Method for rapidly determining multiple anesthetics and metabolites thereof in aquatic products based on QTRAP mass spectrum

Abstract

The invention discloses a method for rapidly determining multiple anesthetics and metabolites thereof in aquatic products based on QTRAP mass spectrometry. The method comprises the following steps: (a) preparing a series of matrix standard working solutions; (b) taking a sample to be detected, adding a formic acid acetonitrile solution and water mixed solution, and performing ultrasonic extraction and centrifugation; enabling the supernate to pass through a PRiME HLB solid-phase extraction column, taking effluent, and fixing the volume by using ammonium formate, so as to obtain a to-be-detected sample solution of the aquatic product; and (c) adopting an MRM-IDA-EPA mode, determining the series of matrix standard working solutions and the aquatic product to-be-detected sample solution through QTRAP mass spectrometry, performing qualitative determination through automatic comparison with a secondary mass spectrum standard spectrum library of nine anesthetics and three anesthetic metabolites, performing quantification according to an MRM data peak area, and calculating the content of each substance. The method disclosed by the invention can effectively meet the detection requirements of anesthetics and metabolites thereof in sudden and large-batch aquatic products, the detection efficiency is improved, and the detection limit is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and more specifically, to a method for rapidly determining multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry. Background Art

[0002] Anesthetics refer to drugs that can temporarily and reversibly cause the entire body or part of the body to lose consciousness and pain. With the continuous development of my country's economy and the continuous improvement of people's living standards, people have put forward higher requirements for high-quality, safe and healthy aquatic products. In order to keep aquatic products alive and fresh, fishery anesthetics have been widely used during transportation and temporary storage to reduce the metabolic intensity of aquatic products, inhibit stress reactions, delay damage and prolong their survival time.

[0003] At present, my country does not pay enough attention to the use of anesthetics in aquatic products. There are gaps in the limit standards and regulatory laws for anesthetics in aquatic products. In order to make huge profits, unscrupulous merchants will illegally add anesthetics in the breeding, fishing and processing processes. At present, the research on the detection methods of anesthetics and their metabolites in aquatic products is still in its infancy. The relevant detection methods mainly include liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry. Due to the complex matrix of aquatic products, high content of protein, fat, phospholipids, etc., and low content of anesthetics and their metabolites in aquatic products, the sample pretreatment requirements are high. The current detection methods mostly use solid phase extraction for sample pretreatment, which has cumbersome steps, time-consuming and labor-intensive, and affects the recovery rate of some target objects. Therefore, it is urgent to re-establish a rapid, accurate and sensitive detection method for multiple anesthetics in aquatic products. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry. This study established a monitoring method for 12 anesthetics and their metabolites in aquatic products by PRiME HLB column purification and combining the advantages of high sensitivity and good quantitative and qualitative accuracy of QTRAP mass spectrometry. The method is fast, simple, green and environmentally friendly, has good purification effect, can effectively respond to the sudden and large-scale detection needs of anesthetics and their metabolites in aquatic products, improves detection efficiency, and reduces the detection limit.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for rapidly determining multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry comprises the following steps:

[0007] (a) Prepare a series of matrix standard working solutions:

[0008] Using methanol as solvent, a mixed standard intermediate working solution was prepared, which contained 9 anesthetics, namely, ethyl m-aminobenzoate methanesulfonate, benzocaine, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, and ropivacaine, at a concentration of 250 ng / mL, and 3 anesthetic metabolites, namely, m-aminobenzoic acid, p-aminobenzoic acid, and p-acetylaminobenzoic acid, at a concentration of 2500 ng / mL.

