CN114487176B - Rapid screening method for dangerous substances in aquatic products - Google Patents
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Abstract
The invention belongs to the technical field of quality safety of aquatic products, and particularly relates to a rapid screening method for dangerous substances in an aquatic product. The 39 kinds of veterinary drug residual samples are respectively extracted by acetonitrile (containing 0.1% of acetic acid) and ethyl acetate (containing 0.1% of acetic acid), purified and concentrated by a dispersion solid phase extraction method, and then are quantitatively determined by a liquid chromatography-mass spectrometry-positive mode multi-reaction monitoring method and an external standard method. The method is simple, has low detection cost, and can remarkably improve the operation timeliness and the detection period and improve the detection efficiency.
Description
Technical Field
The invention belongs to the technical field of quality safety of aquatic products, and particularly relates to a rapid screening method for dangerous substances in an aquatic product.
Background
The nonstandard medication of the aquatic product cultivation easily causes the veterinary drug residue of the aquatic product to exceed the standard, and the pesticide residue of the aquatic product feeding easily causes the pesticide residue of the edible part of the aquatic product. The non-standard discharge of harmful substances easily causes pesticide residues in water quality, thereby causing the safety risk of aquatic product quality. The detection method for market monitoring of veterinary drug residues in aquatic products is a detection method for Guan Nong veterinary drug residues in the national standard recommended use method, namely a GB/T method. The following two problems exist in performing the national standard method: firstly, the method has the applicability problem that the existing national standard method matrix is rarely suitable for aquatic product matrix detection; secondly, the problem of missing in a method for simultaneously detecting multiple kinds of risk substances. The existing agricultural and veterinary drug detection methods are all similar drug residue detection methods, and cannot meet the requirements of efficient screening of various types in the actual screening process. Pretreatment of dangerous materials in aquatic products is a necessary experimental operation step of the materials to be tested before the materials are subjected to instrumental analysis, and the pretreatment method generally comprises extraction, purification, concentration and the like. The pretreatment method of the substance to be tested usually adopts a commercial integrated purification small column. The general purification columns for veterinary drug residues are divided into two categories: one type is direct filtration type, and the other type is adsorption elution type. The direct filtration type purifying column filters impurities in the environment of the sample solution to be detected by utilizing the functions of different molecular sizes, ion exchange and the like, so as to achieve the aim of purification. The adsorption elution type purifying column uses high polymer materials to adsorb the object to be detected in the solution to be detected by utilizing the polarity difference of the object to be detected, and then changes the solution environment to desorb the object to be detected so as to realize the purifying purpose. Above-mentioned two kinds of purifying small column, its detection cost is all relatively high, and the price cost still receives purifying the handling capacity influence of small column, and multiple class analyte purifying effect is not good.
Disclosure of Invention
The invention aims to provide the rapid screening method for the dangerous substances in the aquatic products, which has the advantages of simple method and low detection cost, can obviously improve the operation timeliness and the detection period, and improves the detection efficiency, so that the rapid screening method is better suitable for enterprises and market supervision departments and serves for the safety monitoring of the aquatic products.
The invention establishes a high-throughput rapid screening method for various agricultural and veterinary drug residues under aquatic product (fish, shellfish and shrimp) matrixes, is applicable to aquatic product matrixes, realizes high-throughput screening, and can obviously improve detection efficiency.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a screening method for 39 agricultural and veterinary drug residues in aquatic products, wherein the 39 agricultural and veterinary drugs are as follows: tylosin, tobrazilin, chlorpanaxadiol, clindamycin, norfloxacin, fleroxacin, enoxacin, sparfloxacin, ofloxacin, lomefloxacin, tilmicosin, ractopamine, spray-on-terol, erythromycin, ciprofloxacin, sulfamethazine, sulfachloropyridazine, sulfadoxine, sulfamethoxazole, benzoyl sulfa, sulfamonomethoxine, sulfaquinoxaline, sulfabenzopyrazole, sulfadimethyl pyrimidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfapair-methoxine, sulfamethazine, sulfamethodazine, thiacloprid, ethiprole, piroxicam, lincomycin, salad floxacin, and danofloxacin;
the method comprises the following steps:
(1) Extraction of
Accurately weighing 2.0g of a homogenized aquatic product muscle sample, placing the homogenized aquatic product muscle sample into a 50mL centrifuge tube, sequentially adding 3mL of EDTA buffer solution, 10mL of acetic acid-acetonitrile mixed solution, 4.0g of anhydrous sodium acetate and 1.0g of sodium chloride, carrying out vortex mixing for 1min, carrying out ultrasonic extraction for 10min, standing for 10min, then centrifuging at a speed of 9900r/min at a temperature of 0-4 ℃ for 10min to obtain a first extract, adding 7mL of acetic acid-ethyl acetate mixed solution into the extracted residue, carrying out ultrasonic extraction for 10min, centrifuging at a speed of 9900r/min to obtain a second extract, and combining the two extracts;
(2) Purification of
Adding 0.4g of C18, 0.4g of ethylenediamine-N-Propylsilane (PSA) and 1.2g of anhydrous magnesium sulfate into the extracting solution obtained in the step (1), carrying out vortex mixing for 2min, centrifuging at a rotating speed of 9900r/min for 10min at a temperature of 0-4 ℃, taking all supernatant fluid, adding 5mL of N-hexane into a 50mL centrifuge tube, carrying out vortex mixing for 2min, centrifuging at a rotating speed of 9900r/min at a temperature of 0-4 ℃, and extracting with N-hexane to obtain a purified extracting solution;
(3) Concentrating and redissolving
Accurately measuring 5mL of purified extract, concentrating and redissolving in a 10mL uncovered glass test tube, filtering the extract by a filter membrane, and measuring by high performance liquid chromatography-tandem mass spectrometry.
