Method for simultaneously detecting 25 anesthetics in aquatic product

Abstract

The invention discloses a method for simultaneously detecting 25 anesthetics in an aquatic product. The method comprises the following steps: (1) extraction: weighing a sample, adding water, oscillating and vortex, oscillating and extracting for 3-8 minutes by using an organic solvent, adding anhydrous sodium sulfate, quickly shaking for 3-8 minutes, centrifuging, completely transferring an organic solvent layer into a nitrogen blowing pipe, blowing nitrogen to 0.5 mL, and fixing the volume by acetonitrile; (2) purifying: adding the fixed solution into a purifying agent, wherein the dosage ratio of the fixed solution to the purifying agent is 1mL: (100-200mg); the purifying agent is prepared by mixing MgSO4, C18, PSA and Carb according to the weight ratio of 50: (20-50): (10-50): (10-50), adding water into the supernatant, and filtering to obtain a purified solution, and the purifying agent is prepared by mixing MgSO4, C18, PSA and Carb according to the weight ratio of 50: (20-50): (10-50); and (3) detecting. The method is simple and rapid in pretreatment and high in sensitivity, and can meet the rapid and accurate detection requirements of various anesthetics in aquatic products.

Description

Technical Field

[0001] The invention relates to the technical field of toxic chemical substance detection, and in particular to a method for simultaneously detecting 25 anesthetics in aquatic products. Background Art

[0002] Food safety has always been a focus of people's attention, and the quality and safety of fresh aquatic products with high nutritional value have attracted more attention at home and abroad. Due to the constraints of geographical, environmental and climatic factors, fresh aquatic products usually need to be transported over long distances to reach all parts of the country. During transportation and circulation, aquatic products are prone to stress reactions due to increased density, collisions, and increased oxygen consumption, which can lead to injuries or even death. Therefore, anesthetics are often used to reduce the metabolism of aquatic products, thereby increasing the survival rate of aquatic products. Commonly used anesthetics in the transportation of aquatic products include caine (such as MS-222, benzocaine, etc.), eugenol (such as eugenol, isoeugenol, etc.), quinaldine, and some drugs with sedative effects (such as diazepam), among which caine and eugenol anesthetics are the most widely used.

[0003] At present, Japan allows eugenol as an anesthetic for fishing, while Australia, Chile, New Zealand and other countries have approved isoeugenol as an anesthetic for fishing, while MS-222 is the only legal anesthetic in the United States and Canada. Quinaldine is cheap and is only used for auxiliary transportation of live fish due to its irritation to the mucous membrane, and is widely used abroad. In GB31650-2019 "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food", my country allows lidocaine and procaine to be used in edible animals without the need to set residue limits, and allows diazepam to be used for treatment, but it must not be detected in animal-derived foods. There are no clear regulations on the use limits of other anesthetics, so there is a certain risk in the safety assessment of these unlimited anesthetics. Studies have shown that eugenol anesthetics may cause liver damage, but their carcinogenicity is unclear. Caine anesthetics may affect the central nervous system and cardiovascular system, and quinaldine is irritating to human skin and other parts. Therefore, it is of great significance to establish an analytical method that is efficient, accurate, sensitive and can meet the requirements of detecting a variety of anesthetics.

