CN104678010B - A kind of detection method of nicarbazine - Google Patents
A kind of detection method of nicarbazine Download PDFInfo
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- CN104678010B CN104678010B CN201510044091.3A CN201510044091A CN104678010B CN 104678010 B CN104678010 B CN 104678010B CN 201510044091 A CN201510044091 A CN 201510044091A CN 104678010 B CN104678010 B CN 104678010B
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- UKHWDRMMMYWSFL-UHFFFAOYSA-N Nicarbazin Chemical compound CC=1C=C(C)NC(=O)N=1.C1=CC([N+](=O)[O-])=CC=C1NC(=O)NC1=CC=C([N+]([O-])=O)C=C1 UKHWDRMMMYWSFL-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 57
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 8
- 239000004202 carbamide Substances 0.000 claims abstract description 7
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims abstract description 3
- 239000000273 veterinary drug Substances 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 31
- 238000012360 testing method Methods 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000013068 control sample Substances 0.000 claims description 3
- 239000007943 implant Substances 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
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- LYRFLYHAGKPMFH-UHFFFAOYSA-N octadecanamide Chemical group CCCCCCCCCCCCCCCCCC(N)=O LYRFLYHAGKPMFH-UHFFFAOYSA-N 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- JEZZOKXIXNSKQD-UHFFFAOYSA-N 1,3-bis(4-nitrophenyl)urea Chemical compound C1=CC([N+](=O)[O-])=CC=C1NC(=O)NC1=CC=C([N+]([O-])=O)C=C1 JEZZOKXIXNSKQD-UHFFFAOYSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 4
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- 230000014759 maintenance of location Effects 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
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 230000001165 anti-coccidial effect Effects 0.000 description 2
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- 229940073485 nicarbazin Drugs 0.000 description 2
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- 239000005695 Ammonium acetate Substances 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
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- 229940043376 ammonium acetate Drugs 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000003224 coccidiostatic agent Substances 0.000 description 1
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- 238000010829 isocratic elution Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
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Abstract
The present invention relates to one and measure in veterinary drug nicarbazine 4 simultaneously, 4,-dinitro diphenyl urea and 2-hydroxyl-4, the method of 6-dimethyl pyrimidine, comprises the steps, by sample wiring solution-forming, it is injected in high performance liquid chromatograph and detects, the condition of high performance liquid chromatography is: select bonding alkylamide group chromatography column, column temperature 25 DEG C～40 DEG C, sample size 0.5 μ L～2 μ L；Mobile phase selects water and acetonitrile, adopts gradient elution, and program is, 0～8min, 99%～10% water；8～16min, 10% water；16～17min, 10%～99% water, flow rate of mobile phase is 0.5mL/min～1.5mL/min, and detection wavelength is 260nm～380nm；Theoretical cam curve calculates according to nicarbazine and is not less than 5000, the advantages such as the method has a detection sensitivity height, good separating effect, simple and convenient.
Description
Technical field
The present invention relates to the analysis field of high phase liquid chromatograph, be specifically related in nicarbazine 4,4, the detection of-dinitro diphenyl urea (DNC) and HDP (HDP).
Background technology
Nicarbazine (nicarbazin) has another name called Nicarbazin, and nineteen fifty-five is developed by Merck company of the U.S., has efficient, wide spectrum coccidiostat activity, and preventive effect is fine, and resistance development is extremely slow, remains one of maximally effective anticoccidial drug so far.The chemical name of nicarbazine is 4,4,-dinitro diphenyl urea (DNC) and the molar complex such as grade of HDP (HDP), be abbreviated as DNC HDP, CAS:330-95-0.Independent DNC Anti-human globulin test is more weak, and after forming 1:1 molecular clathrate with HDP, its anticoccidial effect is best.Nicarbazine is a kind of typical clathrate, and the stability of its structure is more weak than chemical bond much can be opened in solution upon dissolution, exists with two kinds of molecular forms.Chemical constitution is as follows:
EINECS:206-359-1
Molecular formula: C19H18N6O6
The chemical constitution of nicarbazine component
DNC content in " veterinary medical quality standard " regulation nicarbazine crude drug of Ministry of Agriculture's version in 2003 should be 67.4%～73.0%, and HDP should be 27.7%～30.0%, and adopts spectrophotography to measure its content (Ministry of Agriculture the 307th bulletin) respectively.But spectrophotography colour developing instability, is subject to impurity or additive interference and affects quantitatively.
