CN106093265B - The method of residual amino urea in a kind of derivatization-chromatography Flour and flour products - Google Patents
The method of residual amino urea in a kind of derivatization-chromatography Flour and flour products Download PDFInfo
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- 235000013312 flour Nutrition 0.000 title claims abstract description 50
- DUIOPKIIICUYRZ-UHFFFAOYSA-N semicarbazide Chemical compound NNC(N)=O DUIOPKIIICUYRZ-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 19
- 150000003349 semicarbazides Chemical class 0.000 claims abstract description 73
- 238000001212 derivatisation Methods 0.000 claims abstract description 61
- 239000000047 product Substances 0.000 claims abstract description 55
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 33
- 238000012360 testing method Methods 0.000 claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 239000000706 filtrate Substances 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 9
- 230000001376 precipitating effect Effects 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 238000005374 membrane filtration Methods 0.000 claims abstract description 4
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 239000012086 standard solution Substances 0.000 claims description 26
- SKDHHIUENRGTHK-UHFFFAOYSA-N 4-nitrobenzoyl chloride Chemical class [O-][N+](=O)C1=CC=C(C(Cl)=O)C=C1 SKDHHIUENRGTHK-UHFFFAOYSA-N 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 10
- 239000012498 ultrapure water Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- DBJUEJCZPKMDPA-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O DBJUEJCZPKMDPA-UHFFFAOYSA-N 0.000 claims description 4
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 4
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- ONIKNECPXCLUHT-UHFFFAOYSA-N 2-chlorobenzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1Cl ONIKNECPXCLUHT-UHFFFAOYSA-N 0.000 claims 1
- 238000007689 inspection Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 19
- 238000010521 absorption reaction Methods 0.000 abstract description 7
- 239000012071 phase Substances 0.000 description 11
- 210000005069 ears Anatomy 0.000 description 9
- 239000004156 Azodicarbonamide Substances 0.000 description 7
- XOZUGNYVDXMRKW-AATRIKPKSA-N azodicarbonamide Chemical compound NC(=O)\N=N\C(N)=O XOZUGNYVDXMRKW-AATRIKPKSA-N 0.000 description 7
- 235000019399 azodicarbonamide Nutrition 0.000 description 7
- 235000013305 food Nutrition 0.000 description 6
- IAIWVQXQOWNYOU-FPYGCLRLSA-N nitrofural Chemical compound NC(=O)N\N=C\C1=CC=C([N+]([O-])=O)O1 IAIWVQXQOWNYOU-FPYGCLRLSA-N 0.000 description 6
- 229960001907 nitrofurazone Drugs 0.000 description 5
- 108010068370 Glutens Proteins 0.000 description 4
- 235000021312 gluten Nutrition 0.000 description 4
- 239000011701 zinc Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- -1 Nitro Nitrofurans Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000000155 isotopic effect Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- ULUZGMIUTMRARO-UHFFFAOYSA-N (carbamoylamino)urea Chemical compound NC(=O)NNC(N)=O ULUZGMIUTMRARO-UHFFFAOYSA-N 0.000 description 1
- CMWKITSNTDAEDT-UHFFFAOYSA-N 2-nitrobenzaldehyde Chemical class [O-][N+](=O)C1=CC=CC=C1C=O CMWKITSNTDAEDT-UHFFFAOYSA-N 0.000 description 1
- YVQVOQKFMFRVGR-VGOFMYFVSA-N 5-(morpholin-4-ylmethyl)-3-[(e)-(5-nitrofuran-2-yl)methylideneamino]-1,3-oxazolidin-2-one Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OC(CN2CCOCC2)C1 YVQVOQKFMFRVGR-VGOFMYFVSA-N 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004088 foaming agent Substances 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 229950000337 furaltadone Drugs 0.000 description 1
- PLHJDBGFXBMTGZ-WEVVVXLNSA-N furazolidone Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OCC1 PLHJDBGFXBMTGZ-WEVVVXLNSA-N 0.000 description 1
- 229960001625 furazolidone Drugs 0.000 description 1
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- NXFQHRVNIOXGAQ-YCRREMRBSA-N nitrofurantoin Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)NC(=O)C1 NXFQHRVNIOXGAQ-YCRREMRBSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of methods of residual amino urea in derivatization chromatography Flour and flour products, comprise the following steps：(1) reacted using semicarbazides and derivatization reagent, detected with HPLC/UV, drafting is obtained using amino urea concentration as abscissa, using derivatization Product characteristics peak area as the standard curve of ordinate；(2) sample to be tested is taken, with extractant ultrasonic extraction, centrifuging and taking supernatant；Derivatization reagent, precipitating reagent are added in into supernatant, supernatant is taken after centrifugation, and passes through organic membrane filtration, obtains filtrate；(3) sample detects the filtrate in step (2) with HPLC/UV, reads derivatization Product characteristics peak area, and reference standard curve obtains the content of semicarbazides in sample to be tested.Compared with the prior art, by derivatization reagent, enhance the UV absorption of derivative compound, facilitate detection；And detecting instrument is simply universal, and testing cost is low, and is conducive to the quick detection of semicarbazides, available for large-scale sample detection, suitable for standardization.
