CN109813820A - A method of identification phoxim metabolite in fresh-water fishes different tissues - Google Patents
A method of identification phoxim metabolite in fresh-water fishes different tissues Download PDFInfo
- Publication number
- CN109813820A CN109813820A CN201910096527.1A CN201910096527A CN109813820A CN 109813820 A CN109813820 A CN 109813820A CN 201910096527 A CN201910096527 A CN 201910096527A CN 109813820 A CN109813820 A CN 109813820A
- Authority
- CN
- China
- Prior art keywords
- phoxim
- metabolite
- mass
- megalobrama amblycephala
- sample
- 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.)
- Granted
Links
- ATROHALUCMTWTB-OWBHPGMISA-N phoxim Chemical class CCOP(=S)(OCC)O\N=C(\C#N)C1=CC=CC=C1 ATROHALUCMTWTB-OWBHPGMISA-N 0.000 title claims abstract description 102
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000013505 freshwater Substances 0.000 title claims abstract description 22
- 229950001664 phoxim Drugs 0.000 claims abstract description 91
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 73
- 150000002500 ions Chemical class 0.000 claims abstract description 48
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000002207 metabolite Substances 0.000 claims abstract description 25
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical group [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 13
- 230000014759 maintenance of location Effects 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 6
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 210000001519 tissue Anatomy 0.000 claims description 46
- 210000004185 liver Anatomy 0.000 claims description 32
- 210000002381 plasma Anatomy 0.000 claims description 31
- 210000003734 kidney Anatomy 0.000 claims description 22
- 239000000284 extract Substances 0.000 claims description 20
- 238000004458 analytical method Methods 0.000 claims description 19
- 210000000936 intestine Anatomy 0.000 claims description 19
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000012071 phase Substances 0.000 claims description 14
- 239000007789 gas Substances 0.000 claims description 11
- 238000001819 mass spectrum Methods 0.000 claims description 11
- 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 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 239000012074 organic phase Substances 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 7
- 238000001269 time-of-flight mass spectrometry Methods 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- LJSQFQKUNVCTIA-UHFFFAOYSA-N Diethyl sulfide Natural products CCSCC LJSQFQKUNVCTIA-UHFFFAOYSA-N 0.000 claims description 4
- 230000007423 decrease Effects 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000010354 integration Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 238000011282 treatment Methods 0.000 claims description 2
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000007654 immersion Methods 0.000 abstract description 4
- 238000001802 infusion Methods 0.000 abstract 1
- 241001275890 Megalobrama amblycephala Species 0.000 description 105
- 210000003205 muscle Anatomy 0.000 description 31
- 239000000523 sample Substances 0.000 description 31
- 238000000605 extraction Methods 0.000 description 20
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 18
- 230000000694 effects Effects 0.000 description 17
- 239000011159 matrix material Substances 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 239000012496 blank sample Substances 0.000 description 11
- 230000008520 organization Effects 0.000 description 8
- 210000003491 skin Anatomy 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- JFDZBHWFFUWGJE-UHFFFAOYSA-N benzonitrile Chemical compound N#CC1=CC=CC=C1 JFDZBHWFFUWGJE-UHFFFAOYSA-N 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000002203 pretreatment Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000276701 Oreochromis mossambicus Species 0.000 description 3
- AFCIMSXHQSIHQW-UHFFFAOYSA-N [O].[P] Chemical compound [O].[P] AFCIMSXHQSIHQW-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000004495 emulsifiable concentrate Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- FZENGILVLUJGJX-NSCUHMNNSA-N (E)-acetaldehyde oxime Chemical compound C\C=N\O FZENGILVLUJGJX-NSCUHMNNSA-N 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 241000252498 Ictalurus punctatus Species 0.000 description 2
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 2
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006298 dechlorination reaction Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960001008 heparin sodium Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000002048 multi walled nanotube Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- -1 phoxim carboxylic acid Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 210000005084 renal tissue Anatomy 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000790794 Argulus Species 0.000 description 1
- 241000252230 Ctenopharyngodon idella Species 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- 241001262815 Dactylogyrus Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 241001523601 Gyrodactylus Species 0.000 description 1
- 108010034145 Helminth Proteins Proteins 0.000 description 1
- 241000252234 Hypophthalmichthys nobilis Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001275898 Mylopharyngodon piceus Species 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- PKUWKAXTAVNIJR-UHFFFAOYSA-N O,O-diethyl hydrogen thiophosphate Chemical compound CCOP(O)(=S)OCC PKUWKAXTAVNIJR-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical compound OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- QCJQWJKKTGJDCM-UHFFFAOYSA-N [P].[S] Chemical compound [P].[S] QCJQWJKKTGJDCM-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 150000001343 alkyl silanes Chemical group 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000003736 gastrointestinal content Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 210000002816 gill Anatomy 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 210000001853 liver microsome Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- CFNHVUGPXZUTRR-UHFFFAOYSA-N n'-propylethane-1,2-diamine Chemical compound CCCNCCN CFNHVUGPXZUTRR-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003986 organophosphate insecticide Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of methods for identifying phoxim metabolite in fresh-water fishes different tissues, it is administered using infusion method, pass through metabolite of ultra performance liquid chromatography series connection level four bars flight time high resolution mass spec (UPLC-QTOFMS) the qualitative detection phoxim in different tissues, comprising the following steps: (1) fresh-water fishes acquire different tissues after the immersion of phoxim solution；(2) sample is extracted, extractant is acetonitrile and ethyl acetate with volume ratio 5:1 mixing, and cleanser is anhydrous magnesium sulfate and octadecylsilane chemically bonded silica with mass ratio 10:1 mixing；(3) UPLC-QTOFMS qualitative analysis: with phoxim, O, O, O’,O’The retention time and mass of ion of two phosphorothionate of ,-tetraethyl and O, O- diethyl thiophosphoric acid are compared, and judge the metabolite of each tissue sample.
Description
Technical field
The invention belongs to aquaculture technology and test and analyze technical field, and in particular to one kind is based on ultra high efficiency liquid phase color
The method of phoxim and its metabolite in spectrum series connection level four bars flight time high resolution mass spec qualitative analysis fresh water fish tissues.
Background technique
Phoxim (Phoxim) is a kind of organophosphorus insecticide, be currently used as veterinary drug control pig and the mite of sheep, louse and
Other vermins.In China phoxim be also approved for kill or drive away parasitize megalobrama amblycephala, Tilapia mossambica, grass carp,
Chinese Sao, anchor head Sao, argulus, Gyrodactylus, Dactylogyrus, nematode in the fresh water fish body such as black carp, silver carp, bighead, carp, crucian etc.