[0009] Different amounts of mixed standard intermediate working solutions were respectively transferred, 500 μL of negative aquatic product matrix extract was added to each, and the solution was fixed to 1 mL with 10 mmol / L ammonium formate solution to prepare a series of matrix standard working solutions;

[0010] (b) Sample pretreatment:

[0011] Take 2.5 g of the sample to be tested, add 10 mL of a mixed solution of 0.1 wt% formic acid acetonitrile solution and water in a volume ratio of 80:20, mix well, perform ultrasonic extraction, and centrifuge; take 2 mL of the supernatant after centrifugation and pass it through a PRiME HLB solid phase extraction column, collect the effluent, take 500 μL of it and dilute it to 1 mL with 10 mmol / L ammonium formate to obtain a sample solution of aquatic products to be tested;

[0012] (c) Detection:

[0013] The MRM-IDA-EPA mode was used to measure a series of matrix standard working solutions by QTRAP mass spectrometry to obtain MRM data and secondary fragment full spectrum data, and a secondary mass spectrum standard library of 9 anesthetics and 3 anesthetic metabolites in aquatic products was established.

[0014] The MRM-IDA-EPA mode was used to measure the sample solutions of aquatic products by QTRAP mass spectrometry. The contents of 9 anesthetics and 3 anesthetic metabolites in the aquatic product samples were obtained by automatic comparison with the secondary mass spectrum standard library and quantitative analysis based on the peak area of ​​the MRM data.

[0015] Furthermore, the concentrations of the matrix standard working solutions prepared in step (a) are: 9 anesthetic concentrations are all 2 ng / mL and 3 anesthetic metabolite concentrations are all 20 ng / mL, 9 anesthetic concentrations are all 4 ng / mL and 3 anesthetic metabolite concentrations are all 40 ng / mL, 9 anesthetic concentrations are all 10 ng / mL and 3 anesthetic metabolite concentrations are all 100 ng / mL, 9 anesthetic concentrations are all 20 ng / mL and 3 anesthetic metabolite concentrations are all 200 ng / mL, 9 anesthetic concentrations are all 40 ng / mL and 3 anesthetic metabolite concentrations are all 400 ng / mL, 9 anesthetic concentrations are all 100 ng / mL and 3 anesthetic metabolite concentrations are all 1000 ng / mL.

[0016] Furthermore, the specific preparation method of the mixed standard intermediate working solution in step (a) is: using methanol as a solvent, first prepare 9 anesthetic standard stock solutions with a concentration of 10 μg / mL and 3 anesthetic metabolite standard stock solutions with a concentration of 100 μg / mL respectively; then absorb the 9 anesthetic standard stock solutions and the 3 anesthetic metabolite standard stock solutions, mix them, and make up to volume with methanol to prepare a mixed standard intermediate working solution.

[0017] Furthermore, the chromatographic conditions for the QTRAP mass spectrometry detection of the series of matrix standard working solutions and the aquatic product sample solutions are:

[0018] Chromatographic column: InfinityLab Poroshell 120Aq-C18 2.1mm×150mm, 2.7μm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.30mL / min; injection volume: 1μL; column temperature: 40℃; gradient elution conditions: 0-3.0min, mobile phase A decreased from 95% to 85%, mobile phase B increased from 5% to 15%; 3.0-6.0min, mobile phase A decreased from 85% to 60%, mobile phase B increased from 1.5% to 2.0%; 15% to 40%; 6.0-12.0min, mobile phase A decreased from 60% to 5%, mobile phase B increased from 40% to 95%; 12.0-14.0min, mobile phase A was maintained at 5%, mobile phase B was maintained at 95%; 14.0-14.1min, mobile phase A increased from 5% to 95%, mobile phase B increased from 95% to 5%; 14.1-17.0min, mobile phase A was maintained at 95%, mobile phase B was maintained at 5%.

[0019] Furthermore, the mass spectrometry conditions for the QTRAP mass spectrometry detection of the series of matrix standard working solutions and the aquatic product sample solutions to be tested are:

[0020] Electrospray ion source; ion source temperature 550°C; electrospray voltage 5500V; scan mode: MRM-IDA-EPI; curtain gas pressure: 30psi; nebulizer pressure 55psi; auxiliary heater pressure: 55.0psi; collision gas: Medium; parent ions, daughter ions and mass spectrometry parameters of 9 anesthetics and 3 anesthetic metabolites are shown in the following table:

[0021]

[0022] The * in the table indicates quantitative ion pairs.

