanghai Sinopharm; ultra-high performance liquid chromatography (ACQUITYTM), from Waters, USA; tandem quadrupole mass spectrometer (ESI) Waters Xevo TQS, from Waters, USA; Waters ACQUITY UPLC BEHAmide chromatographic column, from Waters, USA.

[0017] 1. Linear Relationship and Limit of Quantitation The initial mobile phase was used to dilute the 0.2, 0.5, 2, 10 and 40 ng / mL TTX standard working solutions, and the concentrations were 2, 5, 10, 20, 50 ng / mL 4,9-anhydroTTX standard working solutions. The standard curve was drawn based on the mass concentration corresponding to the detection peak area. The results showed that the linear correlation coefficients of TTX in the mass concentration range of 0.2-40 ng / g were all greater than 0.999; the linear correlation coefficients of 4,9-anhydroTTX in the mass concentration range of 2-50 ng / g were all greater than 0.999; the detection limit of TTX was 0.2 ng / mL, and the quantification limit was 0.65 ng / g. The detection limit of 4,9-anhydroTTX was 1.2 ng / g, and the quantification limit was 3.6 ng / g. The specific results are shown in Table 1.

[0018] Table 1 Regression equation, correlation coefficient, detection limit and quantification limit of TTX and 4,9-anhydroTTX Compound Regression equation <![CDATA[Correlation coefficient (r 2 )]]> Detection limit (ng / g) Limit of quantification (ng / g) TTX Y=646.654x+454.748 0.9996 0.2 0.65 4,9-anhydroTTX Y=430.458x+64.7152 0.9995 1.2 3.60 2. Investigation of Matrix Effect Hairy clams, mud snails, rainbow clams and razor clams were selected as representative shellfish species, and the final optimized conditions were used to investigate the influence of matrix effects on TTX and 4,9-anhydroTTX. The matrix effect was evaluated by calculating the matrix factor (MF = K1 / K2, K1 is the peak area of ​​the target compound at 5 times the quantification limit concentration added to the shellfish matrix, and K2 is the peak area of ​​the target compound at 5 times the quantification limit concentration added to the initial mobile phase). If MF < 1, it means that there is a matrix inhibition effect; if MF = 1, it means that there is no matrix effect; if MF > 1, it means that there is a matrix enhancement effect. It is generally believed that when 0.8 < MF < 1.2, the matrix effect is not significant, which is a weak matrix effect; when 0.5 < MF < 0.8 or 1.2 < MF < 1.5, it is a medium-intensity matrix effect; when MF < 0.5 or MF > 1.5, it is a strong matrix effect. The results are shown in Table 2.

[0019] Table 2 Matrix factors of TTX and 4,9-anhydroTTX in different shellfish Compound Hairy Clam Mud Snail Rainbow Bright Cherry Clam Sinonovacula constricta TTX 0.88 0.82 0.81 0.82 4,9-anhydroTTX 0.85 0.81 0.80 0.81 As shown in Table 2, the matrix factors of the target substances in different shellfish samples are between 0.80 and 0.88, indicating that the shellfish matrix effect is not significant. The purification effect of the currently established method is good, and the standard curve prepared with the initial mobile phase can be directly used to accurately quantify the target substances.