Helminth.If phoxim improper use, it may cause it and generate residual in aquatic animal tissue, and then harmful to human is strong
Health.
Phoxim includes phosphoric acid ester bond hydrolysis and de- alkyl, cyano benzene first in rat, pig and the main degradation step of rabbit
Aldoxime (being hippuric acid in rat and the further detoxification of pig, benzonitrile is degraded in rabbit) and the pungent sulphur of de- ethyl
Phosphorus.In mouse and calf, cyano group oxidation is the important channel of a degradation, in conjunction with phoxim carboxylic acid, the oxidation of phoxim
Analog is the response to high insecticidal activity insecticide, is found in fly extract without in mammals, although this
The intermediate product of the possible of short duration appearance of a compound is temporarily accredited in rat body as de- ethyl phoxim.Phoxim is in pig
Intracorporal metabolism: radiolabeled phoxim fills both ends pig by the single dose mouth of 5mg/kg bw in the form of gelatine capsule.Most
High radioactive concentration appears in after medication for 24 hours, and activity highest is radiated in fat and is equivalent to 1320 μ g/kg, is equivalent to 600 in liver
μ g/kg is equivalent to 350 μ g/kg in kidney, and concentration when being equivalent to 50 μ g/kg, 72h in muscle in tissue is about above-mentioned dense
The half of angle value, only phoxim and cyano benzene first cyanogen oxime can be accredited in tissue, only can be quantified in fat.In fat
Phoxim is found in (radioactive activity for having 90%), loin and muscle, does not find cyano benzene first in loin, muscle and liver
Aldoxime.Percutaneous drug delivery, dosage 100mg/kg are passed through to seven pigs as dashing agent using radiolabeled phoxim
Bw, skin bioavilability are 1.2%~2.9% (European Medicines Agency, 2005).Liu Rongfei etc.
(2013) using high performance liquid chromatography tandem ion-trap mass spectrograph to the metabolite and metabolic pathway of the external hepatomicrosome of crucian
It is studied.Phoxim is directly appended to carry out in hepatomicrosome by the research using the method for preparation crucian hepatomicrosome
Incubated in vitro.This method is different in the actual use situation of fish body from phoxim, and the phoxim used liver particle in vitro
The concentration of body is that the so high concentration of 100 μm of ol/L (35.796mg/L) also not and exists in a practical situation, therefore phoxim
The intracorporal metabolin of fish may be different from the metabolite of phoxim of external liver microsomes incubation high concentration.It is pungent to disclose
Metabolite of the sulphur phosphorus in fish body inner tissue, this research study pungent sulphur according to phoxim actual conditions used in fish
Metabolite of the phosphorus in the tissue such as megalobrama amblycephala blood plasma, muscle, skin, liver, kidney, the gill, intestines, result of study are closer practical
Situation, and this research also provides scientific basic to formulate the maximum residue limit of phoxim in aquatic products.
Ultra performance liquid chromatography series connection level four bars flight time mass spectrum (UPLC-QTOFMS) is by ultra high efficiency liquid phase, ion
Source, mass analyzer, four part of detector composition, using level four bars ion focusing transmission, positive and negative dipulse repulsion, vertical acceleration
The mode detected with reflection time-of-flight mass mass analyzer and microchannel plate carrys out analysis of compounds molecular mass, has height
Effect, highly sensitive, specificity is strong, detection pattern is various, analysis speed is fast, provides the spy of data information abundant and data processing
Point.Ultra performance liquid chromatography partial size is superfine, it has higher separating degree, analysis speed and sensitivity than high performance liquid chromatography,
It is lower to the processing requirement of complex sample biological sample, be conducive to save human and material resources and time；And have with mass spectrum simultaneous well
Capacitive can improve the resolution ratio of chromatography on the whole, reduce ion and inhibit, reduce co-elute phenomenon, improve mass spectrographic sensitive
Degree and reliability, can analyze the substance of low content in complex matrices, and utmostly the interference of matrix is rejected in Shangdi.QTOFMS knot
The advantage for having closed two kinds of instruments of quadrupole rod mass spectrum and flight time mass spectrum, under full scan mode, UPLC effluent is through electron spray
Directly by quadrupole rod through LINAC after ion source ionizationTMIt is linear that collision cell is accelerated to import time-of-flight mass analyzer, it carries out anti-
Accurate mass analysis under emission mode.Use powerful information association data acquisition scheme (IDA) and high-resolution, high precision quality
The scanning of number level-one and second level scan pattern, obtain corresponding high-resolution exact mass number first mass spectrometric figure and second order ms figure.
For QTOFMS in resolution ratio >=25000 (FWHM) of low karyoplasmic ratio (m/z 100), the resolution ratio of high karyoplasmic ratio (m/z 950) is reachable
40000 (FWHM), while the karyoplasmic ratio (m/z) that can accurately measure object considerably increases analysis target to 4 after decimal point
The certainty of object.Even and if the QQ-TOF mass spectrometry of high-resolution (0.1u) is in the resolution ratio of low karyoplasmic ratio (m/z 100)
1000, the resolution ratio of high karyoplasmic ratio (m/z 1000) is only capable of being accurate to for 10000 (FWHM) karyoplasmic ratio (m/z) for measuring object
1 after decimal point.Therefore, can be good at carrying out phoxim metabolite in fresh-water fishes are respectively organized using UPLC-QTOFMS
Separation, identification work.
At present there are no phoxim in aquatic livestock body blood plasma, muscle, skin, liver, kidney, the gill, intestines etc. tissue in
Metabolite identification report, and the present invention using ultra performance liquid chromatography connect level four bars flight time high-resolution
Phoxim and its metabolite in the fresh-water fishes in-vivo tissue that phoxim is administered by immersion way for mass spectrum (UPLC-QTOFMS)
Accurate molecular weight identification is carried out to belong to for the first time.
