CN116953104B - Method for simultaneously measuring various insect repellents of deet and anabasine pesticides and metabolites in urine - Google Patents
Method for simultaneously measuring various insect repellents of deet and anabasine pesticides and metabolites in urine Download PDFInfo
- Publication number
- CN116953104B CN116953104B CN202310823607.9A CN202310823607A CN116953104B CN 116953104 B CN116953104 B CN 116953104B CN 202310823607 A CN202310823607 A CN 202310823607A CN 116953104 B CN116953104 B CN 116953104B
- Authority
- CN
- China
- Prior art keywords
- deet
- imidacloprid
- acetonitrile
- solution
- urine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000002700 urine Anatomy 0.000 title claims abstract description 50
- 239000000077 insect repellent Substances 0.000 title claims abstract description 34
- 239000002207 metabolite Substances 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000000575 pesticide Substances 0.000 title claims abstract description 10
- MTXSIJUGVMTTMU-JTQLQIEISA-N (S)-anabasine Chemical compound N1CCCC[C@H]1C1=CC=CN=C1 MTXSIJUGVMTTMU-JTQLQIEISA-N 0.000 title claims abstract description 8
- 229930014345 anabasine Natural products 0.000 title claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 106
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 239000002917 insecticide Substances 0.000 claims abstract description 20
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 10
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 150000002500 ions Chemical class 0.000 claims description 47
- 239000000243 solution Substances 0.000 claims description 46
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 claims description 20
- 239000011550 stock solution Substances 0.000 claims description 20
- 239000005875 Acetamiprid Substances 0.000 claims description 19
- AYEAUPRZTZWBBF-UHFFFAOYSA-N n'-[(6-chloropyridin-3-yl)methyl]-n-cyanoethanimidamide Chemical compound N#C\N=C(/C)NCC1=CC=C(Cl)N=C1 AYEAUPRZTZWBBF-UHFFFAOYSA-N 0.000 claims description 19
- HOKKPVIRMVDYPB-UVTDQMKNSA-N (Z)-thiacloprid Chemical compound C1=NC(Cl)=CC=C1CN1C(=N/C#N)/SCC1 HOKKPVIRMVDYPB-UVTDQMKNSA-N 0.000 claims description 18
- KLAYVYFJJVHZEM-UHFFFAOYSA-N 3-(ethylcarbamoyl)benzoic acid Chemical compound CCNC(=O)C1=CC=CC(C(O)=O)=C1 KLAYVYFJJVHZEM-UHFFFAOYSA-N 0.000 claims description 18
- 239000005940 Thiacloprid Substances 0.000 claims description 18
- 239000012071 phase Substances 0.000 claims description 18
- PXXLQQDIFVPNMP-UHFFFAOYSA-N 3-(diethylcarbamoyl)benzoic acid Chemical compound CCN(CC)C(=O)C1=CC=CC(C(O)=O)=C1 PXXLQQDIFVPNMP-UHFFFAOYSA-N 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 14
- 150000001412 amines Chemical class 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000005906 Imidacloprid Substances 0.000 claims description 11
- 229940056881 imidacloprid Drugs 0.000 claims description 11
- MATMQDMQFSFQHB-UHFFFAOYSA-N n-[1-[(6-chloropyridin-3-yl)methyl]-5-hydroxy-4,5-dihydroimidazol-2-yl]nitramide Chemical compound OC1CNC(=N[N+]([O-])=O)N1CC1=CC=C(Cl)N=C1 MATMQDMQFSFQHB-UHFFFAOYSA-N 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 claims description 9
- 102000053187 Glucuronidase Human genes 0.000 claims description 7
- 108010060309 Glucuronidase Proteins 0.000 claims description 7
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 7
- PGOOBECODWQEAB-FIBGUPNXSA-N 2-[(2-chloro-1,3-thiazol-5-yl)methyl]-1-nitro-3-(trideuteriomethyl)guanidine Chemical compound [2H]C([2H])([2H])NC(N[N+]([O-])=O)=NCC1=CN=C(Cl)S1 PGOOBECODWQEAB-FIBGUPNXSA-N 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 6
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 6
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- YWTYJOPNNQFBPC-KHORGVISSA-N n-[1-[(6-chloropyridin-3-yl)methyl]-4,4,5,5-tetradeuterioimidazol-2-yl]nitramide Chemical compound [2H]C1([2H])C([2H])([2H])N=C(N[N+]([O-])=O)N1CC1=CC=C(Cl)N=C1 YWTYJOPNNQFBPC-KHORGVISSA-N 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 6
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 5
- 238000001819 mass spectrum Methods 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- -1 acetamiprid-D 3 Chemical compound 0.000 claims description 4
- 230000001442 anti-mosquito Effects 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- FRZJZRVZZNTMAW-UHFFFAOYSA-N n,n-diethyl-3-(hydroxymethyl)benzamide Chemical compound CCN(CC)C(=O)C1=CC=CC(CO)=C1 FRZJZRVZZNTMAW-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 239000011159 matrix material Substances 0.000 abstract description 12
- 238000011084 recovery Methods 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 7
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000010606 normalization Methods 0.000 abstract description 2
- 239000007789 gas Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 6
- 239000000758 substrate Substances 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 239000012491 analyte Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- YWTYJOPNNQFBPC-UHFFFAOYSA-N imidacloprid Chemical compound [O-][N+](=O)\N=C1/NCCN1CC1=CC=C(Cl)N=C1 YWTYJOPNNQFBPC-UHFFFAOYSA-N 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 241000282412 Homo Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 150000001793 charged compounds Chemical class 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000000155 isotopic effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000005871 repellent Substances 0.000 description 2
- 230000002940 repellent Effects 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000013582 standard series solution Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- PGOOBECODWQEAB-UHFFFAOYSA-N (E)-clothianidin Chemical compound [O-][N+](=O)\N=C(/NC)NCC1=CN=C(Cl)S1 PGOOBECODWQEAB-UHFFFAOYSA-N 0.000 description 1
- IDCPFAYURAQKDZ-UHFFFAOYSA-N 1-nitroguanidine Chemical group NC(=N)N[N+]([O-])=O IDCPFAYURAQKDZ-UHFFFAOYSA-N 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 239000005888 Clothianidin Substances 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 239000005592 Penoxsulam Substances 0.000 description 1
- SYJGKVOENHZYMQ-UHFFFAOYSA-N Penoxsulam Chemical compound N1=C2C(OC)=CN=C(OC)N2N=C1NS(=O)(=O)C1=C(OCC(F)F)C=CC=C1C(F)(F)F SYJGKVOENHZYMQ-UHFFFAOYSA-N 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 206010074268 Reproductive toxicity Diseases 0.000 description 1
- 206010040880 Skin irritation Diseases 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 150000001408 amides Chemical group 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000004401 flow injection analysis Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 230000007688 immunotoxicity Effects 0.000 description 1
- 231100000386 immunotoxicity Toxicity 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 229920000831 ionic polymer Polymers 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000009372 pisciculture Methods 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000002728 pyrethroid Substances 0.000 description 1
