CN115494190A - Method for simultaneously determining residual quantity of triadimefon and triadimenol enantiomer serving as metabolite of triadimefon in fruit and vegetable paste - Google Patents
Method for simultaneously determining residual quantity of triadimefon and triadimenol enantiomer serving as metabolite of triadimefon in fruit and vegetable paste Download PDFInfo
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- CN115494190A CN115494190A CN202211162647.5A CN202211162647A CN115494190A CN 115494190 A CN115494190 A CN 115494190A CN 202211162647 A CN202211162647 A CN 202211162647A CN 115494190 A CN115494190 A CN 115494190A
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- triadimenol
- triadimefon
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- BAZVSMNPJJMILC-UHFFFAOYSA-N triadimenol Chemical class C1=NC=NN1C(C(O)C(C)(C)C)OC1=CC=C(Cl)C=C1 BAZVSMNPJJMILC-UHFFFAOYSA-N 0.000 title claims abstract description 57
- WURBVZBTWMNKQT-UHFFFAOYSA-N 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)butan-2-one Chemical compound C1=NC=NN1C(C(=O)C(C)(C)C)OC1=CC=C(Cl)C=C1 WURBVZBTWMNKQT-UHFFFAOYSA-N 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 51
- 235000012055 fruits and vegetables Nutrition 0.000 title claims abstract description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 90
- 238000011084 recovery Methods 0.000 claims abstract description 18
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- 102000007641 Trefoil Factors Human genes 0.000 claims abstract description 13
- 235000015724 Trifolium pratense Nutrition 0.000 claims abstract description 13
- 239000002207 metabolite Substances 0.000 claims abstract description 13
- 101150084500 cel2 gene Proteins 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 9
- 238000010828 elution Methods 0.000 claims abstract description 8
- 238000010812 external standard method Methods 0.000 claims abstract description 5
- 239000005846 Triadimenol Substances 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 34
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 33
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 24
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 20
- 239000011550 stock solution Substances 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 16
- FFSJPOPLSWBGQY-UHFFFAOYSA-N triazol-4-one Chemical compound O=C1C=NN=N1 FFSJPOPLSWBGQY-UHFFFAOYSA-N 0.000 claims description 14
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
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- 239000012224 working solution Substances 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- CLUPOLFGIGLMIQ-UHFFFAOYSA-N heptane;propan-2-ol Chemical compound CC(C)O.CCCCCCC CLUPOLFGIGLMIQ-UHFFFAOYSA-N 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000012086 standard solution Substances 0.000 claims description 7
- 239000012496 blank sample Substances 0.000 claims description 6
- 235000013399 edible fruits Nutrition 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 5
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
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- 239000007788 liquid Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- GHQPBDDZGPAVJP-UHFFFAOYSA-N azanium;methanol;hydroxide Chemical compound N.O.OC GHQPBDDZGPAVJP-UHFFFAOYSA-N 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims 2
- 238000004364 calculation method Methods 0.000 claims 1
- 240000008790 Musa x paradisiaca Species 0.000 abstract description 9
- 235000018290 Musa x paradisiaca Nutrition 0.000 abstract description 9
- 244000099147 Ananas comosus Species 0.000 abstract description 7
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- 238000011002 quantification Methods 0.000 abstract description 6
- CBHOOMGKXCMKIR-UHFFFAOYSA-N azane;methanol Chemical compound N.OC CBHOOMGKXCMKIR-UHFFFAOYSA-N 0.000 abstract description 5
- 239000000447 pesticide residue Substances 0.000 abstract description 3
- 230000001502 supplementing effect Effects 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
- 238000000926 separation method Methods 0.000 description 24
- 239000000575 pesticide Substances 0.000 description 14
- 239000012071 phase Substances 0.000 description 14
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- 150000003852 triazoles Chemical class 0.000 description 8
- 239000006184 cosolvent Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 6
- SBTVLCPCSXMWIQ-UHFFFAOYSA-N (3,5-dimethylphenyl) carbamate Chemical compound CC1=CC(C)=CC(OC(N)=O)=C1 SBTVLCPCSXMWIQ-UHFFFAOYSA-N 0.000 description 5
- 102100033770 Alpha-amylase 1C Human genes 0.000 description 5
- 101000779871 Homo sapiens Alpha-amylase 1A Proteins 0.000 description 5
- 101000779870 Homo sapiens Alpha-amylase 1B Proteins 0.000 description 5
- 101000779869 Homo sapiens Alpha-amylase 1C Proteins 0.000 description 5
- 101000652482 Homo sapiens TBC1 domain family member 8 Proteins 0.000 description 5
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- 238000004458 analytical method Methods 0.000 description 5
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- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 239000003899 bactericide agent Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- FKHIFSZMMVMEQY-UHFFFAOYSA-N talc Chemical compound [Mg+2].[O-][Si]([O-])=O FKHIFSZMMVMEQY-UHFFFAOYSA-N 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
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- 230000000295 complement effect Effects 0.000 description 3
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- 235000019253 formic acid Nutrition 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 238000005557 chiral recognition Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- XQUXKZZNEFRCAW-UHFFFAOYSA-N fenpropathrin Chemical compound CC1(C)C(C)(C)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 XQUXKZZNEFRCAW-UHFFFAOYSA-N 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- -1 (-) -R-triazolone Chemical class 0.000 description 1