Summary of the invention
The object of the present invention is to provide a kind of using ultra performance liquid chromatography series connection level four bars flight time high-resolution matter
Compose the method that (UPLC-QTOFMS) identifies phoxim metabolite in fish different tissues, sample pre-treatments side in this method
Method is suitable for a variety of different tissues (blood, muscle, skin, liver, kidney, the gill, intestines etc.) of fresh water fish body and processing method is grasped
Make simple, quick；Based on ultra performance liquid chromatography series connection level four bars flight time mass spectrum (UPLC-QTOFMS) to phoxim and its
Metabolite separates in fresh water fish tissues, identifies, not only analyzes that speed is fast, but also can obtain phoxim and its metabolite
Accurate mass number, it is ensured that the accuracy of phoxim and its metabolite identification result.
To achieve the above object, the technical solution adopted by the present invention is that:
A method of identification phoxim metabolite in fresh-water fishes different tissues, which is characterized in that including following step
It is rapid:
(1) phoxim impregnates administration
Fresh-water fishes are immersed in the phoxim solution of 1~5mg/L, and (phoxim emulsifiable concentrate acetone solution is simultaneously diluted with distilled water
Be formulated) in, impregnate 2h, 4h, 6h, 8h, 10h, for 24 hours, blood plasma and liver, kidney, the gill, intestines, skin are acquired after 48h respectively
Skin, musculature and homogeneous processing；
(2) sample treatment
The above-mentioned sample respectively organized is weighed respectively, extractant A and cleanser B is sequentially added, is centrifuged after vortex oscillation, is collected
Extracting solution is repeated to extract sample residue with extractant A, and combined extract uses methanol constant volume, through 0.22 μm of filter after being dried with nitrogen
Film filtering, filtrate are detected for UPLC-Q-TOFMS；
The extractant A is acetonitrile and ethyl acetate with volume ratio 5:1 mixing；
The cleanser is anhydrous magnesium sulfate and octadecylsilane chemically bonded silica with mass ratio 10:1 mixing；
(3) UPLC-QTOFMS qualitative analysis
Each tissue sample is detected using ultra performance liquid chromatography series connection level four bars flight time high resolution mass spec technology, is surveyed
The retention time of the compound and mass of ion, with phoxim, O, O, O ', O ', two phosphorothionate of-tetraethyl and O, O- diethyl
The retention time and mass of ion of base thiophosphoric acid are compared, and judge the metabolite of each tissue sample.
Further, UHPLC analysis condition specifically: C18 chromatographic column, 100mm × 2.1mm × 1.8 μm；Column temperature: 30 DEG C,
Flow velocity: 0.25mL/min；Mobile phase: organic phase is methanol, and water phase is 0.1% aqueous formic acid, gradient elution program: starting ladder
The methanol that organic phase is 10% and 0.1% aqueous formic acid that water phase is 90% are spent, maintains 1min, then the organic phase in 5min
The methanol that linear rise is 90% to volumetric concentration, 0.1% aqueous formic acid that water phase linear decline is 10% maintain 4min,
Organic phase volume linearly decreases to 10% methanol in 0.1min, and water phase volume linearly rises to 90%, finally maintains 1.9min；
Sampling volume is 5.0 μ L.
Further, QTOFMS analysis condition specifically: electric spray ion source, positive ion mode, scanning range M/Z
100~1000Da, integration time 0.199957secs；Flight time mass spectrum scanning product ion mass range be 50~
1000Da, removes cluster voltage: 45V, collision energy 35eV, and extension collision energy is 15.0eV；Plasma diffusing W,Mo delay is 67, ion
Discharging width is 25, and spraying gas is 50Psi, and auxiliary heating gas is 55Psi, and transom window gas is 25Psi, and ionization temperature is 400 DEG C,
Cation spray voltage 5500V.
Compared with prior art, the present invention has the following advantages and beneficial effects:
1, in order to disclose metabolite of the phoxim in Fish tissue, fresh-water fishes are soaked in containing phoxim by the present invention
Water body in by fish body itself then different tissues will be acquired and carry out qualitative point after phoxim absorbs, is enriched to metabolism in vivo
Analysis, therefore result is analyzed closer to actual conditions.
2, superfine (the UPLC use of ultra performance liquid chromatography (UPLC) partial size on the basis of high performance liquid chromatography (HPLC)
1.7 μm of two microns of Asia chromatographic column, column length is shorter, compared to 5 μm of traditional HPLC and 3 μm of chromatographic column, separating rate
9 times and 3 times have been respectively increased), UPLC ratio HPLC has higher separating degree, and peak capacity expands 2~3 times, sensitivity compared with
HPLC improves 2~3 times.UPLC is lower to the processing requirement of complex biological sample, is conducive to save human and material resources and time；
And have good compatibility with mass spectrum, the resolution ratio of chromatography can be improved on the whole, reduced ion and inhibited, it is existing to reduce co-elute
As improving mass spectrographic sensitivity and reliability, the substance of low content in complex matrices can be analyzed, utmostly base being rejected in Shangdi
The interference of matter.
3, (qualitative ability is better than triple with powerful qualitative ability for quadrupole rod time-of-flight mass spectrometry (Q-TOFMS)
Quadrupole rod mass spectrum QQQ/MS), it is analyzed at first mass spectrometric MS1 and second order ms MS2 mode, parent ion and fragment ion is provided
Accurate mass, it can accurately measure after compound molecule quality to decimal point 4 and provide element composition, multistage fragment from
Son keeps qualitative results more accurate, and QQQ/MS is only capable of after measurement compound molecule quality to decimal point 1, Q-TOF/MS phase
Compare QQQ/MS, there is higher resolution ratio, more efficient Quality Identification and higher selectivity.Cylinder metabolism-ure is identified
Using HPLC-QQQ/MS, since the accuracy of this tandem mass spectrum is not high, sensitivity is low, resolution ratio is weaker, quality testing width
It is limited, the accurate molecular quality of metabolin cannot be provided, it is difficult to the closer isomer of retention time is differentiated, it is qualitative inaccurate
Really, qualitatively compound amounts are also less for institute.Using UPLC-QTOFMS come qualitative cylinder metabolism-ure, the above problem can be one by one
It solves.
4, the present invention is optimized for the extracting method of phoxim and its metabolin, as shown in Figure 1 acetonitrile and acetic acid
The extractant that ethyl ester is configured to according to volume ratio 5:1 adopts the recovery rate highest of phoxim in fresh water fish tissues and its metabolin
Use MgSO4It is minimum that the mixing cleanser that+C18 is 10:1 according to mass ratio purifies the matrix effect that sample generates.
Detailed description of the invention
Fig. 1: the effect of different extractants compares.
Fig. 2: the effect of different cleansers compares.