In the above technical scheme, further, the volume fraction of acetic acid in the acetic acid-acetonitrile mixed solution is 0.1%.
In the above technical scheme, further, the volume fraction of acetic acid in the acetic acid-ethyl acetate mixed solution is 0.1%.
In the above technical solution, in step (2), further, n-hexane is used for extraction at least 3 times.
In the above technical scheme, in the step (3), N is used at normal temperature 2 Blowing the solvent to dryness for concentration; adding acetic acid-acetonitrile mixed solution with the volume fraction of 0.1% of acetic acid for redissolution.
In the above technical solution, further, in the step (3), the high performance liquid chromatography-tandem mass spectrometry adopts a liquid chromatography-mass spectrometer: AB SCIEX 5500Q-trap, external standard method is used for quantification;
the liquid chromatography-tandem mass spectrometry measurement conditions were:
a. chromatographic column: XDB C18.1X100 mm,1.8 μm;
b. mobile phase: a is 2mM ammonium formate 50mM aqueous formic acid solution, B is 2mM ammonium formate 50mM acetonitrile/water (95/5) solution;
c. ion source: an electrospray ion source;
d. scanning mode: scanning positive ions;
e. the detection mode is as follows: monitoring multiple reactions;
f. ionization voltage: 5500v;
g. atomizing gas: 50psi;
h. auxiliary gas: 60psi;
i. air curtain air pressure: 30psi;
j. atomization temperature: 600 ℃;
k. sample injection amount: 10. Mu.L;
flow rate: 0.3mL/min.
In the above technical scheme, further, the preparation method of the mobile phase comprises the following steps:
(1) Mobile phase a: accurately measuring 0.126g of ammonium formate, dissolving in 1000mL of water, adding 2.3mL of formic acid into the solution, shaking uniformly, and performing ultrasonic treatment for 5min;
(2) Mobile phase B: accurately weighing 0.126g of ammonium formate, dissolving in 50mL of water, adding 950mL of acetonitrile and 2.3mL of formic acid into the solution, shaking uniformly, and performing ultrasonic treatment for 5min.
The beneficial effects of the invention are as follows:
compared with a commercial integrated purification small column, the method of the invention utilizes extraction and salting out in addition to adsorption in the purification principle. The problems that when a purifying small column is used, the passage pressure is too large to screen due to the difference of particle sizes in the solution, the fixed treatment capacity of the filler of the purifying column is limited, the detection period is too long, and the steps are complicated are avoided;
the method has the advantages of simple and convenient operation, cost saving, short time consumption, and improvement of detection efficiency while ensuring recovery rate and precision;
the invention uses acetonitrile (containing 0.1% acetic acid) and ethyl acetate (containing 0.1% acetic acid) to extract respectively, and the extraction efficiency is 100%;
EDTA is added in the extraction process, so that the separation degree of the sarcin substances is better, and the recovery rate is improved;
in the purification process, C18 and n-hexane are used for multi-step degreasing, and the influence on resolution and sensitivity of the liquid chromatography-mass spectrometer is reduced.
