CN114814013B - Method for rapidly screening unknown chemical hazard residues in aquatic products based on high-resolution mass spectrum database - Google Patents

Abstract

The invention relates to a method for rapidly screening unknown chemical hazard residues in aquatic products based on a high-resolution mass spectrum database, which comprises the following steps: (1) Performing pretreatment by using an improved QuECHERS method, repeatedly extracting with formic acid-acidified acetonitrile twice, and purifying by using C18 to obtain a to-be-detected purifying liquid; (2) chromatographic mass spectrometry detection: analyzing and detecting the purified liquid by an analysis method of ultra-high performance liquid chromatography and high resolution mass spectrometry, and (3) matching a database: the method comprises the steps of searching and comparing the acquired information such as retention time, accurate mass numbers of parent ions and ion ions with a constructed high-resolution mass spectrum database to determine unknown chemical hazard in a sample matrix, so that the rapid screening of the chemical hazard in the aquatic product is realized. The method of the invention greatly shortens the pretreatment and detection time of the sample, improves the detection efficiency, obtains the screening detection result in extremely short time, and realizes the simultaneous determination of multiple types of chemical hazard.

Description

Method for rapidly screening unknown chemical hazard residues in aquatic products based on high-resolution mass spectrum database

Technical Field

The application relates to the field of food safety detection, and relates to a method for rapidly screening unknown chemical hazard residues in fish and shellfish based on a high-resolution mass spectrum database.

Background

As one of the important food sources of the protein, the aquatic product can provide enough nutrient components for human bodies, especially a large amount of polyunsaturated fatty acids (such as DHA and EPA) contained in fish and shellfish products, can reduce the occurrence of fatty liver and other diseases, and has good alleviation effect on cardiovascular and cerebrovascular diseases. Research reports that the consumption of aquatic products in China exceeds the yield of the aquatic products, particularly fish and shellfish products, by 2030, and the consumption of the aquatic products per unit time in 1961 is increased to more than 20 kg in 2018. Meanwhile, the aquatic product is also one of food commodities with the largest international trade amount, and the export amount of fishes and shellfishes in developing countries exceeds the sum of coffee, rubber, cocoa, tobacco, meat and rice, wherein the total trade amount of fish products accounts for 10% of the export amount of all agricultural products. Just because the demand of people for aquatic products is higher and higher, the aquatic product cultivation production at present faces a great challenge, and how to avoid the pollution of chemical hazardous substances while ensuring the yield is a problem to be solved urgently.

At present, the aquatic product cultivation safety faces double challenges: on one hand, as agricultural and veterinary drugs and other persistent pollutants can be introduced into the aquatic products during the cultivation, circulation and processing processes, the substances are easy to accumulate in a food chain and finally are eaten by human beings, thereby threatening the health of the human bodies; on the other hand, due to continuous deterioration of the ecological environment and the mutual influence between the ecological environments, particularly the excessive industrial wastewater discharged on land flows into the ocean, the ecological environment of the ocean is seriously influenced, and the growth and propagation processes of fish, shellfish and other aquatic products in the ocean are further influenced, so that a great number of safety problems are caused. These chemical hazards, after entering the human body, can disrupt the endocrine system, obstruct the development and reproductive processes, and damage the central nervous system and immune system. Therefore, to ensure the quality safety of aquatic products, regulatory authorities in all countries of the world have established standards regarding the Maximum Residual Limits (MRLs) of chemically hazardous substances in aquatic products. In order to cope with the dual effects of drug residues added in the cultivation process and ecological environment deterioration, the quality safety of aquatic products at entrances and exits in China is guaranteed, and an accurate and reliable analysis and detection method is needed in the aquatic product cultivation industry to cope with unknown chemical hazard in the aquatic products and ensure the safety of people on tongue tips.

Most detection methods can only detect one substrate or detect a certain chemical hazard in multiple substrates due to the complexity of the substrate of the cultured aquatic products, so that the detection methods have certain limitations and singleness. The traditional gas chromatography and high performance liquid chromatography have certain defects in multi-residue detection, and are particularly easy to generate false positives in qualitative aspect. At present, the high-resolution mass spectrometry technology is to collect full-scan data of a substrate to be detected by using a high-resolution mass spectrometer, and not to select an ion range in advance for specific chemical hazardous substances, so that analysis of hundreds of chemical hazardous substances is completed at one time. The technique is based on the exact mass numbers and retention times of parent and secondary mass spectrometry fragment ions to identify chemically damaging residues, which are more sensitive due to higher mass accuracy and resolution. Meanwhile, the technology can realize the distinguishing identification of the isomer or the substances with similar structures, the confirmation of the unknown substances and the accurate qualitative and quantitative target substances. Therefore, the invention adopts ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to screen unknown chemical hazard substances.

The high-resolution mass spectrum detection technology mainly comprises the steps of comparing data collected by a high-resolution mass spectrometer with an established standard sample mass spectrum database, and completing screening and confirming of unknown chemical dangers. At present, organic mass spectrometry databases are mainly matched with more databases for gas phase mass spectrometry technologies, such as an NIST mass spectrometry library and a Wiley mass spectrometry library of the American institute. However, for chemical hazards that are difficult to volatilize, existing commercial mass spectrometry databases are far from satisfactory for use at the present stage. Meanwhile, the design principles of liquid chromatograph mass spectrometers of different companies are different, the adopted ionization modes are different, and the analysis parameters are different, so that the mass spectrograms obtained by the instruments of different companies have larger differences, and the difficulty is brought to sample detection adopting liquid analysis. However, by adopting the same liquid chromatography condition and mass spectrometry condition, the accurate mass numbers of the parent ions and mass spectrometry fragment ions obtained by different instruments are the same, and the standard substances and the acquired parameters of the sample to be detected are compared, so that the method can be used as a basic condition for rapid screening.

Disclosure of Invention

The invention aims to provide a method for rapidly screening unknown chemical hazard residues in aquatic products (fish and shellfish) based on a high-resolution mass spectrum database, so that simultaneous determination of multiple types of chemical hazards is realized. The method is simple to operate, high in sensitivity and good in reproducibility, can be widely applied to rapid screening of harmful chemical harmful substances in the aquatic products, and provides a technical support method for guaranteeing the quality safety of the aquatic products.

The invention provides a method for rapidly screening unknown chemical hazard residues in fish and shellfish based on a high-resolution mass spectrum database, which comprises the following steps:

(1) Sample extraction and purification: pretreating fish meat and shellfish meat by adopting an improved QuECHERS method;

(2) And (3) chromatographic mass spectrometry detection: carrying out sample analysis by using an analysis method of high-resolution mass spectrometry of ultra-high performance liquid chromatography;

(3) Matching database: and searching and comparing the acquired parameters of the sample to be detected with the established high-resolution mass spectrum database to realize screening and confirming of unknown chemical hazard.

The aquatic product matrix suitable for the invention comprises fish, oyster, scallop and the like.

In the above method, the pretreatment of the shellfish meat substrate by the modified QuECHERS method in step (1) comprises:

s1, performing preliminary pretreatment on the obtained fish meat and shellfish meat; specifically, removing fascia from a muscle tissue sample of an aquatic product, and performing preliminary crushing by using a tissue chopper; and (3) manually removing shells and other tissues of the aquatic product soft tissue samples, and pulping by using a beater. The two types of matrix samples were thoroughly broken up uniformly using a tissue refiner prior to analysis.

S2, extracting the treated substrate twice by using acetonitrile acidified by formic acid to obtain supernatant of the extract;

s3, adding the extract supernatant and the purifying agent into a centrifuge tube for mixing and centrifuging to obtain purified extract supernatant;

s4, drying the supernatant by using nitrogen, re-dissolving, and filtering with a 0.22 mu m filter membrane to obtain a sample to be tested.

Further, step S1 includes:

pretreatment of fish: killing fresh fish, cleaning, removing scale, removing viscera, removing head and tail, peeling, collecting back fish, removing fishbone, stirring with a tissue homogenizer, placing into a clean container, sealing, and marking.

Pretreatment of oyster: thoroughly cleaning Concha Ostreae sample with clear water, cutting off the muscle of the closed casing, opening the casing, washing with distilled water to remove silt and other foreign matters, picking up broken casing and other impurities, taking all soft tissues (including alimentary canal, carnis Ostreae meat quality and skirt edge), fully and uniformly stirring with a tissue homogenizer, loading into a clean container, sealing, and marking.

Pre-treating scallop: thoroughly cleaning scallop sample with clear water, cutting off the adductor muscle, opening the shell, washing the inside with distilled water to remove sediment and other foreign matters, picking up broken shells and other sundries, taking all soft tissues (including alimentary canal, scallop post and skirt edge), fully and uniformly stirring by using a tissue homogenizer, loading into a clean container, sealing, and marking.

Further, step S2 includes:

s21, accurately weighing 2.00g (+ -0.02 g) of homogenized and homogenized fish or shellfish sample into a 10mL centrifuge tube, then adding 8mL of 0.1% formic acid-acidified acetonitrile solution, carrying out vortex mixing, extracting, and centrifuging to obtain supernatant;

s22 the supernatant was transferred to a 50mL centrifuge tube, the residue was repeatedly extracted once with 8mL of 0.1% formic acid acidified acetonitrile, and the supernatants were combined.

Wherein, vortex mixing time is 2min, centrifugal speed of centrifugal treatment is 5 000r/min, centrifugal time is 5min, and centrifugal temperature is 4 ℃.

Further, step S3 includes:

s31, placing 500mg of the C18 dispersible adsorbent into a 50mL graduated polypropylene centrifuge tube;

s32, adding the supernatant into the 50mL polypropylene centrifuge tube, and fully vortex-purifying to obtain a purified mixed solution;

s33, centrifuging the purified mixed solution to obtain purified extract supernatant; wherein, vortex purification time is 1min, centrifugal speed of centrifugal treatment is 6 000r/min, centrifugal time is 10min, and centrifugal temperature is 4 ℃.

In the method, the chromatographic mass spectrometry detection in the step (2) is to analyze a sample by using an analysis method of high-resolution mass spectrometry of ultra-high performance liquid chromatography, in particular to analyze by using ultra-high performance liquid-time-of-flight mass spectrometry.

Further, the liquid chromatography conditions at the time of detection were: chromatographic column: ACQUITY UPLC BEH C18 (100 mm. Times.2.1 mm,1.7 μm) or other column of comparable performance; column temperature: 40 ℃; mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution, and phase B is acetonitrile; flow rate: 0.4mL/min; mobile phase gradient elution procedure: 0 to 0.5min 5 percent of B; linearly rising from 5% B to 15% B within 0.5-3.0 min; 3.0 to 6.0min, linearly increasing from 15 percent B to 40 percent B, and keeping for 3min; linearly increasing from 40% B to 60% B within 9.0-15.0 min; linearly rising from 60.0% B to 99.0% B for 15.0-19.0 min, maintaining for 4.0min, returning to the initial mobile phase, maintaining for 2.0min, and preparing for the next sample injection; sample injection volume: 5.00. Mu.L.

