CN106018602A - Compound detection reagent for detecting sulfonamide compound and detection method thereof - Google Patents
Compound detection reagent for detecting sulfonamide compound and detection method thereof Download PDFInfo
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- CN106018602A CN106018602A CN201610347829.8A CN201610347829A CN106018602A CN 106018602 A CN106018602 A CN 106018602A CN 201610347829 A CN201610347829 A CN 201610347829A CN 106018602 A CN106018602 A CN 106018602A
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- compound
- acetonitrile
- sulfonamides
- phase
- detection
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- 229940124530 sulfonamide Drugs 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 title claims abstract description 57
- -1 sulfonamide compound Chemical class 0.000 title claims abstract description 55
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 31
- 150000001875 compounds Chemical class 0.000 title claims abstract description 17
- 239000000523 sample Substances 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 29
- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- 239000013558 reference substance Substances 0.000 claims abstract description 11
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 238000000622 liquid--liquid extraction Methods 0.000 claims abstract description 6
- 230000006920 protein precipitation Effects 0.000 claims abstract description 6
- 238000000638 solvent extraction Methods 0.000 claims abstract description 6
- 239000012472 biological sample Substances 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 90
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- 229960002597 sulfamerazine Drugs 0.000 claims description 30
- QPPBRPIAZZHUNT-UHFFFAOYSA-N sulfamerazine Chemical compound CC1=CC=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 QPPBRPIAZZHUNT-UHFFFAOYSA-N 0.000 claims description 30
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 22
- 239000007789 gas Substances 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 12
- 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 11
- 235000019253 formic acid Nutrition 0.000 claims description 11
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- NHZLNPMOSADWGC-UHFFFAOYSA-N 4-amino-N-(2-quinoxalinyl)benzenesulfonamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CN=C(C=CC=C2)C2=N1 NHZLNPMOSADWGC-UHFFFAOYSA-N 0.000 claims description 8
- ZZORFUFYDOWNEF-UHFFFAOYSA-N sulfadimethoxine Chemical compound COC1=NC(OC)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ZZORFUFYDOWNEF-UHFFFAOYSA-N 0.000 claims description 8
- GPTONYMQFTZPKC-UHFFFAOYSA-N sulfamethoxydiazine Chemical compound N1=CC(OC)=CN=C1NS(=O)(=O)C1=CC=C(N)C=C1 GPTONYMQFTZPKC-UHFFFAOYSA-N 0.000 claims description 8
- 229960002229 sulfametoxydiazine Drugs 0.000 claims description 8
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 7
- GWBPFRGXNGPPMF-UHFFFAOYSA-N N-[4-[(4-nitrophenyl)sulfamoyl]phenyl]acetamide Chemical compound C1=CC(NC(=O)C)=CC=C1S(=O)(=O)NC1=CC=C([N+]([O-])=O)C=C1 GWBPFRGXNGPPMF-UHFFFAOYSA-N 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 6
- 229950004215 sulfanitran Drugs 0.000 claims description 6
- 150000003456 sulfonamides Chemical class 0.000 claims description 6
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 5
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- VLYWMPOKSSWJAL-UHFFFAOYSA-N sulfamethoxypyridazine Chemical compound N1=NC(OC)=CC=C1NS(=O)(=O)C1=CC=C(N)C=C1 VLYWMPOKSSWJAL-UHFFFAOYSA-N 0.000 claims description 5
- 229960004936 sulfamethoxypyridazine Drugs 0.000 claims description 5
- NHUHCSRWZMLRLA-UHFFFAOYSA-N Sulfisoxazole Chemical compound CC1=NOC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1C NHUHCSRWZMLRLA-UHFFFAOYSA-N 0.000 claims description 4
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 4
- 150000001768 cations Chemical class 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 4
- 238000005374 membrane filtration Methods 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- PBCZLFBEBARBBI-UHFFFAOYSA-N sulfabenzamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC(=O)C1=CC=CC=C1 PBCZLFBEBARBBI-UHFFFAOYSA-N 0.000 claims description 4
- 229960004730 sulfabenzamide Drugs 0.000 claims description 4
- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 claims description 4
- 229960004306 sulfadiazine Drugs 0.000 claims description 4
- 229960000973 sulfadimethoxine Drugs 0.000 claims description 4
- 229960002135 sulfadimidine Drugs 0.000 claims description 4
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 claims description 4
- VACCAVUAMIDAGB-UHFFFAOYSA-N sulfamethizole Chemical compound S1C(C)=NN=C1NS(=O)(=O)C1=CC=C(N)C=C1 VACCAVUAMIDAGB-UHFFFAOYSA-N 0.000 claims description 4
- 229960005158 sulfamethizole Drugs 0.000 claims description 4
- 229960005404 sulfamethoxazole Drugs 0.000 claims description 4
- 229960002211 sulfapyridine Drugs 0.000 claims description 4
- GECHUMIMRBOMGK-UHFFFAOYSA-N sulfapyridine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=CC=N1 GECHUMIMRBOMGK-UHFFFAOYSA-N 0.000 claims description 4
- 229960003097 sulfaquinoxaline Drugs 0.000 claims description 4
- 229960001544 sulfathiazole Drugs 0.000 claims description 4
- JNMRHUJNCSQMMB-UHFFFAOYSA-N sulfathiazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CS1 JNMRHUJNCSQMMB-UHFFFAOYSA-N 0.000 claims description 4
- 229960001975 sulfisomidine Drugs 0.000 claims description 4