Fig. 3: megalobrama amblycephala plasma sample and blank plasma samples chromatogram and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim one in 3-b megalobrama amblycephala plasma sample in 3-a megalobrama amblycephala plasma sample
Grade mass spectrogram, phoxim second order ms figure in 3-c megalobrama amblycephala plasma sample, 3-d megalobrama amblycephala blood plasma blank sample extract ion stream
Chromatogram, 3-e megalobrama amblycephala blood plasma blank sample first mass spectrometric figure, O, O, O in 3-f megalobrama amblycephala plasma sample ', O ' ,-tetraethyl two
Phosphorothionate extracts ion stream chromatogram, and 3-g blank plasma extracts ion stream chromatogram, O, O, O in 3-h megalobrama amblycephala blood plasma ',
O ', two phosphorothionate first mass spectrometric figure of-tetraethyl, 3-i megalobrama amblycephala blood plasma first mass spectrometric figure, O, O in 3-j megalobrama amblycephala blood plasma,
O ', O ', two phosphorothionate second order ms figure of-tetraethyl, O in 3-k megalobrama amblycephala blood plasma, O- diethyl thiophosphoric acid extracts ion
Flow chromatography figure, 3-l megalobrama amblycephala blood plasma blank extract ion stream chromatogram, O in 3-m megalobrama amblycephala blood plasma, O- diethyl thiophosphoric acid
First mass spectrometric figure, 3-n megalobrama amblycephala blood plasma blank first mass spectrometric figure, O in 3-o megalobrama amblycephala plasma sample, O- diethyl thiophosphoric acid
Second order ms figure.
Fig. 4: megalobrama amblycephala muscle samples and blank muscle samples chromatogram and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim one in 4-b megalobrama amblycephala muscle samples in 4-a megalobrama amblycephala muscle samples
Grade mass spectrogram, phoxim second order ms figure in 4-c megalobrama amblycephala muscle samples, 4-d megalobrama amblycephala muscle blank sample extract ion stream
Chromatogram, 4-e megalobrama amblycephala muscle blank sample first mass spectrometric figure, O, O, O in 4-f megalobrama amblycephala muscle samples ', O ' ,-tetraethyl two
Phosphorothionate extracts ion stream chromatogram, and 4-g blank muscle samples extract ion stream chromatogram, 4-h megalobrama amblycephala muscle samples
Middle O, O, O ', O ', two phosphorothionate first mass spectrometric figure of-tetraethyl, 4-i megalobrama amblycephala blank muscle samples extraction ion flow chromatography
Scheme, O, O, O in 4-j megalobrama amblycephala muscle samples ', O ', two phosphorothionate second order ms figure of-tetraethyl, 4-k megalobrama amblycephala muscle sample
O in product, O- diethyl thiophosphoric acid extract ion stream chromatogram, and 4-l megalobrama amblycephala blank muscle samples extract ion stream chromatogram,
O in 4-m megalobrama amblycephala muscle samples, O- diethyl thiophosphoric acid first mass spectrometric figure, 4-n megalobrama amblycephala blank muscle samples first mass spectrometric
Scheme, O in 4-o megalobrama amblycephala muscle samples, O- diethyl sulfide is for phosphoric acid second level mass spectrogram.
Fig. 5: megalobrama amblycephala skin samples and blank skin samples chromatogram and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim one in 5-b megalobrama amblycephala skin samples in 5-a megalobrama amblycephala skin samples
Grade mass spectrogram, phoxim second order ms figure in 5-c megalobrama amblycephala skin samples, 5-d megalobrama amblycephala skin blank sample extract ion stream
Chromatogram, 5-e megalobrama amblycephala skin blank sample first mass spectrometric figure, O, O, O in 5-f megalobrama amblycephala skin histology ', O ' ,-tetraethyl two
Phosphorothionate extracts ion stream chromatogram, O, O, O in 5-g megalobrama amblycephala skin histology ', O ', two phosphorothionate one of-tetraethyl
Grade mass spectrogram, O, O, O in 5-h megalobrama amblycephala skin histology ', O ', two phosphorothionate second order ms figure of-tetraethyl, 5-i megalobrama amblycephala
Skin blank tissue extracts ion stream chromatogram, 5-j megalobrama amblycephala skin blank tissue first mass spectrometric figure.
Fig. 6: megalobrama amblycephala liver specimens and blank liver specimens chromatogram and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim one in 6-b megalobrama amblycephala liver specimens in 6-a megalobrama amblycephala liver specimens
Grade mass spectrogram, phoxim second order ms figure in 6-c megalobrama amblycephala liver specimens, 6-d megalobrama amblycephala blank liver specimens extract ion stream
Chromatogram, 6-e megalobrama amblycephala liver blank sample first mass spectrometric figure, O, O, O in 6-f megalobrama amblycephala liver organization ', O ' ,-tetraethyl two
Phosphorothionate extracts ion stream chromatogram, O, O, O in 6-g megalobrama amblycephala liver organization ', O ', two phosphorothionate one of-tetraethyl
Grade mass spectrogram, O, O, O in 6-h megalobrama amblycephala liver organization ', O ', two phosphorothionate second order ms figure of-tetraethyl, 6-i megalobrama amblycephala
Liver blank tissue extracts ion stream chromatogram, O, O, O in 6-j megalobrama amblycephala liver organization ', O ', two phosphorothionate of-tetraethyl
First mass spectrometric figure, O in 6-k megalobrama amblycephala liver organization, O- diethyl thiophosphoric acid extract ion stream chromatogram, 6-l megalobrama amblycephala liver
O in dirty tissue, O- diethyl thiophosphoric acid first mass spectrometric figure, O in 6-m megalobrama amblycephala liver organization, O- diethyl sulfide is for di(2-ethylhexyl)phosphate
Grade mass spectrogram, 6-n megalobrama amblycephala blank liver organization extract ion stream chromatogram, 6-o megalobrama amblycephala blank liver organization first mass spectrometric
Figure.
