CN113092604B - Rapid detection method for three common anesthetics in aquatic product - Google Patents
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Abstract
The invention discloses a rapid detection method for three common anesthetics in aquatic products, belonging to the technical field of detection. The invention utilizes a single quadrupole gas chromatograph-mass spectrometer (GC-MS) for screening, quantifying and confirming, adopts a Thermo TG 1701MS capillary column as an analytical column, and establishes a method for measuring the contents of three anesthetics of Eugenol (Eugeniol, CAS: 97-53-0), Isoeugenol (Isoeugenol, CAS: 97-57-1) and 3-ethyl aminobenzoate methylsulfonate (MS-222, CAS: 886-86-2) in aquatic products in a positive ion selective ion monitoring mode (SIM). The detection limit of the method is 25.0 mu g/kg of eugenol and isoeugenol, and 50.0 mu g/kg of ethyl 3-aminobenzoate methyl sulfonate. Compared with the traditional instrument analysis method, the detection method is simpler and faster, has good linear correlation, and has the characteristics of high sensitivity, matrix interference resistance, high accuracy and high stability.
Description
Technical Field
The invention belongs to the technical field of product safety and detection, and particularly relates to a rapid detection method for three common anesthetics in aquatic products.
Background
With the improvement of living standard of people, the demand of fresh and live aquatic products is increasing day by day. The long-distance transportation of living aquatic products can easily cause stress reaction of living aquatic animals, and the phenomena of bruising and mass death occur. Anesthesia of live aquatic animals during transport is an effective means to increase the volume of transport and survival, and therefore the use of anesthetics during distribution has become a norm. Several anesthetics have been approved for use in edible aquatic products by countries of the european union, united states, japan, korea, australia, new zealand, chile, etc. Currently, there are the anesthetics approved for use abroad: ethyl 3-aminobenzoate methanesulfonate (MS222), Eugenol (Eugenol) and Isoeugenol (Isoeugenol). MS222 has good anesthesia effect and high metabolism speed, and is allowed to be used in most countries; isoeugenol is the main ingredient of many commercial fishery anesthetics; eugenol is widely used in the domestic aquatic product transportation field due to the characteristics of high efficiency, low price and convenient purchase. Currently, no anesthetic is approved in China for edible aquatic products, and no relevant limit standard is established. The aquatic product dealer is usually experienced in use, and the use amount is not considered as long as the aquatic product dealer does not cause mass death. After the live aquatic product is transported to a destination, a seller sells the live aquatic product only when the fishes and the shrimps are awake, the necessary drug stopping period is not carried out for temporary rearing, and the live fishes sold to the hands of consumers still have high anesthetic residues.
Therefore, a high-efficiency detection method for the anesthetic in the aquatic product needs to be developed and formulated so as to provide a detection means for related scientific research work, and provide technical support for the industry competent department to master the residual condition of the anesthetic in the aquatic product and carry out risk management on the anesthetic for fishing.
CN 202010138679.6 discloses a method for rapidly detecting eugenol anesthetic in aquatic products, which comprises the steps of preparing a separation liquid, preparing a liquid to be detected, detecting an olefin double bond structure, generating a carbamate structure, and detecting and judging the carbamate structure, wherein the method is used for judging whether the eugenol anesthetic is contained in the aquatic products by utilizing the color reaction of potassium permanganate, converting the carbamate structure and detecting the carbamate structure. The method can accurately detect whether the eugenol anesthetic is added in the aquatic product or not under the condition of not using large instruments. The invention has the following disadvantages: (1) detection by color reaction requires a high concentration level of the target substance, and high sensitivity detection is difficult to achieve, and specific values of the sensitivity of the method are not mentioned in the patent. (2) According to the principle of the detection method, the compound with the olefin double bond structure in the extracting solution is actually detected, but the olefin double bond structure is not specific to eugenol, so that the method is easy to generate false positive at a low concentration level. (3) MS222 is currently the most widely tolerated anesthetic agent in the world and this method does not detect this compound.