- PJTGFNAHESRDIO-UHFFFAOYSA-N (3-chloro-4-methylphenyl)carbamic acid Chemical compound CC1=CC=C(NC(O)=O)C=C1Cl PJTGFNAHESRDIO-UHFFFAOYSA-N 0.000 description 1
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 1
- 241001251200 Agelas Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920002160 Celluloid Polymers 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 229960001518 levocarnitine Drugs 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
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- 239000002728 pyrethroid Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
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- 230000001988 toxicity Effects 0.000 description 1
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to the technical field of pesticide residue detection, in particular to a method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer serving as a metabolite of triadimefon in fruit and vegetable paste. The invention establishes a method for separating and measuring triadimefon and the residue of the metabolite enantiomer thereof in the fruit and vegetable puree (banana puree, pineapple puree and grape puree) for supplementing the main infants by ultra-high performance combined phase chromatography. Samples were extracted with acetonitrile by NH 2 Purifying the sample with column, separating with Acquity Trefoil CEL2 chiral chromatographic column, and purifying with supercritical CO 2 Gradient elution with-0.5% ammonia methanol as mobile phase, and quantification by external standard method. The limit of quantification is 0.05mg/kg, the range of the recovery rate of the added standard is 80.1-106%, and the RSD is 3.3-7.6%. By adopting the method, the residues of triadimefon and triadimenol enantiomer, a metabolite thereof, in 30 fruit and vegetable paste samples purchased from the market are measured, and the detected amount is 0.12 mg/kg-0.54 mg/kg.
Description
Technical Field
The invention relates to the technical field of pesticide residue detection, in particular to a method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer serving as a metabolite of triadimefon in fruit and vegetable paste.
Background
Chirality is a ubiquitous phenomenon in nature, and with the development of science, the demand for optically pure substances is increasing, and the separation of chiral enantiomers increasingly draws attention of people. In traditional chiral pesticide research, enantiomers are not distinguished, and all enantiomers are treated as the same substance 1 GB 2763-2021 "maximum limit of pesticide residue in food safety national standard food" only stipulates the limit of pesticide racemate residue and the use standard, and does not precisely stipulate the effective and low-toxicity enantiomer and the non-effective and high-toxicity enantiomer residue limit of each pesticide. However, each enantiomer of chiral pesticide has great difference in biological activity, environmental behavior and ecological toxicology 2-4 . For example, fenpropathrin has 1 chiral center, wherein the insecticidal activity of the (+) -fenpropathrin is higher than that of (-) -fenpropathrin, and the degradation speed is faster 5 . Therefore, the establishment of the separation and analysis method of chiral pesticide enantiomers has great significance for the evaluation and research of the safety of chiral pesticides in the toxicity, activity and ecological environment of the enantiomer level and the development of more optically pure monomers.
Triazole pesticide as one kind of efficient bactericide can protect fruit, soybean, grain and other crops from bacteria damage and has wide application in agricultural production 6 . The existing detection method of triazole bactericide mainly comprises high performance liquid chromatography 7-9 High performance liquid chromatography-mass spectrometry combined method 10,11 And the like, mainly aiming at the racemate of the triazole bactericide.The triazole bactericide contains 1 or 2 chiral centers and exists in 2 or 4 enantiomers 12 . Of the 4 enantiomers of triadimenol, the enantiomer with the structure of 1R and 2S and 1S and 2R has higher bactericidal activity 13 . The structural formulas of the triazolone (A) and the triadimenol (B) are as follows:
an important direction for the research of chiral pesticides is to research and develop and use high-activity high-optical purity pesticide enantiomer 13 . The current chiral separation and analysis method of triazole pesticides is mainly High Performance Liquid Chromatography (HPLC) 14-16 And liquid chromatography-tandem mass spectrometry (LC-MS/MS) 17-19 However, the HPLC separation degree is good, but the separation time is long, and the consumption of organic reagents is large; LC-MS/MS has high accuracy, but the instrument is expensive and the cost is high. Therefore, the development of a rapid and efficient detection method has wide application prospect. Ultra-high performance phase-locked chromatography (UPC) 2 ) The supercritical carbon dioxide is taken as the main mobile phase, and the method has the advantages of higher mass transfer rate and higher separation efficiency compared with the traditional organic mobile phase 20 . The study shows that UPC 2 The technology is more suitable for analyzing isomerides and structural analogues which are difficult to separate by the traditional liquid chromatography, and is successfully applied to triazole pesticides 1,21 Pyrethroid pesticide 22 Stimulant veterinary drug 23,24 Antibiotic veterinary drug 25 Levo-carnitine weight-reducing medicine 26,27 And (3) separating and measuring an isochiral compound.