Further, the four-level rod time-of-flight mass spectrometry conditions at the time of detection are: ion source: electrospray ion source, positive and negative ion modes (esi+, ESI-); ion source temperature: 110 ℃; capillary voltage: 4.0kV; desolventizing gas temperature: 325 deg.c; desolventizing gas flow: 11L/min nitrogen; taper hole voltage: 65V; atomizer pressure: 40psig nitrogen; octopoleRFPeak:750V; using full information tandem Mass Spectrometry (MS) ^E ) Mode, acquisition range: m/z is 50-1500 Da. To ensure mass number accuracy, real-time calibration was performed with leucine enkephalin (mass concentration 50ng/mL, m/z:556.2771 in positive ion mode);

In the above method, the matching database in step (3) includes the following steps:

s1, preparing 756 chemical hazard standard substances into standard liquid with the concentration of 100ng/mL, and injecting according to the analysis method of the ultra-high performance liquid chromatography and high resolution mass spectrometry;

s2, pretreating fish and shellfish according to the improved QuECHERS method to obtain a liquid to be detected, and injecting according to an analysis method of high-resolution mass spectrometry of the ultra-high performance liquid chromatography;

s3, data acquisition is carried out on the 756 kinds of chemical hazard standard substances to obtain information such as chromatographic retention time, accurate mass number of parent ions, secondary ion fragments and the like of target chemical hazard substances, and simultaneously, information such as English names, CAS numbers, single isotope mass numbers and the like is added to establish a high-resolution mass spectrum database;

s4, comparing the actual sample data acquired in the step S2 with a high-resolution mass spectrum database of the common chemical hazard in the step S3, and judging whether the sample contains the unknown chemical hazard. The deviation of the detected chromatographic peak retention time and the retention time in a spectrum library is within +/-2.5%, the deviation of the accurate mass number of parent ions and the theoretical mass number is less than or equal to 5ppm, the deviation of at least 2 or more fragment ions with higher abundance in the secondary fragment ions and the mass number of the corresponding fragment ions in the spectrum library is less than 10ppm, and the abundance of the secondary fragment ions is consistent with the abundance of the corresponding fragment ions in the standard working solution with the concentration close to that of the secondary fragment ions.

The method can utilize the high-resolution mass spectrum database to rapidly screen 756 unknown chemical hazard residues in the fish meat and the shellfish meat.

Furthermore, by utilizing the detection method, the invention also provides a high-resolution mass spectrum database for rapidly screening chemical harmful substances in the aquatic products, and the specific database is shown in table 1. According to the retention time of chemical hazard in a mass spectrum database, the accurate mass number of parent ions and secondary fragment ions, the rapid screening of the chemical hazard is realized, and meanwhile, the substrate matching calibration curve of various substrates is determined according to the method, so that the quantification of 756 chemical hazard is realized.

The 756 chemical hazards are specifically as follows:

the number of the pesticides is 524, including oxadiazon, propyzamide, benomyl, clomazone, aniline, cyclopamine, diphenylamine, chlorpyrifos, buprofezin, methamphetamine, chlorpyrifos, atrazine-deethyl, clomazone, diazinon, dinotefuran, buprofezin, ethirimide, simazine, amifos, zhong Dingtong, desmopride, propyzamide, fenoxacarb, aldrin, dichlormid, coumaphos, prometryn, ciprofen, ethephon, beta-hexasix, metalaxyl, chlorpyrifos, methyl parathion, anthraquinone, delta-hexasix, fenthion, malathion, fenitrothion, ethyl paraoxon, triazolone, parathion, pendimethalin, linuron, chlorfenamide, ethyl bromethion, quinophos, trans-chlordane (gamma), phencyclist, profenofos, fenphos, prometum, plastic Dioding reagent, procymidone, methidathion, fenpropazine, dichlormid, oxadiazon, fenphos, chlorpyrifos, pyrifos, bupirimate, carboxin, flufenamide, 4 '-d, ethion, methiphos, epoxiconazole, myclobutanil, gramoxazole (methyl), propiconazole, fenphos, bifenthrin, imazalil, flubenfol, 4' -methoxam, oxadixyl, tetramethrin, tebuconazole, flubenfomin, pyridaphos, thiophos, trichlorfon, carboxin, cis-permethrin, trans-permethrin, pyrazophos, cypermethrin, fenvalerate, deltamethrin, trichlorfon, dichlorphos, butamol, dichlorvos, clomazone, flufenacet, chlor, avena, chlorbenazolin, clomazone, alpha-hexahexazin, terbutafos, terbutaline, ciprofloxacin, dichlorphos, promethazine, clodinafop-propargyl, chlornitramine, terbuthylazine, chlorfluazuron, chlorpyrifos-methyl, dimethenamid, mechlor, pyrifos-methyl, terbutazone, graminium, prothiotep, triclopyr, metolachlor, oxychlorodane, pyrimidone, methoprene, bromaphos, benfomesal, ethoxyfurafin, isoprothiolane, alpha-thiodane, propanil, isosalazine, benfophos, chlorpyrifos, cis-chlordane (alpha), tolfenfluramid, 4 '-d-methyl, chlorpyrifos, bazachlor, iodized, sulphos, Z-chlorfenamide, chlorpyrifos, profenox, fluazuron, triazamate, 2' -d the present invention relates to a method for the treatment of a disease selected from the group consisting of isodieldrin, hexaconazole, fenbuconazole, 2,4 '-d-running, paclobutrazol, methoprene, imazalil, propyl ester, benfuracarb, methyl, pyriftalid, oxyfluorfen, chlorfenapyr, beta-thiodane, trifloxystrobin, 4' -d-running, trithion, benfop, dichlorphos, triazophos, chlorfenapyr, thiodane sulfate, fenisobromolate, neo-Yanling, fenpropathrin, bromophos, benfophos, hexazinone, valicarb, fenpyrad, chlorpyrifos, prochloraz, coumaphos, dichlorvos, diphenfen, 3, 5-dichloroaniline, gramine, E-fenphos, o-phenylphenol, cis-1, 2,3, 6-tetrahydrophthalimide, fenobuconamide, flubenuron, chlorfenamide, chlorfenapyr, wild wheat dicamba, pyrimethanil, gamma-hexazin, etoposide, atrazine, heptachlor, iprobenfos, chlorzophos, triclosan, fenpropimorph, transfluthrin, clomazone, tolclofos-methyl, iprovalicarb, ametryn, simetryn, bromuron, zinone, thiabendazole, epsilon-hexazin, iprovalicarb, angustifop, diethofencarb, pipradan, bioallethrin, 2,4' -d fenpyroximate, fenbuconazole, chlorthion, propargyl, penconazole, triazophos, tetrachloraz, profenofos, triadimenol, pretilachlor, kresoxim-methyl, fluazifop-butyl, fluazinam, ethaboxam, uniconazole, flusilazole, triazamate, diniconazole, synergistic ether, propargite, fenpyraclostrobin, oxadiazon, tebufenpyrad, fenazaquin phenyl-pyrethroid, fludioxonil, fenoxycarb, sethoxydim, anilofos, flumetofen, fenfluramine, mefenacet, permethrin, pyridate, fluorofluorofen, bitertanol, ethofenprox, thioxanthone, alpha-cypermethrin, flufenvalerate, S-fenvalerate, difenoconazole, flumetsulam, flumetofen, methafop, etoposide, pentachlorobenzene, triisobutyl phosphate, fluazifop, 4-bromo-3, 5-xylyl-N-methyl carbamate, oat ester, benfop, 2,3,5, 6-tetrachloroaniline, tributyl phosphate, 2,3,4, 5-tetramethoxybenzene, pentachloromethoxybenzene, pasture, vegetable phosphorus, methabenzthiazuron, simarone, atran, atrazine-isopropyl, tertiofos, tebufenozide, fluazulene, atrazine, 2, 6-dichlorobenzamide, 2,4' -trichlorobiphenyl, 2,4, 5-trichlorobiphenyl, terbuthylazine-deethyl, 2,3,4, 5-tetrachloroaniline, sunflower seed musk, xylene musk, pentachloroaniline, azido, terbuthylazine, ding Mi amide, 2',5,5' -tetrachlorobiphenyl, musk, benfop-butyl, xylenol thiazate, benfop-butyl, methyl paraoxon, heptylphosphine, tibetan musk, carbochlor, octachlorostyrene, pyrithione, isoeuclidean, bupropion, chlorpyrifos, dimethyl chlorophthalic acid, 4' -dichlorobenzophenone, phthalyl ester, musk ketone, pimidazole, cyprodinil, corncob, oxaisosalate, ifosf, 2',4, 5' -pentachlorobiphenyl, 2 methyl 4-chlorobenzoate ethyl ester, aqueous amine thiophos, methamphetamine, miticidal alcohol, trans-nonachloride, xidectin, desmopride, fludioxonil, brombenephos, etox, triad, sterilized phosphorus, 2,3, 4', 5-pentachlorodiphenyl, 4' -dibromodiphenyl ketone, flutriafol, triazophos, ethyl methidathion, 2', 4',5' -hexachlorodiphenyl, lv triadimenol, ethasone, hexythiazox, 2',3, 4',5' -hexachlorobiphenyl, penoxsulam, benfurthrin, cyproconazole, butyl benzyl phthalate, clodinafop-propargyl, thionyl sulfate, trifluoperazole, isooctyl fluroxypyr, thion sulfone, triphenyl phosphate, oxaziclomefone, 2', 5' -hexachlorobiphenyl, penoxsulam, benfurthrin, cyproconazole, butyl benzyl phthalate, clodinafop-propargyl, thionyl chloride trifluobenzole, fluroxypyr, fosetyl, triphenyl phosphate, metamitron, 2', phosphorus oxychloride, pencycuron, buthiuron, tebufenpyrad-S-methyl, fenphos, phenanthrene, spiroxamine, fenpyroximate, butyl pyrimidyl and jasmone; jasmonic acid inducer, benomyl, chlornitramine, fluquindox, propargyl, triadimefon, brombutamide, acetochlor, benoxacin, terfenazal, penoxsulam, furazamide, alaacil-S-methyl, benfurazafen, dithiopyr, mefenoxam, malamat, simeconazole, dimethyl chlorophthalic acid, thiozamate, methylparaben, butralin, zoxamide, diphenoxylate, fenfenox, allethrin, isofipronil, methoprene, flufenacet, fomesamide, tetrachlorfon, furalachlor, thiamethoxam, cyprodinil, captan, imazamox, imazalil, pyrifos, (E) -benzofenapyr, thiocyan, trifloxystrobin, imazamox, trifloxystrobin, pyrifos, oxazafen, fenpyr, fenbucil, benfurazafen the present invention relates to a pharmaceutical composition comprising fluquindox, chlorfenapyr, trifloxystrobin, imibencarb, bisbenzoxazole acid, fipronil, imazafen, flufenoxaprop-ethyl, pyriftalid, pyrifos, fenbufen, mefenamate, valinate, etoxazole, pyriproxyfen, flupyraflufen, iprodione, pirfenphos, furamide, bifonazate, isodieldrin, clofenamate, fenamidone, napropylamine, pyraclostrobin, lactofen, trimethoprim, pyraclonil, clofos, spirodiclofen, fenazafen, furazolidone, pyriftalid, pyriminobac-methyl, flubenfop-methyl, clofentezine, clofenacet-methyl, methamidone, chlorfenapyr, tebufenozide, fenpyrad, tebufenozide, thifesulfuron, ethoxysulfuron, spinosyn, mepiquat chloride, tricyclazole, isoproturon, pymetrozine, clomazone, methomyl, cinosulfuron, pyrazosulfuron, methomyl, cymoxanil, omethoate, ethoxyquinoline, dacron, imazethapyr, cymoxanil, triamcinolone, terfenacet Ding Linfeng, cyazofamid, florasulam, benomyl, chlormequat, folpet, methomyl, cartap;