- 231100000372 reproductive toxicity Toxicity 0.000 description 1
- 230000007696 reproductive toxicity Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000012865 response to insecticide Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000036556 skin irritation Effects 0.000 description 1
- 231100000475 skin irritation Toxicity 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000007655 standard test method Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for simultaneously measuring various insect repellents of deet and neonicotinoid insecticides and metabolites in urine, belonging to the technical field of biomedical detection. The method comprises the steps of carrying out enzymolysis treatment on a urine sample to be detected, purifying and enriching the urine sample after enzymolysis through solid phase extraction, carrying out redissolution on the urine sample after enrichment by using 25% acetonitrile aqueous solution to finish preparation of a detected sample, separating the urine sample by using an ultra-high performance liquid chromatograph, and carrying out multi-ion reaction monitoring mode detection by using a triple quadrupole tandem mass spectrometer. The correlation coefficient r 2 of 12 target analytes is larger than 0.99, the average recovery rate of samples is 80.0% -120%, the precision is less than 13.6%, the detection limit is 0.001-0.02 mug/L, and the quantitative limit is 0.004-0.07 mug/L. The matrix factor (MF i) after internal standard normalization is 0.80-1.20, and CV <15%. The method is accurate, reliable, simple and quick, and can be used for simultaneously detecting 12 kinds of insect repellents of the deet and anabasine pesticides and metabolites in urine.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a method for simultaneously measuring 12 kinds of deet insect repellents, anabasine pesticides and metabolites in urine, in particular to a method for simultaneously measuring 8 kinds of anabasine pesticides and metabolites of deet, 3- [ (ethylamino) carbonyl ] benzoic acid, deet omega-carboxylic acid and omega-hydroxyl deet 4 kinds of deet insect repellents and metabolites, and imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid, thiacloprid, 5-hydroxyl imidacloprid and N-desmethyl acetamiprid in urine.
Background
The penoxsulam is a very effective insect repellent active ingredient, which is effective against mosquitoes, biting flies, ticks, etc., and achieves its repellent characteristics by blocking the receptors that insects use to detect carbon dioxide and lactic acid produced by humans and animals. 3- [ (ethylamino) carbonyl ] benzoic acid has similar repellent properties and mechanisms of action as that of deet, which may coexist in some commercial insect repellent formulations. When using a product containing the mosquito-repellent amine, the mosquito-repellent amine is contacted and inhaled through the skin into the human body, metabolized in the blood by the liver and discharged with urine. The deet and its metabolites can therefore be detected in urine samples, blood and other body fluids. However, some people may develop skin irritation or allergic reactions such as redness, itching, swelling and urticaria after contacting the deet, and it is reported that the contact of the deet affects the nervous system, which causes people to worry about the safety thereof. The omega-carboxylic acids and omega-hydroxydeems of deem are metabolites of deem, formed in the liver and excreted with urine, and are generally considered biomarkers of deem exposure.
New nicotine is a systematic pesticide, as a substitute for organophosphorus, carbamate and pyrethroid pesticides, and is widely used in various fields such as agricultural pest control, veterinary drugs, bactericides, invertebrate pest control in fish farming, and the like. Humans are widely exposed to such substances through food, water, soil, air, house dust, and the like. Neonicotinoid insecticides have known environmental risks such as insecticide resistance, reduced diversity and distribution of pollination media such as bees, reduced population of insect eating birds. Because of its relatively low affinity and poor blood brain barrier penetration, it is considered to be less toxic in vertebrates. However, recent studies have shown that neonicotinoids have potential toxic effects on mammals and even humans, including cytotoxicity, genotoxicity, reproductive toxicity, neurotoxicity, immunotoxicity, hepatotoxicity and hepatoma carcinogenicity. Imidacloprid, clothianidin, acetamiprid and thiacloprid are commonly used neonicotinoid insecticides, with imidacloprid being the most widely used. The main metabolite of imidacloprid is 5-hydroxy imidacloprid and N-desmethyl acetamiprid is the metabolite of acetamiprid. And detecting imidacloprid, acetamiprid, 5-hydroxy imidacloprid and N-desmethyl acetamiprid in urine of the patient suspected of being poisoned by the neonicotinoid insecticide. Thus, these substances can serve as potential biomarkers of neonicotinoid insecticide exposure.
The concentration of the deet insect repellent, the neonicotinoid insecticide and the metabolite is known by taking the deet insect repellent, the neonicotinoid insecticide and the metabolite as exposure biomarkers, so that the exposure and the use condition of human beings to the deet insect repellent and the neonicotinoid insecticide can be better known, and the potential influence of the human beings on health can be further evaluated.
The chemical structures of the mosquito-repellent amine and the neonicotinoids are very different, the mosquito-repellent amine containing an amide function and a toluene ring, and the neonicotinoids containing a unique nitroguanidine moiety and a variety of other functions. Due to these structural differences, the simultaneous detection of the deet and the neonicotinoid and metabolite is more challenging, requiring more complex extraction and separation methods to ensure that the two classes of compounds are adequately recovered and separated from each other. Therefore, few methods are currently available for detecting mosquito-repellent amines together with neonicotinoid class 2 substances and metabolites. In the prior art, there is a patent in 2020 in China, application number: 202011140211.7, application publication number: CN113009057a, the patent name is "method for detecting neonicotinoid insecticides and metabolites in urine by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry", which simultaneously detects 12 kinds of compounds in total of class 1 substances. The accuracy of quantification of 12 target compounds is compromised using only 1 isotope internal standard.