Description
Technical field
The present invention relates in food safety monitoring field more particularly to a kind of derivatization-chromatography Flour and flour products
The method of residual amino urea can accurately detect remaining semicarbazides in flour or Flour product.
Background technology
Semicarbazides belongs to hydrazides based compound, is normally used as a kind of marker to detect the furan of the violated class illegally used
It mutters XiLin class antibiotic.Recently the study found that using azodicarbonamide as plasticising in the bottle cap of vial for sealing
Contain semicarbazides in the rubber washer of agent, and the flour processing using azodicarbonamide as gluten fortifier is into during Flour product
Semicarbazides can be generated.
Semicarbazides (semicarbazide, SEM) is a kind of research has shown that with carcinogenicity, mutagenesis and possible something lost
Pass the excessive risk substance paid close attention to by food security of toxicity.Semicarbazides source in food mainly has 3, first, animal derived
Semicarbazides, mostly come from the metabolite of the nitrofurazone drug in veterinary drug；Second is that food pack and canned plastic lid
In foaming agent；Third, gluten fortifier azodicarbonamide makes moist, is thermally decomposed in flour, Flour product.
Semicarbazides in animal derived food is essentially from the metabolite of used Nitrofuran antibiotics.Nitro
Nitrofurans mainly includes furazolidone, nitrofurazone, furaltadone, furantoin.Due to Nitrofuran antibiotics and
Its metabolin semicarbazides has the toxic side effects such as stronger carcinogenic, mutagenesis, teratogenesis shape, is forbidden by most countries, European Union
Forbid in nineteen ninety-five in food using such drug, according to european union directive, metabolites of nitrofuran method for detecting residue
Detection limit performance requirement (MRPL) is 1 μ g/kg.Also the ban forbidden using nitrofuran has been promulgated in 2002 in China.
Semicarbazides in flour or Flour product essentially from its gluten fortifier azodicarbonamide (azodicarbonamide,
ADA decomposition product), decomposable process are that ADA is decomposed under conditions of moistening or high temperature and generated semicarbazides, and ADA can also be through
It crosses wheat gluten reduction and is converted into biruea (biurea, HDC), the further Degradation and Transformations of HDC are semicarbazides (SEM).Azo first
Amide is in the country such as China, Canada, U.S. as a kind of additive for being allowed to addition maximum and being no more than 45mg/kg.And
There is no the coherent detection standards and limitation of the metabolite semicarbazides to the azodicarbonamide in flour or Flour product in China
It is required that.