The number of the veterinary medicines is 182, comprising benzoylsulfadiazine, sulfadiazine, sulfamonomethoxine, sulfadimidine, sulfamethyidine, sulfamebendazole, sulfamethidiazine, sulfabenzene pyrazole, sulfapyrazole, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfadimidine, trimethoprim, cinnoxacin, danofloxacin, difloxacin, enfluxacin, flumequine, gatifloxacin, lomefloxacin, bamofoxacin, moxifloxacin, nalidixic acid, ofloxacin, obismic acid, sarafloxacin, sparfloxacin, tosulfloxacin, albendazole-2-amino sulfone, albendazole sulfoxide, aminothiazole, thiabendazole (thiabendazole), dimefzole (dimet-zazole) Fenbendazole (phenylthioimidazole), flubendazole (flubendazole), hydroxyisopropanazole, ipronidazole, mebendazole, thiabendazole (candidazole), 5-hydroxythiabendazole, levamisole, metronidazole, oxfendazole, propoxybendazole, luo Xiao, secnidazole, tinidazole, triclabendazole, clindamycin, doramectin, epromycin, ivermectin, as-column crystal white mold A1, spiramycin, tilmicosin, tylosin, virginiamycin M1, beclomethasone dipropionate, betaminosone valerate, chlordexamethasone acetate, clobetaxosone propionate, clobetasone butyrate, cortisone, deflazacort, dexamethasone, diflorasone diacetate, episterone, fludrocortisone, fludrocort, fluormethone pivalate, fluocinolone acetonide, fludrolide, fluorometholone, fluticasone propionate, halcinonide, hydrocortisone, megestrol, medroxyprogesterone acetate, methylprednisolone, mometasone furoate, prednisolide, testosterone, triamcinolone acetonide, bambuterol, hydroxymethylcycloterol, clenbuterol, isocclenbuterol Pan Te, clenbuterol, fenoterol, formoterol, pirbuterol, ractopamine, ritodrine, salmeterol, sotalol, terbutaline, tobuterol, clenbuterol Pan Te, acetaminophen, chlorpromazine, clopyralid, 4' -diaminodiphenyl sulfone, cabadol, haloperidol, azapirone, propidium, propinzine, carbamazepine, pimelide, imipramine, sulpirone, flupirtine, caffeine, and caffeine 1, 7-dimethylxanthine, chloroprocaine, cinchocaine, lidocaine, procaine, tetracaine, brompheniramine, cetirizine, chlorpheniramine, fluphenazine, hydroxyzine, promethazine, terfenadine, bifonazole, econazole, griseofulvin, ketoconazole, naftifine, flunixin, ketotifen, lornoxicam, melitracin, oxaprozin, antipyrin, sulfamidine, difluorofloxacin hydrochloride, fleroxacin, enoxacin, sarafloxacin hydrochloride, nafofloxacin, norfloxacin, gemifloxacin mesylate, glipizide, repaglinide, hygromycin B, sulfadimefuzole, amikacin, tolbutamide, gliclazide, glibenclamide, 1-aminohydantoin hydrochloride, pioglitazone hydrochloride, metformin hydrochloride, glimepiride, sulfamethazine, m-methoxine hydrochloride, sulfacetamide, sulfachlorpyridazine, sulfamethoxazole, sulfanifedipine, sulfap-methoxypyrimidine, sulfamethoxazole, buformin hydrochloride, phenformin hydrochloride, clarithromycin, ciprofloxacin, kanamycin sulfate, tobramycin;

The persistent organic matter is 32 kinds, including tripropyl phosphate, triisobutyl phosphate, tributoxyethyl phosphate, 2-ethylhexyl diphenyl phosphate, trioctyl phosphate, tricresyl phosphate, p-toluyl phosphate, tricresyl phthalate, tri (1, 3-dichloroisopropyl) phosphate, toluene diphenyl phosphate, triphenyl phosphate, tri (2-chloropropyl) phosphate, tributyl phosphate, trichloroethyl phosphate, triethyl phosphate, trimethyl phosphate, butyl benzyl phthalate, diphenyl phthalate, di (2-butoxyethyl) phthalate, dibutyl phthalate, dicyclohexyl phthalate, dimethyl phthalate, di-n-pentyl phthalate, diethyl phthalate, diisobutyl phthalate, dihexyl phthalate, dioctyl phthalate, di (2-methoxy) ethyl phthalate, di-2-ethoxyethyl phthalate, di-4-methyl-2-pentyl phthalate, dioctyl phthalate, and dinonyl phthalate;

the marine biotoxins are 18: thiocarbamoyl gonyatoxin 2, thiocarbamoyl gonyatoxin 3, descarbomoyl gonyatoxin 2, descarbomoyl gonyatoxin 3, descarbomoyl neosaxitoxin, descarbomoyl gonyatoxin 1, gonyatoxin 2, gonyatoxin 3, gonyatoxin 4, gonyatoxin 6, neosaxitoxin, saxitoxin, tetrodotoxin, microcystins RR, microcystins LR, okadan Tian Ruan sponge acid, and ganglion.

The invention provides a method for rapidly screening unknown chemical hazard residues in aquatic products based on a high-resolution mass spectrum database, which comprises the following steps:

s1, carrying out pretreatment on aquatic product samples by using a modified QuECHERS method;

s2, analyzing chemical hazard by adopting an ultra-high performance liquid chromatography and high resolution mass spectrometry combined method

S3, collecting 756 kinds of related information including English names, CAS numbers and molecular weight information, collecting 756 kinds of chemical hazard standard products of retention time, ionization form, parent ions and accurate molecular weights of the parent ions, wherein 756 kinds of chemical hazard products comprise chemical hazard products in the national standard of current aquatic products, and cover pesticides, veterinary drugs and organic pollutants, and establishing a high-resolution mass spectrum database for rapidly screening the chemical hazard products in the aquatic products, wherein the specific database is shown in Table 1;

s4, collecting data of a sample to be detected and carrying out search comparison with the established mass spectrum database, so as to realize rapid screening of unknown chemical hazard.

Aiming at aquatic product matrixes, particularly fish and shellfish matrixes, the sample has the characteristics of complex matrixes, high lipid and protein content and the like, and a method for rapidly screening unknown chemical hazard residues in fish and shellfish based on a high-resolution mass spectrum database is established. The method has the advantages of no derivatization step for substances which are difficult to volatilize and have stronger polarity, simple operation, good reproducibility and high sensitivity, and can be widely applied to screening and analyzing unknown chemical hazard substances in aquatic product matrixes. According to the invention, acetonitrile acidified by 1% formic acid is used as an extraction reagent, extraction liquid is purified by using C18 as a purifying agent through repeated extraction twice, finally, data acquisition is carried out on liquid to be detected through ultra-high performance liquid chromatography and high resolution mass spectrum, and searching and comparison are carried out by utilizing an established high resolution mass spectrum database, so that the rapid screening of unknown chemical hazard in the aquatic product under the condition of no standard substance is realized. The method has the characteristics of high recovery rate, strong applicability, short time consumption, high precision and the like, and each technical index can meet the requirements of daily detection and analysis of aquatic products such as fish, shellfish and the like, thereby providing technical support for guaranteeing the quality safety of the aquatic products.

The one-step method provided by the invention can be used for rapidly screening 756 kinds of chemical hazard substances, can realize the quality safety analysis of the cultured aquatic products in a short time, especially the fishes and shellfishes with larger daily consumption, and can ensure the safety of the tips of the tongue of people from the source.

Drawings

FIG. 1 is a chromatogram of a detected standard, wherein (a) is a chromatogram retention time profile of the detected standard; (b) To detect standard chromatographic retention times and molecular weight distribution patterns.

FIG. 2 is a graph showing the distribution of SDL and LOQ of 756 chemical hazards in tilapia, grouper, oyster and scallop substrates.

FIG. 3 shows the distribution of the matrix effects of 756 chemical hazards in tilapia, grouper, oyster and scallop matrices.

FIG. 4 is a graph showing the effect and retention time of chemical hazard matrix in tilapia, grouper, oyster and scallop matrix, wherein (a) is tilapia; (b) is grouper; (c) is oyster; (d) is a scallop.

FIG. 5 is the accuracy and sensitivity of 756 chemical hazards in tilapia, grouper, oyster and scallop substrates at 150ng/mL during and between days; wherein:

(a) And (b) accuracy and sensitivity of the tilapia matrix in and between days, respectively;

(c) And (d) accuracy and sensitivity of grouper substrate within and throughout the day, respectively;

(e) And (f) accuracy and sensitivity of oyster matrix in and between days, respectively;

(g) And (h) accuracy and sensitivity of scallop matrix day and day-to-day, respectively;

FIG. 6 is a daily ratio of tilapia, grouper, oyster, and scallop substrates at 10ng/mL and 150ng/mL for 756 chemical hazards accuracy (70% -120%) and sensitivity (0% -20%) during the day; (a) a tilapia matrix; (b) grouper substrate; (c) oyster matrix; (d) scallop matrix.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used, unless otherwise specified, are all commercially available. The following examples relate to test analysis equipment, reagents and conditions as follows:

1. instrument and apparatus: agilent 1290 affinity II ultra-high performance liquid chromatograph (Agilent company, usa), agilent6530Q-TOF time-of-flight mass spectrometer (Agilent company, usa), milli-Q ultra-pure water machine (Millipore company, usa), low temperature centrifuge (beckman coulter company, usa); vortex-Genie 2 Vortex oscillator (us Scientific Industries); electronic balance (Sartorius company, germany); nitrogen weatherer (Zymark company, usa).

2. Reagent: acetonitrile (LC-MS grade, dima technologies); water (LC-MS grade, merck libo limited); formic acid (99% purity, acros, usa); CNWBOND HC-18 purification packing (China Annotation Co.); 756 chemical hazard standards (purity: 95% or more, standard substance manufacturers such as Shimadzu SHIMADZU, tianjin A-Tara, canada national institute of standards).

3. Preparing a standard working solution:

preparing a stock solution: respectively diluting and split charging chemical hazard standard substances into brown sample injection bottles, dissolving with acetonitrile to constant volume to prepare 1.0mg/mL standard stock solution, keeping in dark under-18deg.C, sealing, and keeping for 4 months.

Preparing a mixed working solution: and accurately transferring a proper amount of standard stock solution, and preparing each standard stock solution into a mixed working solution with the concentration of 10 mug/mL by using acetonitrile, wherein the solution needs to be prepared on site.

Example 1

1. Instrument working conditions:

chromatographic conditions of ultra-high performance liquid chromatograph:

sample injection volume: 5.00. Mu.L;

the column was ACQUITY UPLC BEH C (100 mm. Times.2.1 mm,1.7 μm) or other equivalent performance column.

Column temperature: 40 ℃;

mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution, and phase B is acetonitrile;

Flow rate: 0.4mL/min;

mobile phase gradient elution procedure: 0 to 0.5min 5 percent of B; linearly rising from 5% B to 15% B within 0.5-3.0 min; 3.0 to 6.0min, linearly increasing from 15 percent B to 40 percent B, and keeping for 3min; linearly increasing from 40% B to 60% B within 9.0-15.0 min; and (3) linearly rising from 60.0% B to 99.0% B for 15.0-19.0 min, maintaining for 4.0min, returning to the initial mobile phase, maintaining for 2.0min, and preparing for the next sample injection.

Mass spectrometry conditions for a four-bar time-of-flight mass spectrometer:

ion source: an electrospray ion source;

positive and negative ion modes (esi+, ESI-);

ion source temperature: 110 ℃;

capillary voltage: 4.0kV;

desolventizing gas temperature: 325 deg.c;

desolventizing gas flow: 11L/min nitrogen;

taper hole voltage: 65V;

atomizer pressure: 40psig nitrogen;

OctopoleRFPeak：750V；

using full information tandem Mass Spectrometry (MS) ^E ) Mode, acquisition range: m/z is 50-1500 Da. To ensure Mass accuracy, leucine enkephalin (Mass concentration 50ng/mL, m/z:556.2771 in positive ion mode) was used for real-time calibration, and data acquisition processing was performed by Mass Hunter software developed by Agilent corporation.

Specific information in the 756 chemical hazard high resolution mass spectrometry databases established included chinese name, molecular formula, retention time, parent ion exact mass number, collision energy, and secondary fragment ion information, as shown in table 1.