[0023] The beneficial effects of the present invention are:

[0024] The present invention has established a QTRAP mass spectrometry rapid determination method for 9 anesthetics and 3 anesthetic metabolites in aquatic products: the method has a simple and fast pretreatment of the sample to be tested, adopts a new type of solid phase extraction column PRiMEHLB purification, and combines protein precipitation technology to effectively remove most of the matrix interference in the aquatic product sample, and reduce the matrix effect in the later analysis process; the secondary mass spectrogram of the fish matrix of the aquatic product is established by the scanning mode of multiple reaction monitoring-information correlation acquisition-enhanced product ion scanning (MRM-IDA-EPA), which can accurately identify unknown compounds, and at the same time, the peak area information of the compound is obtained by scanning with high selectivity and high sensitivity, and then the compound is accurately quantitatively analyzed. The method of the present invention is fast, simple, green and environmentally friendly, has a good purification effect, has good accuracy and precision, can effectively cope with the sudden and large-scale detection needs of anesthetics and their metabolites in aquatic products, improves the detection efficiency, and reduces the detection limit, and has practical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the chemical combination bonds of the three chromatographic columns in the embodiment;

[0026] Figure 2 It is the quantitative ion chromatogram of the m-aminobenzoic acid ethyl ester methanesulfonate standard product in the embodiment;

[0027] Figure 3 It is the quantitative ion chromatogram of the m-aminobenzoic acid standard in the embodiment;

[0028] Figure 4 The quantitative ion chromatogram of the benzocaine standard in the embodiment;

[0029] Figure 5 The quantitative ion chromatogram of the p-aminobenzoic acid standard in the embodiment;

[0030] Figure 6 is the quantitative ion chromatogram of the acetaminobenzoic acid standard in the embodiment;

[0031] Figure 7 The quantitative ion chromatogram of the chloroprocaine standard in the embodiment;

[0032] Figure 8 It is the quantitative ion chromatogram of the procainamide standard in the embodiment;

[0033] Fig. 9 It is the quantitative ion chromatogram of the lidocaine standard product in the embodiment;

[0034] Fig.10 It is the quantitative ion chromatogram of the dibucaine standard substance in the embodiment;

[0035] Fig.11The quantitative ion chromatogram of the bupivacaine standard in the embodiment;

[0036] Fig.12 It is the quantitative ion chromatogram of the prilocaine standard substance in the embodiment;

[0037] Fig.13 : is the quantitative ion chromatogram of the ropivacaine standard in the embodiment;

[0038] Fig.14 The secondary mass spectrum of ethyl m-aminobenzoate methanesulfonate standard in the embodiment;

[0039] Fig.15 The secondary mass spectrum of the m-aminobenzoic acid standard in the embodiment;

[0040] Fig.16 The secondary mass spectrum of the benzocaine standard in the embodiment;

[0041] Fig.17 The secondary mass spectrum of the p-aminobenzoic acid standard in the embodiment;

[0042] Fig.18 The secondary mass spectrum of the acetaminobenzoic acid standard in the embodiment;

[0043] Fig.19 The secondary mass spectrum of the chloroprocaine standard in the embodiment;

[0044] Fig. 20 The secondary mass spectrum of the procainamide standard in the embodiment;

[0045] Fig.21 The secondary mass spectrum of lidocaine standard in the embodiment;

[0046] Fig. 22 It is the secondary mass spectrum of the dibucaine standard substance in the embodiment;

[0047] Fig.23 The secondary mass spectrum of the bupivacaine standard in the embodiment;

[0048] Fig.24 The secondary mass spectrum of the prilocaine standard in the embodiment;

[0049] Fig.25 is the secondary mass spectrum of the ropivacaine standard in the embodiment;

[0050] Fig.26 It is a comparison diagram of extraction efficiency of different extraction solvents in the embodiment;

[0051] Fig. 27 4 is a comparison chart of the extraction efficiencies of different solid phase extraction columns in the examples. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] A method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry

[0054] 1 Materials and methods

[0055] 1.1 Instruments, reagents and materials

[0056] QTRAP 5500 + Mass spectrometer (product of AB SCIEX, USA); ME204E precision electronic balance (product of Mettler Toledo, USA); vortex mixer (Guangzhou Yike Laboratory Technology Co., Ltd.); Milli-Q ultrapure water machine (Millipore, USA); homogenizer (Guangzhou Yike Laboratory Technology Co., Ltd.).