Disclosure of Invention
In order to solve the problems existing in the separation of triazole pesticide enantiomers by the conventional liquid chromatography, the invention aims to provide a method for simultaneously determining the residual quantity of triazolone and triadimenol enantiomer which is a metabolite of the triazolone in fruit and vegetable puree.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer, which are metabolites of triadimefon and triadimenol enantiomer, in fruit and vegetable paste, wherein the triadimefon and triadimenol enantiomer thereof are (-) - (R) -triadimefon, (+) - (S) -triadimenol, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol and (+) - (R, R) -triadimenol and (-) - (S, S) -triadimenol, respectively; the method comprises the following steps:
1) Sample extraction
Weighing a sample in a plastic centrifuge tube with a plug, adding acetonitrile, homogenizing and extracting, adding sodium chloride, uniformly mixing by vortex, centrifuging, and transferring supernatant into a concentration bottle; adding acetonitrile into the lower-layer residue, repeatedly extracting for 1 time, combining the two acetonitrile extracting solutions, concentrating to near dryness by using a rotary evaporator, adding acetonitrile: toluene (3;
2) Purification
And transferring the solution to be purified to an activated NH2 column, immediately collecting all effluent after loading, and then adding acetonitrile: eluting with toluene (3, 1, V/V) for 2 times, collecting eluate, mixing eluates, drying with nitrogen in water bath at 40 deg.C until the eluate is nearly dry, adding n-heptane-isopropanol mixed solution (8, 2, V/V), dissolving residue by vortex, filtering the constant volume solution with filter membrane, and measuring;
3) Quantitative detection of ultra-high performance phase-combination chromatography by external standard method
And (3) chromatographic column: acquity Trefoil CEL2; mobile phase: a is CO 2 B is 0.5 percent ammonia water methanol by volume percentage concentration;
gradient elution procedure:
and (3) system backpressure: 13.8MPa; the detection wavelength is 220nm; the flow rate is 1.0mL/min; the column temperature is 35 ℃; the sample size was 5. Mu.L.
As a specific embodiment, the specific method in step 1) is as follows: weighing 10g of sample, accurately obtaining 0.01g of sample, adding 20mL of acetonitrile into a 50mL plastic centrifuge tube with a plug, homogenizing and extracting, adding 3.0g of sodium chloride, uniformly mixing by vortex, centrifuging for 5min at 4 000r/min, and transferring supernatant into a concentration bottle; adding 20mL of acetonitrile into the lower-layer residue, repeatedly extracting for 1 time, combining two acetonitrile extracting solutions, concentrating to be nearly dry by using a rotary evaporator, adding 5mL of acetonitrile: toluene (3.
As a specific embodiment, the specific method in step 2) is as follows: transferring the liquid to be purified to activated NH 2 In the column, after loading, the entire effluent was collected immediately, followed by 5ml acetonitrile: toluene (3.
As a specific embodiment, the racemate standard stock solution is prepared as follows: accurately weighing 0.01g of standard triadimefon and triadimenol racemate, accurately measuring the standard content to 0.1mg, accurately metering the volume to a 10mL volumetric flask by using isopropanol, and preparing 1.0g/L standard stock solution of the racemate.
As a specific embodiment, the standard intermediate solution of the triadimefon and triadimenol racemate is prepared as follows: a quantity of the racemate standard stock solution was accurately aspirated, diluted with n-heptane/isopropanol (8,2,v/v) to 100.0mg/L of a standard intermediate solution.
As a specific embodiment, the standard stock solution of enantiomers is prepared as follows: respectively and accurately weighing 0.01g of 6 enantiomer standard substances to be accurate to 0.1mg, dissolving the standard substances by using isopropanol, and fixing the volume to a 10mL volumetric flask to prepare 1.0g/L enantiomer standard stock solution.
As a specific embodiment, the mixed standard working solution of 6 enantiomers: a certain amount of enantiomer standard stock solutions are accurately transferred respectively and are diluted to series of standard working solutions of 0.5, 1.0, 2.0, 4.0, 10.0 and 50.0mg/L by n-heptane-isopropanol (8.
Preferably, the method further comprises the steps of: measuring 0.5, 1.0, 2.0, 4.0, 10.0 and 50.0mg/L triadimefon and triadimenol enantiomer series standard working solution according to optimized chromatographic conditions, drawing a standard curve by taking the corresponding peak area as a vertical coordinate Y and the mass concentration of the standard solution as a horizontal coordinate X, and performing linear regression; adding a standard substance into a fruit paste blank sample which does not contain triazolone and triadimenol, determining according to an optimized method, and calculating the quantitative Limit (LOQ) of the method by using a signal-to-noise ratio (S/N) =10 to obtain the LOQ of each of 6 enantiomers, wherein the LOQ is 0.05mg/kg; in the linear range of 0.5-50 mg/L, the linearity is good, and the correlation coefficient (r) 2 ) Are all greater than 0.9993.