In detection nicarbazine, the research papers of DNC is relatively more at present.In nicarbazine metabolic process in animal body, wherein HDP easily metabolism in animal body, and remain and be substantially DNC.Therefore, all regulation nicarbazine residual label in kidney, meat, egg is DNC both at home and abroad.In feedstuff and animal tissue and fowl egg, the nicarbazine of residual measures and is generally adopted high performance liquid chromatography (HPLC), liquid chromatography-mass spectrography (HPLC-MS) method and biological methods.In the above-mentioned methods, use liquid chromatograph to detect the document of DNC and HDP double-component little simultaneously.And in the quality control of crude drug and preparation manufacturing enterprise, it is necessary to monitoring DNC and HDP simultaneously.
Macy etc. adopt DNC and the HDP that different chromatographic column determines in feedstuff and premix material in nicarbazine respectively, its operability not strong ([J] .JAssocOffAnalChem., 1984,67 (6): 1115-1117).Agricultural University Of South China Shen Xiang extensively etc. adopt DNC and HDP content that C18 chromatographic column measures in nicarbazine simultaneously ([J]. analyze test journal, 2010,29 (5): 523-526).But owing to HDP polarity is relatively big, HDP chromatographic peak, without being effectively retained, uses acetic acid/ammonium acetate buffer saline solution in the document, it is intended that extend appearance time by reducing the pH of buffer, but DeGrain.Mar í aM. et al. ([J] .Talanta, 2011,85:142 150) adopting the method adding ion-pairing agent to improve the reservation of HDP, shortcoming is that ion-pairing agent is bigger to chromatographic column injury, with the fixing generation Irreversible Adsorption that combines, and then the fixing phase avtive spot of impact.And ion-pairing agent is difficult to rinse well chromatographic column, can be greatly shortened the service life of chromatographic column.The concentration of ion-pairing agent and the retention time of its sample have direct impact, and pH value is more sensitive, and during preparation mobile phase, claimed accuracy is higher, otherwise directly affects repeatability and the repeatability of experiment.
Summary of the invention
For prior art in utilizing high performance liquid chromatography to nicarbazine in the content detection of DNC and HDP, HDP chromatographic peak is without being effectively retained, add ion-pairing agent and can shorten the defect in chromatographic column life-span, the present invention provides a kind of method of high performance liquid chromatography using bonding alkylamide group chromatography column detection DNC and HDP, comprises the steps:
1) preparation of standard curve:
Nicarbazine standard substance are made into the solution to be measured of variable concentrations, it is injected in high performance liquid chromatograph and detects, obtain peak area, draw the standard curve of peak area and material concentration, the condition of high performance liquid chromatography is: select bonding alkylamide group chromatography column, column temperature 25 DEG C～40 DEG C, sample size 0.5 μ L～2 μ L；Mobile phase selects water and acetonitrile, adopts gradient elution, and gradient elution program is: 0～8min, and the percentage composition of water is gradient to 10% by 99%；8～16min, the percentage composition of water is 10%；16～17min, the percentage composition of water is gradient to 99% by 10%, described flow rate of mobile phase 0.5mL/min～1.5mL/min, and detector detection wavelength is 260nm～380nm；Theoretical cam curve calculates according to nicarbazine and is not less than 5000；
2) detection of testing sample
Testing sample is made into solution to be measured, is injected in high performance liquid chromatograph and detects, the conditional synchronization rapid 1 of high performance liquid chromatography) described in condition identical, obtain detection peak area, the concentration according to described standard curve substance for calculation.
In the present invention, the reverse chromatograms post that described chromatographic column is preferably implant with octadecyl amide bonded silica gel.This kind of chromatographic column is suitable for the analysis of highly polar compound, by with the highly polar material effect containing N structure, its retention time can be extended.
In the present invention, described column temperature is preferably 40 DEG C.Column temperature improves, and peak type becomes sharp-pointed, and adjusting suitable column temperature is 40 DEG C.
In the present invention, described sample size is preferably 1 μ L.When the intensity of sample introduction solvent is higher than mobile phase, tested substance can be changed by the speed of chromatographic column, makes peak shape distort, deforms.When being 1 μ L, HDP has a better peak type, and when continuing to increase sample size, HDP peak type is distorted deformation.
In the present invention, described flow velocity is preferably 0.8mL/min.Flow velocity reduces, and retention strengthens, and appearance time extends, and flow velocity is too high, and appearance time shortens, and is unfavorable for the reservation at HDP peak.Adjusting suitable flow velocity is 0.8ml/min.
In the present invention, described detector is UV-detector, 4,4,The detection of-dinitro diphenyl urea and HDP all uses the described detection wavelength of 300nm.