According to the literature, domestic and international generally use efficient liquid phase tandem mass spectrometry detection semicarbazides.Use methanol-water solution
It extracts, adds in Isotopic Internal Standard liquid, add in derivatization reagent 2- nitrobenzaldehydes and carry out derivatization reaction, use phosphate-buffered salt
PH is adjusted, centrifugation is crossed SPE pillars, eluted with ethyl acetate, and nitrogen is blown, and redissolves, and crosses film, is detected through high performance liquid chromatography tandem mass spectrum
(Ye,J.,et al.,Assessment of the Determination of Azodicarbonamide and Its
Decomposition Product Semicarbazide:Investigation of Variation in Flour and
Flour Products.Journal of Agricultural and Food Chemistry,2011.59(17):p.9313-
9318.).Semicarbazides is detected using efficient liquid phase tandem mass spectrometry, the instrument price used is expensive, and Isotopic Internal Standard price lattice
Similary expensive, sample pretreatment process is complicated, and instrumentation is strongly professional, and method is not easy to popularize.
The content of the invention
It is an object of the invention to overcome shortcoming and defect of the prior art, a kind of derivatization-chromatography face is provided
The method of residual amino urea in powder and Flour product, the method can easy, accurately measure the content of residual amino urea.
The present invention is achieved by the following technical solutions：It is remained in a kind of derivatization-chromatography Flour and flour products
The method of semicarbazides, comprises the following steps：
(1) drafting of standard curve：Compound concentration is a series of semicarbazides standard solution of 0.01mg/L-2mg/L, so
Compound concentration corresponds to a series of 100-1000 times of derivatization reagent standard solution of semicarbazides standard solution afterwards；Take certain body
Long-pending semicarbazides standard solution, the corresponding derivatization reagent standard solution for adding in 0.1 times of volume, is obtained by the reaction derivatization product,
It is detected with HPLC/UV, drafting is obtained using amino urea concentration as abscissa, using derivatization Product characteristics peak area as the mark of ordinate
Directrix curve；
(2) sample pretreatment：Sample to be tested is taken, with extractant ultrasonic extraction, centrifuging and taking supernatant；It is added in into supernatant
Derivatization reagent, precipitating reagent take supernatant, and pass through organic membrane filtration after centrifugation, obtain filtrate；
(3) semicarbazides concentration mensuration in sample：Filtrate in step (2) is detected with HPLC/UV, testing conditions and step
(1) it is identical, derivatization Product characteristics peak area is read, reference standard curve obtains the content of semicarbazides in sample to be tested.
Compared with the prior art, the method for residual amino urea in derivatization of the invention-chromatography Flour and flour products,
By derivatization reagent, enhance the UV absorption of derivative compound, facilitate detection；And detecting instrument is simply universal, testing cost
It is low, and be conducive to the quick detection of semicarbazides, available for large-scale sample detection, suitable for standardization.
Further, the derivatization reagent is 4- nitrobenzoyl chlorides.The UV absorption of semicarbazides is very weak, it is difficult to pass through
HPLC/UV is directly measured, and by being reacted with derivatization reagent 4- nitrobenzoyl chlorides, the derivatization product of generation has preferably
UV absorption, convenient for detection.
Further, the derivatization reagent concentration in the step (2) is 5.4mmol/L.
Further, the testing conditions of the HPLC/UV are：Chromatographic column is C18 columns or ODS reverse-phase chromatographic columns；Mobile phase is
The acetic acid solution and acetonitrile of volume fraction 0.2%, gradient elution；Flow velocity is 1.0mL/min；Column temperature is 35 DEG C；Sample size is 20 μ
L；The Detection wavelength of UV is λ=261nm.
Further, the gradient elution program is：It is 0min, 13min, 14min, 16min, 17min, 22min in the time
When the acetic acid solution of corresponding mobile phase 0.2% and the volume ratio of acetonitrile be respectively 95:5、70:30、40:60、40:60、95:5、
95:5。
Further, the amount of the substance of the 4- nitrobenzoyl chlorides added in the step (1) into semicarbazides is semicarbazides
50 times of the amount of substance.
Further, extractant is ultra-pure water in the step (2)；The centrifugal condition is 4200r/min, centrifuges 5min.
The semicarbazides in sample is extracted with ultra-pure water, reduces the use of organic reagent.
Further, semicarbazides standard solution is pure water neutral solution in the step (1), and 4- nitrobenzoyl chloride standards are molten
Liquid is acetonitrile solution, and the two mixing after reacting at room temperature.