Preferably, the method further comprises the steps of: measuring the addition recovery rate and the precision of the method by adopting a method of adding standard solution into a fruit and vegetable puree blank sample without triazolone and triadimenol respectively, wherein the addition water of 6 enantiomers such as (-) -R-triazolone, (+) - (S) -triazolone, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol, (+) - (R, R) -triadimenol and (-) - (S, S) -triadimenol is 0.05,0.2,3mg/kg on average, measuring for 6 times in parallel, and calculating the addition recovery rate and RSD; the recovery rate of 6 target compounds ranged from 80.1% to 106%, and RSD (n = 6) ranged from 3.3% to 7.6%.
Due to the adoption of the technical scheme, the invention establishes a method for separating and measuring the residue of the triadimefon and the metabolite enantiomer thereof in the fruit and vegetable puree (banana puree, pineapple puree and grape puree) as the complementary food for the infants through ultra-high performance combined phase chromatography. Samples were extracted with acetonitrile by NH 2 Purifying the sample with column, separating with Acquity Trefoil CEL2 chiral chromatographic column, and purifying with supercritical CO 2 Gradient elution with-0.5% ammonia methanol as mobile phase, and quantification by external standard method. The limit of quantification is 0.05mg/kg, the range of recovery rate by adding standard is 80.1-106%, and the RSD is 3.3-7.6%. By adopting the method, the residues of triadimefon and triadimenol enantiomer, a metabolite thereof, in 30 fruit and vegetable paste samples purchased from the market are measured, and the detected amount is 0.12 mg/kg-0.54 mg/kg.
Drawings
FIG. 1 shows the effect of different chromatographic columns on the separation of triadimefon and triadimenol enantiomers; wherein: (A) AD-3; (B) AMY1; (C) CEL1; and (D) CEL2.
FIG. 2 shows the effect of different cosolvents on the separation of triadimefon and triadimenol enantiomers; wherein: (a) formic acid-methanol solution (0.5, 99.5, V/V); (B) methanol; (C) an aqueous ammonia-methanol solution (0.5.
FIG. 3 shows the effect of different column temperatures on the separation of triadimefon and triadimenol enantiomers; wherein: (A) 31 ℃; (B) 35 ℃; (C) 40 ℃.
FIG. 4 shows the effect of different solid phase extraction columns on the purification effect and recovery of triadimefon and triadimenol enantiomers; wherein: (A) aluminum-N; (B) Florisil; (C) HLB; (D) NH2.
FIG. 5 is a chromatogram obtained by separating triadimefon and triadimenol racemates; wherein: peak 1 (-) - (R) -triazolone; peak 2 (+) - (S) -triazolone; peak 3 (-) - (S, R) -triadimenol; peak 4 (+) - (R, S) -triadimenol; peak 5 (+) - (R, R) -triadimenol; peak 6, (-) - (S, S) -triadimenol.
FIG. 6 is a chromatogram of a positive banana puree sample; wherein: peak 1 (-) - (R) -triazolone; peak 2 (+) - (S) -triazolone; peak 3 (-) - (S, R) -triadimenol; peak 4 (+) - (R, S) -triadimenol; peak 5 (+) - (R, R) -triadimenol; peak 6, (-) - (S, S) -triadimenol.
Detailed Description
1 experimental part
1.1 instruments, materials and reagents
Ultra-high performance phase-compatible chromatographs (Vortex, USA, equipped with a PDA detector); bench centrifuge (Thermo corporation, usa); n-1210BV rotary evaporator (Tokyo, japan, physico-chemical company); JJ500 electronic balance (doujie balance, usa); AE260 electronic balance (Mettler, switzerland); WH-861 vortex mixer (Wahlida Experimental facilities, inc., taicang); synergy185 ultrapure water instrument (Millipore, usa); nitrogen blowing apparatus (tokyo physical & chemical corporation, japan); microfiltration membrane (0.22 μm, organic phase); chromatography column CHIRALPAK AD-3 (xylonite drug chiral technology (shanghai) ltd); chromatography columns Acquity Trefoil AMY1, CEL2 (Waters corporation, usa).
Isopropanol, toluene, methanol, ethanol, n-heptane, acetonitrile, formic acid (chromatographically pure, merck, germany); sodium chloride (analytical pure, shanghai test tetrahexygvin chemical ltd); a polymer solid phase extraction column HLB column (Oasis, 200mg,6 mL) (activated by 6mL methanol and 6mL water in turn before use); NH (NH) 2 Column (Agela, 500mg, 6mL); florisil column (CNW, 1g, 6ml); column Alumina-N (CNW, 1g, 3mL); the water is ultrapure water; the reagents used in other experiments were analytically pure except for the special instructions.