In the present invention, the compound method of described solution to be measured is, uses DMF or dmso solution sample, and it is diluted by one or both addition in water or acetonitrile.In nicarbazine sample, DNC is highly polar sample, only could dissolve under intensive polar solvent DMF or dimethyl sulfoxide fully.
In the present invention, the compound method of described solution to be measured is preferably, and takes standard substance or sample 50mg to be tested, adds 15mL dmso solution, with acetonitrile: it is settled to 50mL by the mixed liquor that water volume ratio is 80:20.
Further, currently preferred technical scheme is as follows:
The outfit of nicarbazine standard solution: take 50mg nicarbazine control sample, adds 15mL dmso solution, with acetonitrile: it is settled to 50mL as storing solution A by the mixed liquor that water volume ratio is 80:20；
Chromatographic condition: chromatographic column is Agilentporoshell120Bonus-RP (4.6 × 150mm, 2.7 μm), and ultraviolet detection wavelength is 300nm, column temperature 40 DEG C, sample size 1 μ L；Mobile phase is the mixed liquor of water and acetonitrile, and employing gradient elution program is: 0～8min, the percentage composition of water is gradient to 10% by 99%；8～16min, the percentage composition of water is 10%；16～17min, the percentage composition of water is gradient to 99% by 10%, flow rate of mobile phase 0.8mL/min；
Described storing solution 1 is carried out the dilution of variable concentrations, with high performance liquid chromatograph, it is detected one by one, draw 4,4 respectively,The peak area of-dinitro diphenyl urea and HDP and the standard curve of material concentration；
2) detection of nicarbazine testing sample
Nicarbazine test liquid is prepared: taking 50mg nicarbazine sample, add 15mL dmso solution, the acetonitrile identical by same step 1) concentration and the mixed diluting liquid of water are settled to 50mL as storing solution B；
Adopt step 1) described in chromatographic test strip part, storing solution B carried out or be not diluted obtaining sample liquid to be measured, described sample liquid to be measured being detected, obtains peak area, according to described standard curve calculate sample concentration.
Detection method of the present invention, both can use in nicarbazine crude drug and preparation thereof and analyze while bi-component, it is also possible to for the analysis of individually analysis and the free HDP of nicarbazine effective ingredient HDP and DNC.
The method of the present invention can detect at one and detect two components of DNC and HDP in nicarbazine crude drug and preparation thereof under wavelength condition simultaneously.Buffer salt solution and ion-pairing agent need not be used, simplify operating process.There is detection sensitivity height simultaneously, good separating effect, the feature such as simple and convenient, it is applicable to the detection to nicarbazine production process, product and preparation of veterinary drug manufacturing enterprise and supervision department.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of nicarbazine standard substance
Fig. 2 is HDP content standard curve chart
Fig. 3 is DNC content standard curve chart
Fig. 4 is the high-efficient liquid phase chromatogram of nicarbazine sample to be tested
Fig. 5 is the mensuration of free HDP in nicarbazine
Fig. 6 octadecylsilane chemically bonded silica is the reverse chromatograms post mensuration figure of implant
Fig. 7 mobile phase adds the mensuration figure of 0.1% formic acid
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
In embodiment 1 nicarbazine, HDP and DNC measures
The present embodiment relates in nicarbazine HDP and DNC detection, and it specifically comprises the following steps that 1) drafting of HDP and DNC directrix curve
Nicarbazine standard solution: take 50mg nicarbazine control sample (DNC content 70.9%, HDP content 29.1%), dissolves with 15mLDMSO, is settled to 50mL as storing solution A with the mixed diluting liquid that the volume ratio of acetonitrile and water is 80:20.
Chromatographic column is Agilentporoshell120Bonus-RP (4.6 × 150mm, 2.7 μm), and ultraviolet detection wavelength is 300nm, column temperature 40 DEG C, sample size 1 μ L；Mobile phase is the mixed liquor of water and acetonitrile, and employing gradient elution program is: 0～8min, the percentage ratio of water is gradient to 10% by 99%；8～16min, the percentage composition of water is 10%；16～17min, the percentage composition of water is gradient to 99% by 10%, flow velocity 0.8mL/min.
Take storing solution A, take 4mL, 4.5mL, 5mL, 5.5mL, 6mL respectively and be settled to 50mL, sample introduction 1 μ L to chromatograph of liquid, measures the peak area of variable concentrations standard substance respectively, such as Fig. 1 (6mL storing solution A be settled to 50mL after the liquid chromatogram that records to chromatograph of liquid of sample introduction 1 μ L).