Fig. 7 is megalobrama amblycephala kidney samples A and blank kidney samples A chromatogram and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim one in 7-b megalobrama amblycephala kidney samples A in 7-a megalobrama amblycephala kidney samples A
Grade mass spectrogram, phoxim second order ms figure in 7-c megalobrama amblycephala kidney samples A, 7-d megalobrama amblycephala blank kidney samples A extract ion stream
Chromatogram, 7-e megalobrama amblycephala blank kidney samples A first mass spectrometric figure, O in 7-f megalobrama amblycephala renal tissue, O- diethyl thiophosphoric acid
Extract ion stream chromatogram, O in 7-g megalobrama amblycephala nephridial tissue, O- diethyl thiophosphoric acid first mass spectrometric figure, 7-h megalobrama amblycephala kidney
O in tissue, O- diethyl sulfide extract ion stream chromatogram, 7-j for phosphoric acid second level mass spectrogram, 7-i megalobrama amblycephala kidney blank tissue
Megalobrama amblycephala kidney blank tissue first mass spectrometric figure.
Fig. 8 is megalobrama amblycephala gill sample and blank gill sample chromatogram figure and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim level-one matter in 8-b megalobrama amblycephala gill sample in 8-a megalobrama amblycephala gill sample
Spectrogram, phoxim second order ms figure in 8-c megalobrama amblycephala gill sample, 8-d megalobrama amblycephala blank gill sample extraction ion stream chromatogram, 8-
E megalobrama amblycephala gill blank sample first mass spectrometric figure, O in 8-f megalobrama amblycephala gill tissue, O- diethyl thiophosphoric acid extract ion flow chromatography
Scheme, O in 8-g megalobrama amblycephala gill tissue, O- diethyl thiophosphoric acid first mass spectrometric figure, O in 8-h megalobrama amblycephala gill tissue, O- diethyl
Thiophosphoric acid second order ms figure, 8-i megalobrama amblycephala gill blank tissue extract ion stream chromatogram, 8-j megalobrama amblycephala gill blank tissue one
Grade mass spectrogram.
Fig. 9 is megalobrama amblycephala in intestines and blank intestines sample chromatogram figure and mass spectrogram.
Phoxim extracts ion stream chromatogram, phoxim level-one matter in 9-b megalobrama amblycephala intestines sample in 9-a megalobrama amblycephala intestines sample
Spectrogram, phoxim second order ms figure in 9-c megalobrama amblycephala intestines sample, 9-d megalobrama amblycephala intestines blank sample extract ion stream chromatogram, 9-
E megalobrama amblycephala intestines blank sample first mass spectrometric figure, 9-f megalobrama amblycephala intestines blank sample second order ms figure.
Figure 10 is phoxim and its metabolite structures formula
10-a phoxim, 10-b O, O, O ', O ', two phosphorothionate of-tetraethyl, 10c-O, O- diethyl thiophosphoric acid.
Specific embodiment
The specific embodiment provided with reference to the accompanying drawing invention elaborates
Embodiment 1:
1 test material and method
1.1 test drugs, test fish and cultivating condition
Phoxim emulsifiable concentrate (content >=98% is provided by Xingtai City pesticide Co., Ltd), acetone (excellent pure grade, Chinese medicines group
Chemical reagent Co., Ltd), phoxim reference substance (content >=98%, German Dr.Ehrenstorfer company, No. CAS:
14816-18-3), O, O, O ', O ', two phosphorothionate of-tetraethyl (content >=94.3%, U.S. Advanced ChemTech
Company, No. CAS: 3689-24-5), O, (content >=98.0%, U.S. Advanced ChemTech are public for O- diethyl thiophosphoric acid
Department, No. CAS: 5871017-0), pungent oxygen phosphorus (content >=96.7%, German Dr.Ehrenstorfer company, No. CAS: 14816-
17-2)。
Healthy megalobrama amblycephala is purchased from the big market of Wuhan City's Baishazhou, and megalobrama amblycephala weight is (340 ± 19) g, is placed in and uses Gao Meng
Temporarily supported 7 days in (160cm × 60cm × 80cm) in aquarium after the disinfection of sour potassium, experimental water be sufficiently aeration and dechlorination from
Water carries out in breeding process continuing oxygenation using oxygen charging pump, and water temperature is 24 DEG C, and the megalobrama amblycephala of health is selected to use after temporarily supporting
In experiment.
1.2 experimental method
1.2.1 administration mode
Administration is impregnated, immersion is 1mg/L with the concentration of phoxim aqueous solution.
1.2.2 the preparation and administration of phoxim solution
It takes 3g phoxim emulsifiable concentrate acetone solution and is settled to 100mL, be configured to the phoxim acetone that concentration is 30mg/mL
Solution.The phoxim solution of preparation is added to be aerated in the tap water with dechlorination and is configured to concentration containing phoxim as 1mg/L
Aqueous solution, the aqueous solution of the 1mg/L of preparation is injected separately into 60cm × 35cm × 40cm fishbowl, each glass jar
40L phoxim aqueous solution is respectively put into 5 tail megalobrama amblycephalas in each glass and is impregnated.Megalobrama amblycephala 2 after immersion, 4,6,8,10,
24,48h takes out the megalobrama amblycephala of administration from glass jar, and body surface is with distilled water flushing 3 times, with filter paper by the water of megalobrama amblycephala body surface
It blots.(each period) acquires the tissue such as fish blood, muscle, skin, liver, kidney, the gill, intestines respectively.Blood use in advance
The blood taken is put in the centrifuge tube through heparin sodium rinse by the syringe of heparin sodium rinse, and 4000r/min is centrifuged 5min,
Take upper plasma, -80 DEG C of freezen protectives.Intestines are used after being rinsed well intestinal contents with the syringe equipped with distilled water after taking out
Scissors shreds, then with homogenizer by intestines it is homogeneous after -80 DEG C of freezen protectives.Muscle, skin and the gill use homogenizer after being shredded with scissors
- 80 DEG C of freezen protectives after homogeneous.- 80 DEG C of freezen protectives are to be measured after liver, renal tissue homogenizer are homogeneous.
1.2.3 ultra performance liquid chromatography (UPLC) analysis condition
Agilent ZORBAX Eclipse Plus C18 chromatographic column (100mm × 2.1mm, 1.8 μm), column temperature: 30 DEG C,
Flow velocity: 0.25mL/min；Mobile phase: organic phase is methanol, and water phase is 0.1% aqueous formic acid, gradient elution program: starting ladder
Degree be volumetric concentration 10% methanol and 90% 0.1% aqueous formic acid, maintain 1min, then linearly risen in 5min
The methanol that volumetric concentration is 90% maintains 4min, the methanol that volumetric concentration is 10% is then linearly decreased in 0.1min, most
After maintain 1.9min.Sampling volume is 5.0 μ L.