A chromatographic column: CHIRALPAK AD-3 (150 mm. Times.3.0mm, 3 μm, amylose-tris (3, 5-dimethylphenylcarbamate) as filler, acquity Trefoil AMY1 (150 mm. Times.3.0mm, 2.5 μm, amylose-tris (3, 5-dimethylphenylcarbamate) as filler), acquity Trefoil CEL1 (150 mm. Times.3.0mm, 2.5 μm, cellulose-tris (3, 5-dimethylphenylcarbamate)) and Acquity Trefoil CEL2 (150 mm. Times.3.0mm, 2.5 μm, cellulose-tris (3-chloro-4-methylphenylcarbamate) as filler)
And (3) standard substance: triazolone racemate (CAS No.: 43121-43-3, purity. Gtoresistor GmbH, germany, dr Ehrenstetorfer), triadimenol racemate (CAS No.: 55219-65-3, purity. Gtoresistor GmbH, germany, dr Ehrentorfer). Triazolone enantiomer: (-) - (R) -triazolone, (+) - (S) -triazolone, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol, (+) - (R, R) -triadimenol, (-) - (S, S) -triadimenol (purity not less than 95%, manufactured by Shanghai Lianlu Biotechnology Co., ltd.).
1.2 preparation of Standard stock solution and working solution
1.2.1 Standard stock solutions of the racemate
Accurately weighing 0.01g (accurate to 0.1 mg) of standard triadimefon and triadimenol racemate, and accurately metering the volume to a 10mL volumetric flask by using isopropanol to prepare 1.0g/L standard stock solution of the racemate.
Standard intermediate solutions of triadimefon and triadimenol racemates: an amount of the racemate standard stock solution was accurately aspirated, and diluted with n-heptane/isopropanol (8,v/v) to 100.0mg/L of a standard intermediate solution.
1.2.2 enantiomeric Standard stock solutions
Respectively and accurately weighing 0.01g (accurate to 0.1 mg) of 6 enantiomer standard substances, dissolving the standard substances by using isopropanol, and fixing the volume to a 10mL volumetric flask to prepare 1.0g/L enantiomer standard stock solution.
Mixed standard working solutions of 6 enantiomers: a certain amount of enantiomer standard stock solutions are accurately transferred and gradually diluted to series of standard working solutions of 0.5, 1.0, 2.0, 4.0, 10.0 and 50.0mg/L by using n-heptane-isopropanol (8.
1.3 sample pretreatment
1.3.1 sample extraction
Weighing 10g (accurate to 0.01 g) of sample in a 50mL plastic centrifuge tube with a plug, adding 20mL acetonitrile, homogenizing and extracting, adding 3.0g sodium chloride, mixing uniformly by vortex, centrifuging for 5min at 4 000r/min, and transferring the supernatant to a concentration bottle. Adding 20mL of acetonitrile into the lower-layer residue, repeatedly extracting for 1 time, combining two acetonitrile extracting solutions, concentrating to be nearly dry by using a rotary evaporator, adding 5mL of acetonitrile: toluene (3, V/V) was dissolved, and purified.
1.3.2 purification
Transferring the solution to be purified to activated NH 2 In the column, after loading, the whole effluent was collected and then treated with 5ml of acetonitrile: and (2) eluting with toluene (3, 1,v/V) for 2 times, collecting eluent of the receiving solution, combining, drying the eluent in water bath at 40 ℃ with nitrogen till the eluent is nearly dry, adding 1mL of mixed solution of n-heptane-isopropanol (8, 2,v/V), dissolving the residue in a vortex manner for 1min, and filtering the volume-fixed solution through a 0.22 mu m filter membrane to be tested.
1.4 chromatographic conditions
A chromatographic column: acquity Trefoil CEL2 (150 mm. Times.3.0 mm,2.5 μm); mobile phase: a is CO 2 B is 0.5% ammonia methanol; gradient elution procedure: 0-0.8 min (5% B), 0.8-1.0 min (5% -8% B1.0-1.4 min (8% B), 1.4-1.8 min (8% -5% B), 1.8-2.1 min (5% B), 2.1-2.3 min (5% B-15% B), 2.3-2.4 min (15% B), 2.4-2.5 min (15% B), 2.5-2.6 min (5% B), 2.6-2.8 min (5% B), 2.8-3.5 min (5% B-15% B), 2.8-3.5 min (15% B), 3.5-4 min (15% B-5B), 4-5 min (5% B), 4-5 min (5B). And (3) system backpressure: 13.8MPa; the detection wavelength is 220nm; the flow rate is 1.0mL/min; the column temperature is 35 ℃; the sample size was 5. Mu.L.
2 results and discussion
2.1 optimization of the chromatography column
The chiral stationary phase based on amylose-tri (3, 5-dimethylphenyl carbamate) and cellulose-tri (3, 5-dimethylphenyl carbamate) is two types of chromatographic separation stationary phases which are most widely applied, has good chiral recognition capability and resolution capability, and is complementary to each other in the aspect of chiral recognition capability 28 . In the experiment, 4 chiral separation chromatographic columns of CHIRALPAK AD-3, acquity Trefoil AMY1, acquity Trefoil CEL1 and Acquity Trefoil CEL2 are selected to investigate the resolution effect of 6 enantiomers. The result shows that when the AD-3, AMY1 and CEL1 chiral chromatographic columns are used for separation, 6 enantiomers of triazolone and triadimenol do not completely peak, and the chromatographic peaks of partial enantiomers overlap; when CEL2 chiral chromatographic column is adopted for separation, chromatographic peaks of 6 enantiomers of triazolone and triadimenol can realize certain separation, wherein 4 peaks do not realize complete baseline separation, and further separation can be realized by optimizing chromatographic conditions (figure 1). Therefore, the experiment selects a CEL2 chiral chromatographic column to separate the target substances.