Further, the precipitating reagent in the step (2) is potassium ferrocyanide solution and acetic acid zinc solution.
Further, the concentration of the potassium ferrocyanide is 106g/L, and the concentration of the acetic acid zinc solution is 220g/L.
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Description of the drawings
Fig. 1 is the standard curve of the present invention, wherein, abscissa is the concentration of semicarbazides, and ordinate is semicarbazides derivatization
The peak area of product.
Fig. 2 is the HPLC-UV test maps of the derivatization product using deep-fried twisted dough sticks as sample to be tested in embodiment 1, wherein, a
For the semicarbazides standard sample of 1mg/L；B is deep-fried twisted dough sticks sample mark-on treated derivatization product；C is after deep-fried twisted dough sticks sample treatment
Derivatization product.
Fig. 3 be using the HPLC-UV test maps of the young derivatization product as sample to be tested of the ears of an ox or cow in embodiment 2, wherein,
A is the semicarbazides standard sample of 1mg/L；B is the ears of an ox or cow son's sample mark-on treated derivatization product；C is at the ears of an ox or cow son's sample
Derivatization product after reason.
Specific embodiment
The invention discloses the method for residual amino urea in a kind of derivatization-chromatography Flour and flour products, including with
Lower step：
(1) drafting of standard curve：Compound concentration is a series of semicarbazides standard solution of 0.01mg/L-2mg/L, so
Compound concentration corresponds to a series of 100-1000 times of derivatization reagent standard solution of semicarbazides standard solution afterwards；Take certain body
Long-pending semicarbazides standard solution, the corresponding derivatization reagent standard solution for adding in 0.1 times of volume, is obtained by the reaction derivatization product,
It is detected with HPLC/UV, drafting is obtained using amino urea concentration as abscissa, using derivatization Product characteristics peak area as the mark of ordinate
Directrix curve；
Wherein, the derivatization reagent be 4- nitrobenzoyl chlorides, the reaction of the 4- nitrobenzoyl chlorides and semicarbazides
Equation is as follows：The UV absorption of semicarbazides is very weak, it is difficult to directly measured by HPLC/UV, and by with derivatization reagent 4-
Nitrobenzoyl chloride reacts, and the derivatization product of generation has preferable UV absorption, convenient for detection；
(2) sample pretreatment：Sample to be tested is taken, with ultra-pure water ultrasonic extraction, centrifuging and taking supernatant；It is added in into supernatant
Derivatization reagent, precipitating reagent take supernatant, and pass through organic membrane filtration after centrifugation, obtain filtrate；
(3) semicarbazides concentration mensuration in sample：Filtrate in step (2) is detected with HPLC/UV, testing conditions and step
(1) it is identical, derivatization Product characteristics peak area is read, reference standard curve obtains the content of semicarbazides in sample to be tested.
Wherein, the amount of the substance of the 4- nitrobenzoyl chlorides added in the step (1) into semicarbazides is semicarbazides object
50 times of the amount of matter.I.e. described a series of 4- nitrobenzoyl chlorides concentration of standard solution is respectively the one of 0.01mg/L-2mg/L
500 times of the semicarbazides standard solution of series.
In the present invention, the optimal conditions of semicarbazides and derivatization reagent 4- nitrobenzoyl chloride derivatizations are：In room temperature
Under, it is reacted in neutral solution, and the amount of the substance of 4- nitrobenzoyl chlorides is 50 times of the amount of the substance of semicarbazides.
Embodiment 1
In the present embodiment, using deep-fried twisted dough sticks sample as sample to be tested, the concrete analysis side of residual amino urea in the deep-fried twisted dough sticks
Method is as follows.
