A method for simultaneously detecting 25 kinds of anesthetics in aquatic products

Abstract

The application discloses a method for simultaneously detecting 25 kinds of anesthetics in aquatic products, comprising the following steps: (1) extraction: weighing the sample and adding water, oscillating and vortexing, oscillating extraction with an organic solvent for 3-8 min, adding anhydrous sodium sulfate, rapidly shaking for 3-8 min, centrifuging, transferring all the organic solvent layer to a nitrogen blowing tube, nitrogen blowing to 0.5 mL, and constant volume with acetonitrile; (2) purification: adding the above constant solution into a purifying agent, the dosage ratio of the constant solution to the purifying agent is 1 mL: 100-200 mg; oscillating and vortexing, centrifuging, taking the supernatant, adding water, filtering to obtain a purified liquid, and the purifying agent is MgSO4, C 18 , PSA, Carb, which are mixed in a weight ratio of 50:20-50:10-50:10-50; and (3) detection. The method has simple and rapid pretreatment, high sensitivity, and can meet the requirements of rapid and accurate detection of various anesthetics in aquatic products.

Description

Technical Field

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

[0002] Food safety has always been a major concern, and the quality and safety of fresh aquatic products with high nutritional value are of particular interest both domestically and internationally. Due to factors such as region, environment, and climate, fresh aquatic products typically require long-distance transportation to reach destinations across the country. During transportation and distribution, aquatic products are prone to stress reactions due to increased organism density, collisions, and increased oxygen consumption, leading to injury or even death. Therefore, anesthetics are often used to reduce the metabolism of aquatic products, thereby improving their survival rate. Commonly used anesthetics in aquatic product transportation include cocaine derivatives (such as MS-222, benzocaine, etc.), eugenol derivatives (such as eugenol, isoeugenol, etc.), quinalidine, and some sedative drugs (such as diazepam), among which cocaine and eugenol derivatives are the most widely used.

[0003] Currently, Japan permits eugenol as a fish anesthetic, while Australia, Chile, and New Zealand have approved isoeugenol. MS-222 is the only legal anesthetic in the United States and Canada. Quinalidine is inexpensive and, due to its irritant effect on mucous membranes, is only used for auxiliary transportation of live fish, and is widely used abroad. In my country, GB31650-2019, the National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food, allows lidocaine and procaine for use in food animals without setting residue limits, and allows diazepam for therapeutic use, but it must not be detected in animal-derived foods. However, there are no clear limits for the use of other anesthetics, thus posing a certain risk to the safety assessment of these anesthetics without limits. Studies have shown that eugenol-based anesthetics may cause liver damage, but their carcinogenicity is unclear. Caine-based anesthetics may affect the central nervous system and cardiovascular system, and quinalidine has a certain degree of irritation to human skin and other parts of the body. Therefore, it is of great significance to establish an efficient, accurate, and sensitive analytical method that can meet the requirements of detecting a variety of anesthetics.

[0004] Methods for detecting anesthetics in aquatic products mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA), and various rapid detection methods. Chromatography is susceptible to matrix interference, has high detection limits, and carries a risk of false positives. ELISA is costly and unsuitable for large-scale testing. GC-MS is often used for detecting eugenol-based anesthetics, but this method allows for the easy degradation of the target compound. LC-MS offers high sensitivity and strong anti-interference capabilities, enabling trace analysis of various target compounds, and is currently used for the qualitative and quantitative confirmation of anesthetics such as cocaine and eugenol. Due to the complex composition of endogenous interfering substances such as proteins, fats, and pigments in aquatic products, appropriate pretreatment is necessary to improve method sensitivity. In their paper "Determination of Six Anesthetics in Aquatic Products by a Novel QuEChERS Combined with Solid-Phase Extraction-High Performance Liquid Chromatography," Gao et al. used phosphate buffer (pH 4.4)-acetonitrile solution for extraction, followed by purification with an enhanced lipid removal and purification agent (EMR-Lipid), then purification using an HLB solid-phase extraction column, and determination by high-performance liquid chromatography with a diode array detector, with external standard method for quantification. This method uses a solid-phase extraction column for purification, which requires activation, sample loading, rinsing, and elution steps, making it time-consuming and only able to detect six anesthetics in aquatic products. If more anesthetics need to be detected, they must be extracted and purified separately, which reduces the testing efficiency to some extent. At the same time, the use of multiple extraction reagents and consumables also causes some environmental pollution. Summary of the Invention