2.2 selection of Co-solvent in the Mobile phase
Using UPC 2 While in the supercritical state CO of the bulk mobile phase 2 A small amount of cosolvents such as methanol and isopropanol are added, so that the elution and dissolution capacities of the mobile phase can be effectively adjusted, and the separation effect and analysis speed of the target substance can be accurately regulated and controlled. In addition, the chromatographic peak shape can be effectively improved by adjusting the pH value of the mobile phase. The viscosity of the isopropanol is large, and overpressure alarm is easy to occur in the instrument in the test process. Therefore, the experiment examines the influence of different cosolvents such as methanol solution containing 0.5% formic acid, methanol solution containing 0.5% ammonia water and the like on the separation effect. The result shows that when the methanol solution containing 0.5 percent of formic acid is used as the cosolvent, the base line of the chromatogram is unstable, and no obvious chromatographic peak is seen; when methanol and a methanol solution containing 0.5% ammonia water were used, complete separation of 6 target compounds was achieved, and the chromatographic peak shape was good. In comparison, the separation of the last 4 chromatographic peaks was better with 0.5% aqueous ammonia in methanol as co-solvent (see FIG. 2). Therefore, the test selects a methanol solution containing 0.5% ammonia as the cosolvent.
2.3 selection of column temperature
UPC 2 Supercritical state CO in the system 2 The density of the bulk mobile phase varies with the temperature of the column. When the temperature of the chromatographic column is increased in the test, the density of the mobile phase becomes lower, the elution capacity is gradually reduced, and the retention time of the target compound is prolonged. The experiments examined the effect of different column temperatures (31, 35, 40 ℃) on the separation of the target compounds, taking into account the maximum recommended temperature of the column (40 ℃) and the temperature required to maintain the system in a supercritical state (> 31 ℃). The results show that the chromatographic peaks of 6 target compounds are more symmetrical and sharp in peak shape and better in resolution at 35 ℃ than at 31 ℃ and 40 ℃ (see fig. 3). Therefore, 35 ℃ was chosen as column temperature for the experiment.
2.4 optimization of purification conditions
Experiments compare aluminum-N 29 、Florisil 30 、HLB 31 、NH 2 32 And the purification effect of different types of solid phase extraction columns on the fruit paste sample extraction solution is obtained. Adding triadimefon and triadimenol enantiomer standard solutions into a negative fruit paste sample respectively, carrying out homogeneous extraction for 2 times by using acetonitrile, and purifying by adopting 4 different solid phase extraction columns after the extract is concentrated, dried and redissolved. The experimental results show that NH is adopted 2 The average recovery of the target compound was 92.6% during column purging. When the purification is carried out by using 3 solid phase extraction columns of aluminum-N, florisil and HLB, the recovery rate of the target compound is low, and the average recovery rates are respectively 28.6%, 15.9% and 18.2% (see figure 4). Thus, NH was selected experimentally 2 The solid phase extraction column is used as a purification column.
2.5 methodological considerations
2.5.1 Linear Range and quantitative limits
Selecting 0.5, 1.0, 2.0, 4.0, 10.0 and 50.0mg/L triadimefon and triadimenol enantiomer series standard working solutions in section 1.2.2, measuring according to optimized chromatographic conditions, drawing a standard curve by taking the corresponding peak area as an ordinate (Y) and the mass concentration of the standard solution as an abscissa (X), and performing linear regression. By adding standard substances to fruit paste blank samples without triazolone and triadimenolThe LOQ of each of the 6 enantiomers was 0.05mg/kg, as measured by the optimized method, and the quantitative Limit (LOQ) was calculated by the signal-to-noise ratio (S/N) = 10. In the linear range of 0.5-50 mg/L, the linearity is good, and the correlation coefficient (r) 2 ) Are all greater than 0.9993.
2.5.2 recovery and precision
The addition recovery rate and the precision of the method were measured by adding a standard solution to a blank sample of fruit and vegetable puree containing no triazolone and triadimenol, respectively, and the average of the added water for 6 enantiomers such as (-) - - (R) -triazolone, (+) - (S) -triazolone, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol, (+) - (R, R) -triadimenol and (-) - (S, S) -triadimenol was 0.05,0.2,3mg/kg, and the results are shown in Table 1 after measuring 6 times in parallel. The recovery rate of 6 target compounds ranged from 80.1% to 106%, and RSD (n = 6) ranged from 3.3% to 7.6%. The recovery rate and precision are in accordance with SANTE/12682/2019 33 The method can meet the analysis requirements of banana puree, pineapple puree and grape puree samples, and can be used for detection of daily analysis.