- YZMCKZRAOLZXAZ-UHFFFAOYSA-N sulfisomidine Chemical compound CC1=NC(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 YZMCKZRAOLZXAZ-UHFFFAOYSA-N 0.000 claims description 4
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 claims description 4
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 claims description 4
- 229960001082 trimethoprim Drugs 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000005864 Sulphur Substances 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 229960005141 piperazine Drugs 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 229960002673 sulfacetamide Drugs 0.000 claims description 3
- SKIVFJLNDNKQPD-UHFFFAOYSA-N sulfacetamide Chemical compound CC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 SKIVFJLNDNKQPD-UHFFFAOYSA-N 0.000 claims description 3
- BRBKOPJOKNSWSG-UHFFFAOYSA-N sulfaguanidine Chemical compound NC(=N)NS(=O)(=O)C1=CC=C(N)C=C1 BRBKOPJOKNSWSG-UHFFFAOYSA-N 0.000 claims description 3
- 229960004257 sulfaguanidine Drugs 0.000 claims description 3
- QWCJHSGMANYXCW-UHFFFAOYSA-N sulfaphenazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=NN1C1=CC=CC=C1 QWCJHSGMANYXCW-UHFFFAOYSA-N 0.000 claims description 3
- 229960004818 sulfaphenazole Drugs 0.000 claims description 3
- 208000035126 Facies Diseases 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 150000003851 azoles Chemical class 0.000 claims description 2
- XECUMCLPUKPRLJ-UHFFFAOYSA-N [O].C1=CN=CN=C1 Chemical compound [O].C1=CN=CN=C1 XECUMCLPUKPRLJ-UHFFFAOYSA-N 0.000 claims 1
- 239000000284 extract Substances 0.000 claims 1
- 239000000155 melt Substances 0.000 claims 1
- 238000012856 packing Methods 0.000 claims 1
- PBMFSQRYOILNGV-UHFFFAOYSA-N pyridazine Chemical compound C1=CC=NN=C1 PBMFSQRYOILNGV-UHFFFAOYSA-N 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 16
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000001212 derivatisation Methods 0.000 abstract description 3
- 238000011282 treatment Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 239000012716 precipitator Substances 0.000 abstract 1
- 230000014759 maintenance of location Effects 0.000 description 24
- 239000012071 phase Substances 0.000 description 20
- 150000002500 ions Chemical class 0.000 description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000000605 extraction Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000005457 optimization Methods 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 229910021642 ultra pure water Inorganic materials 0.000 description 5
- 239000012498 ultrapure water Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 235000006439 Lemna minor Nutrition 0.000 description 4
- 241000209501 Spirodela Species 0.000 description 4
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 235000013364 duck meat Nutrition 0.000 description 4
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003640 drug residue Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- WMPXPUYPYQKQCX-UHFFFAOYSA-N Sulfamonomethoxine Chemical compound C1=NC(OC)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 WMPXPUYPYQKQCX-UHFFFAOYSA-N 0.000 description 2
- 238000004176 ammonification Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 235000013330 chicken meat Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 210000003195 fascia Anatomy 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 229950003874 sulfamonomethoxine Drugs 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- YBUXKQSCKVQATK-UHFFFAOYSA-N 4-amino-n-phenylbenzenesulfonamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=CC=C1 YBUXKQSCKVQATK-UHFFFAOYSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004401 flow injection analysis Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 150000003217 pyrazoles Chemical class 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229950008456 sulfabenz Drugs 0.000 description 1
- 229960000468 sulfalene Drugs 0.000 description 1
- KXRZBTAEDBELFD-UHFFFAOYSA-N sulfamethopyrazine Chemical compound COC1=NC=CN=C1NS(=O)(=O)C1=CC=C(N)C=C1 KXRZBTAEDBELFD-UHFFFAOYSA-N 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 238000003805 vibration mixing 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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 provides a compound detection reagent for detecting a sulfonamide compound and a detection method thereof. The compound detection reagent is prepared from an organic solvent solution containing various sulfonamide compound reference substances and a protein precipitator. The compound detection reagent provided by the invention can be used for simultaneously analyzing and detecting 28 sulfonamide compounds; the detection scope is wide; the scheme of protein precipitation or liquid-liquid extraction is adopted in the process of sample treatment, so that 28 sulfonamide compounds in the biological sample are purified and enriched; the detection method adopting the compound detection reagent provided by the invention has the advantages of simplicity, accuracy, quickness, stability and less sample capacity; the complex steps of derivatization and enzymatic conversion are not required; the chemical reagent used in the pre-treating process is less; the detection method is environment-friendly; the detection result is not interfered by endogenous substances; the false negative/positive rate is low; the cost of the reagent is low; the compound detection reagent is fit for quick analysis for a large amount of samples.