[0005] In view of this, the purpose of this invention is to address the shortcomings of the prior art by providing a method for simultaneously detecting 25 anesthetics in aquatic products. This method has a simple and rapid pretreatment process and high sensitivity, which can meet the requirements for rapid and accurate detection of multiple anesthetics in aquatic products.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0008] (1) Extraction: Weigh the sample and add water, vortex, 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 blow-off tube, blow nitrogen to 0.5 mL, and make up to volume with acetonitrile;

[0009] (2) Purification: Add the above-mentioned solution to the purifying agent, wherein the ratio of the solution to the purifying agent is 1 mL: 100-200 mg; vortex and centrifuge, collect the supernatant, add water and filter to obtain the purified solution, wherein the purifying agent is MgSO4 and C. 18 It is a mixture of PSA and Carb in a weight ratio of 50:20-50:10-50:10-50;

[0010] (3) Detection.

[0011] Furthermore, in 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 sample is extracted with an organic solvent by shaking for 3-8 minutes, anhydrous sodium sulfate is added, and the mixture is shaken rapidly for 3-8 minutes. The mixture is centrifuged for 3-8 minutes, and the entire organic solvent layer is transferred to a nitrogen blow-off tube. The sample is blown with nitrogen at 40°C to a final volume of 0.5 mL, and the volume is adjusted to 1 mL with acetonitrile. 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, or 0.1% formic acid / acetonitrile (acetonitrile with 0.1% formic acid added, volume fraction).

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

[0014] Furthermore, the purifying agent is MgSO4, C 18 It is a mixture of PSA and Carb 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 employed two mobile phases for elution: acetonitrile / 0.1% formic acid water and acetonitrile / water.

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

[0018] The beneficial effects of this invention are:

[0019] 1. To achieve the simultaneous detection of 25 anesthetics in aquatic products, this invention improves and optimizes the existing QuEChERS-ultra-high pressure liquid chromatography-tandem mass spectrometry method. Specifically, it comprehensively optimizes and improves the extraction, purification, and detection processes. Using the optimized process, the 25 anesthetics show good linearity in the range of 1-50.0 ng / mL (10-500 ng / mL for m-aminobenzoic acid, p-aminobenzoic acid, and 6 eugenol compounds), with a limit of quantitation (S / N=10) of 1-10 μg / kg. Except for m-aminobenzoic acid, p-aminobenzoic acid, and the 6 eugenol compounds, the LOD and LOQ are 5 μg·kg. -1 10 μg·kg -1In addition, the LOD and LOQ of the remaining 19 compounds were 0.5 μg·kg⁻¹. -1 1 μg·kg -1 At low, medium, and high spiking levels, the average recovery rate was 66.2%–112.5%, with intra-batch precision of 2.1%–11.2% (n=6) and inter-batch precision of 2.3%–11.3% (n=3). This method features simple and rapid pretreatment, high sensitivity, and can meet the requirements for rapid and accurate detection of various anesthetics in aquatic products. Furthermore, it offers short detection time, low organic solvent consumption, and is fast, environmentally friendly, and efficient.