TABLE 1 spiked recovery and relative standard deviation of triadimefon and its metabolite triadimenol enantiomers in banana puree, pineapple puree, grape puree samples (n = 6)
2.6 application of the method
2.6.1 resolution of racemic Standard
The purchased triadimefon and triadimenol racemate standards are resolved and tested by the method established in the text. As shown in figure 5, the separation effect of 6 enantiomers is good, effective resolution is realized within 4.5min, the separation degrees are respectively 1.6, 7.2, 2.5, 1.5 and 2.4, and the requirement that R is more than or equal to 1.5 for complete separation is met 34 . According to the retention time sequence of chromatographic peaks, sequentially: (-) - (R) -triazolone, (+) - (S) -triazolone, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol, (+) - (R, R) -triadimenol, (-) - (S, S) -triadimenol). According to the drawn standard curve, adopting external calibrationThe content of 2 triadimefon enantiomers in a standard intermediate solution of triadimefon racemate of section 1.2.1, 20.0mg/L and the content of 4 triadimefon enantiomers in a standard intermediate solution of triadimefon racemate of section 50.0mg/L were calculated by a quantitative method, wherein the content of (-) - (R) -triadimefon was 8.9mg/L, the content of (+) - - (S) -triadimefon was 9.3mg/L, the content of (-) -S, R) -triadimefon, (+) - (R, S) -triadimenol, (+) - (R, R) -triadimenol and (-) -S, S) -triadimenol were 19.5,20.8,4.6 and 4.3mg/L, respectively. Content ratio of enantiomer in triadimefon and triadimenol racemate 1,19,35 The proportions of the enantiomers in the racemates of the industrial triadimefon and triadimenol are reported to be substantially the same.
2.6.2 testing of actual samples
In order to examine the effectiveness and the practicability of the method, the established method is applied to detect triadimefon and triadimenol enantiomer serving as a metabolite thereof in 30 randomly-extracted samples of banana puree, pineapple puree and grape puree (10 parts of banana puree, 14 parts of pineapple puree and 6 parts of grape puree). The results showed that 1 sample of banana puree contained 0.20mg/kg, (+) - (S) -triazolone, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol, (+) - (R, R) -triadimenol and (-) - (S, S) -triadimenol, respectively, 0.23mg/kg, 0.54mg/kg, 0.51mg/kg, 0.13mg/kg and 0.12mg/kg of triadimenol, as detected as enantiomeric components of triadimenol and triadimenol (FIG. 6).
3 conclusion
The method for separating and measuring the residue of the triadimefon and the metabolite enantiomer thereof in the fruit and vegetable puree (banana puree, pineapple puree and grape puree) as a complementary food for the main infants by the ultra-high performance synthetic phase chromatography is established. Samples were extracted with acetonitrile by NH 2 Purifying the sample with column, separating with Acquity Trefoil CEL2 chiral chromatographic column, and purifying with supercritical CO 2 Gradient elution with-0.5% ammonia methanol as mobile phase, and quantification by external standard method. The limit of quantification is 0.05mg/kg, the range of the recovery rate of the added standard is 80.1-106%, and the RSD is 3.3-7.6%. By adopting the method, the residues of triadimefon and triadimenol enantiomer, a metabolite thereof, in 30 fruit and vegetable paste samples purchased from the market are measured, and the detected amount is 0.12 mg/kg-0.54 mg/kg.
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Claims (9)
1. A method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer, which are metabolites of triadimefon and triadimenol enantiomer, in fruit and vegetable paste, wherein the triadimefon and triadimenol enantiomer thereof are (-) - (R) -triadimefon, (+) - (S) -triadimenol, (-) - (S, R) -triadimenol, (+) - (R, S) -triadimenol and (+) - (R, R) -triadimenol and (-) - (S, S) -triadimenol, respectively; the method is characterized by comprising the following steps:
1) Sample extraction
Weighing a sample in a plastic centrifuge tube with a plug, adding acetonitrile, homogenizing and extracting, adding sodium chloride, uniformly mixing by vortex, centrifuging, and transferring supernatant into a concentration bottle; adding acetonitrile into the lower-layer residue, repeatedly extracting for 1 time, combining the two acetonitrile extracting solutions, concentrating to near dryness by using a rotary evaporator, adding acetonitrile: toluene (3;
2) Purification
Transferring the solution to be purified to an activated NH2 column, loading, collecting all effluent liquid, and adding acetonitrile: eluting with toluene (3, 1,v/V) for 2 times, collecting eluent of the receiving solution, mixing, drying in water bath at 40 ℃ with nitrogen till the solution is nearly dry, adding an n-heptane-isopropanol mixed solution (8, 2,v/V), dissolving residues in a vortex manner, filtering the constant volume solution with a filter membrane, and measuring;
3) Quantitative detection of ultra-high performance phase-combination chromatography by external standard method
A chromatographic column: acquity Trefoil CEL2; mobile phase: a is CO 2 B is 0.5 percent ammonia water methanol by volume percentage concentration;
gradient elution procedure:
and (3) system backpressure: 13.8MPa; the detection wavelength is 220nm; the flow rate is 1.0mL/min; the column temperature is 35 ℃; and the sample size is 5 muL.
