High-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrum

Abstract

The invention relates to the technical field of detection, and particularly discloses a high-throughput screening and quantifying method for anesthetics in aquatic products based on high-resolution mass spectrometry, which comprises the following steps: constructing a mass spectrometry database containing information of at least 72 anesthetic compounds; the method comprises the following steps: carrying out pretreatment on an aquatic product sample by adopting an improved QuEChERS method; carrying out analysis and acquisition under specific chromatography and mass spectrometry conditions by utilizing an ultra-high performance liquid chromatography-electrostatic field orbitrap high-resolution mass spectrometer; the method comprises the following steps: acquiring data by adopting a Full MS-ddMS2 scanning mode; and finally, analyzing the data through a'non-targeted discovery-targeted confirmation 'strategy, namely, firstly performing non-targeted screening by using Compressed Discovery software, and then performing targeted confirmation and quantification on the basis of the mass spectrum database through TraceFinder software, the method is high in flux, good in sensitivity and strong in anti-interference capability, and the method is suitable for large-scale popularization and application. Various types of anesthetic residues in aquatic products can be rapidly and accurately screened, identified and quantified, and the defects of an existing standard detection method are overcome.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and specifically discloses a high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry. Background Technology

[0002] To reduce stress on fish during farming and transportation, and to improve farming efficiency and survival rates, anesthetics are widely used in the farming and transportation of aquatic products. The use of anesthetics can significantly reduce the activity and oxygen consumption of aquatic products, thereby maintaining their freshness and improving their survival rate. However, their safety is also a concern.

[0003] my country lacks clear guidelines for the use of anesthetics in fisheries, and has not specified the types, dosages, or withdrawal periods of permitted anesthetics. It only regulates the use of sedatives such as methaqualone, chlorpromazine, diazepam, and thiamethoxam in animal-derived foods. Specifically, GB31650-2019, the National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food, stipulates that chlorpromazine, diazepam, and thiamethoxam are permitted for therapeutic use only and must not be detected in animal-derived foods. The Codex Alimentarius Commission (CAC), the European Union, Australia, and other countries and organizations have all issued relevant regulations regarding the detection and maximum limits for sedatives.

[0004] Based on the detection of anesthetics in previous risk monitoring of aquatic products, it can be inferred that there is a certain degree of abuse of anesthetics in actual circulation. Given the current chaotic market for fishery anesthetics, the existence of regulatory blind spots, and the frequent detection of anesthetics in risk monitoring, it is necessary to carry out research on monitoring technologies for anesthetics and their metabolites in the circulation of aquatic products and to establish a high-throughput screening and quantitative method based on high-resolution mass spectrometry. Summary of the Invention

[0005] This invention provides a high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry. It features high throughput, good sensitivity, and strong anti-interference ability, enabling rapid and accurate screening, identification, and quantification of various types of anesthetic residues in aquatic products, thus overcoming the shortcomings of existing standard detection methods.

[0006] In view of the above problems, the present invention provides a high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry, comprising the following steps: S1. Construct a high-throughput screening mass spectrometry database for anesthetics, the database containing the names, CAS numbers, molecular formulas, retention times, adduct ions, and precise mass numbers of secondary fragment ions of various anesthetic compounds; S2. Preparation of aquatic product samples to be tested: Weigh the homogenized aquatic product sample, add acetonitrile solution containing formic acid for extraction, purify using the QuEChERS method, concentrate the purified extract by nitrogen blowing, reconstitute, and then test. S3. Instrumental analysis: The test solution prepared in step S2 was subjected to ultra-high performance liquid chromatography-high resolution mass spectrometry analysis, and data acquisition was performed using Full MS-ddMS2 mode; S4. Data Analysis: The data collected in step S3 is analyzed using a "non-targeted discovery-targeted confirmation" strategy. First, non-targeted compounds are screened and preliminarily identified. Then, targeted confirmation and quantification are performed based on the mass spectrometry database constructed in step S1.

[0007] Specifically, in step S1, the mass spectrometry database covers at least 72 anesthetic compounds, including cocaine, benzodiazepines, phenothiazines, barbiturates, and other sedatives.