With peak area for vertical coordinate (Y), mass concentration is abscissa (X), and the regression equation recording HDP is Y=3.620X-1.085 (such as Fig. 2), and correlation coefficient 0.9998, the range of linearity is 23.3mg/L～34.9mg/L；The regression equation recording DNC is Y=2.736X-1.271 (such as Fig. 3), and correlation coefficient is 0.9996, and the range of linearity is 56.7mg/L～85.1mg/L.
Taking nicarbazine storing solution A appropriate, when 10 times that chromatographic peak peak height is noise (S/N=10), recording HDP lower limit of quantitation is 0.45mg/L；DNC lower limit of quantitation is 0.037mg/L；Gradually dilute with mixed diluting liquid, when 3 times that chromatographic peak peak height is noise (S/N=3), record HDP detection and be limited to 0.12mg/L；DNC detection is limited to 0.011mg/L.2) test of nicarbazine testing sample
Nicarbazine test liquid is prepared: take 50mg nicarbazine sample, adds 15mLDMSO and dissolves, with acetonitrile: water volume ratio is that 80:20 mixed diluting liquid is settled to 50mL as storing solution B.
Select the chromatographic condition identical with step 1), take storing solution B5mL and be settled to 50mL, sample introduction 1 μ L to chromatograph of liquid, measure the peak area (Fig. 4) of two kinds of components of nicarbazine, and bring standard curve calculating content respectively into.
The mensuration of free HDP in embodiment 2 nicarbazine
The present embodiment relates to the mensuration of free HDP in nicarbazine, and it specifically comprises the following steps that
According to Ministry of Agriculture's standard: take crude drug nicarbazine 0.50g, accurately weighed, it is placed in 25mL volumetric flask, adds phosphate buffer (pH=7.0) 20mL, jolting 10min, and be diluted to scale, shake up, filter immediately.Discarding just filtrate, access middle filtrate, precision measures 5mL, is placed in 50mL volumetric flask, with acetonitrile: water volume ratio is the mixed liquor of 80:20, shakes up, and is configured to for test sample solution.
Select the chromatographic condition identical with embodiment 1, the HDP peak area (Fig. 5) recorded, according to standard curve, calculate the content of free HDP.
Comparative example 1
Comparing with embodiment 1, it differs only in, and uses poroshell120ECC18Post (4.6 × 100mm, 2.7 μm) replaces Agilentporoshell120Bonus-RP, and mobile phase is acetonitrile and water, adopts isocratic elution, and the volume ratio of acetonitrile and water is 50:50, and it is tested, and acquired results is shown in Fig. 6.
As seen from Figure 6, HDP goes out cutting edge of a knife or a sword at 1.185min, and the dead time that eluent goes out cutting edge of a knife or a sword is 1.113min, and the two almost goes out cutting edge of a knife or a sword simultaneously, and it is excessively similar to go out the cutting edge of a knife or a sword time, and this can affect the accuracy of HDP detection.
Comparative example 2
Comparing with embodiment 1, it is distinctive in that, adds 0.1% formic acid in mobile phase water, and acquired results is shown in Fig. 7.
As seen from Figure 7, the method adding 0.1% formic acid in mobile phase water, HDP and DNC retention time shortens.Therefore adjusting the pH of mobile phase by acid adding in prior art is the reservation that acidity can not increase HDP, uses chromatographic column of the present invention and eluent can realize HDP and DNC and retains preferably and separate.
Be can be seen that by above example and comparative example, in the process that DNC and HDP detects in nicarbazine, use chromatographic column of the present invention, can effectively extend the appearance time of HDP in nicarbazine, elution program is optimized by the present invention further, DNC and HDP can being carried out good separation, therefore efficient liquid-phase chromatography method of the present invention can realize simultaneously to the accurate detection of DNC and HDP in nicarbazine.
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (9)
1. one kind measures in veterinary drug nicarbazine 4 simultaneously, the method for 4 '-dinitro diphenyl urea and HDP, it is characterised in that comprise the steps:
1) preparation of standard curve:
Nicarbazine standard substance are made into the solution to be measured of variable concentrations, are injected in high performance liquid chromatograph and detect, obtain peak area, draw the standard curve of peak area and material concentration；The condition of high performance liquid chromatography is: select bonding alkylamide group chromatography column, column temperature 25 DEG C～40 DEG C, sample size 0.5 μ L～2 μ L；Mobile phase selects water and acetonitrile, adopts gradient elution, and gradient elution program is: 0～8min, and the percentage composition of water is gradient to 10% by 99%；8～16min, the percentage composition of water is 10%；16～17min, the percentage composition of water is gradient to 99% by 10%, described flow rate of mobile phase 0.5mL/min～1.5mL/min, and detector detection wavelength is 260nm～380nm；；
2) detection of testing sample
Testing sample is made into solution to be measured, is injected in high performance liquid chromatograph and detects, the conditional synchronization rapid 1 of high performance liquid chromatography) described in condition identical, obtain detection peak area, the concentration according to described standard curve substance for calculation.