Description
Technical field
The invention belongs to drug measurement techniques field, be specifically related to a kind of combine detection for detecting sulfonamides compound
Reagent and detection method thereof.
Background technology
Sulfa drugs (SAs) refers to the general name with a class medicine of P-aminobenzene-sulfonamide structure, has antimicrobial spectrum
Extensively, the advantage such as curative effect is strong, be one of antibiotic most-often used in current aquaculture.Due to SAs action time in vivo and
The metabolism time is longer, and the sulfanilamide taken in by any approach is likely in human body accumulation, and concentration exceedes after certain value people
Body function can produce harm.SAs dealed with medicine went is mainly reflected in injury of kidney, oncogenicity effect, anaphylaxis, hematopoietic function
Destruction, digestive system obstacle etc..Owing to the harm of sulfanilamide is relatively big, and use more in cultivation, therefore European Union, the U.S., Japan
Require to improve constantly to horizontal amine drug residue limits in animal derived food in country, many sulfa drugss by
It is classified as disabling veterinary drug.In order to ensure Safety of Food Quality, need in quick, accurate, cheap many residuals method detection food
Sulfa drug residue.The detection method of sulfa drug residue mainly has euzymelinked immunosorbent assay (ELISA), high performance liquid chromatography, gas phase color
Spectrum-mass spectrography, high performance liquid chromatography-tandem mass method, high performance capillary electrophoresis, biological immune method etc..Euzymelinked immunosorbent assay (ELISA) and radiation
Immunization is mainly used in large-scale screening operation, false positive easily occurs, is generally not used for quantitatively；Gas chromatography electronics
Acquisition detector carries out quantitative analysis, has higher specificity, but the method is relatively complicated, is difficult to operation；High-efficient liquid phase color
Spectrum uv detection method and high performance capillary electrophoresis sensitivity are low, poor specificity, particularly the sample matrix interference when low concentration detects
Greatly, it is difficult to the most qualitative confirmation；Although gas chromatography-mass spectrography sensitivity and specificity are the highest, but derivatization process is loaded down with trivial details,
It is not suitable for high throughput analysis.
Summary of the invention
For the present situation of above-mentioned sulfonamides analyte detection, it is an object of the invention to provide a kind of for detecting sulfonamides
The combine detection reagent of compound and detection method thereof, the present invention provide combine detection reagent can detect poultry, Fowl meat, tissue and
Up to 28 kinds sulfa drugss in the humoral bio samples such as its correlated product and poultry, fowl urine, saliva.
For achieving the above object, the present invention is achieved by the following technical solutions:
Present invention provide for detecting the combine detection reagent of sulfonamides compound, described combine detection reagent includes:
Organic solvent solution containing multiple sulfonamides compound reference substance and protein precipitant, every kind contained sulfonamides compound pair
Being 0.1ng-20ng/mL according to product concentration in organic solvent solution, described protein precipitant is methanol, acetonitrile or acetic acid second
Ester.
Further: the described organic solvent solution containing multiple sulfonamides compound reference substance is containing 28 kinds of sulfonamides
The volume ratio of compound is the methanol solution of 50%；Described 28 kinds of sulfonamides compounds are respectively as follows: sulfamerazine, kynix
Piperazine, cistosulfa, sulfabenzamide, sulfadimethoxine, sulphaquinoxaline, sulfaphenazole, sulfanitran, sulfadiazine, sulphur
Amine acetyl, sulfapyridine, sulfathiazole, sulfadimidine, sulfamonomethoxine, sulfamethoxine, Sulfamethoxazole, sulfanilamide
Amidine, trimethoprim, sulfamethizole, arnosulfan pyrimidine, sulfamethoxypyridazine, bensulfa piperazine, sulfasomidine,
Sulfaquinoxaline, sulfameter, sulfanilamide, ganda and bosporon.