[0008] Specifically, in step S2, the QuEChERS method is as follows: add QuEChERS extraction salt pack to the sample extract, centrifuge and take the supernatant into a dispersion solid phase extraction purification tube, centrifuge again and take the supernatant, and concentrate by nitrogen blowing.

[0009] Specifically, in step S3, the conditions for ultra-high performance liquid chromatography include: a C18 column; mobile phase A being an aqueous solution of ammonium acetate containing formic acid, and mobile phase B being acetonitrile; and a gradient elution program.

[0010] Specifically, the gradient elution program is as follows: 0-2 min, 90% A; 2.1-5 min, 65% A; 5.1-7 min, 40% A; 7-11 min, 40%-20% A; 11-12 min, 20%-10% A; 12-12.5 min, 10% A; 12.5-12.6 min, 10%-90% A; 12.6-14 min, 90% A.

[0011] Specifically, in step S3, the high-resolution mass spectrometry is an electrostatic field orbital trap mass spectrometry, and the mass spectrometry conditions include: using a heated electrospray ion source, scanning positive and negative ions simultaneously; first-stage full scan resolution ≥70000 FWHM, and second-stage scan resolution ≥17500 FWHM.

[0012] Specifically, in step S4, the "non-targeted discovery" involves using Compound Discoverer software to process the collected mass spectrometry data and preliminarily identify potential compounds through retention time alignment, peak detection, background subtraction, and mass spectrometry database retrieval.

[0013] Specifically, in step S4, the “targeted confirmation” involves using TraceFinder software to import the mass spectrometry database constructed in step S1, setting confirmation parameters, and performing automated confirmation analysis on suspected compounds identified through non-targeted screening. The confirmation parameters include: a mass deviation tolerance of 5 ppm for the parent ion and daughter ions, a requirement for at least two matching daughter ions, and an isotope distribution similarity threshold of 80%.

[0014] Specifically, in step S4, the quantitative analysis is performed using the matrix-matched standard curve external standard method.

[0015] A high-throughput screening mass spectrometry database for anesthetics in aquatic products based on high-resolution mass spectrometry, used for implementing the above-described high-throughput screening and quantification method for anesthetics in aquatic products, is stored electronically and contains the name, CAS number, molecular formula, retention time, adduct ions and precise mass number information of one or more anesthetic compounds, and can be accessed by TraceFinder or similar mass spectrometry data analysis software for targeted screening and confirmation.

[0016] One or more technical solutions provided in this invention have at least the following technical effects or advantages: A high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry offers advantages such as high throughput, high sensitivity, and strong anti-interference capabilities. It can rapidly and accurately screen, identify, and quantify various types of anesthetic residues in aquatic products, thus overcoming the shortcomings of existing standard detection methods. Attached Figure Description

[0017] Figure 1 Extraction ion currents for each compound in this invention Figure 1 ; Figure 2 Extraction ion currents for each compound in this invention Figure 2 ; Figure 3 Extraction ion currents for each compound in this invention Figure 3 ; Figure 4 Extraction ion currents for each compound in this invention Figure 4 ; Figure 5 Extraction ion currents for each compound in this invention Figure 5 ; Figure 6 Extraction ion currents for each compound in this invention Figure 6 ; Figure 7 Extraction ion currents for each compound in this invention Figure 7 ; Figure 8 Extraction ion currents for each compound in this invention Figure 8 ; Figure 9 Extraction ion currents for each compound in this invention Figure 9 ; Figure 10 This is a chromatographic retention diagram of the isomer MS-222 and benzocaine under a Hillic column in this invention; Figure 11 This is a chromatographic retention diagram of the isomer MS-222 and benzocaine under the T3 column in this invention; Figure 12 This is a chromatographic retention diagram of the isomer MS-222 and benzocaine under a C18 column in this invention; Figure 13 Chromatographic retention behavior of compounds (clozapine, chloroprocaine, prilocaine) with an injection volume of 2 μL; Figure 14 Chromatographic retention behavior of compounds (clozapine, chloroprocaine, prilocaine) with an injection volume of 5 μL; Figure 15 The chromatographic retention behavior of the compounds (clozapine, chloroprocaine, and prilocaine) is shown in the chromatographic graphs at an injection volume of 10 μL. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] First, in the implementation of this invention, liquid chromatography-high resolution mass spectrometry technology, with its unique advantages such as high resolution, high sensitivity, and strong resistance to matrix interference, can, in the absence of standards, combine with a database to achieve rapid screening, identification, and confirmation of potential or unknown organic pollutants in complex samples using primary precise molecular weight, retention time, isotope abundance ratio, and secondary characteristic ions as screening conditions.