Drawings
FIG. 1 is a tylosin standard curve and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 2 is a tilmicosin standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 3 shows a standard curve of the cissamycin and an MRM mass spectrum, wherein a is the standard curve, and b is the MRM mass spectrum;
FIG. 4 is a ractopamine standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 5 is a standard curve of tobuterol and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 6 is a standard curve of the spray Butterflyer and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 7 is a standard curve of chlorpinalin and a MRM mass spectrum, a is a standard curve, and b is a MRM mass spectrum;
FIG. 8 is a lincomycin standard curve and MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 9 is a standard curve of erythromycin and a MRM mass spectrum, a is a standard curve, and b is a MRM mass spectrum;
FIG. 10 is a clindamycin standard curve and MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 11 is a ciprofloxacin standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 12 is a norfloxacin standard curve and an MRM mass spectrum, wherein a is a standard curve and b is an MRM mass spectrum;
FIG. 13 is a standard curve of sarafloxacin and a MRM mass spectrum, a is a standard curve, and b is a MRM mass spectrum;
FIG. 14 is a standard curve of darofloxacin and an MRM mass spectrum, a is a standard curve, b is an MRM mass spectrum;
FIG. 15 is a standard curve of Fluoxacin and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 16 is a standard curve of enoxacin and a MRM mass spectrum, a is a standard curve, and b is a MRM mass spectrum;
FIG. 17 is a sapafloxacin standard curve and MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 18 is a standard curve of ofloxacin and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 19 is a lomefloxacin standard curve and an MRM mass spectrum, wherein a is a standard curve and b is an MRM mass spectrum;
FIG. 20 is a sulfamethoxazole standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 21 is a sulfachloropyridazine standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 22 is a sulfadoxine standard curve and an MRM mass spectrum, wherein a is a standard curve and b is an MRM mass spectrum;
FIG. 23 is a sulfamethoxazole standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 24 is a standard curve of Sulfadiisooxazole and a MRM mass spectrum, a is a standard curve, and b is a MRM mass spectrum;
FIG. 25 is a standard curve of benzoylsulfonamide and a MRM mass spectrum, a is a standard curve, and b is a MRM mass spectrum;
FIG. 26 is a standard curve of sulfamonomethoxine and a MRM mass spectrum, wherein a is the standard curve and b is the MRM mass spectrum;
FIG. 27 is a sulfaquinoxaline standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 28 is a sulfanilamide phenylpyrazole standard curve and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 29 is a standard curve of sulfadimidine and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 30 is a sulfadiazine standard curve and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 31 is a sulfapyridine standard curve and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 32 is a sulfathiazole standard curve and an MRM mass spectrum, wherein a is a standard curve and b is an MRM mass spectrum;
FIG. 33 is a sulfamethoxydiazine standard curve and an MRM mass spectrum, wherein a is a standard curve, and b is an MRM mass spectrum;
FIG. 34 is a sulfamethazine standard curve and MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 35 shows sulfamethoxypyridazine standard curves and MRM mass spectrograms, wherein a is a standard curve, and b is an MRM mass spectrogram;
FIG. 36 is a thiacloprid standard curve and an MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 37 is an imidacloprid standard curve and MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 38 is a diethofencarb standard curve and MRM mass spectrum, a is a standard curve, and b is an MRM mass spectrum;
FIG. 39 shows the standard curve of pirimicarb and the MRM spectrum, a is the standard curve, and b is the MRM spectrum.