2. Extracting and purifying fish and shellfish matrix by improved Queclers method

(1) Pretreatment of experimental substrates

Pretreatment of fish: killing fresh fish, cleaning, removing scale, removing viscera, removing head and tail, peeling, collecting back fish, removing fishbone, stirring with a tissue homogenizer, placing into a clean container, sealing, and marking.

Pretreatment of oyster: thoroughly cleaning Concha Ostreae sample with clear water, cutting off the muscle of the closed casing, opening the casing, washing with distilled water to remove silt and other foreign matters, picking up broken casing and other impurities, taking all soft tissues (including alimentary canal, carnis Ostreae meat quality and skirt edge), fully and uniformly stirring with a tissue homogenizer, loading into a clean container, sealing, and marking.

Pre-treating scallop: thoroughly cleaning scallop sample with clear water, cutting off the adductor muscle, opening the shell, washing the inside with distilled water to remove sediment and other foreign matters, picking up broken shells and other sundries, taking all soft tissues (including alimentary canal, scallop post and skirt edge), fully and uniformly stirring by using a tissue homogenizer, loading into a clean container, sealing, and marking.

(2) Extraction of sample matrix

Accurately weighing 2.00g (+ -0.02 g) of homogenized fish or shellfish sample into a 10mL centrifuge tube, then adding 8mL of acetonitrile acidified by 0.1% formic acid as an extraction solvent, fully vortex-mixing and uniformly extracting for 2min, enabling the sample to fully contact with the extraction solution, centrifuging at 5 000r/min for 5min at 4 ℃, transferring the supernatant into a 50mL centrifuge tube, repeatedly extracting the residual residues once by 8mL of acetonitrile acidified by 0.1% formic acid, and combining the two supernatants for later use.

(3) Purification of sample matrices

The supernatant was poured into a 50mL graduated polypropylene centrifuge tube containing 500mg of C18 dispersible adsorbent, the sample was vortex purified for 1min, thoroughly mixed and centrifuged at 6000r/min for 10min at 5℃followed by transferring the supernatant into a 50mL centrifuge tube and blow-dried with nitrogen at 40 ℃. Finally acetonitrile was used: the water (1:1, V/V) is redissolved to 1mL, and the redissolved solution is filtered by a 0.22 mu m filter membrane for the on-line detection of UHPLC-QTOF-MS.

3. Experimental results

(1) Optimized determination of non-targeted screening chromatographic conditions for chemical hazards in aquatic products

The non-targeted screening of the aquatic product chemical hazard needs to cover four types of hazard, and has the characteristics of multiple types and large polarity difference. In order to achieve effective separation of these four types of hazards in the chromatographic process, it is necessary to perform integrated optimization of chromatographic conditions according to current existing data. According to the current literature report and the existing multi-residue detection national standard, the four substances can be separated by utilizing high performance liquid chromatography. The chromatographic column used for the liquid chromatographic separation of the four substances is mainly an inverse C18 column, and the specification is a general UPLC small column which is suitable for mass spectrum. Therefore, the invention selects ACQUITY UPLC BEH C column (130A, 1.7 μm,2.1mm×100 mm) of Waters company, and uses Agilent 1290 information LC and 6530 accurate mass Q-TOF MS combined system to construct the database.

From the aspect of elution conditions, most pesticides have relatively low polarity, for example, a 23-minute elution procedure is adopted in liquid chromatography-mass spectrometry (GB 23200.14-2016 national food safety Standard fruit and vegetable juice and fruit wine) for measuring 512 pesticides and related chemical residuals, wherein the time period for 40-99% acetonitrile elution is 17 minutes; the polarity distribution of veterinary drugs is relatively wide, for example, an elution program of 23 minutes is adopted in liquid chromatography-high resolution tandem mass spectrometry (LC-high resolution MS) for identifying 150 veterinary drugs and other compounds in livestock and urine of No. 197-9-2019 of the bulletin of agricultural rural area, wherein the time period for eluting 5% -40% of methanol is 10 minutes, and the time period for eluting 40% -100% of methanol is 13 minutes. The 756 kinds of hazardous substances locked by the invention are mainly pesticides and veterinary drugs, so the chromatographic elution conditions used by the invention are obtained by optimizing the two elution conditions. It can be seen from fig. 1 (a) that the chromatographic retention times of these standards are more evenly distributed throughout the elution process, while the number of chemical hazards separated during each time period is more evenly distributed. It can further be seen from fig. 1 (b) by the relationship between molecular weight and retention time that the chemical hazards of different molecular weights are distributed relatively uniformly throughout the elution process, no specificity being present for each time period, either large or small. These indicate that the chromatographic elution conditions used in the studies of the present invention are ideal and enable the efficient separation of all chemically hazardous standard substances.

(2) Screening detection limit and quantitative limit of 756 chemical hazards in fish and shellfish substrates

The sensitivity of the method was assessed by examining the screening limit of detection (SDL) and limit of quantification (LOQ) in the sample matrix. Wherein the determination of SDL is performed by detecting 20 samples after matrix addition, and the target analyte can be detected at least 19 times during 20 experiments, and the corresponding addition concentration is SDL. As shown in fig. 2 (a), about 93.12%, 92.46%, 93.91% and 93.25% of chemical hazards in tilapia, grouper, oyster and scallop substrates, respectively, meet the eu standard, i.e., SDL is lower than the maximum residual level of 0.01mg/kg specified by the eu, which also demonstrates that the established sensitivity of the fish and shellfish non-target detection method meets the requirements. LOQ is the lowest concentration that verifies the accuracy of chemical hazards, and according to EU standards the recovery should be in the range of 70% -120% and the Relative Standard Deviation (RSD) of 0% -30% when the substrate addition concentration is less than or equal to 0.01mg/kg or 70% -120% and the RSD of 0% -20% when the substrate addition concentration is 0.01-0.1 mg/kg. From FIG. 2 (b), it can be seen that 92.72%, 92.59%, 94.45% and 93.79% of the tilapia, grouper, oyster and scallop substrates meet the EU standards, respectively, while the two types of substrates meet the EU standards at the addition levels of 20 and 50mg/kg, except for the undetectable substances. The maximum residual concentration of chemical hazard in the matrix of the fish and shellfish is 10 mug/kg-5 mg/kg, and the data result shows that the screening detection limit and the quantitative limit of the method meet the daily analysis detection requirement in four matrixes of tilapia, grouper, oyster and scallop, and the unknown chemical hazard substances in the matrix of the fish and shellfish can be detected. In a word, the analysis method can accurately measure chemical hazard in the unknown sample, and the sample does not need to be further concentrated to improve the detection sensitivity.

(3) Linear relationship of 756 chemical hazards in fish and shellfish substrates

Linear relationship by determining the relationship between the detection signal of a chemical hazard in a sample extract and the known amount of analyte detection signal produced by a standard solution, the degree of correlation established between the two variables is determined by r ² And (3) representing. Constructing a linear regression equation by taking the peak area of chemical hazard components as an ordinate and the mass concentration rho (ng/mL) of the added standard working solution as an abscissaAnd drawing a standard correlation curve. The linear range of 713 (94.31%), 701 (92.73%), 703 (92.99%) and 696 (92.06%) chemical hazardous substances in tilapia, grouper, oyster and scallop, respectively, is 10-250ng/mL, which is also the maximum concentration range set in the analysis method, the above r of the target analyte ² All greater than 0.990, demonstrating that most chemical hazards exhibit ideal correlation over the linear range of 10-250 ng/mL. The veterinary medicine glimepiride exists in the tilapia matrix, the linear range cannot reach 250ng/mL due to the saturation effect of signals at higher concentration, the linear range is 10-100ng/mL, and 37 kinds of signal values are not detected in the linear range; the maximum value of the linear concentration of 2 target analytes in the grouper matrix, namely pesticide chlorphenamine and sterile phosphorus is 100ng/mL, and the remaining 40 analytes have no signal value detected in the linear range; of the 14 (1.32%) chemical hazards in oyster substrates, eight pesticides and two veterinary drugs cannot reach 250ng/mL due to the saturation effect of the detection signal at higher concentration, resulting in a linear range of 10-150ng/mL and 4 non-linearities in the concentration range; the detection signal of 15 (1.99%) chemical hazards present in the scallop matrix reaches saturation effect at higher concentrations, resulting in a linear range of 12 analytes between 10-150ng/mL, the remaining 3 being non-linear in this concentration range. Through the analysis of the detection results of 756 chemical hazards in the four matrixes, more than 92.0% of chemical hazards in the four matrixes show ideal linear correlation in the detection linear range.

TABLE 2 Linear distribution of 756 chemical hazards in fish and shellfish substrates

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(4) Matrix effects of 756 chemical hazards in fish and shellfish matrix

The matrix effect is the effect of the co-extract in the fish sample matrix on the concentration of the target analyte, and is a common problem when analyzing sample matrices using liquid methods because of ionization competition of matrix components with chemically hazardous substances at the column outlet. The matrix effect of each compound was expressed as either enhancement (> 0%) or inhibition (< 0%) by comparing the slope of the matrix-matched calibration curve to that of the pure solvent calibration curve. The matrix effect was evaluated in detail by dividing the results into six ranges and three groups. The analysis results are shown in fig. 3, when the value of the matrix effect is in the range of-20% -0% or 0% -20%, the corresponding weak matrix effect is a medium matrix effect in the range of-50% -20% or 20% -50%, and a strong matrix effect of-50% or less or 50% or more.

As shown in fig. 3, 641 species (84.79%), 653 species (86.37%), 703 species (92.99%) and 698 species (92.33%) of chemical hazards in the matrix of tilapia, grouper, oyster and scallop, respectively, exhibit weak matrix effects, i.e., between-20% and 20%, and the chemical hazards exhibiting weak matrix effects are not affected by the matrix effects, and can be quantified using a pure solvent calibration curve. The residual chemical hazard in the four matrixes shows medium matrix effect or strong matrix effect, and the two groups are easy to be influenced by biological matrixes, so quantitative data are required to be obtained by means of matching the matrixes with a calibration curve, and the influence of signal enhancement or inhibition caused by the matrix effect on the accuracy of experiments is avoided. From the above analysis, more than about 84% of the chemical hazards are found in the fish and shellfish matrix effects at the weak matrix effect level. Compared with fish matrix, the shellfish matrix is less affected by the matrix, the number of undetected chemicals is less compared with fish matrix, the matrix effect can not be detected by 4 compounds in oyster matrix, and the matrix effect can not be detected by 3 compounds in scallop matrix. The analysis shows that the method for detecting the chemical hazard in the fish and shellfish matrix is less influenced by matrix effect, and can accurately and quantitatively detect the chemical hazard.

To verify the relationship between retention time and matrix effect two variables, the matrix effect data and retention time for 756 chemical hazards are plotted on a scatter plot (fig. 4). Fig. 4 (a) (b) shows in the scatter plot of tilapia and grouper matrices that during the first chemical hazard elution to 15 minutes, more medium-matrix effect chemical hazards appear, and strong-matrix effect substances appear, exhibiting large instrument signal inhibition. However, from 15 minutes to the last chemical elution, the matrix effect of most chemical hazards is in the weak matrix effect zone; FIG. 4 (c) (d) shows in a scatter plot of oyster and scallop matrix that from 15 minutes to the last chemical elution, more chemical hazards are present in the medium or strong matrix effect region, indicating that the instrument signal is suppressed. Whereas during the first 15 minutes, most of the chemically hazardous substances are in the zone of weak matrix effect. By analyzing the four matrixes of the fish and the shellfish, the matrixes of the fish and the shellfish are not identical in appearance, but most of chemical hazards are weak in matrix effect, so that only very individual chemical hazard substances with strong matrix effect need to be quantified by matrix matching calibration in the quantifying process.