[0004] The main methods for detecting anesthetics in aquatic products include high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay and various rapid detection methods. Chromatography is easily interfered by the matrix, has a high detection limit, and has the risk of false positives. The enzyme-linked immunosorbent assay is expensive and not suitable for large-scale detection. Gas chromatography-mass spectrometry is more commonly used for the detection of eugenol anesthetics, but this method makes the target compound easily degraded. Liquid chromatography-tandem mass spectrometry has the characteristics of high sensitivity and strong anti-interference, and can realize trace analysis of a variety of target compounds. It has been used for qualitative and quantitative confirmation of anesthetics such as caine and eugenol. Due to the complexity of endogenous interferences such as proteins, fats, and pigments in aquatic products, appropriate pretreatment is required to improve the sensitivity of the method. Gao et al. used phosphate buffer (pH 4.4)-acetonitrile solution extraction, enhanced lipid removal purifier (EMR-Lipid) purification, HLB solid phase extraction column purification, HPLC-diode array detector determination, and external standard method quantification in "Determination of 6 anesthetics in aquatic products by a new QuEChERS combined with solid phase extraction-high performance liquid chromatography". This method uses solid phase extraction column purification, and the purification process requires activation, sampling, rinsing, elution and other steps, which is time-consuming and can only detect 6 anesthetics in aquatic products. If more anesthetics need to be tested, they can only be extracted and purified separately, which reduces the inspection efficiency to a certain extent. At the same time, the use of multiple extraction reagents and consumables also causes certain pollution to the environment. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for simultaneously detecting 25 anesthetics in aquatic products in view of the deficiencies in the prior art. The method has simple and rapid pretreatment and high sensitivity, and can meet the requirements for rapid and accurate detection of multiple anesthetics in aquatic products.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for simultaneously detecting 25 anesthetics in aquatic products comprises the following steps:

[0008] (1) Extraction: Weigh the sample, add water, vortex and shake, extract with organic solvent for 3-8 min, add anhydrous sodium sulfate, shake rapidly for 3-8 min, centrifuge, transfer the entire organic solvent layer to a nitrogen blowpipe and blow with nitrogen to 0.5 mL, and make up to volume with acetonitrile;

[0009] (2) Purification: Add the above-mentioned fixed solution to the purifier, the ratio of the fixed solution to the purifier is 1 mL: 100-200 mg; vortex and centrifuge, take the supernatant, add water and filter to obtain the purified solution, the purifier is MgSO4, C 18 , PSA, and Carb are mixed in a weight ratio of 50:20-50:10-50:10-50;

[0010] (3) Detection.

[0011] Furthermore, in the step (1), the sample is weighed and placed in a centrifuge tube, water is added, and the mixture is vortexed for 1-5 minutes. The mixture is extracted with an organic solvent for 3-8 minutes. Anhydrous sodium sulfate is added, the mixture is rapidly shaken for 3-8 minutes, and the mixture is centrifuged for 3-8 minutes. The organic solvent layer is completely transferred to a nitrogen blowpipe, and nitrogen is blown to 0.5 mL at 40° C. The volume of acetonitrile is adjusted to 1 mL. The ratio of the sample, water, organic solvent, and anhydrous sodium sulfate is 2 g: 2 mL: 10 mL: 5 g.

[0012] Furthermore, the organic solvent in step (1) is acetonitrile, methanol, ethyl acetate, 0.1% formic acid / acetonitrile (0.1% formic acid added to acetonitrile, volume fraction).

[0013] Furthermore, the organic solvent in step (1) is acetonitrile.

[0014] Furthermore, the purifier is MgSO4, C 18 , PSA and Carb are mixed in a weight ratio of 50:30:20:20.

[0015] Furthermore, in step (3), ultra-high pressure liquid chromatography-tandem mass spectrometry is used for detection, and the chromatographic column is Acquity HSS T3 column.

[0016] Furthermore, the liquid chromatography used two mobile phases for elution, namely, acetonitrile / 0.1% formic acid water mobile phase and acetonitrile / water mobile phase.

[0017] Furthermore, acetonitrile / 0.1% formic acid water was used as the mobile phase, and the positive ion mode was adopted; the acetonitrile / water mobile phase was run once when the positive and negative ions were scanned simultaneously.