Based on the problems, the invention establishes a detection method of ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, and uses 9 isotopic calibration amounts to measure the concentration of 12 insect repellents of the deet and the neonicotinoid insecticides and metabolites in a urine sample, wherein the concentrations comprise 2 insect repellents: the 2 insect repellent metabolites of the group consisting of deet and 3- [ (ethylamino) carbonyl ] benzoic acid: the omega-carboxylic acid of the deet and the omega-hydroxyl deet, 6 kinds of neonicotinoid insecticides: imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid and thiacloprid, 2 neonicotinoid metabolites: 5-hydroxy imidacloprid and N-desmethyl acetamiprid. Wherein, the imidacloprid-1, the imidacloprid-2, the clothianidin-1 and the clothianidin-2 are isomers with each other.
Disclosure of Invention
In view of this, the present invention provides a method for simultaneously determining a plurality of anti-mosquito amine insect repellents and neonicotinoid insecticides and metabolites in urine.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for simultaneously determining a plurality of deet insect repellents, anabasine pesticides and metabolites in urine, comprising the steps of:
(1) Preparing a standard solution;
(2) Freezing and thawing a urine sample to be tested, performing enzymolysis, and purifying and enriching the urine sample after the enzymolysis;
(3) UPLC-MS/MS detection is carried out on the purified and enriched urine sample;
(4) Methodology evaluation.
Further, the preparation method of the standard solution in the step (1) comprises the following steps:
The method comprises the steps of preparing single standard stock solutions with the mass concentration of 100mg/L from the acetonitrile of the deet, 3- [ (ethylamino) carbonyl ] benzoic acid, deet omega-carboxylic acid, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid, thiacloprid, 5-hydroxyl imidacloprid and N-desmethyl acetamiprid respectively, and diluting the single standard stock solutions with acetonitrile to prepare mixed standard intermediate solutions;
Respectively preparing the deet-D 10, 3- [ (ethylamino) carbonyl ] benzoic acid-D 5 and deet omega-carboxylic acid-D 10 into an internal standard single standard stock solution with the mass concentration of 100mg/L by acetonitrile;
Respectively preparing imidacloprid-D 4, acetamiprid-D 3, clothianidin-D 3, thiacloprid-D 4, 5-hydroxy imidacloprid-D 4 and N-desmethyl acetamiprid- 2H3 13C15N2 into internal standard single standard stock solution with the mass concentration of 500mg/L by acetonitrile;
preparing a mixed internal standard intermediate solution with the mass concentration of each internal standard substance of 2000ng/mL by using acetonitrile to dilute an internal standard single standard stock solution;
the mixed internal standard intermediate solution was diluted with acetonitrile to a mixed internal standard use solution having a mass concentration of 200 ng/mL.
Furthermore, the preparation method of the mixed standard intermediate solution comprises the following steps:
Acetonitrile is utilized to prepare mixed standard intermediate solutions of which the mass concentrations of the deet omega-carboxylic acid, the 3- [ (ethylamino) carbonyl ] benzoic acid and the 5-hydroxy imidacloprid are 2500ng/mL, 10000ng/mL and 3000ng/mL respectively, and the mass concentrations of the deet omega-hydroxy deet, the imidacloprid-1, the imidacloprid-2, the acetamiprid, the clothianidin-1, the clothianidin-2, the thiacloprid and the N-desmethyl acetamiprid are 1000 ng/mL.
Further, the internal standard of the deet is deet-D 10, the internal standard of 3- [ (ethylamino) carbonyl ] benzoic acid is 3- [ (ethylamino) carbonyl ] benzoic acid-D 5, the internal standard of deet omega-carboxylic acid is deet omega-carboxylic acid-D 10, the internal standard of omega-hydroxyl deet is deet-D 10, the internal standard of imidacloprid-1 and imidacloprid-2 is imidacloprid-D 4, the internal standard of clothianidin-1 and clothianidin-2 is clothianidin-D 3, the internal standard of acetamiprid is acetamiprid-D 3, the internal standard of thiacloprid is thiacloprid-D 4, the internal standard of 5-hydroxyl imidacloprid-D 4, and the internal standard of N-desmethyl acetamiprid is N-desmethyl acetamiprid- 2H3 13C15N2.
Further, the enzymolysis in the step (2) comprises the following operation steps: performing light-shielding enzymolysis on the frozen and thawed urine sample for 17 hours under the water bath condition of 37 ℃ by using 200ng/mL mixed internal standard use solution, beta-glucuronidase and 0.2mol/L potassium phosphate buffer solution;
further, the beta-glucuronidase is more than or equal to 80000 units; ph=6.8 of potassium phosphate buffer solution;
Further, the volume ratio of the urine sample after freeze thawing, the mixed internal standard use solution, the beta-glucuronidase and the potassium phosphate buffer solution is as follows: 100:2:3:70;
Further, the purifying and enriching operation steps in the step (2) are as follows: after the enzymolysis urine sample passes through the column, washing a sample tube and eluting the solid-phase extraction column by using pure water, and eluting the solid-phase extraction column by using acetonitrile+ethyl acetate solution and 2% acetonitrile formate solution in sequence;
Further, the volume ratio of acetonitrile to ethyl acetate in the acetonitrile+ethyl acetate solution is 2:1.