Detailed Description
The following examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
Example 1
(1) Standard solution preparation
(1) Preparation of standard stock solution: accurately weighing 10mg of each of tylosin, josamycin, tobuterol, chlorpanaxline, clindamycin, norfloxacin, fleroxacin, enoxacin, sparfloxacin, ofloxacin, lomefloxacin, tilmicosin, ractopamine, plagioterol, erythromycin, ciprofloxacin, sulfamethoxazole, sulfachlorpyridazine, sulfadoxine, sulfamethoxazole, benzoyl sulfa, sulfamonomethoxine, sulfaquinoxaline, sulfabenzene pyrazole, sulfadimethyl pyrimidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamethazine, sulfamethoxazole, thiacloprid, imidacloprid, diethofencarb, piroxicam, sarafloxacin and dalofloxacin standard samples, and preparing 1000mg/L single standard stock solution by methanol to 10mL respectively, and storing the single standard stock solution at-18 ℃ for future use in a dark place;
(2) mixing standard working solution: the standard stock solution is diluted step by using 20 percent methanol to prepare mixed working solution with the concentration of 1 mug/L, 2 mug/L, 5 mug/L, 8 mug/L and 10 mug/L, and the linear range is 1 to 100 mug/L;
(2) Extraction of
Accurately weighing 2.0g of a homogenized aquatic product muscle sample, placing the homogenized aquatic product muscle sample into a 50mL centrifuge tube, sequentially adding 3mL of EDTA buffer solution, 10mL of acetic acid-acetonitrile solution containing 0.1 percent, 4.0g of anhydrous sodium acetate and 1.0g of sodium chloride, carrying out vortex mixing for 1min, carrying out ultrasonic extraction for 10min, standing for 10min, centrifuging at a speed of 9900r/min at a temperature of 0-4 ℃ for 10min, transferring supernatant into another 50mL centrifuge tube, adding 7mL of acetic acid-ethyl acetate mixed solution into residues, carrying out ultrasonic extraction for 10min, centrifuging at a speed of 9900r/min to obtain a second extracting solution, and combining the two extracting solutions;
(3) Purification of
Adding 0.4g of C18, 0.4g of PSA adsorbent and 1.2g of anhydrous magnesium sulfate into the extracting solution, mixing the mixture with vortex for 2min at the temperature of 0-4 ℃ at the speed of 9900r/min, centrifuging for 10min, taking all supernatant into a 50mL centrifuge tube, adding 5mL of n-hexane, mixing the mixture with vortex for 2min at the temperature of 0-4 ℃ at the speed of 9900r/min, centrifuging for 4min, sucking the upper n-hexane, discarding the upper n-hexane, and repeatedly extracting the n-hexane for at least 3 times;
(4) Concentrating and redissolving
Accurately measuring 5mL of the lower purified extract in a 10mL uncovered glass test tube, and N at normal temperature 2 Blowing to dryness. 1mL of 0.1% acetic acid-acetonitrile solution was reconstituted. Filtering with 0.22 μm organic microporous membrane, and filling the filtrate into sample bottle for detection.
The detection adopts a liquid chromatography-mass spectrometer: AB SCIEX 5500Q-trap, measurement conditions are:
a. chromatographic column: XDB C18.1X100 mm,1.8 μm or equivalent;
b. mobile phase:
phase A: accurately measuring 0.126g of ammonium formate, dissolving in 1000mL of water, adding 2.3mL of formic acid into the solution, shaking uniformly, and performing ultrasonic treatment for 5min to obtain a 2mM ammonium formate 50mM formic acid aqueous solution;
and B phase: accurately measuring 0.126g of ammonium formate, dissolving in 50mL of water, adding 950mL of acetonitrile and 2.3mL of formic acid into the solution, shaking uniformly, and then performing ultrasonic treatment for 5min to obtain a 2mM ammonium formate 50mM acetonitrile/water (95/5) solution;
c. ion source: an electrospray ion source;
d. scanning mode: positive ion scan (esi+);
e. the detection mode is as follows: multiple Reaction Monitoring (MRM);
f. ionization voltage (IS): 5500v;
g. atomizing Gas (Gas 1): 50psi;
h. assist Gas (Gas 2): 60psi;
i. curtain air pressure (CUR): 30psi;
j. atomization Temperature (TEM): 600 ℃;
k. sample injection amount: 10. Mu.L;
flow rate: 0.3mL/min.
(5) Qualitative analysis
Comparing a liquid chromatographic separation spectrogram of a sample solution obtained under the same liquid chromatographic condition with a liquid chromatographic separation spectrogram of a standard substance, and if a chromatographic peak with the retention time consistent with that of a certain standard substance exists in the sample spectrogram, and the ultraviolet absorption spectrum after background subtraction is consistent with that of the standard substance, preliminarily identifying that the substance exists in the sample.
And respectively sampling the standard working solutions of the matrix, preparing a standard working curve by taking the concentration as an abscissa and the peak area as an ordinate, and quantifying the sample by using an external standard method of the standard working curve of the matrix.
The addition concentration of the target substance is 5-50 mug/kg, the recovery rate is 60.2-118.8%, and the RSD is 0.01-11.14%.
The 39 standard reference retention times and the linear equation are shown in table 1.
TABLE 1
The 39 standard substance standard curves and the MRM mass spectrum are shown in FIG. 1.
The above examples are only preferred embodiments of the present invention and are not limiting of the implementation. The protection scope of the present invention shall be subject to the scope defined by the claims. Other variations or modifications may be made in the various forms based on the above description. Obvious variations or modifications of the embodiments are within the scope of the invention.
