CN1227528C - High-efficient liquid phase chromatographic analysing method for regulating pyrurine and its impurity - Google Patents
High-efficient liquid phase chromatographic analysing method for regulating pyrurine and its impurity Download PDFInfo
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- CN1227528C CN1227528C CN200410014128.XA CN200410014128A CN1227528C CN 1227528 C CN1227528 C CN 1227528C CN 200410014128 A CN200410014128 A CN 200410014128A CN 1227528 C CN1227528 C CN 1227528C
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- forchlorfenuron
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- 239000012535 impurity Substances 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 14
- 239000007791 liquid phase Substances 0.000 title claims description 11
- 230000001105 regulatory effect Effects 0.000 title 1
- GPXLRLUVLMHHIK-UHFFFAOYSA-N forchlorfenuron Chemical compound C1=NC(Cl)=CC(NC(=O)NC=2C=CC=CC=2)=C1 GPXLRLUVLMHHIK-UHFFFAOYSA-N 0.000 claims abstract description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000005979 Forchlorfenuron Substances 0.000 claims abstract description 14
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000012071 phase Substances 0.000 claims description 14
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 17
- BLBDTBCGPHPIJK-UHFFFAOYSA-N 4-Amino-2-chloropyridine Chemical compound NC1=CC=NC(Cl)=C1 BLBDTBCGPHPIJK-UHFFFAOYSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 10
- 239000013558 reference substance Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- GWEHVDNNLFDJLR-UHFFFAOYSA-N 1,3-diphenylurea Chemical compound C=1C=CC=CC=1NC(=O)NC1=CC=CC=C1 GWEHVDNNLFDJLR-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 1
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 1
- 238000009849 vacuum degassing Methods 0.000 description 1
- 229940023877 zeatin Drugs 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The present invention relates to a high performance liquid chromatographic analysis method of forchlorfenuron and impurities thereof. The high performance liquid chromatographic conditions are as follows: a chromatographic column is C18, 5 mum, 150*4.6mm(I. D.); a moving phase is formed by methanol and water in a volume ratio of 60:40; a flow speed is 1.0 mL/min; a column temperature is 30 DEG C; a detection wavelength is UV 254 nm; a sample size is from 5 to 10 muL. The high performance liquid chromatographic analysis method of the present invention for the forchlorfenuron and the impurities thereof can simultaneously and accurately measure the contect of CPPU and impurities ACP and DPU in a CPPU sample. Thus, the present invention provides a simple and reliable method for the quality control analysis during a production process.
Description
Technical field
The present invention relates to high efficiency liquid phase chromatographic analysis method, specifically, relate to content with high-efficient liquid phase chromatogram technique analysis forchlorfenuron and impurity thereof.
Background technology
N-(2-chloro-4-pyridine)-N '-phenylurea (is called for short: forchlorfenuron, N-(2-chloro-4-pyridyl)-N '-phenylurea, CPPU) be a kind of plant growth regulator of cytokinin, can induce the accumulation of anthocyanin and carbohydrates in certain plants such as radish, tomato, apple, berry and the grape.Its physiologically active is higher than the general purine type basic element of cell division, and synthetic easy, with low cost, is considered to the cheap substitute of zeatin.At present about the research of CPPU mainly concentrate on its mechanism of action and application facet [referring to: wait bravely, Malaysian is auspicious, Xia Zhongmei, Ceng Xianbin, plant nutrient and fertilizer journal, 1999,5 (2), 106-114].About the assay method of CPPU, and present domestic existing vapor-phase chromatography [referring to: Jiang Xin, Zhouning County, agricultural chemicals, 1996,35 (9), 21-22; Lei Shaorong, Luo Ling, soil agrochemistry circular, 1997,12 (4), 55-56], high performance liquid chromatography is [referring to Xu Defeng, Wu Sujiang, Jiangsu agricultural chemicals, 1999,4,18-19; Zhu Peifang, Liu Yulin, the 14 national chromatogram symposium collection of thesis, 2003, LC-094,287-289].
Forchlorfenuron by following reaction make [referring to: DE 2843722 (1979); US 4193788 (1980)]:
Thereby (4-Amino-2-chloropyridine, ACP) with accessory substance N, (N, N '-Diphenylurea DPU) etc., must control in the production N '-acardite may raw material 4-amino-2-chloropyridine in product.But the method for measuring coexistent impurity content in the product is not simultaneously appeared in the newspapers as yet.
Summary of the invention
The purpose of this invention is to provide a kind of high performance liquid chromatography that can measure forchlorfenuron content and impurity content thereof.