Further, in the step (3), the UPLC-MS/MS detection is performed, and the chromatographic column is as follows: waters ACQUITYHSS T3, specification: 2.1X100 mm,1.8 μm; the sample injection amount is as follows: 5.0. Mu.L; column temperature: 40 ℃; sample introduction temperature: 10 ℃; the flow rate is: 0.25mL/min; the mobile phase is: phase A is formic acid aqueous solution with the volume ratio of 0.1 percent, and phase B is acetonitrile; the elution procedure was: when the time is 0min to 6.0min, the mobile phase A is 75 percent, and the mobile phase B is 25 percent; when the time is 6.0 to 8.0min, the mobile phase A is 75 to 50 percent, and the mobile phase B is 25 to 50 percent; when 8.0 min-11.0 min, the mobile phase A is 50% -75% and the mobile phase B is 50% -25%; when the time is 11.0 min-15.0 min, the mobile phase A is 75%, and the mobile phase B is 25%;
Further, the UPLC-MS/MS detection in the step (3) has mass spectrum conditions as follows: ESI ion source, positive ion scanning, MRM mode monitoring, ion source temperature 500 ℃, spraying voltage 5500V, collision gas voltage High, gas curtain pressure 30psi, atomization pressure 40psi, auxiliary pressure 40psi, scanning time 30ms; the MRM ion acquisition parameters are: quantitative ion pair of deet: 192.2/119.0; quantitative ion pair of 3- [ (ethylamino) carbonyl ] benzoic acid: 194.0/149.1; quantitative ion pair of the mosquito-repellent amine omega-carboxylic acid: 222.2/149.0; quantitative ion pair of omega-hydroxydee: 208.1/135.0; imidacloprid-1 quantitative ion pair: 256.1/208.8; imidacloprid-2 quantitative ion pair: 256.0/209.1; clothianidin-1 quantitative ion pair: 250.0/169.1; clothianidin-2 quantitative ion pair: 250.0/169.2; acetamiprid quantitative ion pair: 223.0/126.0; thiacloprid quantitative ion pair: 253.0/125.9; quantitative ion pair of 5-hydroxy imidacloprid: 272.0/225.0; quantitative ion pair of N-desmethyl acetamiprid: 209.0/126.2;
further, the UPLC-MS/MS detection in the step (3) comprises the following quantitative methods: and (5) quantifying by an internal standard method.
Compared with the prior art, the invention has the beneficial effects that: the detection limit of the invention is 0.001-0.02 mug/L, the quantitative limit is 0.004-0.07 mug/L, the average standard adding recovery rate is 80.0% -120%, the precision is <13.6%, the matrix factor (MF i) after internal standard normalization is 0.80-1.20, and the CV% <15, thus ensuring no obvious matrix effect. The method is sensitive, accurate and stable, and is suitable for quantitative analysis of various insect repellents of the deet and neonicotinoid insecticides and metabolites in urine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of the operation of the present invention;
Figure 2 is a graph of chromatograms of 12 deet insect repellents and neonicotinoid insecticides and metabolites and isotopic internal standards.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The required medicament is a conventional experimental medicament and is purchased from a commercial channel; the test methods not mentioned are conventional test methods and will not be described in detail herein.
Example 1 preparation of Standard solution
(1) Preparation of standard stock solution: and respectively and accurately weighing 1.0mg (0.0001 g) of each of the deet, the 3- [ (ethylamino) carbonyl ] benzoic acid, the deet omega-carboxylic acid, the omega-hydroxyl deet, the imidacloprid-1, the imidacloprid-2, the clothianidin-1, the clothianidin-2, the acetamiprid, the thiacloprid, the 5-hydroxyl imidacloprid and the N-desmethyl acetamiprid, dissolving the components in acetonitrile, and fixing the volume to 10mL to prepare a single standard stock solution with the mass concentration of 100 mg/L. The basic conditions for the 12 target analytes are shown in Table 1.
(2) Preparing a mixed standard intermediate solution: and respectively taking 250 mu L, 1000 mu L and 300 mu L of the single standard stock solution of the deet omega-carboxylic acid, the 3- [ (ethylamino) carbonyl ] benzoic acid and the 5-hydroxy imidacloprid, and 100 mu L of the single standard stock solution of the deet omega-carboxylic acid, the omega-hydroxy deet omega-carboxylic acid, the imidacloprid-1, the imidacloprid-2, the acetamiprid, the clothianidin-1, the clothianidin-2, the thiacloprid and the N-desmethyl acetamiprid, and preparing mixed standard intermediate solutions with mass concentration of 2500ng/mL of the deet omega-carboxylic acid, 10000ng/mL of the 3- [ (ethylamino) carbonyl ] benzoic acid, 3000ng/mL of the 5-hydroxy imidacloprid and 1000ng/mL of other 9 compounds in a 10mL volumetric flask.
(3) Preparing an internal standard single standard stock solution: 1.0mg (accurate to 0.0001 g) of each of the internal standard substances of the deet-D 10, the deet-omega-carboxylic acid-D 10 and the 3- [ (ethylamino) carbonyl ] benzoic acid-D 5 are accurately weighed, dissolved in acetonitrile and fixed to 10mL, and respectively prepared into internal standard single standard stock solution with the mass concentration of 100 mg/L. And accurately weighing 5.0mg (0.0001 g accurate) of each of the imidacloprid-D 4, acetamiprid-D 3, clothianidin-D 3, thiacloprid-D 4, 5-hydroxy imidacloprid-D 4 and N-desmethyl acetamiprid- 2H3 13C15N2 internal standard substances, dissolving the internal standard substances by acetonitrile, and fixing the volume to 10mL to prepare internal standard single standard stock solutions with the mass concentration of 500mg/L respectively. The basic conditions for the 9 isotope internal standard are shown in Table 2.
(4) Preparation of mixed internal standard use solution: 200 mu L of the internal standard single standard stock solution of the deet-D 10, the deet omega-carboxylic acid-D 10 and the 3- [ (ethylamino) carbonyl ] benzoic acid-D 5, 40 mu L of the internal standard single standard stock solution of the imidacloprid-D 4, the acetamiprid-D 3, the clothianidin-D 3, the thiacloprid-D 4, the 5-hydroxy imidacloprid-D 4 and the N-desmethyl acetamiprid- 2H3 13C15N2 are taken, and acetonitrile is used for volume metering to scale in a 10mL volumetric flask to prepare the mixed internal standard intermediate solution with the mass concentration of each internal standard substance of 2000 ng/mL. Acetonitrile was diluted to a mixed internal standard use solution with a mass concentration of 200 ng/mL.
TABLE 112 target analytes
Table 2, 9 isotope internal standard
Example 2 pretreatment of urine to be tested
(1) Freezing and thawing: the urine sample was frozen at-80℃and thawed slowly at 4 ℃. After equilibrated to room temperature, mix well with vortex mixer, accurately remove 1mL and place in 96-well collection plate.