1.2.4 sample pre-treatments
1) each 1g of liver, kidney, the gill, skin, intestines, blood the pre-treatment of the samples such as blood plasma, liver, kidney, the gill, skin, intestines: are weighed
1mL, adds 5mL extractant A, vortex 30s, ultrasonic 3min, then plus 0.5g cleanser B, vortex 30s, 7000r/min be centrifuged 5min,
Supernatant is shifted into 10mL centrifuge tube, then plus 5mL extractant A repeat to extract primary, merge that extracting solution, 50 DEG C of nitrogen are blown to twice
Dry, 1mL methanol constant volume crosses 0.22 μm of filter filtering, upper UPLC-Q-TOFMS analysis.
2) muscle samples pre-treatment: taking muscle 5.0g in 50mL centrifuge tube, adds 20mL extractant A, vortex 30s, ultrasound
3min, then plus 3.0g cleanser B, vortex 30s, 7000r/min be centrifuged 5min, shift supernatant into 150mL chicken heart bottle, then plus
15mL extractant A repeats to extract once, and 3mL liquid is transferred to by extracting solution, 45 DEG C of rotary evaporations to 3mL or so twice for merging
In 10mL centrifuge tube, chicken heart bottle is washed in two times with 5mL extractant A, merges cleaning solution, and 50 DEG C of nitrogen are blown to dry, and 1mL methanol is fixed
Hold, crosses 0.22 μm of filter filtering, upper UPLC-Q-TOFMS analysis.
1.2.5 QTOFMS analysis condition
Electric spray ion source (ESI), positive ion mode, parent ion TOFMS Masses scanning range be M/Z 100~
1000Da, integration time 0.199957secs.Product ion TOFMS Masses scanning range is 50~1000Da of M/Z, removes cluster
Voltage (DP): 45V, collision energy (CE) are 35eV, and extension collision energy (CES) is 15.0eV；Plasma diffusing W,Mo postpones (IRD)
67, plasma diffusing W,Mo width (IRW) is 25, and spraying gas (GS1) is 50Psi, and auxiliary heating gas (GS2) is 55Psi, transom window gas
It (CUR) is 25Psi, ionization temperature is 400 DEG C, cation spray voltage 5500V.
1.2.6 the screening of extractant
Phoxim, O, O, O are added into megalobrama amblycephala musculature ', O ', two phosphorothionate of-tetraethyl, O, O- diethyl
Thiophosphoric acid, pungent oxygen phosphorus standard mixed solution make its ultimate density 5mg/kg (n=3).Methanol, acetonitrile, 0.1% first are used respectively
Sour methanol, 0.1% formic acid acetonitrile, methylene chloride, acetone, ethyl acetate, acetonitrile+ethyl acetate (v:v, 5:1) are used as extractant,
Using the rate of recovery as evaluation criterion, extract the screening of agent, according to 1.2.4 sample-pretreating method carry out sample pre-treatments and
1.2.5QTOFMS analysis condition is detected, and testing result is shown in Fig. 1, as shown in Figure 1 with acetonitrile+ethyl acetate (v:v, 5:1) work
For extractant, the equal > 85.0% of the extraction recovery of phoxim and its metabolite is better than methanol, acetonitrile, 0.1% formic acid first
Extraction recovery of the extractants such as alcohol, 0.1% formic acid acetonitrile, methylene chloride, acetone, ethyl acetate to phoxim metabolite
(50%~85.2%).Therefore, the preferred acetonitrile+ethyl acetate (v:v, 5:1) of this method is used as extractant.
1.2.7 the screening of cleanser
Matrix effect (ME, %) refers to the component in sample in addition to analyte, since matrix usually divides analyte
Analysis process has significant interference, and the accuracy of impact analysis result, these are influenced and interference is referred to as matrix effect.Matrix effect
Should there are ion inhibition or ion humidification, sound of the matrix to analysis object when showing that ME value is negative value according to its calculation formula
Should be worth it is inhibited, positive value indicate matrix to analysis object response have humidification.To megalobrama amblycephala muscle groups
Knit middle addition phoxim, O, O, O ', O ', two phosphorothionate of-tetraethyl, O, O- diethyl thiophosphoric acid, pungent oxygen phosphorus standard are mixed
Closing solution makes its ultimate density 5mg/kg (n=3).Graphon (GCB), N- propyl ethylenediamine (PSA), ten is respectively adopted
Eight alkyl silane bonded silica gels (C18), anhydrous magnesium sulfate (MgSO4), anhydrous sodium sulfate (Na2SO4), multi-walled carbon nanotube
(MWNT) and anhydrous magnesium sulfate (MgSO4)+octadecylsilane chemically bonded silica (C18) (m:m, 10:1) to the extracting solution of sample into
Row purification evaluates clean-up effect with matrix effect (ME, %), and then is screened and purified agent, and the absolute value of matrix effect is smaller, only
It is better to change effect.As a result Fig. 2 is seen, with the cleanser anhydrous magnesium sulfate (MgSO of optimization4)+octadecylsilane chemically bonded silica
(C18) (m:m, 10:1) is better than the clean-up effect of other above-mentioned cleansers to the clean-up effect that megalobrama amblycephala is respectively organized, using MgSO4
+ C18 (m:m, 10:1) as cleanser to after the purification of megalobrama amblycephala tissue to the matrix effect of phoxim and its metabolin < | 12%
|, and use matrix effect > after other above-mentioned cleansers purification sample | 14% |), therefore, the preferred MgSO of this method4+C18(m:
M, 10:1) it is used as cleanser.
Matrix effect (ME, %) calculation formula is as follows:
1.2.8 phoxim Methanogenesis in megalobrama amblycephala tissue
Using the PeakView of AB SCIEX companyTMMaserView option in menu column, with UPLC-QTOFMS
Total ion current figure be control, the total ion current figure that the total ion current figure of actual sample subtracts blank control sample carries out difference
Compare, obtains the accurate mass number of the object of difference, the position pair that the structural formula and metabolism further according to phoxim may occur
Metabolite of the phoxim in megalobrama amblycephala blood plasma, muscle, skin, liver, kidney, the gill, intestinal tissue is speculated.