[0020] Secondly, based on the structure of each compound, Full MS-ddMS2 mode was used for detection. First, at a resolution of 70,000 ppm and within the set scan range, a 100 ng / mL mixed standard working solution was analyzed using Full MS full scan mode. Based on the precise theoretical mass number of each compound, its extracted ion chromatogram was obtained, and the retention time and precise mass number of each compound were determined. Upon reaching the set conditions, the secondary mass spectrometry mode was automatically triggered. By optimizing the collision energy, the optimal secondary mass spectrum for each compound was obtained, and its characteristic fragment ions were identified. The secondary mass spectrum of each anesthetic and its characteristic fragment ions were correlated with its compound name, chemical formula, precise mass number, and retention time to complete the construction of the TraceFinder targeted screening spectral library.

[0021] Next, based on UHPLC-Q-Orbitrap data, comprehensive non-targeted analysis was performed using Compound Discoverer. Potential compounds were preliminarily identified through high-precision mass spectrometry matching and database searching. Subsequently, for the identified compounds, confirmation was completed using a targeted screening analysis method established in TraceFinder software, with a confirmation standard of 5 ppm mass precision, at least two daughter ion matches, and 80% isotope matching. This achieved a closed loop of "non-targeted screening - targeted confirmation," and quantitative analysis was performed using TraceFinder software.

[0022] Furthermore, in specific implementation, the present invention includes the following steps: The first step was to select the instruments and corresponding materials. For the instruments, the following were selected: Ultimate 3000 high-performance liquid chromatography-Q-Exactive electrostatic field orbital trap high-resolution mass spectrometry system (Thermo Fisher Scientific, USA); Milli-Q ultrapure water system (Millipore, USA); N-EVAP112 nitrogen blowing concentrator (Organomation Associates, USA); analytical balance (Mettler Toledo, Switzerland); ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); refrigerated centrifuge (Thermo Fisher Scientific, USA); and vortex mixer (Shanghai Qingpu Huxi Instrument Factory).

[0023] Regarding materials, 72 anesthetic standard substances were purchased from Dr. Ehrenstorfer GmbH (Germany) and Tianjin Alta Technology Co., Ltd., respectively; formic acid and ammonium acetate (mass spectrometry grade, Sigma Aldrich, USA); acetonitrile and methanol (mass spectrometry grade, Merck, USA); QuEChERS reagent (Shimadzu, Japan); dimethyl sulfoxide and sodium chloride (Sinopharm Group); 75 batches of aquatic products were purchased from local supermarkets or farmers' markets. Edible portions were taken, and samples were prepared according to the requirements of GB / T30891-2014. Fish were washed and the skin and mucus were removed; shrimp were washed, the head, shell, and vein were removed, and the muscle portion was homogenized and stored at -18℃. The samples were thawed and weighed before the experiment.

[0024] The second step is sample pretreatment. For aquatic products such as fish and shrimp: accurately weigh 10g of homogenized sample, add 10mL of acetonitrile solution containing 1% formic acid, shake vigorously for 1min, add QuEChERS extraction salt packet to the above extract, shake vigorously for 1min, centrifuge at 10000r / min for 10min at 4℃, take 6mL of supernatant into dSPE purification tube, shake vigorously for 1min, centrifuge at 10000r / min for 10min at 4℃, take 4mL of supernatant into 10mL centrifuge tube, blow with nitrogen at 40℃ until nearly dry, and make up to 1.0mL with methanol-acetonitrile-water (2+3+5) containing 0.05% formic acid, mix well, filter through a 0.45μm microporous membrane and then proceed with the analysis.