[0020] 2. Regarding extraction, due to the significant structural differences among the 25 anesthetics, some being highly lipid-soluble and others highly water-soluble, the extraction solvent needed to consider the extraction efficiency of all target compounds. Taking into account the properties of each compound, this study investigated 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 (prawn, tilapia, and grass carp). Because aquatic products such as fish and shrimp have high protein content, directly adding highly polar organic solvents such as acetonitrile can disrupt the hydrogen bonds and hydrophobic interactions within protein molecules, causing them to denature and clump together, thus affecting the extraction effect. Therefore, this application first added water, shook the sample to disperse it, and then added the organic solvent. The recovery rates of the 25 compounds extracted by the organic solvent acetonitrile were all between 65% and 115%, demonstrating excellent results.

[0021] Furthermore, acetonitrile is used during volume adjustment to further precipitate proteins and reduce the dissolution of interfering impurities.

[0022] 3. In the purification section, different types of adsorbents are used to adsorb interfering substances and retain the target compound in the solution. This application's research found that adding more types of adsorbents is not necessarily better, nor is adding more quantity necessarily better. Excessive addition of either type or quantity can further increase impurities and interference, thus affecting the purification effect. However, insufficient addition will also affect the purification effect. This study uses C... 18 PSA and Carb are used as adsorbents, among which C 18 Primarily adsorbs nonpolar co-extractants, such as lipophilic compounds like fats; PSA is mainly used to adsorb organic acids and pigments; Carb is often used to remove polar co-extractants, such as sterols; due to the large amount of co-extractants such as proteins, pigments, and fats in the sample extract, C is added in combination. 18 The sample was purified using PSA and Carb, with a combined concentration of 30 mg C. 18 When combined with 20mg PSA and 20mg Carb, not only is the recovery rate guaranteed, but the solution can also effectively remove interfering substances such as proteins, fats and pigments, making the sample meet the instrument detection requirements and demonstrating excellent purification effect.

[0023] 4. During the detection process, separation was performed using a Waters HSS T3 column. Two mobile phases were used: acetonitrile-0.1% formic acid water as the mobile phase in positive ion mode; and acetonitrile-water mobile phase, which ran once during simultaneous positive and negative ion scanning. Ultimately, the separation of 25 compounds was good, and matrix effects in the detection process of different matrix samples were eliminated, reducing interference. Attached Figure Description

[0024] Figure 1 Quantitative ion maps of 25 anesthetic agents;

[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 The recovery rates of 25 target compounds in three matrices are shown in the figure when the organic solvent is acetonitrile.

[0029] Figure 6 The recovery rates of 25 target compounds in three matrices are shown in the figure when methanol is used as the organic solvent.

[0030] Figure 7 The recovery rates of 25 target compounds in three matrices are shown in the figure when the organic solvent is ethyl acetate.

[0031] Figure 8 The recovery rates of 25 target compounds in three matrices are shown in the figure when the organic solvent is 0.1% formic acid acetonitrile.

[0032] Figure 9 The peak shapes of six anesthetic agents—bupivacaine, promocaine, butacaine, cincocaine, tetracaine, and ropivacaine—are shown in two mobile phases: acetonitrile-water and acetonitrile-0.1% formic acid water.

[0033] Figure 10 Matrix effect diagrams for 25 anesthetic agents;

[0034] Figure 11 The graph shows the average recovery rate and precision of 25 anesthetics in different matrices.

[0035] Figure 12 The total ion chromatogram is for positive samples. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments. In the following embodiments, the abbreviations are respectively: 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 includes the following steps:

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

[0040] (2) Purification: Add the above-mentioned solution to the purifying agent, which is 50 mg MgSO4 and 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 (UHPLC-MS / MS) was used for detection. The chromatographic column was Acquity. HSS T3 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). The gradient elution program was as follows: 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 min, 95%–5% A. The first mobile phase was acetonitrile (A) / water (B), with a gradient elution program: 0–0.7 min, 40% A; 0.7–2.0 min, 40%–60% A; 2.0–4.0 min, 60% A; 4.0–7.0 min, 60%–95% A; 7.0–8.0 min, 95% A; 8.0–8.1 min, 95%–40% A; 8.1–9.0 min, 40% A. The column temperature was 40℃; the flow rate was 0.3 mL / min; the injection volume was 2 μL; and the column was a Waters HSS T3 (2.1 mm × 100 mm, 1.8 μm).