(5) Methodology investigation of 756 chemical hazards in fish and shellfish substrates

1) Accuracy and precision: the accuracy of the method is the proximity of the measured value to a known true value, the precision is the proximity between a series of measured values obtained from multiple samplings of the same sample, the accuracy is the mean value of the measured values (n=5) calculated for each horizontal repetition, the precision is expressed in RSD. The 756 chemical hazard substances were plotted as dots in a scatter plot using experimental results at matrix addition concentrations of 150ng/mL during the day and between days, graphically representing the accuracy and precision values for each compound.

As a result, as shown in FIG. 5, at a concentration of 150ng/mL, both in the fish matrix and in the shellfish matrix, most of the chemical hazards were located in a square area, i.e., accuracy was between 70% -120% and RSD was between 0% -20%, with only individual chemical hazards being outside the square range. The compounds outside the square range in the fish and shellfish matrix are not identical, but the substances outside the square in the day or in the daytime are basically identical for the same matrix, such as myclobutanil, amisulbrom and albendazole sulfoxide in tilapia and the pesticides triclosan, 2,4' -drip and epsilon-hexa in oyster matrix, which also verify the stability and reliability of the method.

In the present invention, the sensitivity of the instrument signal, which is the signal-to-noise ratio of 756 chemical hazards, was divided into three quantitative limits (10, 20, 50. Mu.g/kg), and the LOQ value of most of the four substrates was 10. Mu.g/kg. Reference is made to the industrial guidelines: the accuracy value should meet 70% -120% (20% relative standard deviation) at the LOQ level and 80% -115% (15% relative standard deviation) at the higher level, as the standard of the biological analysis method verifies. As shown in fig. 6, the results of the daily and daytime accuracy and precision tests in tilapia, grouper, oyster matrix and scallop matrix, respectively, at a matrix addition concentration of 10ng/mL, more than 88% of the chemical hazards in the daily and daytime tests of the fish matrix met the square range criteria described above, and more than 92% of the chemical hazards in the daily and daytime tests of the shellfish matrix met the square range criteria described above; at a matrix addition concentration of 150ng/mL, more than 92% of the chemical hazards of the fish matrix met the square range criteria described above in both the daily and daytime tests, and more than 97% of the chemical hazards of the shellfish matrix met the square range criteria described above in both the daily and daytime tests. With the improvement of the adding concentration, the accuracy and precision of the chemical hazardous substances are improved, and no obvious difference exists between the daily precision and the daily precision of the same chemical hazardous substances. By analyzing the above results, it is shown that the concentration values of most chemical hazards in the four types of substrates can be accurately and reliably determined and are completely effective within one week.

2) Recovery rate: recovery is the ratio of analyte remaining at the final measurement point after the analyte is added prior to extraction. Recovery rate with fish tissue or shellfish tissue without target compound residue as blank matrix, performing three concentration level addition recovery test, and good recovery rate can improve target analyte sensitivity. Recovery is also a parameter of the authenticity, i.e. accuracy, of the analytical process, and recovery is typically in the range of 70% -120% (RSD. Ltoreq.20%) where the process is acceptable for authenticity. The experimental results of recovery were divided into six different ranges (< 30%, 30-50%, 50-70%, 70-120%, > 120% and indeterminate), and the data results of the relative standard deviation were also divided into two groups (0% -20% and. Gtoreq.20%) to evaluate recovery of all chemical hazards.

As shown in table 3, when the addition concentration was 10 μg/kg, more than 88% of the chemical hazard in the fish matrix satisfied the recovery rate 70% -120% and RSD was in the range of 0% -20%, and more than 92% of the chemical hazard in the shellfish matrix satisfied the recovery rate 70% -120% and RSD was in the range of 0% -20%; when the addition concentration reaches 50 mug/kg, more than 93% of chemical hazard in the fish matrix meets the recovery rate 70% -120% and RSD is in the range of 0% -20%, and more than 98% of chemical hazard in the shellfish matrix meets the recovery rate 70% -120% and RSD is in the range of 0% -20%. As the concentration of the addition increases, the amount of compound that meets the recovery requirement increases. Through the analysis of the results of the recovery rates of the fish and shellfish substrates, it can be seen that the recovery rate range is satisfied by more than 88% of the chemical hazards at the lowest addition concentration, and the recovery rates of the two shellfish substrates are obviously better than those of the two fish substrates at the three addition concentrations, and the chemical hazard number of which is lower than 70% is higher than that of which is higher than 120%. In summary, most of the chemically hazardous substances, except for a few, were recovered well at all levels of addition for the four substrates in the validation of the method of the present invention.

TABLE 3 recovery rates and relative standard deviations for various additive concentrations in four matrices

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(6) Detection and analysis of fish and shellfish practical sample

The utility of this method in the detection of actual samples was further evaluated by detecting commercially available actual samples. The method was applied to non-targeted screening analysis of 64 product samples from farms and retail markets to quantify chemical hazards present by using the matrix-matched calibration curves of the present invention. Details of the detection of chemical hazards in fish and shellfish are shown in table 4 below. The detection result of the actual sample of the tilapia shows that the pesticide fenpropidin and the veterinary drug econazole exist, wherein the pesticide residue level is slightly higher than the maximum residue amount of 0.02mg/kg regulated by European Union. The detection result of the grouper actual sample shows that the pesticides of the metazachlor, the methamphetamine sulfoxide and the veterinary drug of the flubendazole exist, and the content of the metazachlor, the methamphetamine sulfoxide and the veterinary drug of the flubendazole are lower than the maximum residue limit of 0.05mg/kg regulated by European Union. The detection result of the oyster practical sample shows that the pesticide pyraclostrobin and the persistent pollutant dihexyl phthalate exist, and the detected chemical hazardous substances are lower than the maximum residue specified by national standards. The detection result of the scallop actual sample shows that the pesticide nitenpyram and the veterinary drug pyrazole tam are both lower than the maximum residue limit specified by national standards. The practicality of the established method for rapidly screening unknown chemical hazard residues in fish and shellfish is further verified through actual sample detection. Therefore, in the aquatic product cultivation process, safety factors still need to be further paid attention to control, problematic products are prevented from flowing into the market, and the supervision department should further strengthen the control of the quality safety of the products.

TABLE 4 detailed information on detection of chemical hazards in actual samples

Table 1 of the present invention is a database of non-targeted screening mass spectra of 756 chemical hazard substances, and in Table 1, the compound names represented by the numbers 1-756 are as follows:

1. 2, propyzamide, 3, hymexazol, 4, chlormefos, 5, propyzamide, 6, cycloprotic, 7, diphenylamine, 8, acetamiprid, 9, ethaboxam, 10, phorate, 11, methamphetamine, 12, pentachloronitrobenzene, 13, atrazine-deethyl, 14, clomazone, 15, diazinon, 16, dinotefuran, 17, bupirimate, 18, simazine, 19, prophos, 20, zhong Dingtong, 21, desmopraph, 22, propargyl, 23, carbofuran, 24, eistun, 25, dichlorvos, 26, dermatophagofos, 27, prometryne, 28, cyprohexane, 29, ethephon, 30, beta-hexatick, 31, metalaxyl, 32, chlorpyrifos, 33, methylphos, 34, anthraquinone, 35, delta-hexapole, 36, 37 times thiophos, 38, fenitrothion, 39, ethyl paraoxon, 40, triazolone, 41, parathion, 42, pendimethalin, 43, linuron, 44, chlorfenamate, 45, ethyl bromothion, 46, quinothion, 47, trans-chlordane (gamma), 48, fenitrothion, 49, metazachlor, 50, benfuracarb, 51, profos, 52, plastic, 53, dieldrin, 54, procymidone, 55, methidathion, 56, fenpropazine, 57, dichlormate, 58, oxadiazon, 59, benfophos, 60, fenhexamid, 61, acaricidal, 62, bupirimate, 63, carboxin, 64, fluoroamide, 65, 4' -drop, 66, ethion, 67, methiphos, 68, epoxiconazole, 69, myclobutanil, 70, gramineraloxid (methyl), 71, propiconazole, 72, fenprox, 73, fenprox, 74, 75, fenpropineb Triclosan, 76, flubenamal, 77, 4 '-methoprene, 78, oxadixyl, 79, tetramethrin, 80, tebuconazole, 81, fluben, 82, pyridaphethione, 83, iminothiolane, 84, trifloxystrobin, 85, carboxin oxide, 86, cis-permethrin, 87, trans-permethrin, 88, pyrazophos, 89, cypermethrin, 90, fenvalerate, 91, deltamethrin, 92, dichlormate, 93, butachlor, 94, diquat, 95, kefebuxol, 96, clomazone, 97, fasfos, 98, difenoconazole, 99, tetramethrin, 100, heptenaphos, 101, hexachlorobenzene, 102, clomazone, 103, oat, 104, chlorfenapyr, 105, trifluralin, 106, chlorfenapyr, 107, methidathion, 108, oxaprozin, 109, alpha-hexazin, 110, terfenacet, 111, 112, ciprofloxacin, 113, phoxim, 114, chlorphenamine, 115, clodinafop, 116, chlornitramine, 117, terbuthylazine, 118, chlorfluazuron, 119, flufenoxuron, 120, sulfoxaflor, 121, chlorpyrifos, 122, dimethenamid, 123, dimethenamid, 124, mechlorethamine, 125, pyrifos, 126, terbutryn, 127, graminium, 128, prothiotep, 129, triclopyr, 130, metolachlor, 131, oxychlorodane, 132, pyrimidephos, 133, methoprene, 134, bromthiophos, 135, benfomesafen, 136, ethofumesafen, 137, isoproturon, 138, alpha-thiodane, 139, dichlorvos, 140, iso Liu Lin, 141, fos, 142, chlorpyriphos, 143, cis-chlordane (alpha), 144, meflofop, 145, 4' -d, 146, butachlorfenamide, 148, butachlor, 149, 147 Iodophos, 150, Z-cycloxaprid, 151, chlorbenzuron, 152, profenofos, 153, fludioxonil, 154, buprofezin, 155, 2,4' -drop, 156, isodiradiol, 157, hexaconazole, 158, fenpyrad, 159, 2,4' -drop, 160, paclobutrazol, 161, methoprene, 162, imazalil, 163, propyl ester, 164, methyl dicamba, 165, herbicidal ethers, 166, oxyfluorfen, 167, fenphos, 168, beta-endosulfan, 169, fenfluroxypyr, 170, 4' -drop, 171, trithion, 172, benalaxyl, 173, dichlorphos, 174, triazophos, 175, fenphos, 176, chlorfenamide, 177, thiodan sulfate, 178, fenpyrad, 179, neo, 180, fenpropathrin, 181, bromophos, 182, fenphos, 185, fenphos, 184, fenpropimorph, 186, fenarimol, 187, benfophos, 188, prochloraz, 189, coumaphos, 190, cyfluthrin, 191, cyfluthrin, 192, dichlorvos, 193, biphenyl, 194, bentazone, 195, 3, 5-dichloroaniline, 196, molinate, 197, E-fenphos, 198, o-phenylphenol, 199, cis-1, 2,3, 6-tetrahydrophthalimide, 200, fenobucarb, 201, flumetsulam, 202, hexaflumuron, 203, paracetamol, 204, imazalil, 205, pyrimethanil, 206, gamma-hexazin, 207, etoposide, 208, atrazine, 209, heptachlor, 210, iprobenfos, 211, clomazone, 212, triclosamide, 213, fenpropimorph, 214, tebufenpyr, 215, clomazone, 216, tolclomazone, 217, propineb, 218, 219, bromarone, bromafos, 221. oxaziclomefone, 222, thiamethoxam, 223, epsilon-hexa, 224, isopropane, 225, ambrox, 226, diethofencarb, 227, perazone, 228, bioallethrin, 229, 2,4' -d-i, 230, fenpyroximate, 231, bisfenhexamid, 232, chlorpyrifos, 233, propathrin, 234, penconazole, 235, benomyl, 236, tetraconazole, 237, profenofos, 238, fluquindox, 239, triadimenol, 240, pretilachlor, 241, kresoxim, 242, fluazifop-butyl, 243, fluazinam, 244, ethylacaricidal, 245, uniconazole, 246, flusilazole, 247, triflumuron, 248, diniconazole, 249, pyriproxyfen, 250, propargite, 251, fenamid, 252, flufenoxaprop, 253, tebufenpyr, 254, fenacet, 255, phenothrin, 256, 257, flufenamid, 258, sethoxydim, 259, anilofos, 260, flumethrin, 261, lambda-cyhalothrin, 262, mefenacet, 263, permethrin, 264, pyridaben, 265, fluoroethyl, 266, bitertanol, 267, ethofenprox, 268, buprofezin, 269, alpha-cypermethrin, 270, fluvalinate, 271, S-fenvalerate, 272, difenoconazole, 273, flumioxazin, 274, flumetofen-pentyl, 275, methamphetamine, 276, etharight sulfoxide, 277, pentachlorobenzene, 278, triisobutyl phosphate, 279, murill, 280, 4-bromo-3, 5-xylyl-N-methylcarbamate, 281, oat ester, 282, benfophos, 283, 2,3,5, 6-tetrachloroaniline, 284, tributyl phosphate, 285, 2,3,4, 5-tetramethoxybenzene, 286, pentachloromethoxybenzene, 287, prochloraz, 288. vegetable phosphorus, 289, methabenzthiazuron, 290, simaron, 291, atrapassed, 292, atrazine-deisopropyl, 293, terbuthionsulfone, 294, tefluthrin, 295, bromhexine, 296, indac, 297, cyclouron, 298, 2, 6-dichlorobenzamide, 299, 2,4' -trichlorobiphenyl, 300, 2,4, 5-trichlorobiphenyl, 301, terbuthylazine-deethyl, 302, 2,3,4,5, -tetrachloroaniline, 303, sunflower musk, 304, xylene musk, 305, pentachloroaniline, 306, azidine, 307, terbuthylazine, 308, ding Mi amide, 309, 2', 5' -tetrachlorobiphenyl, 310, musk, 311, benfodane, 312, xylenothiazide, 313, oxyphenol, 314, methylparaben, 315, heptamide, 316, tibetan musk, 317, carbochlor, 319, octocrylene, azophos, octopine, 320, isoeuclidean agent, 321, buprofezin, 322, chlorpyrifos, 323, dimethyl chlorophthalide, 324, 4' -dichlorobenzophenone, 325, phthalyl ester, 326, musk ketone, 327, piimidazole, 328, cyprodinil, 329, corncob, 330, oxyiso Liu Lin, 331, triclopyr, 332, 2',4, 5' -pentachlorodiphenyl, 333, 2 methyl 4-chlorobutoxyethyl, 334, fenphos, 335, methamphetamine sulfone, 336, acaricidal alcohol, 337, trans-nine chloride, 338, miticidal agent, 339, desolvated She Lin, 340, fludioxonil, 341, bromophenzene phosphorus, 342, ethylnasal discharge, 343, sterilized phosphorus, 344, 2,3, 4', 5-pentachlorodiphenyl, 345, 4' -dibromobenzophenone, 346, tebuconazole, 347, dimine phosphorus, 348, ethylmethidathion, 349, 2', 4',5' -hexachlorobiphenyl, 350, lv triadimenol, 351. ethaboxam sulfone, 352, hexythiazox, 353, 2',3, 4',5 '-hexachlorobiphenyl, 354, wilsons, 355, bifenthrin, 356, cyproconazole, 357, butyl benzyl phthalate, 358, clodinafop-propargyl, 359, phoxim sulfoxide, 360, trifluoperazole, 361, fluroxypyr, 362, phoxim sulfone, 363, triphenyl phosphate, 364, oxaziclomefone, 365, 2',3, 4',5' -heptachlorobiphenyl, 366, tebufenpyrad, 367, cloquintocet, 368, cycloxaprin, 369, furfuryl azole, 370, trifluralin, 371, benomyl sulfoxide, 372, benomyl sulfone, 373, fenpiclonil, 374, fluquinconazole, 375, fenbuconazole, 376, cyclic ethylene tridecanate, 377, propoxur, 378, isoprocarb, 379, methamidophos, 380, acenaphthene, 381, cycloxaprid, 382, phthalimide, 383, phosphorus oxychloride, 384, pencycuron, 385, buthiuron, 386, endo-phosphorus-S-methyl, 387, fenphos, 388, phenanthrene, 389, spiroxamine, 390, fenpyroximate, 391, butyl pyrimidine phosphorus, 392, jasmone; jasmonic acid inducer, 393, fenpropidin, 394, chlornitramine, 395, fluquindone, 396, propargyl oxamine, 397, pirimicarb, 398, phosphoramidon, 399, benomyl, 400, brombutamide, 401, acetochlor, 402, benoxacin, 403, terfenadine, 404, penoxsulam, 405, furamide, 406, alamic acid benzene-S-methyl, 407, furazafen, 408, dithiopyr, 409, metalaxyl, 410, marathon, 411, simeconazole, 412, dimethyl chlorophthalide, 413, thiabendazole, 414, methylparaben, 415, butralin, 416, zoxamine, 417, diphenox, 418, allethrin, 419, isovalerate, 420, chlorquinone, 421, methoprene, 422, flufenacet, 423, pyrifos, 424, tetrachloraz, 425, furalachlor, 427, thiazate, 426, 428, dimethenamid, 429, pyripyroxad, 430, picoxystrobin, 431, imazamox, 432, imazamox, 433, (E) -benoxaden, 434, thiocyan, 435, methomyl sulfone, 436, imazalil, 437, isoprothiolane, 438, cyflufenamid, 439, pyriminobac-methyl, 440, oxazophos, 441, (Z) -benoxamine, 442, benomyl, 443, thifluzamide, 444, quinoxyfen, 445, chlorfenapyr, 446, trifloxystrobin, 447, imibenconazole-ox-debenzoyl, 448, bisbenzoxazole, 449, fipronil, 450, propathrin, 451, flufenoxaprop, 452, epoxiconazole, 453, pyriproxyfen, 454, barnac, 455, thenalachlor, 456, clethodim, praziram, 458, valicarb, 459, etoxazole, 460, pyriproxyfen, 461, fluazifop-butyl, 463, fluazifop-butyl, 465. bifenazate, 466, isodiradil, 467, clomazone, 468, imidazolone, 469, napropylamine, 470, pyraclostrobin, 471, lactofen, 472, triclopyr, 473, pyraclostrobin, 474, clophos, 475, spirodiclofen, 476, benfenpyr, 477, furazolidone, 478, pyriftalid, 479, flusilafen, 480, pyriminobac, 481, acetamiprid, 482, flumetsulam, 483, propiconazole, 484, fluazinam, 485, exoxyheptachloride, 486, methamidophos, 487, propamocarb, 488, acetamiprid, 489, trichlorfon, 490, cinnating, 491, methamphetamide, 492, carboxin, 493, phoxim, 494, methoprene, hydrazine, diafenthiuron, 496, thifensulfuron, 497, ethoxysulfuron, 498, trifloxystrobin, 499, spinetoram, 500, propiconazole, 500, 502, pymetrozine, 503, clomazone, 504, methomyl, 505, cinosulfuron, 506, pyrazosulfuron, 507, methomyl, 508, cymoxanil, 509, omethoate, 510, ethoxyquinoline, 511, de-methidat, 512, imazethapyr, 513, uniconazole, 514, clomazone, 515, aphidicolinate, 516, te Ding Linfeng, 517, cyazofamid, 518, florasulam, 519, benomyl, 520, chlormequat, 521, sethoxydim, 522, folpet, 523, methomyl, 524, cartap, 525, tripropyl phosphate, 526, triisobutyl phosphate, 527, tributoxyethyl phosphate, 528, 2-ethylhexyl diphenyl phosphate, 529, trioctyl phosphate, 530, tricresyl phosphate, 531, p-toluoyl phosphate, 532, tricresyl phosphate, 533, tri (1, 3-dichloropropyl) phosphate, 534, toluene diphenyl phosphate, 535, triphenyl phosphate, 536, tri (2-chloropropyl) phosphate, 537, tributyl phosphate, 538, trichloroethyl phosphate, 539, triethyl phosphate, 540, trimethyl phosphate, 541, butyl benzyl phthalate, 542, diphenyl phthalate, 543, di (2-butoxyethyl) phthalate, 544, dibutyl phthalate, 545, dicyclohexyl phthalate, 546, dimethyl phthalate, 547, di-n-pentyl phthalate, 548, diethyl phthalate, 549, diisobutyl phthalate, 550, dihexyl phthalate, 551, dioctyl phthalate, 552, di (2-methoxy) ethyl phthalate, 553, di-2-ethoxyethyl phthalate, 554, di-4-methyl-2-pentyl phthalate, 555, dioctyl phthalate, 556, dinonyl phthalate, 557, thiocarbamoyl-saxotoxin 2, 558, thiocarbamoyl-saxotoxin 3, 559, dessaxotoxin, saxitoxin, 560, saxitoxin, 573, sulfadiazepine, 573, sulfadiazine, 573, 575, 35, microcystin, 573, 35, sulfadiazine, 573, 35, microcystin, 573, and other drugs, 579. sulfamethoxine, 580, sulfamethazine, 581, sulfamethazine, 582, sulfamethazine, 583, sulfabenzene pyrazole, 584, sulfapyrazole, 585, sulfapyridine, 586, sulfaquinoxaline, 587, sulfathiazole, 588, sulfadimidine, 589, trimethoprim, 590, cinnoxacin, 591, danofloxacin, 592, difluoro-floxacin, 593, enfluroxacin, 594, flumequine, 595, gatifloxacin, 596, lomefloxacin, 597, bambafloxacin, 598, moxifloxacin, 599, nalidixic acid, 600, ofloxacin, 601, obifloxacin, 602, oxolinic acid, 603, salad floxacin, 604, sparfloxacin, 605, toxacin, 606, albendazole (propylthioimidazole), 607, albendazole-2-amino sulfone, 608, albendazole sulfoxide, 609, aminotoluimidazole, 610, thiabendazole (thiabendazole), 611, dimetizole (dimetizole), 612, fenbendazole (phenylthiobendazole), 613, flubendazole (fipronil), 614, hydroxyipronidazole, 615, ipronidazole, 616, mebendazole, 617, thiabendazole (canbendazole), 618, 5-hydroxythiabendazole, 619, levamisole, 620, metronidazole, 621, oxfendazole, 622, propoxybendazole, 623, luo Xiao, 624, secnidazole, 625, tinidazole, 626, triclabendazole, 627, clindamycin, 628, doramectin, 629, epleromycin, 630, ivermectin, 631, lincomycin A1, 632, spiramycin, 633, tilmicosin, 634, tylosin, 635, jinimesulide M1, 636, beclomethasone, 637, beclomethasone dipropionate, 638. betamethasone dipropionate, 639, betamethasone valerate, 640, chlordydrogesterone acetate, 641, clobetasol propionate, 642, clobetasone butyrate, 643, cortisone, 644, deflazacort, 645, dexamethasone, 646, diflorasone diacetate, 647, epitestosterone, 648, fludrocortisone, 649, flumethasone, 650, fluminosone pivalate, 651, fluocinolone acetate, 652, fludropinol, 653, fluorometholone, 654, fluticasone propionate, 655, halcinonide, 656, hydrocortisone, 657, megestrol, 658, medroxyprogesterone acetate, 659, methylprednisolone, 660, mometasone furoate, 661, prednisolone, 662, testosterone, 663, triamcinolone, 664, banbuterol, 665, hydroxymethylcyclone, 666, clenbuterol, 667, clenbuterol, 35668, isoprotujol, 669, clenbuterol, 670, fenoterol, 671, formoterol, 672, pirbuterol, 673, ractopamine, 674, ritodrine, 675, salmeterol, 676, sotalol, 677, terbutaline, 678, tobuterol, 679, clenbuterol, 680, acetaminophen, 681, chlorpromazine, 682, clopyralid, 683, 4' -diaminodiphenyl sulfone, 684, cabado, 685, haloperidol, 686, azapirol, 687, azapirone, 688, propionylpromazine, 689, celecoxib, 690, carbamazepine, 691, diphenhydramine, 692, imipramine, 693, sulpiride, 694, zolpidem, 695, fluoxetine, 696, caffeine, 697, codeine, 698, 1, 7-dimethylxanthine, 699, chlorprocaine, 700, procaine, 700, 703, 706. cetirizine, 707, chlorpheniramine, 708, fluphenazine, 709, hydroxyzine, 710, promethazine, 711, terfenadine, 712, bifonazole, 713, econazole, 714, griseofulvin, 715, ketoconazole, 716, naftifine, 717, flunixin, 718, ketotifen, 719, lornoxicam, 720, melitracin, 721, oxaprozin, 722, antipyrine, 723, sulfamidine, 724, difluorofloxacin hydrochloride, 725, fluoxacin, 726, enoxacin, 727, sarafloxacin hydrochloride, 728, nafofloxacin, 729, norfloxacin, 730, gemifloxacin mesylate, 731, glipizide, 732, repaglinide, 733, hygromycin B,734, sulfadimeizole, 735, amikacin, 736, tolbutamide, 737, gliclazide, 738, gliquidone, 739, glibenclamide, 740, 1-aminohydantoin hydrochloride, 741, pioglitazone hydrochloride, 742, metformin hydrochloride, 743, glimepiride, 744, sulfamonomethoxine, 745, sulfacetamide, 746, sulfachloropyridazine, 747, sulfadimazole, 748, sulfanifedipine, 749, sulfadimidine, 750, sulfamethoxazole, 751, buformin hydrochloride, 752, phenformin hydrochloride, 753, clotrimide, 754, ciprofloxacin, 755, kanamycin sulfate, 756, tobramycin.