The invention discloses a CN 201811030195.9 method for analyzing and rapidly detecting seven anesthetics in aquatic products, and belongs to the technical field of detection. The method for determining 7 anesthetics such as eugenol, methyl eugenol, isoeugenol, eugenol acetate, methyl isoeugenol, isoeugenol acetate and tricaine in aquatic products is established under an electrospray positive ion multiple reaction monitoring mode (MRM) by utilizing the principle of triple quadrupole rod chromatography mass spectrometry (GC-MS/MS) screening, quantification and confirmation and by adopting a silica gel solid phase extraction column and an HP-5MSUI capillary column as an analysis column. Compared with the traditional instrument analysis method, the detection method is simpler and faster, has good linear correlation and low detection limit of 2.0 mu g/kg, and has the characteristics of high sensitivity, matrix interference resistance, high accuracy and high stability. The deficiency of the invention is in two aspects: (1) the pretreatment process adopts a silica gel solid phase extraction column, the operation is more complicated, and the pretreatment of a large batch of samples is difficult to realize in a short time. (2) The triple quadrupole gas mass spectrometer is expensive in equipment, is not popularized in the equipment of scientific research and detection institutions, and is difficult to popularize and use.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting three common anesthetics in aquatic products.
In order to achieve the purpose, the technical scheme provided by the invention is as follows: a method for detecting three anesthetics in an aquatic product by adopting a single quadrupole gas chromatograph-mass spectrometer, wherein the three anesthetics are ethyl 3-aminobenzoate methyl sulfonate, eugenol and isoeugenol respectively, and the method comprises the following steps:
the first step is as follows: extracting an anesthetic from the aquatic product and purifying to obtain a sample liquid to be detected;
1) placing the homogenized sample in a first centrifuge tube, adding an extracting solution, wherein the mass-volume ratio of the sample to the extracting solution is 1:6.5-7.5g/ml, and the extracting solution is acetonitrile: a mixed solution of 8.5:1.5-7.5:2.5 by volume of dichloromethane; uniformly mixing the extracting solution and the sample, performing ultrasonic extraction for 5-8 min, centrifuging for 5-15min at 3000-;
2) liquid-liquid extraction purification, comprising the following steps:
s1, sequentially adding dichloromethane and 0.005-0.015M NaOH solution into the clear liquid of the second centrifugal tube, fully mixing and standing; the adding amount of the dichloromethane is that the content of the dichloromethane in the organic phase in the supernatant exceeds 35 percent, and the adding amount of the NaOH solution is that the pH value of the supernatant is between 8 and 12;
s2, 3000-4000 rpm centrifugation for 4-10 min; after centrifugal layering, measuring the upper-layer aqueous phase solution by using pH test paper, if the pH is close to neutral, adjusting the pH to 9-10 by using 0.05-0.15M NaOH solution, and then mixing and centrifuging again;
s3, transferring the lower organic clear liquid obtained in the step S2 into a fourth centrifugal tube, wherein the lower organic clear liquid is a liquid to be detected;
the second step is that: and analyzing the sample liquid to be detected obtained in the first step by adopting a single quadrupole gas chromatograph-mass spectrometer.
The preferable technical scheme is as follows: the extracting solution is as follows: acetonitrile + dichloromethane ═ 8+2 (V/V).
The preferable technical scheme is as follows: in a first step 1) of the method for extracting an anesthetic from a water product, the method comprises: placing 1.00g of a homogeneous sample in a first centrifuge tube, adding 5mL of extracting solution, performing vortex mixing, performing ultrasonic extraction for 5-8 min, centrifuging for 10min at 3500 rpm, transferring supernatant into a second centrifuge tube, adding 2mL of extracting solution into the first centrifuge tube, performing vortex mixing, performing ultrasonic extraction for 5-8 min, centrifuging for 10min at 3500rp rpm, combining supernatants in a second centrifuge tube, and performing liquid-liquid extraction purification.
The preferable technical scheme is as follows: in the first step 2), the liquid-liquid extraction purification comprises the following steps:
s1, 3.5mL of dichloromethane and 2mL of 0.01M NaoH solution were sequentially added to the supernatant of the second centrifuge tube, mixed well, and allowed to stand for 5 min.