Technical scheme of the present invention is as follows:
The high efficiency liquid phase chromatographic analysis method of a kind of forchlorfenuron and impurity thereof is characterized in that adopting reversed-phased high performace liquid chromatographic, and high-efficient liquid phase chromatogram condition is:
Chromatographic column: C
18, 5 μ m, 150 * 4.6mm (I.D.),
Moving phase is formed volume ratio: methyl alcohol: water=60: 40,
Flow velocity: 1.0mL/min,
Column temperature: 30 ℃,
Detect wavelength: UV 254nm.
Sample size: 5-10 μ L
The above-mentioned forchlorfenuron and the high efficiency liquid phase chromatographic analysis method of impurity thereof are the content of measuring forchlorfenuron behind 50 times of the sample introduction concentration dilutions of impurity content with measuring.
Adopt the high efficiency liquid phase chromatographic analysis method of forchlorfenuron of the present invention and impurity thereof can accurately measure simultaneously CPPU in the CPPU sample and the content of impurity A CP and DPU wherein.For the Quality Control Analysis in the production process provides simple and reliable method.
Description of drawings
Fig. 1 is the chromatogram of the different phase compositions of flowing, wherein: (a) 60% methyl alcohol-40% water; (b) 70% methyl alcohol-30% water; (c) 80% methyl alcohol-20% water.(1) ACP among the figure; (2) DPU; (3) CPPU.
Fig. 2 is the typical curve of CPPU.
Fig. 3 is the typical curve of ACP.
Fig. 4 is the typical curve of DPU.
Fig. 5 is the chromatogram of major component CPPU assay in the sample.
Fig. 6 is the chromatogram of impurity A CP in the sample, DPU assay.
Embodiment
Experimental apparatus and chromatographic condition
Waters Alliance 2695 type high performance liquid chromatographs are equipped with vacuum degassing machine, the quaternary gradient pump, and automatic sampler, 996 type diode array (PDA) detecting devices (U.S. Waters company, Milford, MA, USA).The control of chromatographic fractionation system and record are by Waters Millinium
32Chromatographic work station is finished.
That chromatographic column is selected for use is Kromasil C
18(Jiangsu Hanbang Sci. ﹠ Tech. Co., Ltd.), 5 μ m, 150 * 4.6mm (I.D.).Moving phase: methyl alcohol: water=60: 40 (volume ratio); Flow velocity: 1.0mL/min; Column temperature: 30 ℃, detecting device: UV 254nm.
Reagent and medicine:
N-(2-chloro-4-pyridine)-N '-phenylurea (CPPU), 4-amino-2-chloropyridine (ACP) and N, N '-acardite (DPU) reference substance is provided by luxuriant auxiliary reagent factory, Jintan City, Jiangsu Province.All reference substances all pass through infrared spectrum (IR), mass spectrum (MS), nuclear magnetic resoance spectrum (
1H NMR) characterize, and compare with the standard spectrogram, structure obtains confirming.Methyl alcohol (HPLC level) is available from Jiangsu Hanbang Sci. ﹠ Tech. Co., Ltd..Moving phase and solution with water are the quartzy distilled water of secondary in the experiment.
The selection of embodiment 1. chromatographic conditions
1.1 moving phase
We compare different moving phase compositions, test from high to lower in the methyl alcohol ratio, and be under the condition of 60: 40 (volume ratio) in methyl alcohol-water ratio, each composition can reach baseline separation, sees Fig. 1.
1.2 detection wavelength
Carry out continuous sweep with diode array detector at 200-400nm, ACP has absorption maximum at the 244nm place as can be known, and DPU and CPPU have stronger absorption at the 254nm place.Take into account the sensitivity of the two mensuration, selecting to detect wavelength is 254nm.
1.3 sample concentration
In order to take into account the mensuration of major component content and impurity content, when measuring impurity, the concentration of sample must be bigger, is enough to make impurity can go out the peak; When measuring major component content, solution concentration too conference causes the main peak ultralinear and makes quantitatively inaccurately, so measure behind 50 times of the diluted samples, it is satisfactory to test accuracy (seeing Table 1).
The preparation of standard solution: accurately take by weighing 100.00mg CPPU reference substance, place the 100mL volumetric flask, with moving phase dissolving and be diluted to scale, shake up, getting concentration is the standard reserving solution of 1.0mg/mL, and precision pipettes an amount of storing solution and is mixed with series standard solution with moving phase respectively.
Accurately take by weighing 50.00mg ACP reference substance and 50.00mg DPU reference substance, place the 100mL volumetric flask respectively, add moving phase dissolving and rare to scale, shake up, getting concentration is the storing solution of 0.50mg/mL.Precision pipettes 10.00ml ACP and 5.00ml DPU storing solution adds in the same 100mL volumetric flask, adds moving phase dissolving and rare to scale, shakes up, and getting concentration is the hybrid standard storing solution that ACP and DPU concentration are respectively 0.05mg/mL, 0.025mg/mL.Stepwise dilution gets series standard solution again.