[0025] It should be noted that the instrument conditions set during the implementation of this invention are as follows: First, the chromatographic conditions are: Column: Zorbax Eclipse Plus C18 (150mm × 3.0 mm ID, 1.8μm); Column temperature: 30℃; Flow rate: 1mL / min; Injection volume: 5μL; Mobile phase A is an aqueous solution of ammonium acetate (5mmol / L) containing 0.05% formic acid, and mobile phase B is acetonitrile. Gradient elution: 0~2 min, 90% A; 2.1~5 min, 65% A; 5.1~7 min, 40% A; 7~11 min, 40%~20% A; 11~12 min, 20%~10% A; 12~12.5 min, 10% A; 12.5~12.6 min, 10%~90% A; 12.6~14 min, 90% A.

[0026] Secondly, the mass spectrometry conditions were as follows: The ion source was a HESI (heated ESI) source; the spray voltage was 3.5 kV(+) / 3.2 kV(−); the lens voltage was 50 V; the ion transfer tube temperature was 320°C; the sheath gas flow rate was 40 arb; the auxiliary gas flow rate was 10 arb; the auxiliary gas temperature was 350°C; the scanning mode was simultaneous positive and negative ion scanning; the acquisition mode was Full MS / ddMS2 mode, with the following settings: First-stage full scan resolution: 70000 FWHM; Automatic gain control (AGC): 3 × 10⁶; maximum residence time: 100 ms; Second-stage scan resolution: 17500 FWHM; Automatic gain control (AGC): 3 × 10⁶; maximum residence time: 50 ms; Mass-to-charge ratio window width (Isolation Window): m / z 2.0; Exclude isotopes set to "on"; Target ion list (inclusion): on; Dynamic Exclusion: 5.0. s; Normalized collision energy (NCE) is 20%, 40%, 60%; Cycle count: 5; MSX count: 1.

[0027] The third step involves establishing a mass spectrometry database. A standard solution with a concentration of 100 ng / mL is prepared and collected according to the instrument's chromatographic mass spectrometry conditions. The precise mass number of the first-order theoretical mass is determined based on the molecular formula of the target compound, and information such as the precise mass number of the adduct ion and the retention time of the compound is obtained. At the same time, the precise mass number of the adduct ion of the compound is added to the inclusion list. A secondary mass spectrum with rich fragment ion information is obtained at three different normalized collision energies of 20%, 40%, and 60%. The precise mass numbers of two secondary fragment ions are selected. Finally, a database covering the precise mass numbers of the adduct ions, retention times, molecular formulas, and precise mass numbers of secondary fragment ions of 72 target compounds is established, as shown in Table 1. Table 1 shows the molecular formula, retention time, parent ion, and secondary fragment ion of each compound. Step 4: Non-targeted screening: Non-targeted screening was performed. The raw data acquired by UHPLC-Q-Orbitrap was processed using Compound Discoverer 3.3 software. The "E and L w Stats Unknown ID wOnline and Local Database Searches" workflow was selected for non-targeted compound screening and identification. This workflow mainly includes the following key steps: First, retention time alignment of all samples was performed with a time window of 0.2 min to correct chromatographic drift; then, compound peak detection and grouping were performed, with a mass deviation tolerance of 5 ppm and a minimum signal intensity of 1 × 10⁻⁶. 5 The signal-to-noise ratio (S / N) threshold is 3, and the adduct ion is [M+H]. + With [MH] - In ion mode, to eliminate system background interference, the solvent blank was set as the Blank group and the experimental samples as the Sample group, and automatic background subtraction was performed. Compound structure identification was achieved by jointly searching the mzCloud high-resolution mass spectrometry database and the ChemSpider chemical structure database, and the identification results were intelligently sorted and optimized based on the mzLogic algorithm.

[0028] Targeted Validation: In TraceFinder 4.1 software, a targeted screening method is established based on the accurate mass number of the target compounds. Specifically, the name of each target compound, the precise mass-to-charge ratio of the parent ion and characteristic fragment ions, the adsorption form and charge number, etc. are entered into the database. Validation parameters are set: the mass deviation tolerance of the parent ion and daughter ions is 5 ppm, at least 2 daughter ions must be matched, and the isotopic distribution similarity threshold is set to 80%. Subsequently, this method is used to perform automated screening and validation analysis on the sample data.