S2, 3500 rpm/min centrifugation for 5 min. After centrifugal layering, using pH test paper to detect that the upper layer solution is alkalescent (the pH is more than 9, if the pH is close to neutral, using 0.1M NaoH solution to adjust the pH to be 9-10, and mixing and centrifuging again);
s3, transferring the upper aqueous phase solution to another 15mL plastic centrifuge tube. Transferring the lower organic clear liquid into a 15mL glass centrifuge tube;
s4, adding 4mL of dichloromethane into the plastic centrifuge tube filled with the upper aqueous phase solution, fully mixing, standing for 3min, centrifuging for 3min at 3500 rpm, removing the upper aqueous phase by using a disposable plastic pipette, and combining the lower organic phase clear liquid in the glass centrifuge tube.
In a preferred technical scheme, the first step 2) further comprises the following steps: s5, heating at 40 ℃ by using a nitrogen blower, blowing nitrogen to about 1mL, adding 200uL (ethyl acetate) of 2ug/mL internal standard solution, diluting to 2mL by using ethyl acetate, carrying out vortex mixing, then passing through a 0.22-micron organic microporous filter membrane, and analyzing the filtrate by using a single-quadrupole gas chromatograph-mass spectrometer.
The preferable technical scheme is as follows: the liquid-liquid extraction adopts dichloromethane and 0.01M NaoH solution, and the pH of an aqueous phase is more than 9 during extraction;
the preferred technical scheme is as follows: the internal standard liquid is a mixture of methyl eugenol and 3-aminobenzoate.
The preferred technical scheme is as follows: during liquid-liquid extraction, the content of dichloromethane in the organic phase is more than 35 percent; for example 40%, 45%, 50%, 60%, 70%. Preferably 35 to 50%.
The preferable technical scheme is as follows: the gas chromatographic analysis conditions were: a chromatographic column: thermo TG 1701MS capillary column (30 m.times.0.320 mm.times.0.25 μm); GC conditions were as follows: carrier gas: high-purity helium (99.9995%), constant flow rate 1.0 mL/min; sample inlet temperature: 270 ℃; temperature rising procedure: the initial temperature is 80 deg.C, maintaining for 0.5min, then raising to 122 deg.C at 7 deg.C/min, maintaining for 15min, then raising to 150 deg.C at 7 deg.C/min, maintaining for 8.5min, raising to 240 deg.C at 35 deg.C/min, and maintaining for 4 min; and (3) sample introduction mode: no flow diversion; sample introduction volume: 1.0. mu.L.
The preferable technical scheme is as follows: the analysis conditions of the single-quadrupole gas chromatograph-mass spectrometer are as follows: selecting an ion monitoring mode; an ion source: an EI source; ion source temperature: 270 ℃; ionization energy: 70 eV; solvent retardation: 15.0 min; transmission line temperature: at 260 ℃.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the pretreatment of the detection of the three anesthetic residues in the aquatic products adopts a purification mode of small-volume liquid-liquid extraction, and the operation is simple, convenient and quick. The solid phase extraction column with higher cost and complex operation is avoided, the detection of a large batch of samples is realized, the period is short, and the cost is low.
2. The method simultaneously detects the residues of three anesthetics in the aquatic products, and adopts a single quadrupole gas chromatograph-mass spectrometer for analysis. The detection by using an expensive tandem quadrupole mass spectrometer is avoided. The single quadrupole mass spectrometer is equipped in most scientific research departments and detection institutions for relevant research on the fishing anesthetic, and the method is easy to popularize and use.
3. The volume ratio of acetonitrile to dichloromethane is 8:2, so that three anesthetics are extracted by using the extracting solution, gel formation during layering can be avoided, the polarity of the extracting solution is considered to be different from that of eugenol, isoeugenol and MS222, and the extraction efficiency is high;
4. during liquid-liquid extraction, the content of dichloromethane in the organic phase is more than 35 percent, and effective layering can be realized.
Ambient conditions of pH >8 are effective to inhibit MS222 from entering the aqueous phase, thus controlling the pH of the aqueous phase at delamination to > 9.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a total ion flow diagram of a single-quadrupole gas chromatograph-mass spectrometer for detecting three anesthetic standard solutions
FIG. 2 is a single-quadrupole gas chromatograph-mass spectrometer ion flow diagram for selecting three anesthetic standard solutions
FIG. 3 is a diagram of a quadrupole GC-MS detection ion flow diagram for the selection of three anesthetics in an aquatic product (blank fish flesh matrix theory quantitative limit level addition)
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
The equipment, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: method for detecting three common anesthetics in aquatic product by single-quadrupole gas chromatograph-mass spectrometer
1 materials and methods
(1) Instruments and reagents
Single quadrupole mass spectrometer (thermolfisher DSQ); nitrogen blower (Tianjin Hengao HGC-24); vortex mixer (IKA MS 2); centrifuge (Beijing medical); ultrapure water Milli-Q (Milliporeco); electronic balance (METTLER).