Table 1 is as follows:

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Claims (6)

1. a method for rapidly screening unknown chemical hazard residues in aquatic products based on a high-resolution mass spectrometry database, which is characterized by comprising the following steps:

(1) Sample extraction and purification: pretreating fish meat and shellfish meat by adopting an improved QuECHERS method;

(2) And (3) chromatographic mass spectrometry detection: carrying out sample analysis by using an analysis method of high-resolution mass spectrometry of ultra-high performance liquid chromatography;

(3) Matching database: searching and comparing the acquired parameters of the sample to be detected with the established high-resolution mass spectrum database to realize screening and confirming of unknown chemical hazard;

the pretreatment of fish and shellfish meat using the modified QuECHERS method comprises the following steps:

s1, carrying out preliminary treatment on an obtained aquatic product sample, and homogenizing;

s2, extracting the treated sample matrix twice by using acetonitrile acidified by formic acid to obtain supernatant of the extract;

s3, adding the extract supernatant and the purifying agent into a centrifuge tube, fully mixing, and centrifuging to obtain the purified extract supernatant;

s4, drying the purified supernatant with nitrogen, re-dissolving, and filtering with a 0.22 mu m filter membrane to obtain a sample to be tested;

The step S2 specifically includes the following steps:

s21, accurately weighing a homogenized and homogenized muscle sample of 2.00+/-0.02 g fish or a shellfish sample into a 10 mL centrifuge tube, then adding 8 mL of acetonitrile acidified by 0.1% formic acid, carrying out vortex mixing, extracting, and centrifuging to obtain a supernatant;

s22, transferring the supernatant obtained by centrifugation in the step S21 into a 50 mL centrifuge tube, repeatedly extracting the residual residues once by 8 mL of acetonitrile acidified by 0.1% formic acid, and combining the supernatants of the two times;

the step S3 specifically comprises the following steps:

s31, placing 500 mg of the C18 dispersible adsorbent into a 50 mL graduated polypropylene centrifuge tube;

s32, adding the extracted supernatant into the 50 mL polypropylene centrifuge tube, and fully swirling to obtain a mixed treatment solution;

s33, carrying out centrifugal treatment on the mixed treatment solution to obtain purified extract supernatant;

the liquid chromatography conditions were: chromatographic column: ACQUITY UPLC BEH C18 chromatographic column; column temperature: 40 ℃; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; flow rate: 0.4 mL/min; mobile phase gradient elution procedure: 0-0.5 min 5% B; linearly rising from 5% B to 15% B within 0.5-3.0 min; 3.0-6.0 min, linearly increasing from 15% B to 40% B, and keeping for 3 min; linearly rising from 40% B to 60% B within 9.0-15.0 min; linearly rising from 60.0% B to 99.0% B for 15.0-19.0 min, maintaining for 4.0 min, returning to the initial mobile phase, maintaining for 2.0 min, and preparing for the next sample injection; sample injection amount: 5.00 Mu L;

The chemical hazards include the following 756 types, the specific types are as follows:

the number of the pesticides is 524, including oxadiazon, propyzamide, benomyl, chlormequat, anil, cycloprotic, diphenylamine, chlorpyrifos, ethaenflurane, phorate, methyiphos, pentachloronitrobenzene, atrazine-deethyl, clomazone, diazinon, dinotefuran, buprofezin, bupirifos, simazine, amifos, zhong Dingtong, desmopride, propyzamide, fenoxacarb, aldrin, dichlormid, coumaphos, prometryn, ciprofen, captalide, beta-hexahexa, metalaxyl, chlorpyrifos, methyl parathion, anthraquinone, delta-hexa, betamethasone, fenitrothion, ethyl paraoxon, triazolone, parathion, pendimethalin, linuron, chlorfenamide, ethyl bromethion, gamma-chlordane, oryzalin, fenpyrad, pyroxafen, benfop, prometryn, prochloraz, and varrozen methidathion, cyanazine, dichlormid, oxadiazon, benfophos, chlorpyrifos, ethirimol sulfonate, carboxin, flutolanil, 4 '-drop, ethionine, methidathion, epoxiconazole, myclobutanil, methyl graminearum, propiconazole, fenphos, bifenthrin, benomyl, mefenoxam, flubenomyl, 4' -methoxy drop, oxadixyl, tetramethrin, tebuconazole, fenbuconazole, fenpropiconazole, fenpropimorph, fenitrothion, fenpropimorph, fenpropiconazole, fenitrothion, and other compounds Fluochlorpyrifos, pyridaphethione, imiphos, trichlorfon, carboxin, cis-permethrin, trans-permethrin, pyrazophos, cypermethrin, fenvalerate, deltamethrin, dichlormid, dichlornil, fipronil, clomazone, fashion, difenoconazole, tetrachloronitrobenzene, heptylphosphine, hexachlorobenzene, acephate, avenanthramide, chlorprophos, trifluralin, chlorpyrifos, for the treatment of fenitrothion, oxaden, alpha-hexazin, terbutafos, terbutaline, ciprofloxacin, dichlorphos, chlorphenazine, clodinafop-propargyl, chlorpyrifos, terbutryn, chlorfluazuron, fenitrothion, chlorpyrifos-methyl, dimethenamid, methrin, pyrifos-methyl, terbutryn, prosulfocarb, triclopyr, metolachlor, oxychlorodane, pyrimidone, methoprene, bromethion, benfomesal, ethofumesate, trifluralin, alpha-thiodan, propanil, isoxaphos, fenitrothion, alpha-chlordan, tolfenphos, 4 '-d-i, butachlor, ethion, bazafion, iodiphos, Z-dicamba, chlorfenuron, profenofos, fludioxonil, buprofezin, 2,4' -d-i, isoxaprop-2, 4 '-d-i, fluzachlor, fluben-ethyl, flubenfot-2' -d-ethyl, 4 '-d-ethyl paclobutrazol, methoprene, flubenfot, propyl ester acaricidal alcohol, methyl pyribac-methyl, pyrithiobac-sodium, oxyfluorfen, chlorfenphos, beta-thiodane, fenfluralid, 4' -D-alditol, trithion, benalaxyl, fenoxanil, triazophos, fenbucin, chlorfenapyr, thiodane sulfate, fenpyroxim, neoyan, fenpropathrin, bromophenophos, fenphos, hexazinone, valicarb-methyl, fenpyrad Baofushi, chlorpyrifos, yigossyphos, prochloraz, coumaphos, cyfluthrin, fluvalinate, dichlorvos, biphenyls, dichlorphos, 3, 5-dichloroaniline, molinate, E-chlorfenphos, o-phenylphenol, cis-1, 2,3, 6-tetrahydrophthalimide, fenobucarb, flubendiamide, hexaflumuron, chlorfenapyr, chlormequat, pyrimethanil, gamma-hexa, ethametin, atrazine, tebufenozide, clomazone, triclosan, fenpropimorph, tebufenpyrad, halofop-butyl, tolclofos-methyl, iprovalicarb, ametryn, simetryn, bromuron, metribuzin, thiamethoxam, epsilon-hexahexazinone, iprovalicarb, buprofezin, diethofencarb, perambulator, bioallethrin, 2,4 '-d-fenpropidin, fenisobenzoate, fenpropiram, chlorpyrifos, propargyl penconazole, cyromazine, tetraconazole, profenofos, flumetrazine, triadimenol, pretilachlor, kresoxim-methyl, fluazifop-butyl, chlorfluazuron, fenbuconazole, uniconazole, flusilazole, trinitrofen, diniconazole, synergistic ether, propargite, fenpyrad, oxazachlor, tebufen, fenazaquin, phenothrin, fludioxonil, fenoxycarb, sethoxydim, anilofos, fenpropidin Profipronil, lambda-cyhalothrin, mefenacet, permethrin, pyridaben, fluorofluoroethyl, bitertanol, ethofenprox, thioxanthone, alpha-cypermethrin, flufenvalerate, S-fenvalerate, difenoconazole, flumioxazin, flumetofen-pentyl, methafop, etoposide, pentachlorobenzene, triisobutyl phosphate, triclopyr, 4-bromo-3, 5-xylyl-N-methyl carbamate, oat ester, benfop, 2,3,5, 6-tetrachloroaniline, tributyl phosphate, 2,3,4, 5-tetramethoxybenzene, pentachloromethoxybenzene, pasture, vegetable and fruit phosphorus, mebendazole, simatron, atrazine-deisopropyl, terbutafos sulfone, tefluthrin, bromeflutide, indac, cyclouron, 2, 6-dicarboxamide, 2,4' -trichlorobiphenyl, 2,4, 5-trichlorobiphenyl, terbuthylazine-deethyl, 2,3,4,5, -tetrachloroaniline, sunflower seed musk, xylene musk, pentachloroaniline, azidine, terbuthylazine, ding Mi amide, 2', 5' -tetrachlorobiphenyl, musk, benomyl, xylenol thiabendazole, oxyphenolide, methylparaben, heptylpyridinium, tibetan musk, carbochlor, octachlorostyrene, pyrithione, isoxaben, buprofezin, clomazone, dimethyl chlorophthalic acid, 4 '-dichlorobenzene, phthalyl, ketone musk, piimidazole, cyprodinil, corning, isoxaben, isochlorophos, 2',4,5 '-pentachlorodiphenyl, 2-methyl-4-chlorobutyloxyethyl ester, fenpyr-methyl, methamphetamine sulfone, acaricidal alcohol, trans-nine chlorine, miticidal agent, defoliated phosphorus, fludioxonil, brombenomyl, ethyl acetate, sterilizing phosphorus, 2,3, 4', 5-pentachlorodiphenyl, 4 '-dibromodiphenyl ketone, flutriafol, triazophos, ethyl methidathion, 2', 4',5' -hexachlorodiphenyl, lv triadimenol, ethasone, hexythiazox, 2',3, 4',5 '-hexachlorobiphenyl, penoxsulam, benfurthrin, cyproconazole, butyl benzyl phthalate, clodinafop-propargyl, thionyl sulfate, trifluoperazole, isooctyl fluroxypyr, thion sulfone, triphenyl phosphate, oxaziclomefone, 2',3, 4',5' -heptachlorobiphenyl, tebufenpyrad, cloquintocet-mexyl, cyprodinil, furfuryl azole, trifluralin, fenphos sulfoxide, fenphos sulfone, fenpiclonil, fluquinconazole, fenbuconazole, cyclic ethylene tridecanate, propoxur, isoprocarb, methamidophos, acenaphthene, cycloxaprid, phthalimide, phosphorus oxychloride, pencycuron, buthiuron, inner phosphorus-S-methyl, thiotepa, phenanthrene, spiroxamine, fenpyroximate, butyl pyrifos, jasmone; jasmonic acid inducer, fenpropidin, chlornitramine, fluquindone, propargyl oxamide, pirimicarb, phosphoramidon, clomazone, bromobutachlor, acetochlor, mefenacet, terfenacet, penoxsulam, dinotefuran, alamic acid type benzene-S-methyl, furfur yellow, dithiopyr, mefenoxam, malamat, simeconazole, dimethyl chlorophthalic acid, thiofide, methylparaben, butralin, zoxamide Bifenacet, allethrin, isovaleryl, methoprene, flufenacet, fenoxanil, citalopram, furalaxyl, thiamethoxam, cyprodinil, captan, pyripyroxad, imazamox, (E) -phenoxyjunan, benthiavalicarb-isopropyl, methoprene, imazamox, isoprothiolane, trifloxystrobin, cyflufenamid, pyriminox, oxazachlor, (Z) -phenoxyjunan the composition comprises benomyl, thifluzamide, quinoxyfen, chlorfenapyr, trifloxystrobin, metazachlor, fenbuconazole, mefenoxam, fipronil, imazamox, flufenoxaprop-ethyl, pyrifos, fenbufen, clethodim, trifloxystrobin, fenpyrad, valicarb-methyl, valicarb-isopropyl, pyriproxyfen, iprodione, pirfenphos, furamide, bifenazate, isodieldrin, clofenamate, fenamidone, napropylamine, pyraclostrobin, lactofen, triclopyr, pyraclonil, chlorpyrifos, spirodiclofen, benomyl, furbenfurone, pyrifos, flusilafen, pyriminobac-methyl, fenpyrad, fluazinam, triclopyr, chlorfenapyr, triad, pephos, methamidone, methidathion, carboxin, phoxim, methoxyfenozide, diafenthiuron, thifensulfuron methyl, ethoxysulfuron, spinosyn, mepiquat chloride, tricyclazole, isoproturon, pymetrozine, clomazone, metolcarb, cinosulfuron, pyrazosulfuron-ethyl, methomyl, cymoxanil, omethoate, ethoxyquinoline, dactyloxazine, imazethapyr, uniconazole, clomazone, ter Ding Linfeng, cyazofamid, florasulam, benomyl, chlormequat, sethoxydim, folpet, methoprene;