[0020] Example 1 (1) Pretreatment: After cleaning the shellfish, the shells are peeled off, and the whole shellfish sample (all soft tissues and body fluids) is collected and homogenized to obtain a homogenous slurry; (2) Extraction: Accurately weigh 5±0.01 g of homogenized slurry into a 50 mL centrifuge tube, the mass ratio of the homogenized slurry to 1% by volume acetic acid aqueous solution is 1:1, add 1% by volume acetic acid aqueous solution, vortex and oscillate for 1 min for one extraction, boil in hot water for 10 min after extraction and cool to room temperature, centrifuge at 4°C and 10,000 rpm for 5 min, retain the supernatant, add 1% by volume acetic acid aqueous solution to the centrifugal residue and repeat the previous steps for a second extraction, combine the supernatants, dilute to 10 mL with purified water to obtain an extract, mix 0.5 mL of the extract with 0.1 mL, 25 mmol / L ion pair reagent perfluoroheptanoic acid to obtain a loading solution; (3) Enrichment and purification: 2.5 mL of acetonitrile, 2.5 mL of purified water, 2.5 mL and 5 mmol / L ion pair reagent were used to activate the CNW bond carbon GCB graphitized carbon black solid phase extraction column in sequence, and the sample solution was loaded with a sample volume of 0.5 ml. After loading, it was rinsed with 2 mL of purified water and eluted with acetonitrile containing 0.4% by volume of formic acid. The eluate was blown to near dryness with nitrogen at 40°C and then reconstituted with the initial mobile phase, which was 5% by volume of 5 mmol / L ammonium acetate solution containing 0.1% formic acid and 95% by volume of acetonitrile. Filtered with a 0.22 μm filter membrane, the filtrate was the machine solution; (4) Sample loading and detection: The loading solution was extracted with a sampling needle, loaded according to the set chromatographic-mass spectrometric conditions, and detected by ultra-high performance liquid chromatography-tandem mass spectrometry. The ion spectrum peaks of TTX and 4,9-anhydroTTX were obtained, and the mass concentrations of TTX and 4,9-anhydroTTX were obtained by conversion based on the ion spectrum peak areas and the standard curves of TTX and 4,9-anhydroTTX. The chromatographic conditions were: Waters ACQUITY UPLC BEH Amide column, column size: 50mm×2.1mm, 1.7μm; sample chamber temperature: 4℃; column temperature: 40℃; injection volume: 5μL; mobile phase A: 5mmol / L ammonium acetate solution containing 0.1% formic acid; mobile phase B: acetonitrile; flow rate: 0.3mL / min, gradient elution conditions: 0min, 5% mobile phase A, 95% mobile phase B→0.5min, 5% mobile phase A, 95% mobile phase B→6.0min, 55% mobile phase A, 45% mobile phase B→8.0min, 5 5% mobile phase A, 45% mobile phase B→8.1min, 5% mobile phase A, 95% mobile phase B→10.0min, 5% mobile phase A, 95% mobile phase B; mass spectrometry conditions: electrospray ion source positive ion scanning; monitoring mode: multiple reaction monitoring mode; ion source temperature: 120℃; capillary voltage: 1.5kV; desolvation gas temperature: 600℃; cone gas: 3.5kV; desolvation: high-purity nitrogen, flow rate 800L / h; the mass spectrometry monitoring parameters of TTX and 4,9-anhydroTTX are shown in Table 3.

[0021] Table 3 Mass spectrometry monitoring parameters of TTX and 4,9-anhydroTTX * Qualifier ion Example 2 The difference between this embodiment and embodiment 1 is that the sample is the digestive gland of razor clams, and the rest is the same as embodiment 1.

[0022] Example 3 The difference between this embodiment and embodiment 1 is that the sample is the gills of razor clams, and the rest is the same as embodiment 1.

[0023] Example 4 The difference between this embodiment and embodiment 1 is that the sample is the muscle tissue of razor clams, and the rest is the same as embodiment 1.

[0024] Neither TTX nor 4,9-anhydroTTX was detected in the samples of Examples 1 to 4. Three-level spike experiments were conducted using the samples of Examples 1 to 4 as blank matrices, with TTX spiked at 0.5, 2, and 10 ng / g in the blank matrix and 4,9-anhydroTTX spiked at 2.5, 5, and 20 ng / g in the shellfish matrix. The experimental results are shown in Table 4.

[0025] Table 4 Method recovery, inter-day (n=6) and intra-day (n=3) precision As can be seen from Table 4, the recovery rate of TTX is 79.3-89.5%, the relative standard deviation between days is 2.7-9.2%, and the relative standard deviation within days is 2.6-9.3%; the recovery rate of 4,9-anhydroTTX is 74.0-82.3%, the relative standard deviation between days is 2.7-9.2%, and the relative standard deviation within days is 2.4-9.3%. Both the precision and sensitivity can meet the detection requirements of TTX and 4,9-anhydroTTX in experimental sample tissues.