2. method according to claim 1, it is characterised in that the reversed phase chromatographic column that described chromatographic column is is implant with octadecyl amide bonded silica gel.
3. method according to claim 1 and 2, it is characterised in that described column temperature is 40 DEG C.
4. method according to claim 1 and 2, it is characterised in that sample size is 1 μ L.
5. method according to claim 1 and 2, it is characterised in that described flow velocity is 0.8mL/min.
6. method according to claim 1 and 2, it is characterised in that described detector is UV-detector, 4,4 '-dinitro diphenyl urea and HDP all use the described detection wavelength of 300nm.
7. method according to claim 1 and 2, it is characterized in that, the compound method of described solution to be measured is, uses N, standard substance or testing sample are dissolved by dinethylformamide or dimethyl sulfoxide, and it is diluted by one or both addition in water or acetonitrile.
8. method according to claim 7, it is characterised in that the compound method of the solution to be measured of described testing sample is take testing sample 50mg, adds 15mL dmso solution, with acetonitrile: it is settled to 50mL by the mixed liquor that water volume ratio is 80:20.
9.According toMethod described in claim 1, it is characterised in that comprise the steps:
1) drafting of standard curve；
The outfit of nicarbazine standard solution: take 50mg nicarbazine control sample, adds 15mL dmso solution, with acetonitrile: it is settled to 50mL as storing solution A by the mixed liquor that water volume ratio is 80:20；
Chromatographic condition: chromatographic column is Agilentporoshell120Bonus-RP, its specification is 4.6 × 150mm, 2.7 μm, and ultraviolet detection wavelength is 300nm, column temperature 40 DEG C, sample size 1 μ L；Mobile phase is the mixed liquor of water and acetonitrile, and employing gradient elution program is: 0～8min, the percentage composition of water is gradient to 10% by 99%；8～16min, the percentage composition of water is 10%；16～17min, the percentage composition of water is gradient to 99% by 10%, flow rate of mobile phase 0.8mL/min；
Described storing solution A is carried out the dilution of variable concentrations, with high performance liquid chromatograph, it is detected one by one, draw 4 respectively, the peak area of 4 '-dinitro diphenyl urea and HDP and the standard curve of material concentration；
2) detection of nicarbazine testing sample
Nicarbazine test liquid is prepared: taking 50mg nicarbazine sample, add 15mL dmso solution, the acetonitrile identical by same step 1) concentration and the mixed diluting liquid of water are settled to 50mL as storing solution B；
Adopt step 1) described in chromatographic test strip part, storing solution B carried out or be not diluted obtaining sample liquid to be measured, described sample liquid to be measured being detected, obtains peak area, according to described standard curve calculate sample concentration.
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| CN101450044A (en) * | 2008-12-29 | 2009-06-10 | 天津瑞普生物技术股份有限公司 | Anti-coccidium suspension agent containing nicarbazin and preparation technique thereof |
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2015
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101450044A (en) * | 2008-12-29 | 2009-06-10 | 天津瑞普生物技术股份有限公司 | Anti-coccidium suspension agent containing nicarbazin and preparation technique thereof |
Non-Patent Citations (4)
| Title |
|---|
| A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: Chemometric tools for improving the optimization and validation;María M. De Zan et al.;《Talanta》;20110331;第85卷(第1期);第142-150页 * |
| High-performance liquid chromatography-based determination of nicarbazin excretion in waterfowl;Randal S. Stahl et al.;《Journal of Chromatography B》;20020725;第775卷(第1期);第103-108页 * |
| 尼卡巴嗪原料药及制剂中4,4"-二硝基均苯二脲与2-羟基-4,6-二甲基嘧啶的HPLC法同时测定;沈祥广 等;《分析测试学报》;20100531;第29卷(第5期);第523-526页 * |
| 液质联用快速检测饲料中尼卡巴嗪含量;曹文卿 等;《食品研究与开发》;20131231;第34卷(第23期);第87-91页 * |
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| CN104678010A (en) | 2015-06-03 |
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