2.UPLC-QTOFMS identifies phoxim and its metabolite in megalobrama amblycephala in-vivo tissue
Blood plasma, muscle, phoxim major metabolite is O, O, O in liver ', O ', two phosphorothionate of-tetraethyl and O, O-
Metabolite is in diethyl thiophosphoric acid, the gill and kidney with O, and based on O- diethyl thiophosphoric acid, major metabolite is in skin
O, O, O ', O ', two phosphorothionate of-tetraethyl.Phoxim is mainly that the form of prototype medicine phoxim exists in intestines.QTOFMS is surveyed
The retention time of phoxim and its metabolite, molecular composition, accurate molecular weight M in fixed megalobrama amblycephala tissueA, theoretical molecular weight
MTWith with Mass accuracy, daughter ion (being shown in Table 1).Blood plasma in megalobrama amblycephala body, muscle, skin, liver, kidney, the gill, in intestinal tissue
Phoxim and its metabolite extract chromatography of ions figure, firsts and seconds mass spectrogram is shown in Fig. 3~Fig. 9.Phoxim and its metabolism produce
The structural formula of object is shown in Figure 10.
1 phoxim of table and its metabolin molecular composition, accurate molecular weight MA, theoretical molecular weight MTAnd Mass accuracy
Based on above-mentioned identification method, applicant is also to phoxim in the fresh-water fishes different tissues such as Tilapia mossambica and channel catfish
Metabolite identified, the results showed that, phoxim is in fresh-water fishes (including megalobrama amblycephala, Tilapia mossambica, channel catfish)
Metabolite type is identical in different tissues, but content difference.Therefore, the present invention is phoxim in fresh water fish tissues
Middle residue detection provides detection object, and also for phoxim, maximum residue limit provides technical foundation and section in fresh water fish tissues
Learn foundation.
Claims (3)
1. a kind of method for identifying phoxim metabolite in fresh-water fishes different tissues, which comprises the following steps:
(1) phoxim impregnates administration
Fresh-water fishes are immersed in the phoxim solution of 1~5mg/L, 2h, 4h, 6h, 8h, 10h, for 24 hours, adopt respectively after 48h are impregnated
Collect blood plasma and liver, kidney, the gill, intestines, skin, musculature and homogeneous processing；
(2) sample treatment
The above-mentioned sample respectively organized is weighed respectively, extractant A and cleanser B is sequentially added, is centrifuged after vortex oscillation, is collected and is extracted
Liquid is repeated to extract sample residue with extractant A, and combined extract uses methanol constant volume, through 0.22 μm of filter membrane mistake after being dried with nitrogen
Filter, filtrate are detected for UPLC-Q-TOFMS；
The extractant A is acetonitrile and ethyl acetate with volume ratio 5:1 mixing；
The cleanser is anhydrous magnesium sulfate and octadecylsilane chemically bonded silica with mass ratio 10:1 mixing；
(3) UPLC-QTOFMS qualitative analysis
Each tissue sample, measuring are detected using ultra performance liquid chromatography series connection level four bars flight time high resolution mass spec technology
The retention time and mass of ion for closing object, with phoxim, O, O, O ', O ', two phosphorothionate of-tetraethyl and O, O- diethyl sulfide
It is compared for the retention time and mass of ion of phosphoric acid, judges the metabolite of each tissue sample.
2. the method for identification phoxim metabolite in fresh-water fishes different tissues according to claim 1, feature exist
In UHPLC analysis condition specifically: C18 chromatographic column, 100mm × 2.1mm × 1.8 μm；Column temperature: 30 DEG C, flow velocity: 0.25mL/
min；Mobile phase: organic phase is methanol, and water phase is 0.1% aqueous formic acid, gradient elution program: start gradient organic phase is
0.1% aqueous formic acid that 10% methanol and water phase is 90% maintains 1min, and then organic phase linear rise arrives in 5min
The methanol that volumetric concentration is 90%, 0.1% aqueous formic acid that water phase linear decline is 10% maintain 4min, have in 0.1min
Machine phase volume linearly decreases to 10% methanol, and water phase volume linearly rises to 90%, finally maintains 1.9min；Sampling volume is
5.0μL。
3. the method for identification phoxim metabolite in fresh-water fishes different tissues according to claim 1 or 2, feature
It is, QTOFMS analysis condition specifically: electric spray ion source, positive ion mode, scanning range are 100~1000Da of M/Z,
Integration time 0.199957secs；The product ion mass range of flight time mass spectrum scanning is 50~1000Da, removes cluster voltage:
45V, collision energy 35eV, extension collision energy are 15.0eV；Plasma diffusing W,Mo delay is 67, and plasma diffusing W,Mo width is 25, from
Component: double esi ion sources, spraying gas are 50Psi, and auxiliary heating gas is 55Psi, and transom window gas is 25Psi, and ionization temperature is
400 DEG C, cation spray voltage 5500V.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910096527.1A CN109813820B (en) | 2019-01-30 | 2019-01-30 | Method for identifying metabolite of phoxim in different tissues of freshwater fish |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910096527.1A CN109813820B (en) | 2019-01-30 | 2019-01-30 | Method for identifying metabolite of phoxim in different tissues of freshwater fish |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109813820A true CN109813820A (en) | 2019-05-28 |
| CN109813820B CN109813820B (en) | 2022-02-18 |
Family
ID=66606123
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910096527.1A Active CN109813820B (en) | 2019-01-30 | 2019-01-30 | Method for identifying metabolite of phoxim in different tissues of freshwater fish |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109813820B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376296A (en) * | 2019-06-06 | 2019-10-25 | 张云 | Pacify the quantitative detecting method that sieve replaces Buddhist nun's medicament residue in blood |
| CN113624869A (en) * | 2021-07-30 | 2021-11-09 | 上海市疾病预防控制中心 | Method for rapidly determining organic matter conversion substance in real sample with high flux and high accuracy |
| CN114002345A (en) * | 2021-10-12 | 2022-02-01 | 中国水产科学研究院黑龙江水产研究所 | Fish gill metabonomics sample pretreatment optimization method |