Further: described combine detection reagent also include Mobile Phase Additives, described Mobile Phase Additives be formic acid and
Being combined of ammonium formate.
Further: described protein precipitant is acetonitrile.
Present invention also offers the detection method of the combine detection reagent for detecting sulfonamides compound described in utilization,
Described detection method comprises the following steps:
(1) biological sample pretreatment: detected sample utilizes liquid-liquid extraction method, precipitation of protein or filter membrane filtration method enter
Row pretreatment；
(2) the described organic solvent solution direct injected containing multiple sulfonamides compound reference substance is taken；Obtain every kind of sulphur
The standard curve of aminated compounds；Obtain linear equation, correlation coefficient and the range of linearity；
(3) pretreated detected sample is carried out Liquid Chromatography-Tandem Mass Spectrometry detection:
(4) peak area of each sulfonamides compound Mass Spectrometer Method obtained, substitutes into the mark that in step (2), reference substance obtains
Directrix curve, obtains the content of corresponding sulfonamides compound.
Further: in described step (1), liquid-liquid extraction method is: solid detected sample to be pulverized, adds water, homogenate, add
The water volume entered and example weight are than for 1:1-6:1；Homogenate is extracted with ethyl acetate afterwards, centrifugal, under conditions of 35-45 DEG C
Organic facies is dried up, adds methanol and melt again, centrifugal, take supernatant for analyzing.
Further: in described step (1), precipitation of protein is: solid detected sample to be pulverized, adds water, homogenate；It
Rear homogenate adds methanol or acetonitrile precipitation, and the addition volume of described methanol or acetonitrile and example weight ratio is for 1:1-6:1；From
The heart, takes supernatant, filtering with microporous membrane, takes filtrate for analyzing.
Further: in described step (1), filter membrane filtration method is: by liquid detected sample filtering with microporous membrane, filter
Liquid adds methanol or acetonitrile precipitation, and the addition volume of described methanol or acetonitrile and example weight ratio is for 1:1-6:1；Centrifugal, take
Clear liquid is used for analyzing.
Further: in described step (3), chromatographic condition is: using silica gel bonded phase filling post, flowing is by A phase+B phase
Composition: A phase is acetonitrile water, is the formic acid of 0.002%-0.1% containing percent by volume, and B phase is acetonitrile, containing percent by volume is
The formic acid of 0.002%-0.1%, A phase: the volume ratio of B phase is 1-99%:99-1%.
Further: in described step (3), Mass Spectrometry Conditions is: electron spray ionisation source ESI, cation multiple-reaction monitoring scans
MRM, gas curtain gas 35～40psi, ionizing voltage+5500V, temperature 450～550 DEG C, gas 40～55psi of spraying, auxiliary heating
Gas 40～60psi.
Advantages of the present invention and having the technical effect that
1, the detectable combination that the present invention provides can analyze 28 kinds of sulfonamides compounds of detection simultaneously, and detection range is wide
General；
2, based on detection method optimize result it is not necessary to the detection consistent bare substrate of sample, reference substance solvent without
Direct injection analysis need to be processed and can build standard curve；
3, sample treatment uses albumen precipitation or the scheme of liquid-liquid extraction, to 28 kinds of sulfonamides chemical combination in biological sample
Thing purifies and is enriched with；
4, in the chromatographic isolation of sample, chromatograph short column and the linear gradient elution method of thin bore is used, to 28 kinds of sulfonamides
Compound carries out sharp separation；
5, use the complex flow phase additive of formic acid+ammonium formate, while eliminating matrix effect, improve analysis method
Sensitivity；In the case of ensureing the correctness of measurement result, simplify operating procedure.
6, apply the detection method of composite reagent of the present invention have simplicity, accurately, quickly, stable, sample size is few
Advantage, and without the complex steps such as derivatization, enzymatic conversion, the chemical reagent of pretreatment process application is few, environmental protection, detection
Result is not disturbed by endogenous material, and false negative false positive rate is low, and reagent cost is cheap, it is adaptable to quickly dividing of great amount of samples
Analysis.
Accompanying drawing explanation
Fig. 1 is the extraction ion flow chromatography figure of 28 kinds of sulfonamides compounds in the present invention；Fig. 1 a, Fig. 1 b, Fig. 1 c are described
The extraction ion flow chromatography figure of 28 kinds of sulfonamides compounds.
Fig. 2 is extraction ion flow chromatography sulfanitran being detected in the embodiment of the present invention 2 in duck meat sample.
Detailed description of the invention
Below in conjunction with specific embodiment, technical solution of the present invention is described further.This example is only exemplary applications.No
It is understood that as a kind of restriction to the claims in the present invention protection domain.