Claims (5)
1. A screening method for 39 agricultural and veterinary drug residues in aquatic products is characterized by comprising the following steps:
the 39 agricultural and veterinary drugs are: tylosin, tobrazilin, chlorpanaxadiol, clindamycin, norfloxacin, fleroxacin, enoxacin, sparfloxacin, ofloxacin, lomefloxacin, tilmicosin, ractopamine, spray-on-terol, erythromycin, ciprofloxacin, sulfamethazine, sulfachloropyridazine, sulfadoxine, sulfamethoxazole, benzoyl sulfa, sulfamonomethoxine, sulfaquinoxaline, sulfabenzopyrazole, sulfadimethyl pyrimidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfapair-methoxine, sulfamethazine, sulfamethodazine, thiacloprid, ethiprole, piroxicam, lincomycin, salad floxacin, and danofloxacin;
the method comprises the following steps:
(1) Extraction of
Accurately weighing 2.0g of a homogenized aquatic product muscle sample, placing the homogenized aquatic product muscle sample into a 50mL centrifuge tube, sequentially adding 3mL of EDTA buffer solution, 10mL of acetic acid-acetonitrile mixed solution, 4.0g of anhydrous sodium acetate and 1.0g of sodium chloride, carrying out vortex mixing for 1min, carrying out ultrasonic extraction for 10min, standing for 10min, then centrifuging at a speed of 9900r/min at a temperature of 0-4 ℃ for 8min to obtain a first extract, adding 7mL of acetic acid-ethyl acetate mixed solution into the extracted residue, carrying out ultrasonic extraction for 10min, centrifuging at a speed of 9900r/min to obtain a second extract, and combining the two extracts;
(2) Purification of
Adding 0.4g of C18, 0.4g of ethylenediamine-N-propylsilane and 1.2g of anhydrous magnesium sulfate into the extracting solution obtained in the step (1), carrying out vortex mixing for 2min, then centrifuging at the speed of 9900r/min for 10min at the temperature of 0-4 ℃, taking all supernatant liquid, adding 5mL of N-hexane into a 50mL centrifuge tube, carrying out vortex mixing for 2min, centrifuging at the speed of 9900r/min for 4min at the temperature of 0-4 ℃, and extracting with N-hexane to obtain a purified extracting solution;
(3) Concentrating and redissolving
Accurately measuring 5mL of purified extract in a 10mL uncovered glass test tube, concentrating, redissolving, filtering by an extract filtering membrane, and measuring by high performance liquid chromatography-tandem mass spectrometry;
the detection adopts a liquid chromatography-mass spectrometer: AB SCIEX 5500Q-trap, measurement conditions are:
a. chromatographic column: XDB C18.1X100 mm,1.8 μm or equivalent;
b. mobile phase:
phase A: accurately measuring 0.126g of ammonium formate, dissolving in 1000mL of water, adding 2.3mL of formic acid into the solution, shaking uniformly, and performing ultrasonic treatment for 5min to obtain a 2mM ammonium formate 50mM formic acid aqueous solution;
and B phase: accurately measuring 0.126g of ammonium formate, dissolving in 50mL of water, adding 950mL of acetonitrile and 2.3mL of formic acid into the solution, shaking uniformly, and then performing ultrasonic treatment for 5min to obtain a 2mM ammonium formate 50mM acetonitrile/water (95/5) solution;
c. ion source: an electrospray ion source;
d. scanning mode: positive ion scan (esi+);
e. the detection mode is as follows: multiple Reaction Monitoring (MRM);
f. ionization voltage (IS): 5500v;
g. atomizing Gas (Gas 1): 50psi;
h. assist Gas (Gas 2): 60psi;
i. curtain air pressure (CUR): 30psi;
j. atomization Temperature (TEM): 600 ℃;
k. sample injection amount: 10. Mu.L;
flow rate: 0.3mL/min.
2. The screening method of claim 1, wherein: the volume fraction of acetic acid in the acetic acid-acetonitrile mixed solution is 0.1%.
3. The screening method of claim 1, wherein: the volume fraction of acetic acid in the acetic acid-ethyl acetate mixed solution is 0.1%.
4. The screening method of claim 1, wherein: in the step (2), the extraction is performed at least 3 times by using n-hexane.
5. The screening method of claim 1, wherein: in the step (3), N is used at normal temperature 2 Blowing the solvent to dryness for concentration; adding acetic acid-acetonitrile mixed solution with the volume fraction of 0.1% of acetic acid for redissolution.
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