Eugenol (Eugenol, CAS: 97-53-0, purity 99.5%), Methyl Eugenol (Methyl Eugenol, CAS: 93-15-2, purity 99.5%), Isoeugenol (Isoeugenol, CAS: 97-57-1, purity 98.8%), available from Dr. Ehrenstontorfer; ethyl 3-aminobenzoate methanesulfonate (MS-222, CAS: 886-86-2, 98% purity), 3-aminobenzoate (Methyl-3-aminobenzoate, CAS: 4518-10-9, 98.5% purity) purchased from Merck; acetonitrile, dichloromethane and ethyl acetate are used as chromatographic purity, NaoH is analytical purity, and all water is ultrapure water.
(2) Solution preparation
Standard stock solution (1000. mu.g/mL): accurately weighing eugenol, isoeugenol, 3-ethyl aminobenzoate methylsulfonate, methyl eugenol and 3-methyl aminobenzoate standard substances respectively 10.0mg, dissolving with acetonitrile respectively, metering to 10mL, preparing into 1000 μ g/mL standard stock solution, and refrigerating at-18 deg.C.
Standard dilution solution (10.0. mu.g/mL): accurately sucking 100 μ L standard stock solution of three anesthetics such as eugenol, isoeugenol, 3-ethyl aminobenzoate methylsulfonate, etc., diluting to 10mL with ethyl acetate to obtain 10.0 μ g/mL standard diluted solution, and refrigerating at 4 deg.C.
Internal standard dilution solution (10.0. mu.g/mL): accurately sucking 100 mu L of two anesthetic standard stock solutions of methyl eugenol and methyl 3-aminobenzoate, diluting to 10mL with ethyl acetate to obtain 10.0 mu g/mL internal standard diluted solution, and refrigerating at 4 ℃ for storage.
Extracting solution: 400mL of acetonitrile was mixed with 100mL of dichloromethane.
(3) Pretreatment method
Weighing 1.00g of a homogeneous sample, placing the homogeneous sample in a first centrifuge tube, adding 5mL of extracting solution, performing vortex mixing, performing ultrasonic extraction for 5min, performing 3500 r/min centrifugation for 10min, transferring supernatant into a second centrifuge tube after centrifugation is finished, adding 2mL of extracting solution into the first centrifuge tube, performing vortex mixing, performing ultrasonic extraction for 5min, performing 3500 r/min centrifugation for 10min, combining the supernatants in the second centrifuge tube, and performing liquid-liquid extraction purification.
3.5mL of dichloromethane and 2mL of 0.01M NaoH solution are sequentially added into the clear liquid of the second centrifuge tube, fully mixed, kept stand for 5min, and centrifuged for 5min at 3500 rpm. After centrifugal layering, using pH test paper to detect that the upper-layer aqueous phase solution is alkaline (pH is greater than 9, if the pH is close to neutral, using 0.1M NaoH solution to adjust the pH to be 9-10, and mixing and centrifuging again); transferring the upper aqueous phase solution into another 15mL plastic centrifuge tube, and transferring the lower organic clear liquid into a 15mL glass centrifuge tube; adding 4mL of dichloromethane into a plastic centrifuge tube filled with the upper aqueous phase solution, fully mixing, standing for 3min, centrifuging for 3min at 3500 rpm, removing the upper aqueous phase by using a disposable plastic pipette, and combining the lower organic phase clear liquid in a glass centrifuge tube; heating at 40 deg.C with nitrogen blower, blowing nitrogen gas to about 1mL, adding 2ug/mL internal standard solution 200uL (diluted with ethyl acetate), diluting to 2mL with ethyl acetate, vortex mixing, and filtering with 0.22 μm organic microporous membrane to obtain filtrate, and analyzing with single quadrupole gas chromatograph-mass spectrometer.