[0018] The beneficial effects of the present invention are:

[0019] 1. In order to simultaneously detect 25 anesthetics in aquatic products, the present invention improves and optimizes the existing QuEChERS-ultra-high pressure liquid chromatography-tandem mass spectrometry, specifically, the extraction, purification and detection processes are comprehensively optimized and improved. The optimized process is used for detection. The 25 anesthetics have a good linear relationship in the range of 1-50.0 ng / mL (among which m-aminobenzoic acid, p-aminobenzoic acid and 6 eugenol compounds are 10-500 ng / mL), and the limit of quantification (S / N=10) is 1-10 μg / kg. Except for m-aminobenzoic acid, p-aminobenzoic acid and 6 eugenol compounds, the LOD and LOQ are 5 μg·kg -1 , 10μg·kg -1The LOD and LOQ of the remaining 19 compounds were 0.5 μg kg -1 , 1μg·kg -1 . At the three spike levels of low, medium and high, the average recovery was 66.2%-112.5%, the intra-batch precision was 2.1%-11.2% (n=6); the inter-batch precision was 2.3%-11.3% (n=3). This method has simple and rapid pretreatment and high sensitivity, which can meet the requirements of rapid and accurate detection of various anesthetics in aquatic products. In addition, the detection time is short, the amount of organic solvent used is small, and it is fast, environmentally friendly and efficient.

[0020] 2. In terms of extraction, since the structures of the 25 anesthetics are quite different, some are highly fat-soluble and some are highly water-soluble, the extraction solvent must take into account the extraction efficiency of all target substances. Taking into account the properties of each compound, this study studied the effects of four extraction solvents, acetonitrile, methanol, ethyl acetate, and 0.1% formic acid acetonitrile, on the extraction efficiency of 25 target compounds in three matrices (shrimp, tilapia, and grass carp). Due to the high protein content of aquatic products such as fish and shrimp, the direct addition of strong polar organic solvents such as acetonitrile can destroy the hydrogen bonds and hydrophobic interactions within the protein molecules, causing them to denature and agglomerate, thereby affecting the extraction effect. Therefore, this application first adds water, oscillates and disperses the sample, and then adds an organic solvent. The recovery rates of the 25 compounds extracted with the organic solvent acetonitrile are all between 65% and 115%, with excellent results.

[0021] In addition, acetonitrile is used during volume determination to further precipitate proteins and reduce the dissolution of impurities and interferences.

[0022] 3. Purification: By using different types of adsorbents, the purpose of adsorbing interferences and retaining the target compound to be detected in the solution is achieved. The research of this application found that the more types of adsorbents added, the better. The more the amount of adsorbent added, the better. Too many types and amounts of adsorbents added will further increase the interference of impurities, thereby affecting the purification effect. However, too little will also affect the purification effect. This study uses C 18 , PSA, Carb as adsorbents, among which C 18 It mainly adsorbs non-polar co-extracts, such as lipophilic compounds such as fats; PSA is mainly used to adsorb organic acids, pigments, etc.; Carb is often used to remove polar co-extracts, such as sterols; Since the sample extract contains a large amount of co-extracts such as proteins, pigments, and fats, the compound addition of C 18 , PSA, Carb to purify the sample, the combined content is 30mg C 18 When +20mg PSA+20mg Carb, it can not only ensure the recovery rate but also effectively remove interfering substances such as protein, fat and pigment in the solution, so that the sample meets the instrument detection requirements and the purification effect is excellent.

[0023] 4. During the detection process, Waters HSS T3 chromatographic column was used for separation. Two mobile phases were used: acetonitrile-0.1% formic acid water as the mobile phase, using the positive ion mode; acetonitrile-water mobile phase, which was run once when positive and negative ions were scanned simultaneously. Finally, the separation effect of the 25 compounds was good, and the matrix effect in the detection process of different matrix samples was eliminated to reduce interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the quantitative ion map of 25 anesthetics;

[0025] Figure 2 C 18 The effect of dosage on the recovery rate of 25 anesthetics;

[0026] Figure 3 The effect of PSA dosage on the recovery rate of 25 anesthetics;

[0027] Figure 4 The effect of Carb dosage on the recovery rate of 25 anesthetics;

[0028] Figure 5 This is the recovery rate graph of 25 target compounds in three matrices when the organic solvent is acetonitrile;

[0029] Figure 6 This is the recovery rate graph of 25 target compounds in three matrices when the organic solvent is methanol;

[0030] Figure 7 This is the recovery rate graph of 25 target compounds in three matrices when the organic solvent is ethyl acetate;