[0057] Ethyl m-aminobenzoate methanesulfonate (MS-222), m-aminobenzoic acid, benzocaine, p-aminobenzoic acid, p-acetaminobenzoic acid, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, ropivacaine (purity 99.0%, Tianjin Alta Technology Co., Ltd.); formic acid (chromatographically pure, Shanghai Anpu Experiment Technology Co., Ltd.); acetonitrile (mass spectrometry pure, Merck, Germany); ammonium formate (chromatographically pure, Shanghai Anpu Experiment Technology Co., Ltd.); PRiME HLB solid phase extraction column (150 mg / 3 mL, Waters, USA).

[0058] 1.2 Preparation of standard working solution

[0059] (1) Standard stock solution:

[0060] Take ethyl m-aminobenzoate methanesulfonate (MS-222), benzocaine, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, ropivacaine anesthetic standard products and m-aminobenzoic acid, p-aminobenzoic acid, p-acetylaminobenzoic acid anesthetic metabolite standard products in volumetric flasks, dilute to the mark with methanol, and prepare 9 anesthetic standard solutions with a concentration of 100 μg / mL and 3 anesthetic metabolite standard solutions with a concentration of 1000 μg / mL respectively;

[0061] Accurately pipette 1.0 mL each of 100 μg / mL standard solutions of m-aminobenzoic acid ethyl ester methanesulfonate (MS-222), benzocaine, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, and ropivacaine anesthetics and 1.0 mL each of 1000 μg / mL standard solutions of m-aminobenzoic acid, p-aminobenzoic acid, and p-acetylaminobenzoic acid anesthetics into 10 mL volumetric flasks, dilute to the mark with methanol, and obtain 9 anesthetic standard stock solutions with a concentration of 10 μg / mL and 3 anesthetic metabolite standard stock solutions with a concentration of 100 μg / mL, and store them at -20°C in the dark.

[0062] Prepare the mixed standard intermediate working solution: pipette 0.25 mL of each of the 12 standard stock solutions into the same 10 mL volumetric flask, dilute to the mark with methanol, and obtain a mixed standard intermediate working solution containing 9 anesthetics at a concentration of 250 ng / mL and 3 anesthetic metabolites at a concentration of 2500 ng / mL. Store at 4 °C in the dark.

[0063] A series of matrix standard working solutions: accurately pipette an appropriate amount of the mixed standard intermediate working solution, add 500 μL of the same type of negative aquatic product matrix extract and dilute to 1 mL with 10 mmol / L ammonium formate solution to obtain a series of matrix standard working solutions. The concentrations of the matrix standard working solutions are: 9 anesthetic concentrations are 2 ng / mL and 3 anesthetic metabolite concentrations are 20 ng / mL, 9 anesthetic concentrations are 4 ng / mL and 3 anesthetic metabolite concentrations are 40 ng / mL, 9 anesthetic concentrations are 10 ng / mL and 3 anesthetic metabolite concentrations are 100 ng / mL, 9 anesthetic concentrations are 20 ng / mL and 3 anesthetic metabolite concentrations are 200 ng / mL, 9 anesthetic concentrations are 40 ng / mL and 3 anesthetic metabolite concentrations are 400 ng / mL, 9 anesthetic concentrations are 100 ng / mL and 3 anesthetic metabolite concentrations are 1000 ng / mL.

[0064] 1.3 Sample pretreatment

[0065] Accurately weigh 2.5g of the crushed aquatic product sample to be tested into a clean centrifuge tube, add 10mL of a mixed solution of 0.1wt% formic acid acetonitrile solution and water at a volume ratio of 80:20, vortex mix, ultrasonically extract for 20min, centrifuge at 8000r / min for 5min, and wait for purification. Take 2mL of the above supernatant and pass it through the PRiMEHLB solid phase extraction column, collect the effluent, take 500μL of it and dilute it to 1mL with 10mmol / L ammonium formate to obtain the aquatic product sample solution to be tested, and put it on the machine.