[0043] Mass spectrometry conditions: Electrospray ionization (ESI) source, acetonitrile / 0.1% formic acid water mobile phase in positive ion mode, acetonitrile / water mobile phase scanned once simultaneously with positive and negative ions; scanning mode: multiple reaction monitoring (MRM); ion source temperature 550℃; 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 cocaine derivatives, 6 eugenol derivatives, quinacrine, and diazepam. Specific compound information is shown in Table 1. Ion pairs of the selected 25 target compounds were monitored, and their declustering voltage (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 (methanol as solvent) were purchased from Tianjin Alta Company, each with a mass concentration of 1000 μg / mL; accurately transfer 0.01 mL of each of the above 25 standard solutions (0.10 mL each of m-aminobenzoic acid, p-aminobenzoic acid, and 6 eugenol compounds) into a 10 mL volumetric flask, and dilute to the mark with methanol to prepare a 1 μg / mL mixed standard working solution (10 μg / mL of m-aminobenzoic acid, p-aminobenzoic acid, and 6 eugenol compounds), and store in a -20℃ refrigerator.

[0049] Examples 2-4

[0050] In Examples 2-4, the amount of C18 was 20, 40, and 50 mg, respectively, and the other processes were the same as in Example 1.

[0051] Examples 5-8

[0052] In Examples 5-8, the amount of PSA was 10, 30, 40, and 50 mg, respectively, and the other processes were the same as in Example 1.

[0053] Examples 9-12

[0054] In Examples 9-12, the amount of Carb was 10, 30, 40, and 50 mg, respectively, and the other processes were the same as in Example 1.

[0055] Comparative Examples 1-3

[0056] Comparative Examples 1-3 used Acquity columns, respectively. 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 added to acetonitrile, by volume). Other detection processes were the same as in Example 1.

[0059] Performance testing

[0060] 1. The separation of 25 compounds using the chromatographic columns of Example 1 and Comparative Examples 1-3 was studied. Experiments revealed that the 25 anesthetics could be separated using four different 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, and response value on RRHD (2.1 mm × 100 mm, 1.8 μm) were analyzed. The results showed that procaine, procainamide, and quinalidine exhibited good performance in terms of accuracy. BEH C 18 Agilent SB-Aq C 18 RRHD, Agilent Eclipse Plus C 18 RRHD exhibited poor peak shape and low sensitivity on all three chromatographic columns. The two metabolites, m-aminobenzoic acid and p-aminobenzoic acid, showed weak peak retention, early elution times, and poor peak shape on all three columns. Six eugenol compounds could not be completely separated. However, Acquity... The HSS T3 column was able to completely separate 25 compounds with sharp peaks (see chromatogram). Figure 1 Therefore, the Acquity is determined. The HSST3 column is the optimal column.

[0061] 2. The effects of C18 concentrations of 20, 30, 40, and 50 mg in Examples 1 and 2-4 on the recovery rate of the target compound were investigated respectively. See [reference needed]. Figure 2 The effect of PSA concentrations of 10, 20, 30, 40, and 50 mg in Examples 1 and 5-8 on the recovery rate was investigated. The results are shown in [link to relevant documentation]. Figure 3 The effect of Carb concentrations of 10, 20, 30, 40, and 50 mg in Examples 1 and 9-12 on the recovery rate was investigated. The results are shown in [reference needed]. Figure 4 .