(2) Enzymolysis: to a 96-well collection plate containing 1mL of urine sample, 20. Mu.L of 200ng/mL of the mixed internal standard use solution, 30. Mu.L of beta-glucuronidase (. Gtoreq.80000 unit), 700. Mu.L of 0.2mol/L potassium phosphate buffer solution (pH=6.8) were sequentially added, mixed well, and subjected to enzymolysis in a water bath at 37℃for 17 hours in the absence of light.
(3) Purifying and enriching an enzymolysis urine sample: sequentially activated with 1.5mL of methanol and 1mL of pure waterWAX 96-well extraction plate (extraction plate filler mass 30mg, particle size 30 μm). Transferring the urine sample after enzymolysis to each solid phase extraction column of a 96-well plate, passing the urine sample through the column under the condition of no pressure by means of natural gravity, after the urine sample passes through the column, rinsing a sample tube with 200 mu L of pure water, loading the urine sample, quickly leaching the solid phase extraction column with 1.0mL of pure water, and drying under reduced pressure for 30min. The solid phase extraction column was eluted sequentially with 800 μl acetonitrile + ethyl acetate (v: v=2:1) and 800 μl 2% acetonitrile formate and the eluate was collected using a 96 well collection plate. After the elution is finished, nitrogen is blown to near dryness at 40 ℃, 200 mu L of 25% acetonitrile water solution is added for redissolution, and a 96-well plate centrifuge is used for centrifuging at 4000rpm/min for 10min to be tested.
Example 3 UPLC-MS/MS detection
(1) Chromatographic conditions
In order to allow detection of 12 target analytes and 9 internal standards in example 1 and to achieve optimal separation, the present invention examined the separation of methanol-water, acetonitrile-water, 0.1% (v/v) formic acid aqueous solution-methanol, 0.1% (v/v) formic acid aqueous solution-acetonitrile, and 0.1% (v/v) formic acid aqueous solution-0.1% (v/v) formic acid acetonitrile as mobile phases. When 0.1% (v/v) aqueous formic acid-acetonitrile was used as the mobile phase, the chromatographic peak shape was optimal and the response was highest, see FIG. 2.
Chromatographic conditions: chromatographic column: waters ACQUITYHSS T 3 (1.8 μm, 2.1X100 mm); separation column: waters isolator column (2.1X150 mm); column temperature: 40 ℃; sample tray temperature: 10 ℃; sample injection amount: 5. Mu.L; flow rate: 0.25mL/min; mobile phase: 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B), gradient elution, parameters are shown in Table 3.
TABLE 3 gradient elution parameters
(2) Mass spectrometry conditions
The single standard stock solutions of 12 target analytes and 9 internal standards in example 1 were diluted with acetonitrile to single standard use solutions with a mass concentration of 500 μg/L, respectively, and in the simple substance spectrum mode, an ion source was injected by a flow injection method using an ESI source at a flow rate of 10 μl/min, and full scanning was performed in the positive ion mode and the negative ion mode, respectively, to select an appropriate ionization method and molecular ion peak. The results indicate that in the positive ion mode, the molecular ion [ M+H ] + of the target analyte full scan is optimal. And (3) taking [ M+H ] + of the target analyte as a parent ion to carry out ion scanning, selecting two child ions with highest abundance and least interference, optimizing Collision Energy (CE) and declustering voltage (DP), and selecting CE and DP values which can enable the two child ions to reach the highest abundance. In the liquid chromatography tandem mass spectrometry mode, a two-way connection liquid chromatography and tandem mass spectrometry are used, 100% acetonitrile is used as a mobile phase, and optimized parent ions, child ions, scanning time, collision energy and declustering voltage are used as mass spectrometry conditions (the mass spectrometry conditions are shown in table 2), so that an analysis method is established. And under a positive ion mode, using a mixed standard solution with the concentration of 12 target analytes and 9 internal standard substances of 100 mug/L, and automatically optimizing parameters such as gas curtain gas (CUR), spray voltage (IS), ion source Temperature (TEM), atomizing gas (GS 1), auxiliary gas (GS 2) and the like in an MRM mode to obtain final mass spectrum parameters.
The optimal mass spectrum conditions are as follows:
Ionization mode: an ESI ion source; scanning mode: positive ionization; the detection mode is as follows: polyion reaction monitoring (MRM); ion source Temperature (TEM): 500 ℃; spray voltage (IS): 5500V; collision gas (CAD): high; curtain gas (CUR): 30psi; atomizing gas (GS 1): 40psi; assist gas (GS 2): 40psi; scanning time: 30ms. Other mass spectral parameters are shown in table 4.
Table 4, 12 target analytes and retention times for internal standards, monitoring ion, collision voltage (CE) and declustering voltage (DP)
* To quantify ions
(3) Drawing a standard curve:
1) Preparing standard series intermediate liquid: the mixed standard intermediate solution is diluted step by using 25% acetonitrile water solution to prepare standard series intermediate solution. Wherein the mass concentration of the deet, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, acetamiprid, clothianidin-1, clothianidin-2, thiacloprid and N-desmethyl acetamiprid is sequentially 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, the mass concentration of the omega-carboxylic acid of the deet is sequentially 1250ng/mL, 625ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 12.5ng/mL, 6.25ng/mL, 1.25ng/mL,3- [ (ethylamino) carbonyl ] benzoic acid is sequentially 5000ng/mL, 2500ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 50ng/mL, 25ng/mL, 5ng/mL, the mass concentration of 5-hydroxyl imidacloprid is sequentially 1500ng/mL, 300ng/mL, 7.5ng/mL, and 5.5 ng/mL, and the final assay of each sample is sequentially obtained, and the final assay of the sample is sequentially 1.8 ng, the sample is sequentially equal to the final assay.