[0066] 1.4 Experimental conditions

[0067] 1.4.1 Chromatographic conditions

[0068] Chromatographic column: InfinityLabPoroshell120Aq-C182.1mm×150mm, 2.7μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; flow rate: 0.30mL / min; injection volume: 1μL; column temperature: 40℃; gradient elution conditions are shown in Table 1.

[0069] Table 1 Gradient elution conditions of anesthetics and their metabolites

[0070]

[0071] 1.4.2 Mass spectrometry conditions

[0072] Electrospray ion source; ion source temperature: 550°C; electrospray voltage: 5500 V; scan mode: MRM-IDA-EPI; curtain gas pressure: 30 psi; nebulizer pressure: 55 psi; auxiliary heater pressure: 55.0 psi; collision gas: Medium; mass spectrometry conditions are shown in Table 2.

[0073] Table 2 Mass spectrometry conditions of anesthetics and their metabolites

[0074]

[0075]

[0076] Note: * indicates quantitative ion pair.

[0077] 2 Results and discussion

[0078] 2.1 Optimization of chromatographic column and mobile phase

[0079] Anesthetics and their metabolites have similar structures and different polarities, so a C18 column was selected for separation. The metabolites of anesthetics, m-aminobenzoic acid and p-aminobenzoic acid, are isomers, and the monitored mass spectrometry quantitative ion information is the same, so chromatographic separation is the prerequisite for accurate characterization and quantification.

[0080] The present invention selects three chromatographic columns, Agilent ZORBA XSB-C18 (3.0 mm × 100 mm, 3.5 μm), Agilent Infinity Lab Poroshell 120Aq-C18 (2.1 mm × 150 mm, 2.7 μm), and Agilent Infinity Lab Poroshell 120EC-C18 (2.1 mm × 100 mm, 2.7 μm) for detection. The chemical combination keys of the three chromatographic columns are as follows: Figure 1. Tested under the mobile phase gradient elution conditions of 1.4.1, the results showed that Agilent Infinity Lab Poroshell 120 Aq-C18 had good separation of 12 target compounds, and the chromatographic peaks were symmetrical and sharp. This chromatographic column can improve the retention of polar compounds, can analyze polar and non-polar compounds at the same time, is compatible with 100% aqueous mobile phase, and has low pH stability. When using the ZORBAX SB-C18 column, there are interferences and split peaks near p-aminobenzoic acid, and the peak shape of acetylaminobenzoic acid is poor; when using the Poroshell 120 EC-C18 column, m-aminobenzoic acid has split peaks and poor response, and there are interferences near p-aminobenzoic acid.

[0081] Anesthetics and their metabolites all contain -NH2 or C=O in their structural formulas, and have lone pairs of electrons that can form [M+H] through a proton. + , in the form of positive ions, so the positive ion mode was used. In order to obtain the lowest baseline response and higher target response in this scanning mode, when 0.1% formic acid water-acetonitrile was used as the mobile phase, the target substances were completely separated, with sharp and symmetrical peaks and high sensitivity. Quantitative ion chromatograms of 9 anesthetics and 3 anesthetic metabolites are shown in Figure 2. Figure 2-Figure 13 .