[0062] Depend on Figure 2 It can be seen that in C 18 At a concentration of 30 mg, the recoveries of 25 compounds were optimal, ranging from 61% to 116%; with increasing C... 18 With increasing content, the recovery rates of procaine, procainamide, chloroprocaine, quinalidine, and butacaine decreased significantly. When 30 mg was determined to be C... 18 Based on the optimal addition amount, the effect of PSA concentrations of 10, 20, 30, 40, and 50 mg on the recovery rate was compared. Figure 3It can be seen that 48% of the 25 compounds showed the best recovery rate at a PSA concentration of 20 mg, with recoveries ranging from 70% to 107%. The recoveries of the remaining 52% of compounds showed little difference at PSA concentrations of 10 mg and 20 mg, but the recoveries of all 25 compounds gradually decreased as the PSA concentration increased. Based on the determination that 20 mg is the optimal PSA addition, the effects of Carb concentrations of 10, 20, 30, 40, and 50 mg on the recovery rate were compared. Figure 4 It can be seen that the recovery rate of 80% of the 25 compounds first increases and then decreases with increasing Carb content. When the Carb content is 20 and 30 mg, the recovery rate is not significantly different, ranging from 70% to 101%. From an environmentally friendly perspective, the optimal Carb addition amount is 20 mg. Therefore, the optimal combination of the three adsorbents is 30 mg C. 18 +20mg PSA +20mg Carb.

[0063] 3. The effect 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 (prawn, tilapia, and grass carp) was investigated. The results are shown in [link to results]. Figure 5-8 .

[0064] Since this application aims to detect 25 anesthetics with significant structural differences—some highly lipid-soluble, others highly water-soluble—the organic solvents used for extraction must consider the extraction efficiency of all target compounds. Eugenol-based anesthetics can be extracted using acetonitrile, methanol, or n-hexane; cocaine-based anesthetics commonly use acetonitrile, phosphate buffer, or aqueous acetonitrile solution as extraction solvents; diazepam and other sedatives, being lipid-soluble compounds, are often extracted using acetonitrile or ethyl acetate; and quinalidine is often extracted using acetonitrile. Taking into account the properties of each compound, this application compared 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 (prawns, tilapia, and grass carp). Because aquatic products such as fish and shrimp have high protein content, directly adding highly polar organic solvents such as acetonitrile can disrupt the hydrogen bonds and hydrophobic interactions within protein molecules, causing them to denature and clump together, thus affecting the extraction effect.

[0065] Therefore, in this experiment, water was added first, and the sample was shaken and dispersed before adding the organic solvent. The experiment showed that the methanol extract was relatively turbid, and the protein precipitation effect was poor, with the recovery rate of all 25 compounds below 60%. The ethyl acetate extract contained 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%. The acetonitrile and 0.1% formic acid acetonitrile extracts were clear and had the lowest matrix effect, with the recovery rates of all 25 compounds ranging from 65% to 115%. Therefore, considering 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 four mobile phases—acetonitrile-water, methanol-water, acetonitrile-0.1% formic acid solution, and methanol-0.1% formic acid solution—on the separation performance of the target compound were investigated. See the results below. Figure 9 , Figure 9 The peak shapes of six anesthetics—bupivacaine, promocaine, butacaine, cincocaine, tetracaine, and ropivacaine—were compared using acetonitrile-water and acetonitrile-0.1% formic acid aqueous solutions as mobile phases. The acetonitrile-water system showed a higher response value, and baseline separation was achieved between MS-222 and benzocaine, m-aminobenzoic acid and p-aminobenzoic acid, and the three eugenol isomers. However, the peaks of bupivacaine, promocaine, butacaine, cincocaine, tetracaine, and ropivacaine were broadened and showed poor peak shapes. Adding 0.1% formic acid aqueous solution to the water helped to improve the quasi-molecular ion peak [M+H]. + The formation of acetonitrile can make the peaks of the above compounds sharp and symmetrical. The addition of formic acid will completely suppress eugenol and isoeugenol in negative ion mode. Therefore, considering sensitivity, resolution 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. In the specific detection, acetonitrile / 0.1% formic acid water is used as the mobile phase for elution first, and then acetonitrile-water is used for elution to achieve the separation of target compounds.