Embodiment 1
1. experimental apparatus and reagent
1.1 experimental apparatus
AcQuityTMUPLC ultrahigh-pressure liquid chromatograph, Oasis MCX Solid-Phase Extraction (SPE) pillar (3ml, 60mg) are equal
Purchased from U.S. Waters, API5500Qtrap mass spectrograph purchased from American AB, eddy mixer, TTL-DC II type Nitrogen evaporator, IKA is high
Speed tissue refiner, ultra-pure water instrument (Simplicity, Merck Millipore Corp.), ultrasonic extraction instrument (KQ-250B, city of Kunshan
Ultrasonic instrument company limited), IKA T18Basic high speed dispersor (German).
1.2 experiment reagent
Methanol (chromatographically pure), acetonitrile (chromatographically pure) formic acid purity is 99%, 28 kinds of sulfonamides compounds (purity all >=
99%): sulfamerazine, sulfamethoxypyridazine, cistosulfa, sulfabenzamide, sulfadimethoxine, sulphaquinoxaline, sulfabenz
Between pyrazoles, sulfanitran, sulfadiazine, sulfacetamide, sulfapyridine, sulfathiazole, sulfadimidine, sulfanilamide, methoxy is phonetic
Pyridine, sulfamethoxine, Sulfamethoxazole, sulfaguanidine, trimethoprim, sulfamethizole, arnosulfan pyrimidine, kynix are rattled away
Piperazine, bensulfa piperazine, sulfasomidine, sulfaquinoxaline, sulfameter, sulfanilamide, ganda, sulfanilamide two
First azoles is purchased from Germany Dr.Ehrenstorfer GmbH.
2. detection method
2.1 detected sample pre-treatments
Choose chicken meat sample, stripping and slicing, remove fascia, accurately weigh 1.00g chicken meat sample, add 3ml water, through tissue point at a high speed
Scattered machine is uniformly smashed to pieces and is made homogenised sample, accurately weighs 1ml homogenised sample, is placed in 5mL EP pipe, adds 3mL second with liquid-transfering gun
Nitrile, 2000rpm vortex 5min, 14000g is centrifuged 5min, is transferred to by supernatant in EP pipe, and residue adds 1mL acetonitrile,
2000rpm vortex 5min, 14000g is centrifuged 5min, repeats to extract 1 time, merges supernatant.By Waters Oasis MCX (3cc/
After solid phase extraction column 60mg) first activates with the acetonitrile of 3mL and the ultra-pure water of 3mL, take united extraction liquid loading and cross post,
Add 5mL ultra-pure water drip washing, then with 5mL 4% ammonification acetonitrile eluting, at eluent 40 DEG C, N2Dry up, be settled to 10% acetonitrile
After 200 μ L, 2000rpm vortex 60s, cross the microporous filter membrane of 0.2 μm, treat that UPLC-MS/MS analyzes.
2.2 Liquid Chromatography-Tandem Mass Spectrometry analyses
Chromatographic condition: chromatographic column: ACQUITY UPLCTMBEH C18 post (50mm × 2.1mm, 1.7 μm)；Column temperature 40 DEG C；
Sampling volume 5 μ l；Using silica gel bonded phase filling post, flowing is by A phase+B phase composition: A phase is acetonitrile water (second eyeball and the body of water
Long-pending ratio is 5:95), it is the formic acid of 0.03% containing volume ratio；B phase is acetonitrile, containing the formic acid that volume ratio is 0.03% or acetic acid；A
Phase: the volume ratio of B phase is (1-99%): (99-1%), flow velocity 0.3ml/min；Use gradient elution, flowing phase composition proportioning with
Elution time graded.Eluent gradient elution program is shown in Table 1.
The liquid phase elution requirement of 1 28 kinds of sulfonamides compounds of table
Mass Spectrometry Conditions: use the ionization mode of electron spray ionisation source ESI cation, carry out multiple-reaction monitoring (MRM) and sweep
Retouching, gas curtain gas 35～40psi, ionizing voltage+5500V, temperature 450～550 DEG C, gas 40～55psi of spraying, auxiliary adds steam
40～60psi, interface adds steam On, collides gas Medium.Multiple-reaction monitoring pattern (MRM) is planted, and each compound test two is right
Ion, for quantitatively, is used for qualitative a pair a pair.
The optimization of 3 technical schemes
3.1, chromatographic condition optimization
The present invention selects 2.1 × 50mm, and the C18 chromatographic column of 1.7 μm ensures 28 kinds of sulfonamides within the relatively short time
Efficiently separating of compound.Experiment compare methanol-water, acetonitrile-water system as flow relative 28 kinds of sulfonamides compounds separation
The impact of effect, reduces analysis time to improve the efficiency of analysis, and the acetonitrile-water flowing using eluting power stronger is mutually
System, experiment uses pulsed gradient type of elution, in conjunction with Mobile Phase Additives 0.03% formic acid after optimizing and 0.03% ammonium formate,
Eliminate the ion inhibitory action (matrix effect) flowing out composition in sample altogether to analysans, substantially increase sensitivity, single
The individual running time only needs 6min, and also has the leeway shortening analysis time.