[0029] Step 5: Quantitative methods. For the screened anesthetics, quantitative analysis was performed using the matrix-matched standard curve external standard method. The lower limit of quantification (LOQ) is the minimum spiked concentration at which both recovery and precision are satisfactory. To evaluate the accuracy and precision of the method, six recovery experiments were conducted at three spiking levels: 1x LOQ, 2x LOQ, and 10x LOQ.

[0030] It should be noted that this invention relates to a wide variety of compounds with broad polarity, and some drug molecules contain functional groups such as hydroxyl, amino, carboxyl, and alkyl groups, resulting in significant differences in their chemical properties. To meet the high-throughput requirements of experimental analysis and achieve accurate characterization, the following comparisons were made between Waters ACQUITY UPLC HSS T3 (2.1 × 100 mm ID, 1.8 µm), Thermo Accucorea Q (2.1 × 100 mm ID, 2.6 µm), and Agilent Eclipse plus C. 18 The separation performance of a (150 mm × 3.0 mm ID, 1.8 μm) chromatographic column for 72 compounds was demonstrated. MS-222 and benzocaine are isomers. When using a T3 column for separation, the separation performance of MS-222 and benzocaine was poor. (Agilent Eclipse plus C) 18 The chromatographic column effectively retains a variety of compounds, with sharp and symmetrical peaks exhibiting no obvious tailing. Its separation performance is significantly improved compared to the other two columns; therefore, the more versatile Agilent Eclipse plus C column was chosen. 18 Analytical column, wherein the chromatographic retention behavior of isomer MS-222 and benzocaine under Hillic column is shown in [reference needed]. Figure 10 The chromatographic retention behavior of isomer MS-222 and benzocaine under T3 column conditions is shown in the figure. Figure 11 C 18 The chromatographic retention behavior of the isomer MS-222 and benzocaine under chromatographic column is shown in the figure. Figure 12 .

[0031] Considering that the target compounds are mostly polar compounds, this invention investigated the effects of methanol-water, acetonitrile-water, acetonitrile-0.05% formic acid solution, acetonitrile-5 mmol / L ammonium acetate, and acetonitrile-5 mmol / L ammonium acetate (containing 0.05% formic acid) as mobile phases. The results showed that acetonitrile had a better response and peak shape than methanol when used as the organic phase. The addition of ammonium acetate improved the peak shape. Adding 0.05% formic acid to acetonitrile-5 mmol / L ammonium acetate improved the response of each compound in the positive ion mode. In the negative ion mode, the compounds were not significantly inhibited by the addition of formic acid. Therefore, acetonitrile-5 mmol / L ammonium acetate (containing 0.05% formic acid) was finally selected as the mobile phase system. In mass spectrometry, the recommended concentration of ammonium acetate is generally no more than 20 mM. Excessive salt concentration will not only affect the column life but also inhibit the compound response. Therefore, higher concentrations of salt solution were not tried. Based on this, the gradient elution program was further optimized to obtain better resolution and mass spectrometry response.

[0032] Meanwhile, the responses and peak shapes of various compounds were compared at injection volumes of 2, 5, and 10 μL. At an injection volume of 10 μL, compounds with good responses, such as clozapine and promethazine sulfoxide, showed peak fronting, while compounds like oxybuprocaine and tetracaine broadened their peaks, increasing from 0.15–0.2 minutes at 5 μL to 0.3–0.4 minutes. Chloroprocaine even exhibited a double-peak phenomenon. At an injection volume of 2 μL, the peak shapes were sharp and symmetrical. However, considering the sensitivity of barbiturate compounds collected in negative ion mode, 5 μL was ultimately determined to be a relatively suitable injection volume. The chromatographic retention behaviors of compounds at injection volumes of 2, 5, and 10 μL are shown in [reference needed]. Figure 13-15 Under these chromatographic conditions, the extracted ion chromatograms of 72 compounds are shown below. Figure 1-9 .