[0031] Figure 8 This is the recovery rate graph of 25 target compounds in three matrices when the organic solvent is 0.1% formic acid and acetonitrile;

[0032] Fig. 9 The peak shapes of six anesthetics, namely bupivacaine, pramoxine, butacaine, cinchocaine, tetracaine and ropivacaine, in two mobile phases, acetonitrile-water and acetonitrile-0.1% formic acid water;

[0033] Fig.10 This is the matrix effect diagram of 25 anesthetics;

[0034] Fig.11 The average recovery and precision of 25 anesthetics in different matrices are shown;

[0035] Fig.12 This is the total ion current of the positive sample. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and examples. The abbreviations in the following examples are: N-propylethylenediamine solid phase adsorbent (PSA), octadecylsilane bonded silica gel (C 18 ): 40μm, Agilent Technologies, Inc., USA; Graphitized carbon (Carb): 100μm, Yuexu Technology (Shanghai) Co., Ltd.

[0037] Example 1

[0038] A method for simultaneously detecting 25 anesthetics in aquatic products comprises the following steps:

[0039] (1) Extraction: Weigh 2 g (accurate to 0.01 g) of sample into a 50 mL polypropylene centrifuge tube, add 2 mL of water, vortex for 2 min, extract with 10 mL of acetonitrile for 5 min, add 5 g of anhydrous sodium sulfate, shake rapidly for 5 min, centrifuge at 8000 r / min for 5 min, transfer the acetonitrile layer to a nitrogen blowpipe, blow nitrogen to 0.5 mL at 40 °C, dilute the acetonitrile to 1 mL, and pour the dilute solution into a purification tube;

[0040] (2) Purification: Add the above solution to the purifier, the purifier is 50 mg MgSO4, 30 mg C 18 , 20 mg PSA, 20 mg Carb, vortex for 1 min, centrifuge at 8000 r / min for 5 min, take 0.5 mL of supernatant, add 0.5 mL of water, and filter through a 0.22 μm organic filter membrane;

[0041] (3) Detection: Ultra-high pressure liquid chromatography-tandem mass spectrometry was used for detection, and the chromatographic column was Acquity HSS T3 chromatographic column (2.1 mm×100 mm, 1.8 μm).

[0042] Liquid chromatography conditions: two mobile phases were used, the first mobile phase was acetonitrile (A) / 0.1% formic acid aqueous solution (B), gradient elution program: 0-0.5 min, 5% A; 0.5-1.0 min, 5%-45% A; 1.0-1.5 min, 45% A; 1.5-3.5 min, 45%-95% A; 3.5-5.5 min, 95% A; 5.5-5.6 min, 95%-5% A; 5.6-7 .0min, 5% A; the second mobile phase is acetonitrile (A) / water (B), gradient elution program: 0-0.7min, 40% A; 0.7-2.0min, 40%-60% A; 2.0-4.0min, 60% A; 4.0-7.0min, 60%-95% A; 7.0-8.0min, 95% A; 8.0-8.1min, 95%-40% A; 8.1-9.0min, 40% A. Column temperature 40°C; flow rate 0.3mL / min; injection volume: 2μL; chromatographic column: WatersHSS T3 (2.1mm×100mm, 1.8μm).

[0043] Mass spectrometry conditions: electrospray ionization (ESI) source, acetonitrile / 0.1% formic acid water mobile phase in positive ion mode, acetonitrile / water mobile phase scans positive and negative ions simultaneously; scanning mode: multiple reaction monitoring (MRM); ion source temperature 550°C; ion source voltage 5500V; curtain gas pressure 0.24MPa; nebulizer (GAS1) 0.38MPa; auxiliary gas (GAS2) 0.38MPa.

[0044] The 25 target compounds include 17 caines, 6 eugenols, quinaldine, and diazepam. The specific compound information is shown in Table 1. The 25 target compounds were selected for monitoring ion pairs, and their declustering potential (DP) and collision energy (CE) are shown in Table 1.