(4) Conditions of the apparatus
Gas chromatography conditions: a chromatographic column: thermo TG 1701MS capillary column (30 m.times.0.32 mm.times.0.25 μm); GC conditions were as follows: carrier gas: high-purity helium (99.9995%), constant flow rate 1.0 mL/min; sample inlet temperature: 270 ℃; temperature rising procedure: the initial temperature is 80 deg.C, maintaining for 0.5min, then raising to 122 deg.C at 7 deg.C/min, maintaining for 15min, then raising to 150 deg.C at 7 deg.C/min, maintaining for 8.5min, raising to 240 deg.C at 35 deg.C/min, and maintaining for 4 min; and (3) sample introduction mode: no flow diversion; sample introduction volume: 1.0. mu.L.
Single quadrupole mass spectrometry conditions: selecting an ion monitoring mode; an ion source: an EI source; ion source temperature: 270 ℃; ionization energy: 70 eV; solvent retardation: 15.0 min; transmission line temperature: at 260 ℃. The monitoring ions are shown in Table 1
TABLE 1 monitoring ions of three anesthetics and their internal standards
Note: quantitative Ion Note and Quantitative Ion.
2. Results
2.1 Total ion flowgram
Under the above instrument conditions, the total ion flow pattern of the three anesthetics is shown in FIG. 1. It can be seen from the figure that the target compound not only achieves complete separation and good peak shape, but also has reasonable retention time and no impurity interference at the peak position of the target compound.
2.2 specificity analysis
The mass spectra of the three anesthetics and the two internal standards in the Selective Ion Monitoring (SIM) mode are shown in fig. 2. Under the experimental condition, the mass spectrum data adopts a segmented selection ion monitoring mode, and is divided into 5 segments according to the peak output time of the target object, so that the cycle time required by scanning is shortened, the number of data points obtained on each peak is increased, and the stability and the sensitivity of the result are ensured. After 35min, the column temperature is quickly raised to 240 ℃, so that impurities with high boiling point in the column are left to flow out, and the impurities are prevented from interfering the analysis of the next sample. As can be seen from the selected ion current (SIM) spectra of each target, although different targets have the same monitoring ions, the chromatographic retention times of the targets are greatly different, and the qualitative and quantitative properties of each target are not influenced.
2.3 Linear relationship and detection and quantitation limits
And (3) analyzing the standard solution according to the instrument condition in the step (4), drawing a standard curve by taking the concentration of the target as an abscissa and the peak area of the target as an ordinate, and calculating a regression equation and a correlation coefficient (see table 2 in detail). The correlation coefficients R are all larger than 0.999, which shows that the linear relation between the concentration and the peak area of the three anesthetics in the range of 0.005-1.0 mu g/mL is good.
According to the fact that the signal-to-noise ratio (S/N) of a quantitative ion chromatographic peak is larger than 10, and the recovery rate and the relative standard deviation are determined to be within the range, the quantitative Limit (LOQ) of the method is determined, the eugenol and the isoeugenol are 25.0 mu g/kg, and the MS222 is 50.0 mu g/kg; the signal-to-noise ratio (S/N) of the quantitative ion chromatographic peak is greater than 3 and is the detection Limit (LOD), the eugenol and isoeugenol are 10.0 mu g/kg, and the MS222 is 20.0 mu g/kg. FIG. 3 shows that the blank fish meat matrix is added at the theoretical quantitative limit level, and the samples have no impurity peak interference and good peak patterns near the peak position of the target object, so that the qualitative and quantitative determination can be carried out.
TABLE 2 Linear regression equation for three anesthetics
2.4 accuracy and precision
Adding substances to be tested into blank samples respectively for weever, eel, penaeus vannamei and blue crab matrixes to enable the theoretical contents of eugenol and isoeugenol to be 25.0 mug/kg, 100.0 mug/kg and 250.0 mug/kg; the theoretical MS222 contents of 50.0. mu.g/kg, 200.0. mu.g/kg and 500.0. mu.g/kg were subjected to the above experimental procedure and recovery tests were carried out at 3 levels (see Table 3), with 6 replicates for each blank matrix and each concentration, each for 6 days. The intra-and inter-batch precision was calculated as Relative Standard Deviation (RSD). The result shows that the recovery rate of the method is between 80.8% and 109%, the relative standard deviation is between 3.24% and 14.2%, the GB/T27404-2008 requirement is met, and the method is high in accuracy and precision and capable of meeting the detection requirement.