[0026] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A method for detecting TTX and 4,9-anhydroTTX in shellfish by ion-pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: (1) Pretreatment: After cleaning the shellfish, peeling the shells, collecting the samples and homogenizing them to obtain a homogenous slurry; (2) Extraction: Accurately weigh the homogenized slurry into a centrifuge tube, add 1% by volume acetic acid aqueous solution, vortex and oscillate for extraction, boil the mixture with hot water after extraction and cool to room temperature, centrifuge, retain the supernatant, add 1% by volume acetic acid aqueous solution to the centrifugal residue and repeat the previous steps for secondary extraction, combine the supernatants, make up to volume, obtain the extract, mix the extract with the ion pair reagent, and obtain the loading solution; (3) Enrichment and purification: Activate the graphitized carbon black solid phase extraction column with acetonitrile, purified water and ion pair reagent in sequence, load the sample solution, rinse with purified water after loading, elute with acetonitrile containing 0.4% formic acid by volume, blow the eluate to near dryness with nitrogen, re-dissolve with the initial mobile phase, filter, and the filtrate is the loading solution; (4) Sample loading and detection: Use an injection needle to draw the loading solution, load the sample according to the set chromatography-mass spectrometry conditions, and use ultra-performance liquid chromatography-tandem mass spectrometry for detection. The ion spectrum peaks of TTX and 4,9-anhydroTTX are obtained. The mass concentrations of TTX and 4,9-anhydroTTX are calculated based on the ion spectrum peak areas and the standard curve of TTX and 4,9-anhydroTTX.

2. The method for detecting TTX and 4,9-anhydroTTX in shellfish by ion-pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (2), 5±0.01 g of homogenized slurry is weighed into a 50 mL centrifuge tube, and the mass ratio of the homogenized slurry to 1% by volume acetic acid aqueous solution is 1:1; vortex oscillation is performed for 1 to 3 min, and hot water is boiled for 10 to 12 min; centrifugation is performed at 3 to 5 ° C and 10,000 rpm for 5 to 6 min; the mass ratio of the centrifugal residue to 1% by volume acetic acid aqueous solution is 1:0.4; the supernatant is diluted to 10 mL with purified water; the ion pair reagent is perfluoroheptanoic acid, and 0.5 mL of the extract is mixed with 0.1 mL of 25 mmol / L ion pair reagent.

3. The method for detecting TTX and 4,9-anhydroTTX in shellfish by ion-pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (3), the graphitized carbon black solid phase extraction column is activated with 2.5 mL of acetonitrile, 2.5 mL of purified water, and 2.5 mL and 5 mmol / L ion pair reagent in sequence; the sample loading volume is 0.5 ml; after the sample loading is completed, it is rinsed with 2 mL of purified water and eluted with 2 mL of acetonitrile containing 0.4% by volume of formic acid; the eluent is blown to near dryness with nitrogen at 40°C; the initial mobile phase is 5% by volume of 5 mmol / L ammonium acetate solution containing 0.1% formic acid and 95% by volume of acetonitrile; and filtered with a 0.22 μm filter membrane.

4. The method for detecting TTX and 4,9-anhydroTTX in shellfish by ion-pair solid phase extraction column purification-liquid chromatography-tandem mass spectrometry according to claim 1 or 3, characterized in that: The graphitized carbon black solid phase extraction column is a CNW bondcarbon GCB graphitized carbon black solid phase extraction column.

5. The method for detecting TTX and 4,9-anhydro TTX in shellfish by solid phase extraction column purification-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (4), the chromatographic conditions are: Waters ACQUITY UPLC BEHAmide column, column size 50 mm×2.1 mm, 1.7 μm; sample chamber temperature: 4°C; column temperature: 40°C; injection volume: 5 μL; mobile phase A: 5 mmol / L ammonium acetate solution containing 0.1% formic acid; mobile phase B: acetonitrile; flow rate: 0.3 mL / min, and the gradient elution conditions are: 0 min, 5% mobile phase A, 95% mobile phase B→0.5 min, 5% mobile phase A, 95% mobile phase B→6.0 min, 55% mobile phase A, 45% mobile phase B→8.0 min, 55% mobile phase A, 45% mobile phase B→8.1 min, 5% mobile phase A, 95% mobile phase B→10.0 min, 5% mobile phase A, 95% mobile phase B.

6. The method for detecting TTX and 4,9-anhydro TTX in shellfish by solid phase extraction column purification-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (4), the mass spectrometry conditions are: electrospray ion source positive ion scanning; monitoring mode: multiple reaction monitoring mode; ion source temperature: 120°C; capillary voltage: 1.5 kV; desolvation gas temperature: 600°C; cone gas: 3.5 kV; desolvation: high-purity nitrogen, flow rate 800 L / h.

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