[0045] Table 1 Names, CAS numbers and mass spectrometry parameters of 25 compounds

[0046]

[0047]

[0048] Preparation of mixed standard working solution: 25 single standard solutions (solvent is methanol) were purchased from Tianjin Alta Company, and the mass concentration of each was 1000 μg / mL; accurately pipette 0.01 mL of each of the above 25 standard solutions of 1000 μg / mL (including: 0.10 mL each of m-aminobenzoic acid, p-aminobenzoic acid, and 6 eugenol compounds) and place them in a 10 mL volumetric flask, dilute to the scale with methanol, and prepare a 1 μg / mL mixed standard working solution (including: 10 μg / mL of m-aminobenzoic acid, p-aminobenzoic acid, and 6 eugenol compounds), which was stored in a -20°C refrigerator.

[0049] Embodiment 2-4

[0050] The amounts of C18 in Examples 2-4 are 20, 40, and 50 mg, respectively, and the other processes are the same as in Example 1.

[0051] Embodiment 5-8

[0052] The amounts of PSA in Examples 5-8 are 10, 30, 40, and 50 mg, respectively, and the other processes are the same as in Example 1.

[0053] Examples 9-12

[0054] The amounts of Carb in Examples 9-12 are 10, 30, 40, and 50 mg, respectively, and the other processes are the same as in Example 1.

[0055] Comparative Examples 1-3

[0056] Comparative Examples 1-3 chromatographic columns are Acquity BEH C 18 (2.1mm×100mm, 1.7μm), Agilent SB-Aq C 18 RRHD (2.1mm×100mm, 1.8μm), Agilent Eclipse Plus C 18 RRHD (2.1mm×100mm, 1.8μm).

[0057] Comparative Examples 4-6

[0058] The organic solvents used for extraction in Comparative Examples 4-6 were methanol, ethyl acetate, and 0.1% formic acid acetonitrile (0.1% formic acid was added to acetonitrile, by volume), respectively. Other detection processes were the same as those in Example 1.

[0059] Performance Testing

[0060] 1. The separation of 25 compounds by the chromatographic columns of Example 1 and Comparative Examples 1-3 was studied. It was found that the 25 anesthetics were separated by 4 chromatographic columns (Acquity BEH C18 (2.1mm×100mm, 1.7μm), Acquity HSS T3 (2.1mm×100mm, 1.8μm), Agilent SB-Aq C 18 RRHD (2.1mm×100mm, 1.8μm), AgilentEclipse Plus C 18 The peak shape, resolution, response and other properties on RRHD (2.1 mm × 100 mm, 1.8 μm) were found to be procaine, procainamide and quinaldine in Acquity BEH C 18 、Agilent SB-Aq C 18 RRHD, Agilent Eclipse Plus C 18 The 3 RRHD columns all showed poor chromatographic peak shape and low sensitivity. The chromatographic peak retention of the two metabolites, m-aminobenzoic acid and p-aminobenzoic acid, was weak on the above 3 columns, with early elution time and poor peak shape. The 6 eugenol compounds could not be completely separated, while Acquity The HSS T3 column can completely separate 25 compounds with sharp peaks. See the chromatogram. Figure 1 , so determine Acquity HSST3 column is the best column.

[0061] 2. The effects of 20, 30, 40, and 50 mg of C18 on the recovery rate of the target compound in Example 1 and Examples 2-4 were investigated respectively. Figure 2 ; Investigate the effect of PSA with a content of 10, 20, 30, 40, 50 mg on the recovery rate in Example 1 and Examples 5-8, and the results are shown in Figure 3 ; Investigate the effect of Carb content of 10, 20, 30, 40, 50 mg on the recovery rate in Example 1 and Examples 9-12, and the results are shown in Figure 4 .