[0082] 2.2 Establishment of MRM-IDA-EP1 scanning mode

[0083] The 9 anesthetic concentrations were 20 ng / mL, and the 3 anesthetic metabolite concentrations were 200 ng / mL. The anesthetic and metabolite matrix standard working solutions were taken respectively. The optimal DP and CE voltages were optimized and obtained by finding suitable parent ions and daughter ions, and finally the MRM parameters were obtained. Then, using the MRM-IDA-EPI mode, when a chromatographic peak appeared in a certain channel during the sampling process of several MRMs, the peak status was judged in real time by IDA Criteria. When the intensity of the acquisition signal exceeded the preset value, the software guided the mass spectrometer to automatically switch Q3 to a linear ion trap within 1 ms, and then the enhanced secondary ion full scan mass spectrum of the corresponding compound was obtained. The EPI mass spectral library of standard substances with different matrices was established to assist in its qualitative analysis, and the data collected by MRM can also be used as data for quantitative analysis. Compared with the MRM mode, the EPI spectrum is collected by an ion trap, which enhances the scanning of secondary fragment ions and improves sensitivity. It is also a full-scan mass spectrum with richer confirmation information. It can effectively assist in the qualitative confirmation of trace level or detection limit concentration samples and the identification of drug metabolites, making up for the qualitative difficulties of the traditional quadrupole MRM mode for low-concentration samples or complex matrices.

[0084] 2.3 Establishment of the secondary mass spectrum of anesthetics

[0085] The present invention solves the false positive problem caused by the complex matrix of aquatic products through the MRM-IDA-EPI scanning mode of the QTRAP mass spectrometry system, and establishes the secondary mass spectra of aquatic product anesthetics and their metabolites by preparing standard working solutions through the matrix. The EPI mode is the enhanced daughter ion scanning mode, which can obtain a secondary fragment spectrum with higher sensitivity and a full mass range of different energy fragmentation. The software automatically compares with the spectral library of the matrix standard working solution to help more accurate screening and qualitative analysis. The secondary mass spectra of 9 anesthetics and their 3 anesthetic metabolites are shown in Figure 14-Figure 25 .

[0086] 2.4 Optimization of extraction solvent

[0087] In animal-derived aquatic products, the commonly used extraction solvents for veterinary drug residues are acetonitrile, acetonitrile-water, methanol-water, acidified acetonitrile, acidified acetonitrile-water, etc. The extraction solvent is a strong polar solvent, and the matrix of aquatic products is complex and rich in protein. After adding a strong polar solvent, it is easy to denature into agglomerates, which is not conducive to the extraction of anesthetics and their metabolites. Adding a homogenous proton first and then adding an organic solvent can improve the extraction efficiency. Considering that some anesthetics and their metabolites are easily soluble in water and are primary amine compounds, adding formic acid to acetonitrile helps to protonate the amino group, so acidified acetonitrile water is selected as the extraction solvent, and different concentrations of 0.1% formic acid acetonitrile: water (volume ratio 80:20), 0.2% formic acid acetonitrile: water (volume ratio 80:20), 0.5% formic acid acetonitrile: water (volume ratio 80:20), and 1.0% formic acid acetonitrile: water (volume ratio 80:20) are compared as extraction solvents. The experiment found that when 0.5% formic acid acetonitrile: water (volume ratio 80:20) was used as the extraction solvent, the recovery rate of aminobenzoic acid and procainamide was higher; when 1.0% formic acid acetonitrile: water (volume ratio 80:20) was used as the extraction solvent, the recovery rate of lidocaine was higher; considering comprehensively, 0.1% formic acid acetonitrile: water (volume ratio 80:20) had the best extraction efficiency. Fig.26 .

[0088] 2.5 Selection of pass-through solid phase extraction columns

[0089] The best purification effect of anesthetics and their metabolites in aquatic products is by HLB purification, but traditional HLB purification requires activation, loading, elution, concentration, and re-dissolution, which is cumbersome and time-consuming, and requires high standardized operation of the inspectors. As a new type of column passing method, the pass-through solid phase extraction column is becoming more and more popular among inspectors. Therefore, choosing a suitable pass-through HLB solid phase extraction column can not only improve work efficiency, but also ensure the accuracy of detection. The more common pass-through HLB solid phase extraction columns on the market are OasisPRiMEHLB, CNWBOND phospholipid-free PPR columns, and HLBLim columns. Studies have shown that OasisPRiMEHLB has the best purification effect, and the treated samples are relatively clean and have the highest recovery rate. It can effectively remove various interferences such as fats, phospholipids, pigments, etc. in the samples and reduce the matrix effect; at the same time, it can greatly simplify the pretreatment process, eliminating the activation and equilibration steps. After extraction, the sample can be directly passed through the column, saving a lot of time and reagents, making the pretreatment simpler and more efficient. The extraction efficiency of the three pass-through solid phase extraction columns is shown in Figure 2. Fig. 27 .