(1) drafting of standard curve：Precise semicarbazides 0.0500g with ultra-pure water constant volume in 50mL volumetric flasks, matches somebody with somebody
The semicarbazides storing solution of 1000mg/L is made.With ultra-pure water step by step dilute prepare 0.01mg/L, 0.05mg/L, 0.1mg/L,
A series of semicarbazides standard solution of 0.5mg/L, 1mg/L, 2mg/L.Precise 4- nitrobenzoyl chloride 0.0500g, use second
Nitrile constant volume is configured to the 4- nitrobenzoyl chloride storing solutions of 1000mg/L, dilutes preparation step by step with acetonitrile in 50mL volumetric flasks
A series of 4- nitrobenzoyl chlorides standard solution of 5mg/L, 25mg/L, 50mg/L, 250mg/L, 500mg/L, 1000mg/L.Point
The semicarbazides standard solution of 1mL is not taken to add in the sample injection bottle of 2mL, the 4- nitrobenzoyl chloride standards of 100 μ L of corresponding addition are molten
Liquid obtains derivatization product after normal-temperature reaction, is detected with HPLC/UV, and drafting is obtained using amino urea concentration as abscissa, with derivative
Change the standard curve that Product characteristics peak area is ordinate.Referring to Fig. 1, it is the standard curve of the present invention, the standard is bent
The relational expression of line is：Y=107660.55*X-31.10, RSD<1.9%, detection is limited to：1.8 μ g/L, coefficient R2=
0.9991 is linear good, the content available for semicarbazides in quantitative sample；
Wherein, the testing conditions of the HPLC/UV are：Chromatographic column is C18 columns or ODS reverse-phase chromatographic columns；Mobile phase is body
The acetic acid solution and acetonitrile of fraction 0.2%, gradient elution；Flow velocity is 1.0mL/min；Column temperature is 35 DEG C；Sample size is 20 μ L；
The Detection wavelength of UV is λ=261nm.The gradient elution corresponds to volume ratio such as the following table 1 of mobile phase：
The volume ratio of the corresponding mobile phase of 1 gradient elution program difference elution time of table
(2) sample pretreatment：Precise 2.0000g (being accurate to 0.1mg) deep-fried twisted dough sticks add in super in the centrifuge tube of 50mL
Pure water 20mL is vortexed uniform, and ultrasonic 30min centrifuges 5min under the conditions of 4200r/min, pipettes 16mL upper liquids, to upper liquid
The 4- nitrobenzoyl chlorides of the 5.4mmol/L of 2mL are added in, add in the K for the 1mL that concentration is 106g/L4Fe(CN)6It is with concentration
Zn (the CH of the 1mL of 220g/L3COO)2, 5min is centrifuged under the conditions of 4200r/min, supernatant 1mL after centrifugation is taken to cross 0.22
μm organic filter membrane, obtains filtrate.In step (2), the precipitating reagent used in fried class Flour product is the 1mL that concentration is 106g/L
K4Fe(CN)6It is the Zn (CH of the 1mL of 220g/L with concentration3COO)2, the precipitating reagent used in the Flour product of flour and non-fried class is
Concentration is the K of the 0.5mL of 106g/L4Fe(CN)6It is the Zn (CH of the 1mL of 220g/L with concentration3COO)2。
(3) semicarbazides concentration mensuration in sample：Filtrate is subjected to HPLC-UV tests, test condition is identical with step (1).
Referring to Fig. 2, it is the HPLC-UV test maps of the derivatization product using deep-fried twisted dough sticks as sample to be tested in the present embodiment.It reads
The content of semicarbazides in deep-fried twisted dough sticks sample is calculated in derivatization object product characteristic peak area, reference standard curve.In the present embodiment
In, it is 8.19-9.18mg/kg, recovery of standard addition 95.5%-105% that semicarbazides content in deep-fried twisted dough sticks, which is calculated,.
Compared with the prior art, the method for residual amino urea in derivatization of the invention-chromatography Flour and flour products,
The semicarbazides in sample is extracted with ultra-pure water, reduces the use of organic reagent, by derivatization reagent, enhances derivative compound
UV absorption facilitates detection；And detecting instrument is simply universal, and testing cost is low, and is conducive to the quick detection of semicarbazides, can
For large-scale sample detection, suitable for standardization.
Embodiment 2
The present embodiment is roughly the same with embodiment 1, and difference essentially consists in sample to be tested difference, in the present embodiment,
The sample to be tested is young for the ears of an ox or cow, and the specific analytical method of residual amino urea is as follows in the ears of an ox or cow son.