3.2, Mass Spectrometry Conditions optimization
Use flow injection mode, under cation and negative ion mode, every kind of compound is carried out first mass spectrometric scanning and obtains
Obtain its molecular ion peak；After determining the quasi-molecular ion peak of classes of compounds, respectively it is carried out second mass analysis, it is thus achieved that
Its fragment ion information, determines quota ion and qualitative ion.By optimizing fragmentation voltage (Fragmentor, V), collision voltage
Parameters such as (Collision Energy, eV), makes the quasi-molecular ion of 28 kinds of sulfonamides compounds produce with fragments characteristic ion
Ion pair sensitivity reach optimum；Capillary voltage, atomizing pressure, dry temperature, dry gas stream amount etc. are optimized,
The Ionization Efficiency making every kind of testing compound reaches optimal.Mass ion source part parameters optimization is shown in Table 2.
The mass ion source partial condition (band * person is quota ion) of 2 28 kinds of sulfonamides compounds of table
3.3, sample pre-treatments optimization
Owing to sulfa drugs is insoluble in water, relatively it is soluble in diluted acid, acetonitrile, ethyl acetate, dichloromethane and chloroform
Deng organic solvent.In view of the compatibility of most of sample-pretreating methods, through comparing, selection acetonitrile is extractant, due to side
Method is sensitive, and required sample size is few, and needed for every sample, chemical reagent amount is few, environmental protection；Compared with acetonitrile with ethyl acetate, two
The extraction efficiency such as chloromethanes, chloroform is poor, and in extracting solution, fat content is higher, is unfavorable for purified treatment, and extraction efficiency is relatively low.
After sample adds acetonitrile, supersound extraction 30min after vibration mixing, cross sample detection after the 0.2 organic filter membrane of μm, result display acetonitrile
The extraction efficiency of 28 kinds of sulfonamides compounds is up to more than 90%.And sample is without purifying further, is directly used in UPLC-
MS/MS detects, and illegally adds medicine in can preferably extracting in sample.
4, the range of linearity and detection limit
Benefit from the optimization of analysis method, by the standard solution direct injected of 28 kinds of sulfonamides compounds in UPLC-MS/MS
Be analyzed can standard curve needed for creation analysis, take concentration be respectively 0.078,0.156,0.313,0.625,1.25,
2.5,5,10, the standard solution of 20ng/mL after testing after, draw the standard curve of 28 kinds of sulfonamides compounds, correlation coefficient is equal
More than 0.99 (being shown in Table 3).Standard curve linearly good r >=0.99 (n=5).The representativeness of 28 kinds of sulfonamides compounds in Carnis Gallus domesticus
Chromatogram is shown in Fig. 1.
28 kinds of sulfonamides compound total ion current figures as shown in Figure 1, from figure 1 it appears that sulfamerazine retain time
Between be 2.31min；Sulfamethoxypyridazine sulfamerazine retention time is 2.41min；When cistosulfa sulfamerazine retains
Between be 2.49min；Sulfabenzamide sulfamerazine retention time is 2.64min；Sulfadimethoxine sulfamerazine retention time
For 2.64min；Sulphaquinoxaline sulfamerazine retention time is 2.63min；Sulfaphenazole sulfamerazine retention time
For 2.66min；Sulfanitran sulfamerazine retention time is 2.77min；Sulfadiazine sulfamerazine retention time is
2.21min；Sulfacetamide sulfamerazine retention time is 0.86min；Sulfapyridine sulfamerazine retention time is
2.25min；Sulfathiazole sulfamerazine retention time is 2.36min；Sulfadimidine sulfamerazine retention time is
2.37min；Sulfamonomethoxine sulfamerazine retention time is 2.42min；Sulfamethoxine sulfamerazine retention time
For 2.49min；Sulfamethoxazole sulfamerazine retention time is 2.53min；Sulfaguanidine sulfamerazine retention time is
0.84min；Trimethoprim sulfamerazine retention time is 2.20min；Sulfamethizole sulfamerazine retention time is
2.06min；Arnosulfan pyrimidine sulfamerazine retention time is 2.28min；Sulfamethoxypyridazine sulfamerazine retains
Time is 2.43min；Bensulfa piperazine sulfamerazine retention time is 2.63min；Sulfasomidine sulfamerazine
Retention time is 2.69min；Sulfaquinoxaline sulfamerazine retention time is 2.36min；Sulfameter sulfalene
Pyrimidine retention time is 2.37min；Sulfanilamide sulfamerazine retention time is 2.14min；Ganda sulfamerazine is protected
Staying the time is 2.36min；Bosporon sulfamerazine retention time is 2.75min.