| CN118800338A (en) * | 2024-06-17 | 2024-10-18 | 中国水产科学研究院长江水产研究所 | Glycerophospholipid markers of wild and farmed bighead carp and their applications |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080045734A1 (en) * | 2006-01-13 | 2008-02-21 | Dow Agrosciences Llc | 6-(poly-substituted aryl)-4-aminopicolinates and their use as herbicides |
| CN103656505A (en) * | 2013-12-11 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Fish saprolegnia resisting traditional Chinese medicine extractive water aqua and preparation method thereof |
| CN106018589A (en) * | 2016-05-13 | 2016-10-12 | 东北农业大学 | Detection method for residual quantity of phoxim in animal tissue |
| CN106841589A (en) * | 2016-12-31 | 2017-06-13 | 沈阳金诚科技有限公司 | A kind of ELISA detection kit and detection method for being applicable detection Determination of Phoxim Residues analysis |
| CN107607645A (en) * | 2017-09-13 | 2018-01-19 | 山东出入境检验检疫局检验检疫技术中心 | A method for simultaneous detection of multiple types of veterinary drug residues in fish meat |
| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
-
2019
- 2019-01-30 CN CN201910096527.1A patent/CN109813820B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080045734A1 (en) * | 2006-01-13 | 2008-02-21 | Dow Agrosciences Llc | 6-(poly-substituted aryl)-4-aminopicolinates and their use as herbicides |
| CN103656505A (en) * | 2013-12-11 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Fish saprolegnia resisting traditional Chinese medicine extractive water aqua and preparation method thereof |
| CN106018589A (en) * | 2016-05-13 | 2016-10-12 | 东北农业大学 | Detection method for residual quantity of phoxim in animal tissue |
| CN106841589A (en) * | 2016-12-31 | 2017-06-13 | 沈阳金诚科技有限公司 | A kind of ELISA detection kit and detection method for being applicable detection Determination of Phoxim Residues analysis |
| CN107607645A (en) * | 2017-09-13 | 2018-01-19 | 山东出入境检验检疫局检验检疫技术中心 | A method for simultaneous detection of multiple types of veterinary drug residues in fish meat |
| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
Non-Patent Citations (7)
| Title |
|---|
| JUNG HAN LEE 等: "Simultaneous determination of phoxim and its photo-transformation metabolite residues in eggs using liquid chromatography coupled with electrospray ionization tandem mass spectrometry", 《ANALYTICA CHIMICA ACTA》 * |
| 侯晓慧 等: "超高效液相色谱-飞行时间质谱法测定蔬菜中辛硫磷残留量", 《食品安全质量检测学报》 * |
| 刘荣飞 等: "高效液相色谱和液相色谱-电喷雾质谱联用法鉴定辛硫磷在鲫鱼肝微粒体中代谢产物及途径", 《分析化学》 * |
| 施耐德 等: "《现代液相色谱技术导论》", 31 July 2012, 人民卫生出版社 * |
| 朱雨田: "辛硫磷对鲫鱼体内CYP450酶系的影响及其体内代谢研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 王权 等: "《淡水鱼高产新技术操作手册》", 31 July 2015, 金盾出版社 * |
| 边照阳 等: "《QuEChERS技术及应用》", 31 December 2017, 中国轻工业出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376296A (en) * | 2019-06-06 | 2019-10-25 | 张云 | Pacify the quantitative detecting method that sieve replaces Buddhist nun's medicament residue in blood |
| CN113624869A (en) * | 2021-07-30 | 2021-11-09 | 上海市疾病预防控制中心 | Method for rapidly determining organic matter conversion substance in real sample with high flux and high accuracy |
| CN114002345A (en) * | 2021-10-12 | 2022-02-01 | 中国水产科学研究院黑龙江水产研究所 | Fish gill metabonomics sample pretreatment optimization method |
| CN114002345B (en) * | 2021-10-12 | 2024-05-03 | 中国水产科学研究院黑龙江水产研究所 | Pretreatment optimization method for fish gill metabonomics sample |
| CN118800338A (en) * | 2024-06-17 | 2024-10-18 | 中国水产科学研究院长江水产研究所 | Glycerophospholipid markers of wild and farmed bighead carp and their applications |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109813820B (en) | 2022-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109813820A (en) | A method of identification phoxim metabolite in fresh-water fishes different tissues | |
| Matin et al. | Pharmacokinetics of tolbutamide: prediction by concentration in saliva | |
| Watanabe et al. | Fate of [14C] vinyl chloride after single oral administration in rats | |
| JP2005539069A (en) | A biochemical method for measuring the metabolic adaptability of a tissue or organism | |
| Gabrielsson et al. | Analysis of methadone disposition in the pregnant rat by means of a physiological flow model | |
| Brown et al. | Metabolism of Chloroform—I. The Metabolism of [14C] Chloroform by Different Species | |
| Kalivas et al. | Cross-sensitization between foot shock stress and enkephalin-induced motor activity | |
| Tokuma et al. | Absorption, distribution and excretion of nilvadipine, a new dihydropyridine calcium antagonist, in rats and dogs | |
| Afzal et al. | Moxidectin: absorption, tissue distribution, excretion, and biotransformation of 14C-labeled moxidectin in sheep | |
| Stern et al. | Elimination kinetics of sevoflurane and halothane from blood, brain, and adipose tissue in the rat | |
| Feng et al. | Pharmacokinetics, tissue distribution and excretion of coumarin components from Psoralea corylifolia L. in rats | |
| Sepehr et al. | Pharmacokinetics of the estrogen receptor subtype-selective ligands, PPT and DPN: quantification using UPLC-ES/MS/MS | |
| Adér et al. | On the origin of vanillylmandelic acid and 3‐methoxy‐4‐hydroxy‐phenylglycol in the rat brain | |
| Gasier et al. | A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus | |
| Ye et al. | Identification of the major metabolites of (R)‐salbutamol in human urine, plasma and feces using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry | |
| Wikell et al. | Pharmacokinetic and pharmacodynamic responses to chronic administration of the selective serotonin reuptake inhibitor citalopram in rats | |
| Kabir et al. | Metabolism of octacosanol in liver and muscle of rat | |
| CN109709225A (en) | A method for qualitative and quantitative analysis of militarine and its metabolites in plasma | |
| Bose et al. | Fate of pentachlorophenol in the blue crab, Callinectes sapidus | |
| Sisenwine et al. | The conversion of d-norgestrel-3-oxime-17-acetate to d-norgestrel in female rhesus monkeys | |
| Jaworski et al. | The metabolism of chlorpromazine N-oxide in the rat | |
| Stepanov et al. | Development of a series of novel carbon‐11 labeled PDE10A inhibitors | |
| Rickert et al. | Determination of the tissue distribution and excretion by accelerator mass spectrometry of the nonadecapeptide 14C-Moli1901 in beagle dogs after intratracheal instillation | |
| Christensen | Determination of dextropropoxyphene and norpropoxyphene in autopsy material | |
| HAGINAKA et al. | Chromatographic analysis and pharmacokinetic investigation of cephaloglycin and its metabolites in man |
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 |