[0033] Furthermore, the present invention also requires optimization of pretreatment conditions. For drug multi-residue screening, it is necessary to establish an extraction method with high recovery rate, speed and simplicity to extract multiple compounds with large differences in physicochemical properties at the same time, so as to meet the needs of high-throughput screening.

[0034] Currently, the main methods for pretreatment of anesthetics in aquatic products are solid-phase extraction (SPE) column purification after extraction or direct acetonitrile extraction. However, SPE column purification is cumbersome and time-consuming, and it is difficult to find universally packed columns for compounds with significant differences in properties. Direct extraction, concentration, and loading of these compounds results in a dirty sample matrix and a significant matrix effect. This invention uses the QuEChERS pretreatment method and compares it with HLB column purification and direct acetonitrile extraction, concentration, and loading. The results show that the sample purified by the HLB column appears cleaner, but after injection analysis, except for barbiturates and a small number of para-aminobenzoic acid esters with relatively ideal recoveries, the recovery rates of most compounds are low. Compared with QuEChERS pretreatment, direct acetonitrile extraction and concentration has similar recovery rates, but the obtained sample is dirtier. Therefore, this invention has advantages in terms of recovery rate, matrix effect, and economic time consumption.

[0035] Acetonitrile, methanol, ethyl acetate, and water are commonly used extraction solvents for drug residue detection. Acetonitrile has good solubility for most compounds and can denature proteins without dissolving fats in the sample, effectively reducing interference and simplifying post-processing steps. Therefore, acetonitrile is used as the extraction solvent. Some compounds in aquatic products such as fish and shrimp may be bound to proteins. Therefore, it is considered to add a certain amount of formic acid to the extraction solvent. When pure acetonitrile precipitates proteins, it will cause rapid denaturation of proteins on the outside of the sample, resulting in an encapsulation effect, and drugs inside the tissue cannot be fully extracted. Adding an appropriate amount of water helps to disperse the sample and improve extraction efficiency. However, considering that aquatic product samples themselves contain a certain proportion of water and acetonitrile with a concentration of less than 60% cannot completely denature proteins, this invention selects an acetonitrile-water solution containing 1% formic acid as the extraction solvent.

[0036] QuEChERS extraction salt packs are mainly composed of MgSO4 and NaCl, which can promote the separation of acetonitrile and water phases. Aquatic products contain impurities such as pigments, fats, and water-soluble proteins; adding PSA and C... 18 Dispersed solid materials can effectively remove substances such as fatty acids, sugars, organic acids, and pigments.

[0037] Considering that the solubility of each compound varies in different solvents, some compounds such as diazepam, estazolam, and midazolam have good solubility in methanol, while lorazepam and nitrazepam have better solubility in acetonitrile. In order to avoid the ratio of the compound to the mobile phase being too different, after experimental investigation, a methanol-acetonitrile-water (2+3+5) solution containing 0.05% formic acid was finally selected as the redissolution solution.

[0038] Among them, the matrix effect (ME) is caused by the ionization of the target compound by the co-extract. The matrix working curve and solvent working curve were prepared with concentrations of 2, 5, 10, 20, 50 and 100 ng / mL using yellow croaker matrix. The matrix effect was determined by comparing the slope of the solvent standard curve and the matrix standard curve.

[0039] Matrix effect (ME) = slope of matrix working curve / slope of solvent working curve × 100%. When ME is greater than 120%, it indicates the presence of matrix enhancement effect; when ME is less than 80%, it indicates the presence of matrix inhibition effect; when ME is between 80% and 120%, it indicates no matrix effect or a very weak matrix effect. The results show that aquatic products, as a complex matrix, have a significant impact on most compounds during actual detection. 60.8% of the compounds exhibit a matrix inhibition effect, 8.86% of the compounds, mainly some benzodiazepines, exhibit a matrix enhancement effect, and 30.4% of the compounds, including barbiturates, a small number of benzodiazepines, and cocaine, are less affected by the matrix. Among them, barbiturates are collected in negative ion mode. ESI negative ion mode is sometimes slightly less inhibited than positive ion mode, thus making the analytes less affected by the matrix. It is necessary to establish purification methods according to the characteristics of different types of aquatic matrices to reduce the impact of matrix effect on detection results. Therefore, this invention uses a sample blank matrix solution to prepare a standard curve for quantitative detection.