2) Preparation of standard series of solutions: 400. Mu.L of 25% acetonitrile aqueous solution, 50. Mu.L of standard series intermediate solutions in step 1) (the order of addition here corresponds one by one to the order of 8 standard series intermediate solutions of different mass concentrations in step 1) and 50. Mu.L of mixed internal standard use solution of 200ng/mL in example 1 are added respectively, and mixed uniformly to prepare a total volume of 500. Mu.L of standard series solutions each having an internal standard concentration of 20 ng/mL. Wherein, the mass concentration of the deet, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, acetamiprid, clothianidin-1, clothianidin-2, thiacloprid, N-desmethyl acetamiprid is sequentially 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, 0.05ng/mL, and the concentration converted into the sample is 10 mug/L, 5 mug/L, 2 mug/L, 1 mug/L, 0.5 mug/L, 0.1 mug/L, 0.05 mug/L and 0.01 mug/L; the mass concentration of the mosquito-repellent amine omega-carboxylic acid is 125ng/mL, 62.5ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 1.25ng/mL, 0.625ng/mL, 0.125ng/mL, and the concentration converted into the sample is 25 mug/L, 12.5 mug/L, 5 mug/L, 2.5 mug/L, 1.25 mug/L, 0.25 mug/L, 0.125 mug/L and 0.025 mug/L; the mass concentration of 3- [ (ethylamino) carbonyl ] benzoic acid is 500ng/mL, 250ng/mL, 100ng/mL, 50.0ng/mL, 25.0ng/mL, 5.00ng/mL, 2.50ng/mL, 0.50ng/mL in sequence, and the concentration converted into the sample is 100 mug/L, 50 mug/L, 20 mug/L, 10 mug/L, 5 mug/L, 1 mug/L, 0.5 mug/L and 0.1 mug/L; the mass concentration of 5-hydroxy imidacloprid is 150ng/mL, 75ng/mL, 30ng/mL, 15ng/mL, 7.5ng/mL, 1.5ng/mL, 0.75ng/mL, 0.15ng/mL, and the concentration converted into the sample is 30 mug/L, 15 mug/L, 6 mug/L, 3 mug/L, 1.5 mug/L, 0.3 mug/L, 0.15 mug/L, 0.03 mug/L. Finally, 8 standard series of solutions of different mass concentrations were obtained, each containing 12 target analytes and 9 internal standards. The blank solution was prepared from 450. Mu.L of 25% acetonitrile in water and 50. Mu.L of the 200ng/mL mixed internal standard use solution of example 1.
3) Drawing a standard curve: and (3) utilizing the mass spectrum and chromatographic conditions of the step (1) and the step (2) to sequentially sample and measure the prepared standard series solution according to the sequence from low concentration to high concentration. And (3) carrying out linear regression analysis on the ratio (y) of peak areas of 12 target analytes and corresponding internal standard ion peaks and the ratio (x) of target analytes and corresponding internal standard mass concentrations, and drawing a calibration curve to obtain a linear equation and a correlation coefficient (r) of the 12 target analytes, wherein r is larger than 0.99. The results are shown in Table 5.
Example 4, methodological evaluation
(1) Detection limit and quantitative limit
Referring to the U.S. environmental protection agency (US EPA) detection limit determination program file version 2, two methods (first considering method blank, calculating method blank average value running in 6 months plus 3 times standard deviation; second considering instrument sensitivity, calculating 3 times signal to noise ratio) were used to examine the detection limit, and take its high value as the detection limit of the present invention, and then take the 3.3 times detection limit as the quantitative limit. 1/X 2, the results are shown in Table 5.
Table 5, linear ranges, regression equations, correlation coefficients, detection limits and quantification limits for 12 target analytes
(2) Yield and precision of standard addition
Accuracy and selectivity are determined by the addition of standard recovery and precision. The actual urine sample is used for the labeling recovery test of 12 target analytes at low, medium and high 3 levels, each level is used for 6 parallel tests, and the labeling recovery rate and precision are calculated. The average recovery rate of 12 target analytes is 80.0% -120%, and the precision is less than 13.6%. The results are shown in Table 6.
Table 6, 12 target analytes for standard recovery and precision in urine samples (n=6)
(3) Matrix effect
When complex substrate samples are detected by the UPLC-MS/MS method, the substrate co-extract has substrate enhancement or substrate inhibition effect on ionization of target analytes, thereby affecting quantitative determination of the target analytes. The invention uses 6 different individual urine samples (3 men and 3 women), 6 urine samples from different sources are treated by the example 2, and then the inner standards of the deet, 3- [ (ethylamino) carbonyl ] benzoic acid, deet omega-carboxylic acid, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid, thiacloprid, 5-hydroxyl imidacloprid and N-desmethyl acetamiprid 12 target analytes and deet-D 10, deet omega-carboxylic acid-D 10, 3- [ (ethylamino) carbonyl ] benzoic acid-D 5, imidacloprid-D 4, acetamiprid-D 3, clothianidin-D 3, thiacloprid-D 4, 5-hydroxyl imidacloprid-D 4 and N-desmethyl acetamiprid- 2H3 13C15N2 are added as controls, and the same concentration of the 12 target analytes and the 25% acetonitrile aqueous solution of 9 internal standards are added for determination. The matrix factor for each analyte of interest and internal standard is calculated by calculating the ratio of the peak area in the presence of the matrix to the corresponding peak area of the solvent. The internal standard normalized matrix factor (MF i) is then calculated by dividing the matrix factor of the target analyte by the matrix factor of the internal standard. It is generally believed that MF i in the range of 0.80-1.20 is acceptable, and the Coefficient of Variation (CV) measured for different batches of matrix must not be greater than 15%. The invention examines the substrate effect to meet the requirements, and the results are shown in Table 7.