TABLE 3 recovery and precision of three anesthetics for different matrix blanks
Eugenol and isoeugenol have large polarity difference with MS222, and if dichloromethane with weaker polarity is used for extraction, the recovery rate of MS222 is lower; the methanol with stronger polarity is used for extraction, and the layered extraction and purification cannot be realized. Extraction with pure acetonitrile allows the organic phase to be separated from the aqueous phase by addition of dichloromethane. However, pure acetonitrile is completely adopted for extraction, and when liquid-liquid layering extraction is carried out, a thick gel layer can be generated between two phases for some aquatic products, so that the recovery rate of a target object is influenced. The volume ratio of acetonitrile to dichloromethane is respectively adopted as follows: carrying out ultrasonic extraction on mixed liquor in four proportions of 9:1, 8:2, 7:3 and 6: 4. The result shows that the volume ratio of acetonitrile to dichloromethane is more appropriate to be 8:2, not only can the formation of gel layer during layering be overcome, but also the polarity of the extracting solution is considered to be the difference between eugenol, isoeugenol and MS222, and the extraction efficiency is high.
The liquid-liquid extraction process requires ensuring that the organic phase (acetonitrile + dichloromethane) and the aqueous phase are separated, the ratio of dichloromethane to acetonitrile being important. Experimental results show that the content of dichloromethane in the organic phase exceeds 35%, and effective layering can be realized. In this example, 7mL of the extract (1.4 mL of methylene chloride) was used for extraction, and 3.5mL of methylene chloride was added for liquid-liquid purification, and the total volume of the organic phase was 10.5mL, the volume of methylene chloride was 4.9mL, and the content of methylene chloride in the organic phase was about 46%, thereby ensuring the effect of separation.
The pH value of the water phase has great influence on the recovery rate of the target substance in the liquid-liquid extraction process. Eugenol and isoeugenol are not easy to ionize in weak acid and alkali environments, while MS222 enters a water phase due to ionization in neutral and acidic environments. In the liquid-liquid extraction process, the aqueous phase is respectively prepared by adding standard substances into solutions with pH values of 5, 6, 7, 7.6, 8, 9, 10, 11 and 12, and the concentrations of the three anesthetics in the organic phase are measured. The result shows that the pH is 5-12, and both the eugenol and the isoeugenol are kept in the organic phase; the MS222 will partially enter the aqueous phase under acidic and neutral conditions, and environmental conditions with pH >8 are effective to inhibit the MS222 from entering the aqueous phase, thus controlling the pH of the aqueous phase to >9 upon stratification.
The method is based on a single-quadrupole gas chromatography-mass spectrometry technology, adopts a selective ion monitoring mode, can simultaneously screen, quantify and confirm three anesthetics in aquatic products such as fish, shrimps and crabs according to retention time, characteristic ions and abundance ratios thereof, has the characteristics of high sensitivity, matrix interference resistance, high accuracy and high stability, can meet the detection requirements of residual amounts of eugenol, isoeugenol and MS-222 anesthetics in the aquatic products, and has certain significance for guaranteeing the health of consumers and maintaining the safety of the aquatic product industry in China.