[0062] Depend on Figure 2 It can be seen that in C 18 When the content was 30 mg, the recovery rate of 25 compounds was the best, which was 61%-116%. 18 As the content increased, the recovery rates of procaine, procainamide, chloroprocaine, quinaldine, and butacaine decreased significantly. 18 Based on the optimal addition amount, the effects of PSA with a content of 10, 20, 30, 40, and 50 mg on the recovery rate were compared. Figure 3It can be seen that 48% of the 25 compounds have the best recovery rate when the PSA content is 20mg, with a recovery rate of 70%-107%. The recovery rates of the remaining 52% of the compounds are not much different when the PSA content is 10mg and 20mg, but as the PSA content gradually increases, the recovery rates of the 25 compounds slowly decrease. On the basis of determining 20mg as the optimal addition amount of PSA, the effects of Carb content of 10, 20, 30, 40, and 50mg on the recovery rate are compared. Figure 4 It can be seen that the recovery rate of 80% of the 25 compounds first increases and then decreases with the increase of Carb content. When the Carb content is 20 and 30 mg, the recovery rate is not much different, and the recovery rate is 70%-101%. From the perspective of environmental friendliness, the optimal addition amount of Carb is 20 mg. Therefore, the optimal combination content of the three adsorbents is 30 mg C 18 +20mg PSA+20mg Carb.

[0063] 3. The effects of the organic solvents used in the extraction of Example 1 and Comparative Examples 4-6 on the recovery rates of 25 target compounds in three matrices (shrimp, tilapia, grass carp) were examined. The results are shown in Figure 5-8 .

[0064] Since this application is to detect 25 anesthetics, their structures are quite different, some are highly fat-soluble, and some are highly water-soluble, so the organic solvent used for extraction needs to take into account the extraction efficiency of all target substances. Eugenol anesthetics can be extracted using acetonitrile, methanol, and n-hexane, and caine anesthetics often use acetonitrile, phosphate buffer, acetonitrile aqueous solution, etc. as extraction solvents. Sedatives such as diazepam are fat-soluble compounds, so they are often extracted using acetonitrile and ethyl acetate, while quinaldine is often extracted using acetonitrile. Taking into account the properties of each compound, this application compares the effects of four extraction solvents, acetonitrile, methanol, ethyl acetate, and 0.1% formic acid acetonitrile, on the extraction efficiency of 25 target compounds in three matrices (shrimp, tilapia, and grass carp). Due to the high protein content of aquatic products such as fish and shrimp, directly adding strong polar organic solvents such as acetonitrile can destroy the hydrogen bonds and hydrophobic interactions within the protein molecules, causing them to denature into agglomerates, thereby affecting the extraction effect.

[0065] Therefore, in this experiment, water was first added, the sample was shaken and dispersed, and then the organic solvent was added. The test showed that the methanol extract was turbid, the protein precipitation effect was poor, and the recovery rates of 25 compounds were all below 60%; the ethyl acetate extract had more impurities, which affected the subsequent purification effect. Except for 6 eugenol compounds with a recovery rate of 50%-70%, the recovery rates of the other 19 compounds were all below 65%; acetonitrile and 0.1% formic acid acetonitrile extracts were clear and had the lowest matrix effect, and the recovery rates of 25 compounds were all between 65% and 115%. Therefore, taking into account the extraction efficiency, purification effect, and environmental friendliness of the target compounds, this study used acetonitrile as the extraction solvent.

[0066] 4. The effects of acetonitrile-water, methanol-water, acetonitrile-0.1% formic acid water, and methanol-0.1% formic acid water on the separation of target compounds were investigated. Fig. 9 , Fig. 9 The peak shape comparison of six anesthetics, bupivacaine, pramoxine, butacaine, cinchocaine, tetracaine, and ropivacaine, in two mobile phases, acetonitrile-water and acetonitrile-0.1% formic acid water, was shown. In the acetonitrile-water system, the response value was higher, and MS-222 and benzocaine, m-aminobenzoic acid and p-aminobenzoic acid, and three pairs of eugenol isomers were able to achieve baseline separation. However, the peaks of bupivacaine, pramoxine, butacaine, cinchocaine, tetracaine, and ropivacaine were broadened and had poor peak shapes. The addition of 0.1% formic acid water to water helped to improve their quasi-molecular ion peaks [M+H] + The formation of can make the peaks of the above compounds sharp and symmetrical, while the addition of formic acid will completely suppress eugenol and isoeugenol in negative ion mode. Therefore, considering the sensitivity, separation and peak shape, acetonitrile-water is used as the mobile phase for 6 eugenol compounds, and acetonitrile / 0.1% formic acid water is used as the mobile phase for the remaining 19 compounds. During the specific detection, acetonitrile / 0.1% formic acid water is first used as the mobile phase for elution, and then acetonitrile-water is used for elution, which can achieve the separation of the target compounds.