The number of the veterinary medicines is 182, including benzoylsulfadiazine, sulfadiazine, sulfamonomethoxine, sulfadimidine, sulfadiazine, sulfathiodiazole, sulfadiazine, sulfabenzene pyrazole, sulfapyrazole, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfadimidine, trimethoprim, cinnoxacin, danofloxacin, difloxacin, enfluxacin, flumethazine, gatifloxacin, lomefloxacin, bamofloxacin, moxifloxacin, nalidixic acid, ofloxacin, obiferin, oxaquinic acid, sarafloxacin, sparfloxacin, tosulfloxacin, albendazole-2-amino sulfone, albendazole sulfoxide, aminothiazole, thiabendazole, dimet-zene, fenbendazole, hydroxyisopropyl oxazole, ipronidazole, tolimidazole, thiabendazole ester, ofloxacin, and other drugs 5-hydroxythiabendazole, levamisole, metronidazole, oxfendazole, propoxybendazole, luo Xiao, secnidazole, tinidazole, triclabendazole, clindamycin, doramectin, epleromycin, ivermectin, columniform-A1, spiramycin, tilmicosin, tylosin, virginiamycin M1, beclomethasone dipropionate, betamethasone valerate, megestrol acetate, clobetasol propionate, clobetasone butyrate, cortisone, deflazacort, dexamethasone, diflorasone diacetate, epitestosterone, fludrocortisone, flumethasone, flucinolone acetonide, fludrolone, flumidon, fluticasone, halcinolone, megestrol acetate, methylprednisolone, mometasone furoate, prednisolide, testosterone, triamcinolone acetonide, bambuterol, hydroxymethyl clenbuterol, isocclenbuterol Pan Te, clenbuterol, fenoterol, formoterol, pirbuterol, ractopamine, ritodrine, salmeterol, sotalol, terbutaline, tobuterol, clenbuterol Pan Te, acetaminophen, chlorpromazine, chloropyridine, 4' -diaminodiphenyl sulfone kabat, haloperidol, azapiprazole, azapirone, propidium, thiamethoxam, carbamazepine, diphenhydramine, imipramine, sulpiride, zolpidem, fluoxetine, caffeine, codeine, 1, 7-dimethylxanthine, chloroprocaine, cinchocaine, lidocaine, procainamine, procaine, tetracaine, brompheniramine, cetirizine, chlorpheniramine, fluphenazine, hydroxyzine promethazine, terfenadine, bifonazole, econazole, griseofulvin, ketoconazole, naftifine, flunixin, ketotifen, lornoxicam, melitracin, oxaprozin, antipyrine, sulfamidine, difloxacin hydrochloride, fleroxacin, enoxacin, sarafloxacin hydrochloride, nadifloxacin, norfloxacin, gemifloxacin mesylate, glipizide, repaglinide, hygromycin B, sulfadimidine, and the like amikacin, tolbutamide, gliclazide, gliquidone, glibenclamide, 1-aminohydantoin hydrochloride, pioglitazone hydrochloride, metformin hydrochloride, glimepiride, sulfamonomethoxine, sulfacetamide, sulfachloropyridazine, sulfadimidine, sulfanifedipine, sulfap-methoxypyrimidine, sulfamethoxazole, buformin hydrochloride, phenformin hydrochloride, clotrimide, ciprofloxacin, kanamycin sulfate, amitraz, tobramycin;

The persistent organic matter is 32, including tripropyl phosphate, triisobutyl phosphate, tri (butoxyethyl) phosphate, 2-ethylhexyl diphenyl phosphate, trioctyl phosphate, tricresyl phosphate, p-toluoyl phosphate, tricresyl phthalate, tris (1, 3-dichloroisopropyl) phosphate, toluene diphenyl phosphate, triphenyl phosphate, tris (2-chloropropyl) phosphate, tributyl phosphate, trichloroethyl phosphate, triethyl phosphate, trimethyl phosphate, butyl benzyl phthalate, diphenyl phthalate, bis (2-butoxyethyl) phthalate, dibutyl phthalate, dicyclohexyl phthalate, dimethyl phthalate, di-n-pentyl phthalate, diethyl phthalate, diisobutyl phthalate, dihexyl phthalate, dioctyl phthalate, di (2-methoxy) ethyl phthalate, di-2-ethoxyethyl phthalate, di-4-methyl-2-pentyl phthalate, dioctyl phthalate, dinonyl phthalate;

marine biotoxins are 18: thiocarbamoyl gonyatoxin 2, thiocarbamoyl gonyatoxin 3, descarbomoyl gonyatoxin 2, descarbomoyl gonyatoxin 3, descarbomoyl neosaxitoxin, descarbomoyl gonyatoxin 1, gonyatoxin 2, gonyatoxin 3, gonyatoxin 4, gonyatoxin 6, neosaxitoxin, saxitoxin, tetrodotoxin, microcystins RR, microcystins LR, okadan Tian Ruan sponge acid, and ganglion.

2. The method according to claim 1, wherein step S1 comprises the steps of:

pretreatment of fish: slaughtering fresh fish, cleaning, removing scale, removing viscera, removing head and tail, peeling, collecting back fish, removing fishbone, stirring with a tissue homogenizer, placing into a clean container, sealing, and marking;

pretreatment of oyster: thoroughly cleaning the outer surface of oyster sample with clear water, cutting off the muscle of the shell closure, opening the shell, washing the interior with distilled water to remove sediment and other foreign matters, picking up broken shell impurities, taking all soft tissues, fully and uniformly stirring by using a tissue refiner, loading into a clean container, sealing, and marking;

pre-treating scallop: thoroughly cleaning scallop sample with clear water, cutting off the adductor muscle, opening the shell, washing the inside with distilled water to remove sediment and other foreign matters, picking up broken shell impurities, taking all soft tissues, fully and uniformly stirring by using a tissue refiner, loading into a clean container, sealing, and marking.

3. The method of claim 1, wherein the vortex mixing time is 2 min, the centrifugation speed is 5 000 r/min, the centrifugation time is 5 min, and the centrifugation temperature is 4 ℃.

4. The method of claim 1, wherein the vortex clean-up time is 1 min, the centrifugation speed is 6 000 r/min, the centrifugation time is 10 min, and the centrifugation temperature is 4 ℃.

5. The method of claim 1, wherein the high resolution mass spectrometry conditions are: ion source: an electrospray ion source; ion source temperature: 110 ℃; capillary voltage: 4.0kV; desolventizing gas temperature: 325 deg.c; desolventizing gas flow: 11L/min nitrogen; taper hole voltage: 65V; atomizer pressure: 40 Nitrogen at psig; octopoleRFPeak:750 V, V; the acquisition range is as follows: m/z is 50-1500 Da.

6. The method of claim 1, wherein matching the database comprises the steps of:

s1, preparing 756 chemical hazard standard substances into standard liquid with the concentration of 100ng/mL, and injecting according to the analysis method of the ultra-high performance liquid chromatography and high resolution mass spectrometry; the 756 chemical hazards include chemical hazards in the national standard of current aquatic products, and encompass pesticides, veterinary drugs, and organic pollutants;

s2, pretreating fish meat and shellfish meat according to the improved QuECHERS method to obtain a liquid to be detected, and injecting sample according to an analysis method of high-resolution mass spectrometry of the ultra-high performance liquid chromatography;

S3, data acquisition is carried out on the 756 kinds of chemical hazard standard substances to obtain information such as chromatographic retention time, accurate mass number of parent ions, secondary ion fragments and the like of target chemical hazard substances, and simultaneously English names, CAS numbers and single isotope mass numbers are added to establish a high-resolution mass spectrum database;

s4, comparing the actual sample data acquired in the step S2 with the high-resolution mass spectrum database of the common chemical hazard in the step S3, and judging whether the unknown chemical hazard exists in the sample, wherein the deviation between the retention time of the detected chromatographic peak and the retention time in the spectrum library is within +/-2.5%, the deviation between the accurate mass number of the parent ion and the theoretical mass number is less than or equal to 5ppm, the deviation between at least 2 or more fragment ions with higher abundance in the secondary fragment ions and the mass number of the corresponding fragment ions in the spectrum library is less than 10ppm, and the abundance of the secondary fragment ions is consistent with the abundance of the corresponding fragment ions in the standard working solution with the concentration close to the standard working solution.

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