[0090] 2.6 Methodological evaluation

[0091] 2.6.1 Linear range, detection limit and quantification limit

[0092] In order to reduce the influence of matrix effect, the negative fish sample matrix was treated as described in "1.3". 500 μL of matrix extract was taken, and an appropriate amount of standard intermediate working solution was added. The volume was adjusted to 1 mL with 10 mmol / L ammonium formate to prepare a series of matrix standard working solutions. The method detection limit and quantification limit of 12 anesthetics and their metabolites were calculated with the signal-to-noise ratio S / N ≥ 3 and S / N ≥ 10. The results are shown in Table 3. As can be seen from Table 3, the correlation coefficients are all greater than 0.99, indicating that within the appropriate range, the 9 anesthetics and the 3 anesthetic metabolites all have a good linear relationship.

[0093] Table 3 Linear equations, correlation coefficients, linear ranges, detection limits and quantification limits of 12 anesthetics and their metabolites

[0094]

[0095]

[0096] 2.6.2 Recovery and precision

[0097] Within the range of the standard curve, three concentrations of mixed standard solutions of low, medium and high concentrations were added to the negative fish meat for spiked test. Each concentration point was measured 6 times in parallel, and the matrix standard curve was used for quantitative calculation. The recovery and precision were calculated. The results are shown in Table 4. The results showed that the average spiked recoveries of 9 anesthetics and 3 anesthetic metabolites were 84.3-109.2%, and the precision was 0.61-4.76%, which proved that the recovery and precision of this method were good and met the detection requirements of anesthetics and their metabolites in aquatic products.

[0098] Table 4 Recovery and precision

[0099]

[0100]

[0101] 2.7 Actual sample measurement

[0102] The method of the present invention was applied to 30 samples (including eel, grass carp, bream, black carp, sea bass, crucian carp, tango fish, loach, white shrimp, grass shrimp, river shrimp, silver carp, turbot, carp, salmon) randomly purchased from supermarkets and farmers' markets. The results showed that ethyl m-aminobenzoate methanesulfonate (MS-222) was detected in one grass carp and lidocaine was detected in one river shrimp, and no anesthetics and their metabolites were detected in all other samples. According to the software results, the retention time and secondary fragment ion ratio of the sample met the requirements, and the purity score of the measured secondary spectrum matching the database was more than 90 points, which reflected the EPI advantage of QTRAP mass spectrometry. Highly sensitive secondary fragment spectra can still be obtained for low-concentration compounds in complex aquatic product matrices, which helps to better distinguish false positives and false negatives and ensures that the qualitative results are accurate. Quantitative analysis using MRM data revealed that the content of ethyl m-aminobenzoate methanesulfonate (MS-222) in grass carp was 16.2 μg / kg, and the content of lidocaine in river shrimp was 20.1 μg / kg.