[0067] 5. Matrix Effect

[0068] Endogenous interferences such as proteins in aquatic products and exogenous interferences such as salts introduced during the pretreatment process will inhibit or enhance the ionization of the target compounds, thereby affecting the accuracy of the analysis results. Therefore, it is necessary to examine the matrix effect of the method in this application. This study used negative matrix extracts and pure solvents to prepare standard curves, and used matrix matching and the slope of the solvent standard curve to calculate the matrix effect (ME). When ME is negative, it is a matrix inhibition effect, and when ME is positive, it is a matrix enhancement effect. When |ME|<20%, there is a weak matrix effect; when 20%<|ME|<50%, there is a moderate matrix effect; when |ME|>50%, there is a strong matrix effect. Taking the shrimp and grass carp matrices as examples, from Fig.10 It can be seen that the 25 anesthetics have different degrees of matrix effects in the two matrices, and the matrix effects of eugenol and isoeugenol are the largest, both greater than 50%. The matrix effect of grass carp is higher than that of shrimp. In order to correct the influence of ME on the results, the matrix matching standard curve is used in the quantitative analysis.

[0069] 6. Optimization of mass spectrometry conditions

[0070] According to the structural characteristics of the 25 compounds, a mixed standard solution with a concentration of 200 ng / mL was subjected to a primary mass spectrometry scan to determine its quasi-molecular ion peak. Since eugenol and isoeugenol contain phenolic hydroxyl groups, they are prone to lose a proton to obtain [MH] - Quasi-molecular ion peak has high sensitivity in negative ion detection mode, and other compounds are easy to obtain protons to form [M+H] + The quasi-molecular ion peak is detected, so the positive ion mode is adopted for detection. During the specific detection, the acetonitrile / 0.1% formic acid water mobile phase adopts the positive ion mode. The acetonitrile / water mobile phase scans the positive and negative ions at the same time to increase the sensitivity of the detection.

[0071] In addition, the five pairs of isomers involved in the detection of this application (including tricaine and benzocaine, m-aminobenzoic acid and p-aminobenzoic acid, eugenol and isoeugenol, methyl eugenol and methyl isoeugenol, eugenol acetate and acetyl isoeugenol) need to be completely separated by optimizing the chromatographic conditions. Fragment ions are generated by collision gas collision for secondary mass spectrometry scanning, and the declustering voltage (DP) and collision energy (CE) are optimized. The qualitative and quantitative characteristic ions of the 25 compounds and the optimized mass spectrometry parameters are shown in Table 1, and finally the 25 compounds can be detected simultaneously.

[0072] 7. Linear range and sensitivity

[0073] An appropriate amount of 25 kinds of compound mixed standard working solutions were added to the extracts of two blank matrices (taking shrimp and grass carp as examples) to prepare matrix spiked solutions with concentrations of 1, 2, 5, 10, 20, and 50 ng / mL (m-aminobenzoic acid and p-aminobenzoic acid, and 6 eugenol compounds were prepared with concentrations of 10, 20, 50, 100, 200, and 500 ng / mL). The matrix spiked solutions were injected sequentially, and the mass concentration (X) of the target substance and the corresponding peak area (Y) were linearly regressed, with 1 / x as the weight. The external standard method was used for quantification, and 3 times the signal-to-noise ratio (S / N≥3) was used as the method detection limit (LOD), and 10 times the signal-to-noise ratio (S / N≥10) was used as the method quantification limit (LOQ). The results showed that the linear relationship of 25 anesthetics in two matrices was good, with correlation coefficient (r) greater than 0.995. Except for m-aminobenzoic acid and p-aminobenzoic acid, the LOD and LOQ of 6 eugenol compounds were 5 μg kg -1, 10μg·kg -1 The LOD and LOQ of the remaining 19 compounds were 0.5 μg kg -1 , 1μg·kg -1 , see Table 2 for details.