[0067] 5. Matrix effect

[0068] Endogenous interfering substances such as proteins in aquatic products, as well as exogenous interfering substances such as salts introduced during pretreatment, can inhibit or enhance the ionization of target compounds, thus affecting the accuracy of analytical results. Therefore, it is necessary to investigate the matrix effect of the method in this application. This study used negative matrix extract and pure solvent to prepare a standard curve. The matrix effect (ME) was calculated using the slope of the matrix-matched and solvent standard curves. A negative ME indicates a matrix inhibition effect, while a positive ME indicates a matrix enhancement effect. When |ME| < 20%, a weak matrix effect exists; when 20% < |ME| < 50%, a moderate matrix effect exists; and when |ME| > 50%, a strong matrix effect exists. Taking shrimp and grass carp as examples, from... Figure 10 It can be seen that the 25 anesthetics exhibited varying degrees of matrix effect in the two matrices, with eugenol and isoeugenol showing the largest matrix effects, both exceeding 50%. The matrix effect of grass carp was higher than that of prawn. To correct for the influence of ME on the results, a matrix-matched standard curve was used in the quantitative analysis.

[0069] 6. Optimization of mass spectrometry conditions

[0070] Based on the structural characteristics of 25 compounds, a primary mass spectrometry scan was performed on a mixed standard solution with a concentration of 200 ng / mL to determine its quasi-molecular ion peak. Eugenol and isoeugenol, containing phenolic hydroxyl groups, readily lose a proton to obtain [MH]. - The quasi-molecular ion peak exhibits high sensitivity under negative ion detection methods. ， All other compounds readily undergo proton formation [M+H]. + The quasi-molecular ion peak is detected, so positive ion mode is used for detection. Specifically, the acetonitrile / 0.1% formic acid water mobile phase is scanned in positive ion mode, and the acetonitrile / water mobile phase is scanned once for both positive and negative ions to increase the detection sensitivity.

[0071] In addition, the five pairs of isomers involved in the detection in this application (including tricaine and benzocaine, m-aminobenzoic acid and p-aminobenzoic acid, eugenol and isoeugenol, methyleugenol and methyl isoeugenol, eugenol acetate and acetyl isoeugenol) require optimized chromatographic conditions for complete separation. Fragment ions generated by collisions with a collision gas were used for secondary mass spectrometry scanning. The cluster removal voltage (DP) and collision energy (CE) were optimized. The qualitative and quantitative characteristic ions of the 25 compounds and the optimized mass spectrometry parameters are shown in Table 1, ultimately enabling the simultaneous detection of 25 compounds.

[0072] 7. Linear range and sensitivity

[0073] Add appropriate amounts of mixed standard working solutions of 25 compounds to the extracts of two blank matrices (taking prawn 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). Inject the matrices sequentially. Perform linear regression between the mass concentration (X) of the target analyte and the corresponding peak area (Y), with 1 / x as the weight. Quantify using the external standard method. The limit of detection (LOD) is set at 3 times the signal-to-noise ratio (S / N≥3), and the limit of quantitation (LOQ) is set at 10 times the signal-to-noise ratio (S / N≥10). The tests showed that the 25 anesthetics exhibited good linearity in the two matrices, with correlation coefficients (r) greater than 0.995. Except for m-aminobenzoic acid and p-aminobenzoic acid, the LOD and LOQ of the six eugenol compounds were 5 μg·kg⁻¹.-1 10 μg·kg -1 In addition, the LOD and LOQ of the remaining 19 compounds were 0.5 μg·kg⁻¹. -1 1 μg·kg -1 See Table 2 for details.

[0074] Table 2. Linearity, Limit of Detection, and Limit of Quantification of 25 Compounds

[0075]

[0076]

[0077] 8. Recovery rate and precision

[0078] Mixed standard working solutions containing 1, 2, and 10 μg / kg (10, 20, and 100 μg / kg for m-aminobenzoic acid and p-aminobenzoic acid, and 10, 20, and 100 μg / kg for six eugenol compounds) were added to three negative control matrices: prawn, tilapia, and grass carp. The assays were performed in parallel six times over three consecutive days, and the recoveries and intra- and inter-batch standard deviations were calculated. The recoveries of the 25 target compounds in the three matrices ranged from 65% to 112%, with intra-batch RSDs of 2.1% to 11.2% and inter-batch RSDs of 2.3% to 10.5% (see [link to study]). Figure 11 This method exhibits good sensitivity, high accuracy, and high precision, meeting the requirements of laboratory quality control standards.