TABLE 7 matrix Effect results
Example 5 measurement of actual sample
And collecting 20 parts of actual urine samples, and detecting 12 target analytes of the deet, 3- [ (ethylamino) carbonyl ] benzoic acid, deet omega-carboxylic acid, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid, thiacloprid, 5-hydroxyl imidacloprid and N-desmethyl acetamiprid, wherein other 9 substances except the deet, the omega-hydroxyl deet and the thiacloprid are detected. 3- [ (ethylamino) carbonyl ] benzoic acid at a concentration of 0.72-6.68. Mu.g/L, a concentration of 0.21-7.39. Mu.g/L of the deet omega-carboxylic acid, a concentration of 0.11-0.27. Mu.g/L of imidacloprid-1, a concentration of 0.09-0.26. Mu.g/L of imidacloprid-2, a concentration of 0.007-0.10. Mu.g/L of acetamiprid, a concentration of 0.39-6.78. Mu.g/L of clothianidin-1, a concentration of 0.31-4.73. Mu.g/L of clothianidin-2, a concentration of 0.01-1.22. Mu.g/L of 5-hydroxy imidacloprid, and a concentration of 0.48-2.10. Mu.g/L of N-desmethyl acetamiprid. Because no standard test method or internationally accepted gold standard for the anti-mosquito amine insect repellent and the neonicotinoid insecticide and metabolite in urine exists at present, the comparison of the detection results cannot be performed. However, the method has higher accuracy and selectivity according to the standard recovery rate experiment, the precision experiment, the repeated analysis and the matrix effect evaluation.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A method for simultaneously determining a plurality of deet insect repellents, anabasine pesticides and metabolites in urine, comprising the steps of:
(1) Preparing a standard solution;
(2) Freezing and thawing a urine sample to be tested, performing enzymolysis, and purifying and enriching the urine sample after the enzymolysis;
(3) UPLC-MS/MS detection is carried out on the purified and enriched urine sample;
the UPLC-MS/MS detection comprises the following chromatographic columns: waters ACQUITY HSS T3; the flow rate is: 0.25mL/min; the mobile phase is: phase A is formic acid aqueous solution with the volume ratio of 0.1 percent, and phase B is acetonitrile;
The mass spectrum conditions of the UPLC-MS/MS detection are as follows: ESI ion source, positive ion scan, deet quantitative ion pair: 192.2/119.0; quantitative ion pair of 3- [ (ethylamino) carbonyl ] benzoic acid: 194.0/149.1; quantitative ion pair of the mosquito-repellent amine omega-carboxylic acid: 222.2/149.0; quantitative ion pair of omega-hydroxydee: 208.1/135.0; imidacloprid-1 quantitative ion pair: 256.1/208.8; imidacloprid-2 quantitative ion pair: 256.0/209.1; clothianidin-1 quantitative ion pair: 250.0/169.1; clothianidin-2 quantitative ion pair: 250.0/169.2; acetamiprid quantitative ion pair: 223.0/126.0; thiacloprid quantitative ion pair: 253.0/125.9; quantitative ion pair of 5-hydroxy imidacloprid: 272.0/225.0; quantitative ion pair of N-desmethyl acetamiprid: 209.0/126.2;
(4) Methodology evaluation;
The purifying and enriching operation steps in the step (2) are as follows: after passing through a column, the urine sample subjected to enzymolysis is subjected to solid phase extraction treatment, a sample tube is rinsed by pure water, the solid phase extraction column is leached, and then the solid phase extraction column is eluted by acetonitrile + ethyl acetate solution and 2% acetonitrile formate solution in sequence;
the volume ratio of acetonitrile to ethyl acetate in the acetonitrile+ethyl acetate solution is 2:1;
The solid phase extraction adopts WAX 96 well extraction plate;
the various insect repellents of the deet and the anabasine pesticides and metabolites are deet, 3- [ (ethylamino) carbonyl ] benzoic acid, deet omega-carboxylic acid, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid, thiacloprid, 5-hydroxyl imidacloprid and N-desmethyl acetamiprid.
2. The method for simultaneously measuring a plurality of insect repellents of the deet and neonicotinoid insecticides and metabolites in urine as set forth in claim 1, wherein the standard solution of step (1) is prepared by the following steps: the method comprises the steps of preparing single standard stock solutions with the mass concentration of 100mg/L from the acetonitrile of the deet, 3- [ (ethylamino) carbonyl ] benzoic acid, deet omega-carboxylic acid, omega-hydroxyl deet, imidacloprid-1, imidacloprid-2, clothianidin-1, clothianidin-2, acetamiprid, thiacloprid, 5-hydroxyl imidacloprid and N-desmethyl acetamiprid respectively, and diluting the single standard stock solutions with acetonitrile to prepare mixed standard intermediate solutions;
Respectively preparing the deet-D 10, deet omega-carboxylic acid-D 10 and 3- [ (ethylamino) carbonyl ] benzoic acid-D 5 into an internal standard single standard stock solution with the mass concentration of 100mg/L by acetonitrile;
Respectively preparing imidacloprid-D 4, acetamiprid-D 3, clothianidin-D 3, thiacloprid-D 4, 5-hydroxy imidacloprid-D 4 and N-desmethyl acetamiprid- 2H3 13C15N2 into internal standard single standard stock solution with the mass concentration of 500mg/L by acetonitrile;
preparing a mixed internal standard intermediate solution with the mass concentration of each internal standard substance of 2000ng/mL by using acetonitrile to dilute an internal standard single standard stock solution;
the mixed internal standard intermediate solution was diluted with acetonitrile to a mixed internal standard use solution having a mass concentration of 200 ng/mL.
3. The method for simultaneously measuring a plurality of insect repellents of the deet and neonicotinoid insecticides and metabolites in urine as claimed in claim 2, wherein the method for preparing the mixed standard intermediate solution is as follows: acetonitrile is utilized to prepare mixed standard intermediate solutions of which the mass concentrations of the deet omega-carboxylic acid, the 3- [ (ethylamino) carbonyl ] benzoic acid and the 5-hydroxy imidacloprid are 2500ng/mL, 10000ng/mL and 3000ng/mL respectively, and the mass concentrations of the deet, the omega-hydroxy deet, the imidacloprid-1, the imidacloprid-2, the acetamiprid, the clothianidin-1, the clothianidin-2, the thiacloprid and the N-desmethyl acetamiprid are 1000 ng/mL.
4. The method for simultaneously measuring a plurality of insect repellents of the deet and neonicotinoid insecticides and metabolites in urine as set forth in claim 2, wherein the step of enzymatic hydrolysis in step (2) comprises the steps of: performing light-shielding enzymolysis on the frozen and thawed urine sample for 17 hours under the water bath condition of 37 ℃ by using 200ng/mL mixed internal standard use solution, beta-glucuronidase and 0.2mol/L potassium phosphate buffer solution;
the beta-glucuronidase is more than or equal to 80000unit; pH of potassium phosphate buffer=6.8;
The volume ratio of the urine sample after freeze thawing to the mixed internal standard use solution to the beta-glucuronidase to the potassium phosphate buffer solution is as follows: 100:2:3:70.