[0074] Table 2 Linear relationships, detection limits, and quantification limits of 25 compounds

[0075]

[0076]

[0077] 8. Recovery and precision

[0078] Mixed standard working solutions with the contents of 1, 2, and 10 μg / kg (10, 20, and 100 μg / kg for m-aminobenzoic acid and p-aminobenzoic acid and 6 eugenol compounds) were added to three negative matrices of shrimp, tilapia, and grass carp, respectively. The method was used for 6 parallel determinations and continuous determination for 3 days. The spike recovery rate and the intra-batch and inter-batch standard deviations were calculated. After testing, the recovery rates of 25 target compounds in the three matrices were 65%-112%, the intra-batch RSD was 2.1%-11.2%, and the inter-batch RSD was 2.3%-10.5% (see Fig.11 ). This method has good sensitivity, high accuracy and precision, and meets the requirements of laboratory quality control standards.

[0079] 9. Actual sample testing

[0080] The method established in the study was used to determine the anesthetic content of 90 commercially available aquatic products (including seven species: grass carp, crucian carp, turbot, tilapia, shrimp, lobster, and prawn). The results showed that eugenol was detected in 4 grass carp matrices among the 90 samples, with a content of 0.034-0.21 mg / kg. Fig.12 This is the ion flow diagram of the positive sample, indicating that the detection of the present application can be used for the detection of anesthetics in aquatic products.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for simultaneously detecting 25 anesthetics in aquatic products, characterized in that: The following steps are involved: (1) Extraction: Weigh the sample, add water, vortex and shake, extract with organic solvent for 3-8 min, add anhydrous sodium sulfate, shake rapidly for 3-8 min, centrifuge, transfer the entire organic solvent layer to a nitrogen blowpipe and blow with nitrogen to 0.5 mL, and make up to volume with acetonitrile; (2) Purification: Add the above-mentioned fixed solution to the purifier, the ratio of the fixed solution to the purifier is 1 mL: 100-200 mg; vortex and centrifuge, take the supernatant, add water and filter to obtain the purified solution, the purifier is MgSO4, C 18 , PSA, and Carb are mixed in a weight ratio of 50:20-50:10-50:10-50; (3) Detection.

2. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 1, characterized in that: In the step (1), a sample is weighed and placed in a centrifuge tube, water is added, and the mixture is vortexed for 1-5 minutes. The sample is extracted with an organic solvent for 3-8 minutes. Anhydrous sodium sulfate is added, the mixture is rapidly shaken for 3-8 minutes, and the mixture is centrifuged for 3-8 minutes. The organic solvent layer is completely transferred to a nitrogen blowpipe, and nitrogen is blown to 0.5 mL at 40° C. The volume of acetonitrile is adjusted to 1 mL. The ratio of the sample, water, organic solvent, and anhydrous sodium sulfate is 2 g: 2 mL: 10 mL: 5 g.

3. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 1, characterized in that: The organic solvent in step (1) is acetonitrile, methanol, ethyl acetate, or 0.1% formic acid / acetonitrile.

4. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 1, characterized in that: The organic solvent in step (1) is acetonitrile.

5. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 1, characterized in that: The purifier is MgSO4, C 18 , PSA and Carb are mixed in a weight ratio of 50:30:20:

20.

6. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 1, characterized in that: In the step (3), ultra-high pressure liquid chromatography-tandem mass spectrometry is used for detection, and the chromatographic column is Acquity HSS T3 column.

7. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 6, characterized in that: Liquid chromatography used two mobile phases for elution, namely acetonitrile / 0.1% formic acid water mobile phase and acetonitrile / water mobile phase.

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