[0040] The sixth step involved methodological validation. To evaluate the screening capability based on the TraceFinder software database, screening analyses were conducted at three matrix spiking levels: 0.2, 0.5, and 1 μg / kg. The results showed that at a concentration of 0.2 μg / kg, 70 compounds were detected, accounting for 97.2% of the total compounds; at a concentration of 0.5 μg / kg, 71 compounds were detected, accounting for 98.6% of the total compounds; and at a concentration of 1 μg / kg, all 72 compounds were correctly screened, achieving a 100% detection rate, which effectively meets the requirements for anesthetic screening.

[0041] Blank yellow croaker samples were weighed and pretreated according to the fish and shrimp pretreatment method. The samples were then redissolved in standard curve solutions with concentrations of 2, 5, 10, 20, 50, and 100 ng / mL to obtain matrix-matched standard curves. Standard curves were plotted using the peak area of ​​the precursor ion of the analyte and its corresponding mass concentration (ng / mL). All compounds showed good linearity in the range of 2–100 ng / mL, with linear correlation coefficients (r0, r ... 2 All values ​​were above 0.99. Following the requirements of the EU guideline document SANTE / 12682 / 2019, the limits of quantitation (LOQ), linearity, recovery, and precision were investigated. The LOQ refers to the lowest point at which the target compound, after sample pretreatment according to this invention, meets the requirements for recovery and RSD, as shown in Table 2. Table 2 lists the regression equations, correlation coefficients, matrix effects, LOQ, recovery, and precision for each compound. The LOQs determined in this invention are all between 0.5 and 5 μg / kg, except for zopiclone, whose LOQ is 5 μg / kg; the others are between 0.5 and 2.5 μg / kg. Spiking recovery experiments were conducted at three levels: LOQ, 2-fold LOQ, and 10-fold LOQ. Six parallel samples were analyzed at each level, and the recovery rate and relative standard deviation (RSD) were calculated. All compounds achieved good recovery and precision at the three spiking levels (n=6). Step 7: Actual sample determination. The samples were screened non-targeted using Compound Discover software. Based on an mzCloud matching score greater than 60, a variety of compounds were preliminarily identified. Analysis showed that the main compounds with sedative or anesthetic functions were promethazine and its metabolites promethazine sulfoxide, diazepam, valproamide, eticaine, and chlorpheniramine. Valproamide and eticaine were not validated because standard substances were unavailable. The others were validated using standard substances. The results were consistent with the results of the targeted screening by Tracefinder, confirming the accuracy of the non-targeted screening results.

[0042] Based on the previous verification of quantitative methods, the detected compounds were preliminarily quantified. In addition, considering that eugenol compounds have a high detection rate in the supervision and spot checks, and that a widely validated standard method has been established for their determination, eugenol compounds were quantitatively analyzed in the actual sample test according to BJS201908. At the same time, eugenol in three fish preparations was determined according to this method. Table 3 shows the detection of various anesthetics in 75 batches of aquatic products.

[0043] Diazepam and eugenol had the highest detection rates. Diazepam, due to its good mass spectrometry response, had a high detection rate but relatively low content, all below the limit of quantification, ranging from 0.062 to 0.33 μg / kg. Eugenol was detected in 15 samples, with contents ranging from 19.8 to 250 μg / kg, a detection rate of 22.1%. Two of the three aquatic preparations tested positive for high concentrations of eugenol and methyl eugenol, with eugenol contents of 124.3 and 130.4 mg / kg, respectively, and methyl eugenol contents of approximately 0.1 mg / kg. Methyl eugenol may have originated from a byproduct of eugenol synthesis and extraction, indicating that eugenol is the primary active ingredient. Based on the maximum residue limit of eugenol allowed in aquatic products of 50 μg / kg according to relevant Japanese standards, 12 out of 75 batches of aquatic products were substandard, indicating a serious problem of anesthetic residues in aquatic products. This specification and accompanying drawings are merely illustrative examples of the invention and are intended to cover any and all modifications, variations, combinations, or equivalents within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its scope. Therefore, if such modifications and variations fall within the scope of the invention and its equivalents, the invention is intended to include these modifications and variations.