Claims (6)
1. A rapid detection method for three common anesthetics in aquatic products, wherein the three anesthetics are ethyl 3-aminobenzoate methyl sulfonate, eugenol and isoeugenol respectively, and the method comprises the following steps:
the first step is as follows: extracting anesthetic from aquatic products and purifying to obtain a sample solution to be detected:
1) placing the homogenized sample in a first centrifuge tube, adding an extracting solution, wherein the mass-volume ratio of the sample to the extracting solution is 1:6.5-7.5g/ml, and the extracting solution is acetonitrile: a mixed solution of 8.5:1.5-7.5:2.5 by volume of dichloromethane; uniformly mixing the extracting solution and the sample, performing ultrasonic extraction for 5-8 min, centrifuging for 5-15min at 3000-;
2) liquid-liquid extraction purification, comprising the following steps:
s1, sequentially adding dichloromethane and 0.005-0.015M NaOH solution into the clear liquid of the second centrifugal tube, fully mixing the organic phase with the dichloromethane content of more than 35%, and standing; the addition amount of the NaOH solution is that the pH value of the supernatant is 9-12;
s2, 3000-4000 rpm centrifugation for 4-10 min; after centrifugal layering, measuring the upper-layer aqueous phase solution by using pH test paper, if the pH is close to neutral, adjusting the pH to 9-10 by using 0.05-0.15M NaOH solution, and then mixing and centrifuging again;
s3, transferring the lower organic clear liquid obtained in the step S2 into a fourth centrifugal tube, and concentrating the lower organic clear liquid to obtain a sample liquid to be detected;
the second step is that: analyzing the sample liquid to be detected obtained in the first step by adopting a single quadrupole gas chromatograph-mass spectrometer;
the gas chromatographic analysis conditions were: a chromatographic column: thermo TG 1701MS capillary column, 30m × 0.32mm × 0.25 μm; GC conditions were as follows: carrier gas: high-purity helium with the purity of 99.9995 percent and the constant flow rate of 1.0 mL/min; sample inlet temperature: 270 ℃; temperature rising procedure: the initial temperature is 80 deg.C, maintaining for 0.5min, then raising to 122 deg.C at 7 deg.C/min, maintaining for 15min, then raising to 150 deg.C at 7 deg.C/min, maintaining for 8.5min, raising to 240 deg.C at 35 deg.C/min, and maintaining for 4 min; and (3) sample introduction mode: no flow diversion; sample introduction volume: 1.0 μ L;
selecting an ion monitoring mode; an ion source: an EI source; ion source temperature: 270 ℃; ionization energy: 70 eV; solvent retardation: 15.0 min; transmission line temperature: at 260 ℃.
2. The method for rapidly detecting three common anesthetics in aquatic products according to claim 1, which is characterized in that: the first step is that the extracting solution is prepared by mixing acetonitrile: dichloromethane ═ 8: 2.
3. the method for rapidly detecting three common anesthetics in aquatic products according to claim 1, which is characterized in that: the first step of the process for extracting an anesthetic from an aquatic product comprises: placing 1.00g of a homogeneous sample in a first centrifuge tube, adding 5mL of extracting solution, performing vortex mixing, performing ultrasonic extraction for 5-8 min, centrifuging for 10min at 3500 rpm, transferring supernatant into a second centrifuge tube, adding 2mL of extracting solution into the first centrifuge tube, performing vortex mixing, performing ultrasonic extraction for 5-8 min, centrifuging for 10min at 3500 rpm, combining supernatants in a second centrifuge tube, and performing liquid-liquid extraction purification.
4. The method for rapidly detecting three common anesthetics in aquatic products according to claim 3, characterized in that: the liquid-liquid extraction purification comprises the following steps:
s1, sequentially adding 3.5mL of dichloromethane and 2mL of 0.01M NaoH solution into the clear liquid of the second centrifuge tube, fully mixing, and standing for 5 min;
s2, centrifuging at 3500 rpm for 5 min; after centrifugal layering, using pH test paper to detect that the upper-layer water phase solution is alkalescent and has a pH value of more than 9, if the pH value is close to neutral, using 0.1M NaoH solution to adjust the pH value to 9-10, and then mixing and centrifuging again;
s3, transferring the upper aqueous phase solution to another 15mL plastic centrifuge tube; transferring the lower organic clear liquid into a 15mL glass centrifuge tube;
s4, adding 4mL of dichloromethane into the plastic centrifuge tube filled with the upper aqueous phase solution, fully mixing, standing for 3min, centrifuging at 3500 rpm for 3min, and removing the upper aqueous phase by using a disposable plastic pipette; the lower organic phase supernatant was pooled in glass centrifuge tubes.
5. The method for rapidly detecting three common anesthetics in aquatic products according to claim 4, characterized in that: also comprises the following steps:
s5, heating at 40 ℃ by using a nitrogen blower, blowing nitrogen to 1mL, adding 200uL of 2ug/mL internal standard solution, fixing the volume to 2mL by using ethyl acetate, carrying out vortex mixing, then passing through a 0.22 mu m organic microporous filter membrane, and analyzing the filtrate by using a single quadrupole gas chromatograph-mass spectrometer.
6. The method for rapidly detecting three common anesthetics in aquatic products according to claim 5, characterized in that: the internal standard liquid is a mixture of methyl eugenol and methyl-3-aminobenzoate.
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