Be respectively 0.0001～0.06mg/mL and 0.00005～0.03mg/mL at ACP and DPU concentration of standard solution scope, get and mix reference substance solution 5 μ L sample sizes, to concentration mapping (seeing Fig. 3 and 4), regression equation is respectively with peak area:
A
ACP(area)＝-445.90292+1.39318E7 C
ACP，r＝0.99911；
A
DPU(area)＝75.39214+4.32183E7 C
DPU，r＝0.99970
When CPPU concentration of standard solution scope is 0.0001～0.4mg/mL, get 10 μ L solution sample sizes, to the concentration (see figure 2) of mapping, regression equation is with peak area:
A
CPPU(area)＝-13313.94609+6.88958E7 C
CPPU，r＝0.99944
When signal to noise ratio (S/N ratio) S/N=3, record ACP, DPU, the lowest detectable limit of CPPU is respectively 0.00001mg/mL, 0.000005mg/mL, 0.00001mg/mL.
3.1 main Determination on content
The preparation of sample solution: accurately take by weighing about 100mg CPPU sample, place the 100mL volumetric flask, add the 10mL dissolve with methanol, add moving phase again, shake up standby to scale.Pipette the above-mentioned solution of 2.0mL and place the 100mL volumetric flask, add moving phase, shake up standby to scale.
Select the sample of any 5 lot numbers, prepare sample solution as stated above, carry out HPLC and analyze under optimum test condition, sample size is 10 μ L (see figure 5)s.
3.2 impurity determination
The preparation of sample solution: accurately take by weighing about 100mg CPPU sample, place the 100-mL volumetric flask, add the 10mL dissolve with methanol, add moving phase again, shake up standby to scale.
Select the sample of any 5 lot numbers, prepare sample solution as stated above, carry out HPLC and analyze under optimum test condition, sample size is 5 μ L (see figure 6)s.
The testing result of major component CPPU and impurity sees Table 1 in the sample
Table 1. testing result
| Lot number | CPPU | ACP | DPU | |||
| Content (%) | R.S.D(％) | Content (%) | R.S.D (％) | Content (%) | R.S.D(％) | |
| 1 | 99.71 | 0.68 | 0.274 | 1.05 | 0.00823 | 3.96 |
| 2 | 99.59 | 0.22 | 0.274 | 1.02 | 0.00876 | 2.10 |
| 3 | 99.66 | 0.27 | 0.278 | 1.10 | 0.00889 | 2.22 |
| 4 | 99.72 | 0.57 | 0.274 | 0.58 | 0.00893 | 1.07 |
| 5 | 99.68 | 0.21 | 0.274 | 0.64 | 0.00873 | 1.37 |
Claims (2)
1. the high efficiency liquid phase chromatographic analysis method of forchlorfenuron and impurity thereof is characterized in that adopting reversed-phased high performace liquid chromatographic, and high-efficient liquid phase chromatogram condition is:
Chromatographic column: C
18, 5 μ m, I.D. are 150 * 4.6mm,
Moving phase is formed volume ratio: methyl alcohol: water=60: 40,
Flow velocity: 1.0mL/min,
Column temperature: 30 ℃,
Detect wavelength: UV 254nm,
Sample size: 5-10 μ L.
2. the high efficiency liquid phase chromatographic analysis method of forchlorfenuron according to claim 1 and impurity thereof is characterized in that and will measure 50 times of content of measuring forchlorfenuron of sample introduction sample concentration dilution of impurity content.
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| CN200410014128.XA CN1227528C (en) | 2004-02-23 | 2004-02-23 | High-efficient liquid phase chromatographic analysing method for regulating pyrurine and its impurity |
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| CN200410014128.XA CN1227528C (en) | 2004-02-23 | 2004-02-23 | High-efficient liquid phase chromatographic analysing method for regulating pyrurine and its impurity |
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| CN1227528C true CN1227528C (en) | 2005-11-16 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293202B (en) * | 2013-05-29 | 2015-06-17 | 扬州大学 | Method for rapidly detecting forchlorfenuron residue in fruit juice |
| CN103604846A (en) * | 2013-11-19 | 2014-02-26 | 大连杰信生物科技有限公司 | Rapid detection method for expanding agent |
| CN104926718B (en) * | 2015-04-03 | 2017-03-01 | 青岛科技大学 | A kind of forchlorfenuron sulfonate and application thereof |
| CN104865334B (en) * | 2015-05-19 | 2016-04-20 | 山东省产品质量检验研究院 | A kind of method detecting growth regulator in foliar fertilizer |
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