Claims

1. A high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry, characterized in that, Includes the following steps: S1. Construct a high-throughput screening mass spectrometry database for anesthetics, the database containing the names, CAS numbers, molecular formulas, retention times, adduct ions, and precise mass numbers of secondary fragment ions of various anesthetic compounds; S2. Preparation of aquatic product samples to be tested: Weigh the homogenized aquatic product sample, add acetonitrile solution containing formic acid for extraction, purify using the QuEChERS method, concentrate the purified extract by nitrogen blowing, reconstitute, and then test. S3. Instrumental analysis: The test solution prepared in step S2 was subjected to ultra-high performance liquid chromatography-high resolution mass spectrometry analysis, and data acquisition was performed using Full MS-ddMS2 mode; S4. Data Analysis: The data collected in step S3 is analyzed using a "non-targeted discovery-targeted confirmation" strategy. First, non-targeted compounds are screened and preliminarily identified. Then, targeted confirmation and quantification are performed based on the mass spectrometry database constructed in step S1.

2. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 1, characterized in that, In step S1, the mass spectrometry database covers at least 72 anesthetic compounds, including cocaine, benzodiazepines, phenothiazines, barbiturates, and other sedatives.

3. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 1, characterized in that, In step S2, the QuEChERS method is as follows: add QuEChERS extraction salt pack to the sample extract, centrifuge, take the supernatant into a dispersion solid phase extraction purification tube, centrifuge again, take the supernatant, and concentrate by nitrogen blowing.

4. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 1, characterized in that, In step S3, the conditions for ultra-high performance liquid chromatography include: a C18 column; mobile phase A being an aqueous solution of ammonium acetate containing formic acid, and mobile phase B being acetonitrile; and a gradient elution program.

5. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 4, characterized in that, The gradient elution program is: 0-2 min, 90% A; 2.1-5 min, 65% A; 5.1-7 min, 40% A; 7-11 min, 40%-20% A; 11-12 min, 20%-10% A; 12-12.5 min, 10% A; 12.5-12.6 min, 10%-90% A; 12.6-14 min, 90% A.

6. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 5, characterized in that, In step S3, the high-resolution mass spectrometry is an electrostatic field orbital trap mass spectrometry, and the mass spectrometry conditions include: using a heated electrospray ion source, scanning positive and negative ions simultaneously; first-stage full scan resolution ≥70000 FWHM, and second-stage scan resolution ≥17500 FWHM.

7. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 1, characterized in that, In step S4, the "non-targeted discovery" involves using Compound Discoverer software to process the collected mass spectrometry data and preliminarily identify potential compounds through retention time alignment, peak detection, background subtraction, and mass spectrometry database retrieval.

8. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 1 or 7, characterized in that, In step S4, the "targeted confirmation" is to use TraceFinder software to import the mass spectrometry database constructed in step S1, set confirmation parameters, and perform automated confirmation analysis on suspected compounds screened out without targeting. The confirmation parameters include: the mass deviation tolerance of the parent ion and daughter ion is 5 ppm, at least 2 daughter ions are required to match, and the isotope distribution similarity threshold is set to 80%.

9. The high-throughput screening and quantification method for anesthetics in aquatic products based on high-resolution mass spectrometry as described in claim 1, characterized in that, In step S4, quantitative analysis was performed using the matrix-matched standard curve external standard method.

10. A high-throughput screening mass spectrometry database for anesthetic agents, used to implement the high-throughput screening and quantification method for anesthetic agents in aquatic products based on high-resolution mass spectrometry as described in any one of claims 1-9, characterized in that, The database is stored electronically and contains the name, CAS number, molecular formula, retention time, and precise mass number information of adduct ions and secondary fragment ions of one or more anesthetic compounds. It can be accessed by TraceFinder or similar mass spectrometry data analysis software for targeted screening and confirmation.

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