(1) drafting of standard curve：Precise semicarbazides 0.0500g with ultra-pure water constant volume in 50mL volumetric flasks, matches somebody with somebody
The semicarbazides storing solution of 1000mg/L is made.With ultra-pure water step by step dilute prepare 0.01mg/L, 0.05mg/L, 0.1mg/L,
A series of semicarbazides standard solution of 0.5mg/L, 1mg/L, 2mg/L.Precise 4- nitrobenzoyl chloride 0.0500g, use second
Nitrile constant volume is configured to the 4- nitrobenzoyl chloride storing solutions of 1000mg/L, dilutes preparation step by step with acetonitrile in 50mL volumetric flasks
A series of 4- nitrobenzoyl chlorides standard solution of 5mg/L, 25mg/L, 50mg/L, 250mg/L, 500mg/L, 1000mg/L.Point
The semicarbazides standard solution of 1mL is not taken to add in the sample injection bottle of 2mL, the 4- nitrobenzoyl chloride standards of 100 μ L of corresponding addition are molten
Liquid obtains derivatization product after normal-temperature reaction, is detected with HPLC/UV, and drafting is obtained using amino urea concentration as abscissa, with derivative
Change the standard curve that Product characteristics peak area is ordinate.Referring to Fig. 1, it is the standard curve of the present invention, the standard is bent
The relational expression of line is：Y=107660.55*X-31.10, RSD<1.9%, detection is limited to：1.8 μ g/L, coefficient R2=
0.9991 is linear good, the content available for semicarbazides in quantitative sample.Wherein, the testing conditions of the HPLC/UV are：Chromatography
Column is C18 columns or ODS reverse-phase chromatographic columns；Mobile phase be volume fraction 0.2% acetic acid solution and acetonitrile, gradient elution；Flow velocity
For 1.0mL/min；Column temperature is 35 DEG C；Sample size is 20 μ L；The Detection wavelength of UV is λ=261nm.The gradient elution corresponds to stream
Volume ratio such as the following table 2 of dynamic phase：
The volume ratio of the corresponding mobile phase of 2 gradient elution program difference elution time of table
(2) sample pretreatment：Precise 2.0000g (being accurate to 0.1mg) the ears of an ox or cow sons add in the centrifuge tube of 50mL
Ultra-pure water 20mL is vortexed uniform, and ultrasonic 30min centrifuges 5min under the conditions of 4200r/min, pipettes 16mL upper liquids, to upper strata
Liquid adds in the 4- nitrobenzoyl chlorides of the 5.4mmol/L of 2mL, adds in the K for the 1mL that concentration is 106g/L4Fe(CN)6It is with concentration
Zn (the CH of the 1mL of 220g/L3COO)2, 5min is centrifuged under the conditions of 4200r/min, supernatant 1mL after centrifugation is taken to cross 0.22
μm organic filter membrane, obtains filtrate.
(3) semicarbazides concentration mensuration in sample：Filtrate is subjected to HPLC-UV tests, test condition is identical with step (1).
Referring to Fig. 3, it is using the HPLC-UV test maps of the young derivatization product as sample to be tested of the ears of an ox or cow in the present embodiment.It reads
Take derivatization object product characteristic peak area, the content of semicarbazides in the ears of an ox or cow son's sample is calculated in reference standard curve.In this reality
It applies in example, it is 1.27-1.37mg/kg, recovery of standard addition 76.6%-92.1% that semicarbazides content in the ears of an ox or cow son, which is calculated,.
The invention is not limited in the above embodiment, if the various changes or deformation to the present invention do not depart from the present invention
Spirit and scope, if these changes and deformation belong within the scope of the claim and equivalent technologies of the present invention, then this hair
It is bright to be also intended to comprising these changes and deformation.