[0103] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry, characterized in that: The following steps are involved: (a) Prepare a series of matrix standard working solutions: Using methanol as solvent, a mixed standard intermediate working solution was prepared, which contained 9 anesthetics, namely, ethyl m-aminobenzoate methanesulfonate, benzocaine, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, and ropivacaine, at a concentration of 250 ng / mL, and 3 anesthetic metabolites, namely, m-aminobenzoic acid, p-aminobenzoic acid, and p-acetylaminobenzoic acid, at a concentration of 2500 ng / mL. Different amounts of mixed standard intermediate working solutions were respectively transferred, 500 μL of negative aquatic product matrix extract was added to each, and the solution was fixed to 1 mL with 10 mmol / L ammonium formate solution to prepare a series of matrix standard working solutions; (b) Sample pretreatment: Take 2.5 g of the sample to be tested, add 10 mL of a mixed solution of 0.1 wt% formic acid acetonitrile solution and water in a volume ratio of 80:20, mix well, perform ultrasonic extraction, and centrifuge; take 2 mL of the supernatant after centrifugation and pass it through a PRiME HLB solid phase extraction column, collect the effluent, take 500 μL of it and dilute it to 1 mL with 10 mmol / L ammonium formate to obtain a sample solution of aquatic products to be tested; (c) Detection: The MRM-IDA-EPA mode was used to measure a series of matrix standard working solutions by QTRAP mass spectrometry to obtain MRM data and secondary fragment full spectrum data, and a secondary mass spectrum standard library of 9 anesthetics and 3 anesthetic metabolites in aquatic products was established. The MRM-IDA-EPA mode was used to measure the sample solutions of aquatic products by QTRAP mass spectrometry. The contents of 9 anesthetics and 3 anesthetic metabolites in the aquatic product samples were obtained by automatic comparison with the secondary mass spectrum standard library and quantitative analysis based on the peak area of ​​the MRM data.

2. According to claim 1, a method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry, characterized in that: The concentrations of the matrix standard working solutions prepared in step (a) are: 9 anesthetic concentrations are all 2 ng / mL and 3 anesthetic metabolite concentrations are all 20 ng / mL, 9 anesthetic concentrations are all 4 ng / mL and 3 anesthetic metabolite concentrations are all 40 ng / mL, 9 anesthetic concentrations are all 10 ng / mL and 3 anesthetic metabolite concentrations are all 100 ng / mL, 9 anesthetic concentrations are all 20 ng / mL and 3 anesthetic metabolite concentrations are all 200 ng / mL, 9 anesthetic concentrations are all 40 ng / mL and 3 anesthetic metabolite concentrations are all 400 ng / mL, 9 anesthetic concentrations are all 100 ng / mL and 3 anesthetic metabolite concentrations are all 1000 ng / mL.

3. The method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry according to claim 1, characterized in that: The specific preparation method of the mixed standard intermediate working solution in step (a) is as follows: using methanol as a solvent, first preparing 9 anesthetic standard stock solutions with a concentration of 10 μg / mL and 3 anesthetic metabolite standard stock solutions with a concentration of 100 μg / mL respectively; then taking the 9 anesthetic standard stock solutions and the 3 anesthetic metabolite standard stock solutions and mixing them, and making up the volume with methanol to prepare a mixed standard intermediate working solution.

4. The method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry according to claim 1, characterized in that: The chromatographic conditions for the QTRAP mass spectrometry detection series matrix standard working solutions and aquatic product sample solutions are: Chromatographic column: InfinityLab Poroshell 120Aq-C18 2.1mm×150mm, 2.7μm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.30mL / min; injection volume: 1μL; column temperature: 40℃; gradient elution conditions: 0-3.0min, mobile phase A decreased from 95% to 85%, mobile phase B increased from 5% to 15%; 3.0-6.0min, mobile phase A decreased from 85% to 60%, mobile phase B increased from 15% to 40%; 6.0-12.0min, mobile phase A decreased from 60% to 5%, and mobile phase B increased from 40% to 95%; 12.0-14.0min, mobile phase A was maintained at 5%, and mobile phase B was maintained at 95%; 14.0-14.1min, mobile phase A increased from 5% to 95%, and mobile phase B increased from 95% to 5%; 14.1-17.0min, mobile phase A was maintained at 95% and mobile phase B was maintained at 5%.

5. The method for rapid determination of multiple anesthetics and their metabolites in aquatic products based on QTRAP mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions for the QTRAP mass spectrometry detection of the matrix standard working solutions and the aquatic product sample solutions are: Electrospray ion source; ion source temperature 550°C; electrospray voltage 5500V; curtain gas pressure: 30psi; nebulizer pressure 55psi; auxiliary heater pressure: 55.0psi; collision gas: Medium; parent ions, daughter ions and mass spectrometry parameters of 9 anesthetics and 3 anesthetic metabolites are shown in the following table: The * in the table indicates quantitative ion pairs.

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