[0079] 9. Actual sample testing

[0080] The established method was used to determine the anesthetic content in 90 commercially available aquatic products (including grass carp, crucian carp, turbot, tilapia, prawns, crayfish, and shrimp). The results showed that eugenol was detected in 4 grass carp samples, with levels ranging from 0.034 to 0.21 mg / kg. Figure 12 The positive ion chromatogram indicates that the detection method described in this 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 solutions of the present invention and are not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A method for simultaneously detecting 25 anesthetics in aquatic products, characterized in that: Includes the following steps: (1) Extraction: Weigh the sample and add water, vortex, 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 blow-off tube and blow nitrogen to 0.5 mL, and make up to volume with acetonitrile. The organic solvent is acetonitrile. (2) Purification: Add the fixed solution to the purifying agent, wherein the ratio of the fixed solution to the purifying agent is 1 mL: 100-200 mg; vortex and centrifuge, take the supernatant, add water and filter to obtain the purified solution, wherein the purifying agent is MgSO4 and C. 18 It is a mixture of PSA and Carb in a weight ratio of 50:20-50:10-50:10-50; (3) Detection: Ultra-high pressure liquid chromatography-tandem mass spectrometry was used; 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; 5.6~7.0 min, 5% A; Second mobile phase is acetonitrile A / water B, gradient elution program: 0~0.7 min, 40% A; 0.7~2.0 min, 40%~60% A; 2.0~4.0 min, 60% A; 4.0~7.0 min, 60%~95% A; 7.0~8.0 min, 95% A; 8.0~8.1 min, 95%~40%A; 8.1~9.0 min, 40% A; column temperature 40 ℃; flow rate 0.3 mL / min; injection volume: 2 μL; column: Waters HSST3; Mass spectrometry conditions: Electrospray ionization source; acetonitrile / 0.1% formic acid water mobile phase in positive ion mode; acetonitrile / water mobile phase scanned once simultaneously with both positive and negative ions; scanning mode: multiple reaction monitoring; ion source temperature: 550 ℃; ion source voltage: 5500 V; curtain gas pressure: 0.24 MPa; nebulizer pressure: 0.38 MPa; auxiliary gas pressure: 0.38 MPa. The 25 anesthetics include procaine, procainamide, chloroprocaine, lidocaine, prilocaine, ethyl m-aminobenzoate methanesulfonate, ropivacaine, bupivacaine, butacaine, tetracaine, benzocaine, promocaine hydrochloride, cincocaine, lisocaine, 3-aminobenzoic acid, aminobenzoic acid, para-acetaminobenzoic acid, 2-methylquinoline, disipan, eugenol, isoeugenol, methyleugenol, methyl isoeugenol, eugenol acetate, and acetyl isoeugenol.

2. The method for simultaneously detecting 25 anesthetics in aquatic products according to claim 1, characterized in that: In step (1), the sample is weighed and placed in a centrifuge tube, water is added, and the mixture is vortexed for 1-5 minutes. Acetonitrile is used for extraction for 3-8 minutes, anhydrous sodium sulfate is added, and the mixture is shaken rapidly for 3-8 minutes. The mixture is centrifuged for 3-8 minutes, and the entire acetonitrile layer is transferred to a nitrogen blow-off tube. Nitrogen is blown down to 0.5 mL at 40°C, and the volume is adjusted to 1 mL with acetonitrile. The ratio of the sample, water, acetonitrile, 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 purifying agent is MgSO4, C 18 It is a mixture of PSA and Carb in a weight ratio of 50:30:20:20.

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