The 3 28 kinds of sulfonamides compounds of table quantitative limit, the range of linearity and correlation coefficient in Carnis Gallus domesticus
5, the response rate and precision
The present invention has investigated the response rate and the precision of the method, add in blank sample 3 varying levels (1,6,
28 kinds of sulfonamides compound empirically methods 20ng/mL) measure the response rate, each horizontal replication 6 times.Result shows,
The average recovery rate of 28 kinds of sulfonamides compounds is 85.0%～107%, and relative standard deviation is respectively less than 2.25% and (the results are shown in Table
4)。
Table 4 method recovery of standard addition and precision
Embodiment 2, detected sample specifically detect analysis
Actual detected sample is bought from market, Shandong Province, after process, is stored in the refrigerator of 4 DEG C standby.Use the present invention
The 10 parts of duck meat samples bought are detected by detection method, find 1 example positive, detect, sulfanitran content is
0.52ng/mL, is shown in Fig. 2.
The concrete detection method of the present embodiment is as follows:
(1) biological sample pretreatment: the present embodiment pretreatment uses precipitation of protein (to use in described combine detection reagent
Protein precipitant) process.Choose duck meat sample, stripping and slicing, remove fascia, accurately weigh 1.00g duck meat sample, add 3mL
Water, uniformly smashs to pieces through tissue dispersion machine at a high speed and makes homogenised sample, accurately weigh 1mL homogenised sample, be placed in 5mL EP pipe, use
Liquid-transfering gun adds 3mL acetonitrile, and 2000rpm vortex 5min, 14000g is centrifuged 5min, is transferred to by supernatant in EP pipe, and residue is again
Adding 1mL acetonitrile, 2000rpm vortex 5min, 14000g is centrifuged 5min, repeats to extract 1 time, merges supernatant.
(2) organic solvent solution containing multiple sulfonamides compound reference substance in described combine detection reagent is taken direct
Sample introduction；Obtain the standard curve of every kind of sulfonamides compound；Obtain linear equation, correlation coefficient and the range of linearity.
(3) pretreated testing sample is carried out Liquid Chromatography-Tandem Mass Spectrometry analysis: by Waters Oasis MCX
(3cc/60mg), after solid phase extraction column first activates with the acetonitrile of 3mL and the ultra-pure water of 3mL, united extraction liquid loading is taken
Cross post, add 5mL ultra-pure water drip washing, then with 5mL 4% ammonification acetonitrile eluting, at eluent 40 DEG C, N2Dry up, fixed with 10% acetonitrile
Hold to 200 μ L, after 2000rpm vortex 60s, cross the microporous filter membrane of 0.2 μm, treat that UPLC-MS/MS analyzes.
Liquid chromatograph, Mass Spectrometry Conditions are shown in embodiment 1 with 2.2 in embodiment, mass ion source part parameters optimization
Table 2.
(4) peak area of each compound Mass Spectrometer Method obtained, substitutes into the standard curve in step (2), obtains correspondence
The content of sulfonamides compound.
The present invention uses high performance liquid chromatography tandem mass spectrum technology to establish the analysis method of 28 kinds of sulfonamides compounds.Should
Method easy and simple to handle quick, highly sensitive, specificity is strong, reproducible, 28 kinds of sulfonamides compounds can be measured simultaneously, it is achieved that
Its high throughput analysis, the monitoring requirement of 28 kinds of sulfonamides compounds in can meeting.
Although foregoing combines accompanying drawing and is described the detailed description of the invention of the present invention, but not to invention protection
The restriction of scope, one of ordinary skill in the art should be understood that on the basis of technical scheme, those skilled in the art
Need not to pay various amendments that creative work can make or deformation is the most within the scope of the present invention.
Claims (10)
1. for detecting the combine detection reagent of sulfonamides compound, it is characterised in that described combine detection reagent includes: contain
The organic solvent solution of multiple sulfonamides compound reference substance and protein precipitant, every kind contained sulfonamides compound reference substance
Concentration in organic solvent solution is 0.1 ng-20 ng/mL, and described protein precipitant is methanol, acetonitrile or acetic acid second
Ester.