Claims (9)
1. the method for residual amino urea in a kind of derivatization-chromatography Flour and flour products, it is characterised in that：Including following step
Suddenly：
(1) drafting of standard curve：Compound concentration be 0.01mg/L-2mg/L a series of semicarbazides standard solution, Ran Houpei
Concentration processed corresponds to a series of derivatization reagent standard solution of 100-1000 times of semicarbazides standard solution；Take certain volume
Semicarbazides standard solution, the corresponding derivatization reagent standard solution for adding in 0.1 times of volume, is obtained by the reaction derivatization product, uses
HPLC/UV is detected, and drafting is obtained using amino urea concentration as abscissa, using derivatization Product characteristics peak area as the standard of ordinate
Curve；
(2) sample pretreatment：Sample to be tested is taken, with extractant ultrasonic extraction, centrifuging and taking supernatant；It adds in and derives into supernatant
Change reagent, precipitating reagent, supernatant is taken after centrifugation, and pass through organic membrane filtration, obtain filtrate；The derivatization reagent is 4- nitre
Base chlorobenzoyl chloride；
(3) semicarbazides concentration mensuration in sample：Filtrate in step (2) is detected with HPLC/UV, testing conditions and step (1)
It is identical, derivatization Product characteristics peak area is read, reference standard curve obtains the content of semicarbazides in sample to be tested.
2. the method for residual amino urea, feature in derivatization according to claim 1-chromatography Flour and flour products
It is：Derivatization reagent concentration in the step (2) is 5.4mmol/L.
3. the method for residual amino urea, feature in derivatization according to claim 1-chromatography Flour and flour products
It is：The testing conditions of the HPLC/UV are：Chromatographic column is C18 columns or ODS reverse-phase chromatographic columns；Mobile phase is volume fraction
0.2% acetic acid solution and acetonitrile, gradient elution；Flow velocity is 1.0mL/min；Column temperature is 35 DEG C；Sample size is 20 μ L；The inspection of UV
Survey wavelength is λ=261nm.
4. the method for residual amino urea, feature in derivatization according to claim 3-chromatography Flour and flour products
It is：The gradient elution program is：The corresponding stream when the time is 0min, 13min, 14min, 16min, 17min, 22min
The acetic acid solution of dynamic phase 0.2% and the volume ratio of acetonitrile are respectively 95:5、70:30、40:60、40:60、95:5、95:5.
5. the method for residual amino urea, feature in derivatization according to claim 1-chromatography Flour and flour products
It is：The amount of the substance of the 4- nitrobenzoyl chlorides added in the step (1) into semicarbazides is the amount of semicarbazides substance
50 times.
6. the method for residual amino urea, feature in derivatization according to claim 1-chromatography Flour and flour products
It is：Extractant is ultra-pure water in the step (2)；The centrifugal condition is 4200r/min, centrifuges 5min.
7. the method for residual amino urea, feature in derivatization according to claim 1-chromatography Flour and flour products
It is：Semicarbazides standard solution is pure water neutral solution in the step (1), and 4- nitrobenzoyl chlorides standard solution is molten for acetonitrile
Liquid, and the two mixing after reacting at room temperature.
8. the method for residual amino urea, feature in derivatization according to claim 1-chromatography Flour and flour products
It is：Precipitating reagent in the step (2) is potassium ferrocyanide solution and acetic acid zinc solution.
9. the method for residual amino urea, feature in derivatization according to claim 8-chromatography Flour and flour products
It is：The concentration of the potassium ferrocyanide is 106g/L, and the concentration of the acetic acid zinc solution is 220g/L.
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| US6156704A (en) * | 1988-12-29 | 2000-12-05 | Novartix Ag | Auxin transport inhibitor compounds |
| CN103487529A (en) * | 2013-10-15 | 2014-01-01 | 谱尼测试科技(天津)有限公司 | Method for detecting residual amount of semi carbazide in flour and flour products by high performance liquid chromatography |
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| US6156704A (en) * | 1988-12-29 | 2000-12-05 | Novartix Ag | Auxin transport inhibitor compounds |
| CN103487529A (en) * | 2013-10-15 | 2014-01-01 | 谱尼测试科技(天津)有限公司 | Method for detecting residual amount of semi carbazide in flour and flour products by high performance liquid chromatography |
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