2. The method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer, a metabolite thereof, in fruit and vegetable puree according to claim 1, is characterized in that the specific method in step 1) is as follows: weighing 10g of sample, accurately obtaining the sample to 0.01g, adding 20mL of acetonitrile into a 50mL plastic centrifuge tube with a plug, homogenizing and extracting, adding 3.0g of sodium chloride, uniformly mixing in a vortex manner, centrifuging at 4 000r/min for 5min, and transferring supernatant into a concentration bottle; adding 20mL of acetonitrile into the lower-layer residue, repeatedly extracting for 1 time, combining two acetonitrile extracting solutions, concentrating to be nearly dry by using a rotary evaporator, adding 5mL of acetonitrile: toluene (3.
3. The method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer, a metabolite thereof, in fruit and vegetable puree according to claim 1, wherein the specific method in step 2) is as follows: transferring the liquid to be purified to activated NH 2 In the column, after loading, the whole effluent was collected and then treated with 5ml of acetonitrile: and (2) eluting with toluene (3, 1,v/V) for 2 times, collecting eluent of the receiving solution, combining, drying the eluent in water bath at 40 ℃ with nitrogen till the eluent is nearly dry, adding 1mL of mixed solution of n-heptane-isopropanol (8, 2,v/V), dissolving the residue in a vortex manner for 1min, and filtering the volume-fixed solution through a 0.22 mu m filter membrane to be tested.
4. The method for simultaneously determining the residual quantity of triadimefon and triadimenol enantiomer, a metabolite thereof, in fruit and vegetable puree according to claim 1, characterized in that the preparation method of the racemate standard stock solution is as follows: accurately weighing 0.01g of standard triadimefon and triadimenol racemate, accurately measuring the standard content to 0.1mg, accurately metering the volume to a 10mL volumetric flask by using isopropanol, and preparing 1.0g/L standard stock solution of the racemate.
5. The method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer, a metabolite of triadimenol, in fruit and vegetable puree according to claim 4, wherein the preparation method of the standard intermediate solution of triadimefon and triadimenol racemate is as follows: a quantity of the racemate standard stock solution was accurately aspirated, diluted with n-heptane/isopropanol (8, 2, v/v) to 100.0mg/L of standard intermediate solution.
6. The method for simultaneously determining the residual quantity of triadimefon and triadimenol enantiomer thereof in fruit and vegetable puree according to claim 1, characterized in that the enantiomer standard stock solution is prepared by the following steps: respectively and accurately weighing 0.01g of 6 enantiomer standard substances, accurately weighing the standard substances to 0.1mg, dissolving the standard substances by using isopropanol, and diluting the solution to a volume of 10mL in a volumetric flask to prepare 1.0g/L enantiomer standard stock solution.
7. The method for simultaneously determining the residual quantity of the triadimefon and the triadimenol enantiomer thereof in fruit and vegetable puree according to claim 6, characterized in that the mixed standard working solution of 6 enantiomers: a certain amount of the enantiomeric standard stock solutions were accurately removed, and purified with n-heptane-isopropanol (8, v /v) Diluting the solution to 0.5, 1.0, 2.0, 4.0, 10.0 and 50.0 mg/L.
8. The method for simultaneously determining the residual quantity of triadimefon and triadimenol enantiomer thereof in fruit and vegetable puree as claimed in claim 1, characterized in that the method further comprises the following steps: at a ratio of 0.5, 1.0, 2.0, 4.0,10.0 and 50.0mg/L triazolone and triadimenol enantiomer series standard working solutions, determining according to optimized chromatographic conditions, and taking corresponding peak areas as ordinateYMass concentration of the standard solution is on the abscissaXDrawing a standard curve and performing linear regression; adding a standard substance into a fruit paste blank sample which does not contain triazolone and triadimenol, and determining according to an optimized method by using a signal-to-noise ratio (S/N) =10 limit of quantitation (LOQ) of calculation method, LOQ for 6 enantiomers all 0.05mg/kg; in the linear range of 0.5-50 mg/L, the linearity is good, and the correlation coefficient is (r 2 ) Are all greater than 0.9993.
9. The method for simultaneously determining residual amounts of triadimefon and triadimenol enantiomer, a metabolite thereof, in fruit and vegetable puree according to claim 1, characterized in that the method further comprises the following steps: measuring the addition recovery rate and the precision of the method by adopting a method of adding a standard solution into a blank sample of the fruit and vegetable puree which does not contain triazolone and triadimenol respectively, wherein the average addition amount of water of 6 enantiomers such as (-) - - (R) -triazolone, (+) - (S) -triazolone, (-) - (S, R) -triadimenol, (+) - (R, S) -triazolol, (+) - (R, R) -triazolol and (-) - (S, S) -triazolol is 0.05,0.2,3mg/kg, and the like is measured for 6 times in parallel, and the addition recovery rate and RSD are calculated;
the recovery rate of 6 target compounds is 80.1-106%, RSD (R)n= 6) is 3.3% to 7.6%.
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