Combine detection reagent the most according to claim 1, it is characterised in that: described compare containing multiple sulfonamides compound
The organic solvent solution of product be the volume ratio containing 28 kinds of sulfonamides compounds be the methanol solution of 50%；Described 28 kinds of sulfonamides
Compound be respectively as follows: sulfamerazine, sulfamethoxypyridazine, cistosulfa, sulfabenzamide, sulfadimethoxine, sulphaquinoxaline,
First between sulfaphenazole, sulfanitran, sulfadiazine, sulfacetamide, sulfapyridine, sulfathiazole, sulfadimidine, sulfanilamide
Oxygen pyrimidine, sulfamethoxine, Sulfamethoxazole, sulfaguanidine, trimethoprim, sulfamethizole, arnosulfan pyrimidine, kynix
Pyridazine, bensulfa piperazine, sulfasomidine, sulfaquinoxaline, sulfameter, sulfanilamide, ganda and sulphur
Amine diformazan azoles.
Combine detection reagent the most according to claim 1, it is characterised in that: described combine detection reagent also includes the phase that flows
Additive, described Mobile Phase Additives is the compound of formic acid and ammonium formate.
Combine detection reagent the most according to claim 1, it is characterised in that: described protein precipitant is acetonitrile.
5. utilize the detection method of the combine detection reagent for detecting sulfonamides compound described in claim 1, its feature
It is that described detection method comprises the following steps:
(1) biological sample pretreatment: detected sample utilizes liquid-liquid extraction method, precipitation of protein or filter membrane filtration method carry out pre-
Process；
(2) the described organic solvent solution direct injected containing multiple sulfonamides compound reference substance is taken；Obtain every kind of sulfonamides
The standard curve of compound；Obtain linear equation, correlation coefficient and the range of linearity；
(3) pretreated detected sample is carried out Liquid Chromatography-Tandem Mass Spectrometry detection:
(4) peak area of each sulfonamides compound Mass Spectrometer Method obtained, substitutes into the standard that in step (2), reference substance obtains bent
Line, obtains the content of corresponding sulfonamides compound.
Detection method the most according to claim 5, it is characterised in that: in described step (1), liquid-liquid extraction method is: by solid
Detected sample is pulverized, and adds water, homogenate, and the water volume of addition and example weight ratio is for 1:1-6:1；Homogenate acetic acid second afterwards
Ester extracts, centrifugal, organic facies is dried up under conditions of 35-45 ° of C, adds methanol and melts again, centrifugal, takes supernatant for analyzing.
Detection method the most according to claim 5, it is characterised in that: in described step (1), precipitation of protein is: by solid
Detected sample is pulverized, and adds water, homogenate；Homogenate adds methanol or acetonitrile precipitation, the addition body of described methanol or acetonitrile afterwards
Long-pending with example weight than for 1:1-6:1；Centrifugal, take supernatant, filtering with microporous membrane, take filtrate for analyzing.
Detection method the most according to claim 5, it is characterised in that: in described step (1), filter membrane filtration method is: by liquid
Detected sample filtering with microporous membrane, filtrate adds methanol or acetonitrile precipitation, the addition volume of described methanol or acetonitrile and sample
Product weight ratio is 1:1-6:1；Centrifugal, take supernatant for analyzing.
Detection method the most according to claim 5, it is characterised in that: in described step (3), chromatographic condition is: use silica gel
Bonded phase packings post, flowing is by A phase+B phase composition: A phase is acetonitrile water, is the formic acid of 0.002%-0.1% containing percent by volume,
B phase is acetonitrile, is the formic acid of 0.002%-0.1% containing percent by volume, A phase: the volume ratio of B phase is 1-99%:99-1%.
Detection method the most according to claim 5, it is characterised in that: in described step (3), Mass Spectrometry Conditions is: electron spray
Ionization source ESI, cation multiple-reaction monitoring scanning MRM, gas curtain gas 35 ~ 40 psi, ionizing voltage+5500 V, temperature 450 ~
550 ° of C, spray gas 40 ~ 55 psi, and auxiliary adds steam 40 ~ 60 psi.
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| CN106546671A (en) * | 2016-10-17 | 2017-03-29 | 浙江省食品药品检验研究院 | Method based on sulfa drugs is remained in three posts two dimension HPLC/MS-MS meat products |
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| CN110618215A (en) * | 2018-10-22 | 2019-12-27 | 嘉兴迈维代谢生物科技有限公司 | Quality control product for metabonomics detection and quality control method thereof |
| CN111458417A (en) * | 2019-01-21 | 2020-07-28 | 深圳华大生命科学研究院 | Method and kit for combined detection of multiple antibiotics in sample to be detected |
| CN111650298A (en) * | 2020-06-09 | 2020-09-11 | 武汉市农业科学院 | Method for simultaneously detecting 5 sulfonamides in cow dung by solid-phase extraction-high performance liquid chromatography |
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