5. A method for simultaneously assaying a plurality of anti-mosquito amine insect repellents and neonicotinoid insecticides and metabolites in urine according to claim 1, wherein the detection limit LOD of the anti-mosquito amine insect repellents and neonicotinoid insecticides and metabolites is 0.001-0.02 μg/L and the quantitative limit LOQ is 0.004-0.07 μg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310823607.9A CN116953104B (en) | 2023-07-06 | 2023-07-06 | Method for simultaneously measuring various insect repellents of deet and anabasine pesticides and metabolites in urine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310823607.9A CN116953104B (en) | 2023-07-06 | 2023-07-06 | Method for simultaneously measuring various insect repellents of deet and anabasine pesticides and metabolites in urine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116953104A CN116953104A (en) | 2023-10-27 |
| CN116953104B true CN116953104B (en) | 2024-08-13 |
Family
ID=88454022
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310823607.9A Active CN116953104B (en) | 2023-07-06 | 2023-07-06 | Method for simultaneously measuring various insect repellents of deet and anabasine pesticides and metabolites in urine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116953104B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117805289A (en) * | 2024-02-27 | 2024-04-02 | 中国疾病预防控制中心环境与健康相关产品安全所 | Method for simultaneously detecting 72 antibiotics in human urine |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4549288B2 (en) * | 2005-12-08 | 2010-09-22 | 日本製粉株式会社 | Analysis method of 2,4,5-T using liquid chromatography tandem mass spectrometry (LC-MS / MS) |
| CN108254481B (en) * | 2016-12-29 | 2020-07-14 | 深圳先进技术研究院 | Method for rapidly detecting multi-class medicines, personal care products and pesticides in water |
| CN110749667B (en) * | 2019-09-20 | 2022-07-15 | 青岛理工大学 | Analysis and detection method of four neonicotinoid insecticides in water |
| CN113092619B (en) * | 2021-04-06 | 2022-08-09 | 浙江省农业科学院 | Method for simultaneously detecting thiamethoxam and clothianidin in fish |
-
2023
- 2023-07-06 CN CN202310823607.9A patent/CN116953104B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| Quantification of DEET and neonicotinoid pesticide biomarkers in human urine by online solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry;Samuel E. Baker et al.;Analytical and Bioanalytical Chemistry;20191231;第671-673页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116953104A (en) | 2023-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ueyama et al. | Biological monitoring method for urinary neonicotinoid insecticides using LC‐MS/MS and its application to Japanese adults | |
| CN116953104B (en) | Method for simultaneously measuring various insect repellents of deet and anabasine pesticides and metabolites in urine | |
| Garí et al. | Analysis of metabolites of organophosphate and pyrethroid pesticides in human urine from urban and agricultural populations (Catalonia and Galicia) | |
| Cazorla-Reyes et al. | Single solid phase extraction method for the simultaneous analysis of polar and non-polar pesticides in urine samples by gas chromatography and ultra high pressure liquid chromatography coupled to tandem mass spectrometry | |
| Baker et al. | Isotope dilution high-performance liquid chromatography–tandem mass spectrometry method for quantifying urinary metabolites of synthetic pyrethroid insecticides | |
| Wiltshire et al. | Development of a high-performance liquid chromatographic–mass spectrometric assay for the specific and sensitive quantification of Ro 64-0802, an anti-influenza drug, and its pro-drug, oseltamivir, in human and animal plasma and urine | |
| Gao et al. | Development of an analytical method based on solid-phase extraction and LC-MS/MS for the monitoring of current-use pesticides and their metabolites in human urine | |
| Urakawa et al. | Simultaneous determination of nicotine and cotinine in various human tissues using capillary gas chromatography/mass spectrometry | |
| Gosselin et al. | Quantification of methadone and its metabolite 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine in third instar larvae of Lucilia sericata (Diptera: Calliphoridae) using liquid chromatography-tandem mass spectrometry | |
| CN113899834B (en) | Method for detecting nitrosamine impurities in medicine | |
| Scheidweiler et al. | A validated gas chromatographic–electron impact ionization mass spectrometric method for methylenedioxymethamphetamine (MDMA), methamphetamine and metabolites in oral fluid | |
| WO2023102741A1 (en) | Method for detecting nitrosamine impurities in drug | |
| Godage et al. | Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations | |
| DeArmond et al. | QuEChERS-based approach toward the analysis of two insecticides, methomyl and aldicarb, in blood and brain tissue | |
| CN113933419A (en) | Method for determining concentration of 5 immunosuppressive agents in human whole blood | |
| Picó et al. | Rapid and sensitive ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry for the quantification of amitraz and identification of its degradation products in fruits | |
| Perry et al. | Urinalysis of atrazine exposure in farm pesticide applicators | |
| Dawson et al. | Analysis of selected drug formulations for volatile nitrosamines | |
| Pommier et al. | Simultaneous determination of imipramine and its metabolite desipramine in human plasma by capillary gas chromatography with mass-selective detection | |
| PASTORELLI et al. | 3, 4 Dichloroaniline-haemoglobin adducts in humans: preliminary data on agricultural workers exposed to propanil | |
| Plasencia et al. | Analysis of zearalenone and a-zearalenol in urine of ruminants using gas chromatography-tandem mass spectrometry | |
| Swiner et al. | A proof-of-concept, two-tiered approach for ricin detection using ambient mass spectrometry | |
| Aviram et al. | Dried urine spot and dried blood spot sample collection for rapid and sensitive monitoring of exposure to ricin and abrin by LC–MS/MS analysis of ricinine and l-abrine | |
| Pon et al. | Validated method for the detection and quantitation of synthetic cannabinoids in whole blood and urine, and its application to postmortem cases in Johannesburg, South Africa | |
| WO2022084892A1 (en) | Method of quantifying lysergic acid diethylamide (lsd) and 2,3-dihydro-3-hydroxy-2-oxo lysergide (o-h-lsd) in human plasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |