CN114200050B - HPLC detection method for content of related substances in p-bromoanisole - Google Patents
HPLC detection method for content of related substances in p-bromoanisole Download PDFInfo
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- QJPJQTDYNZXKQF-UHFFFAOYSA-N 4-bromoanisole Chemical compound COC1=CC=C(Br)C=C1 QJPJQTDYNZXKQF-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 25
- 239000000126 substance Substances 0.000 title claims abstract description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 102
- HTDQSWDEWGSAMN-UHFFFAOYSA-N 1-bromo-2-methoxybenzene Chemical compound COC1=CC=CC=C1Br HTDQSWDEWGSAMN-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000000243 solution Substances 0.000 claims abstract description 39
- GZFGOTFRPZRKDS-UHFFFAOYSA-N 4-bromophenol Chemical compound OC1=CC=C(Br)C=C1 GZFGOTFRPZRKDS-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000013558 reference substance Substances 0.000 claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000012488 sample solution Substances 0.000 claims abstract description 11
- 238000002347 injection Methods 0.000 claims abstract description 9
- 239000007924 injection Substances 0.000 claims abstract description 9
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 9
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 9
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000741 silica gel Substances 0.000 claims abstract description 3
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 3
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 3
- 239000002904 solvent Substances 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 238000010828 elution Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 5
- 238000003908 quality control method Methods 0.000 abstract description 2
- 230000002349 favourable effect Effects 0.000 abstract 1
- 239000012535 impurity Substances 0.000 description 12
- 239000011550 stock solution Substances 0.000 description 12
- 238000007865 diluting Methods 0.000 description 8
- 239000012071 phase Substances 0.000 description 7
- 238000012417 linear regression Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005251 capillar electrophoresis Methods 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000004853 microextraction Methods 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000004761 fibrosis Effects 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 229930185605 Bisphenol Natural products 0.000 description 1
- -1 Bisphenol compound Chemical class 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 201000009594 Systemic Scleroderma Diseases 0.000 description 1
- 206010042953 Systemic sclerosis Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000275 quality assurance Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012795 verification Methods 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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Abstract
The invention discloses an HPLC detection method for the content of related substances in p-bromoanisole, which comprises the steps of respectively injecting sample solution and reference substance solution, detecting the content of p-bromophenol and o-bromoanisole in the p-bromoanisole by an HPLC method, wherein the HPLC chromatographic conditions are as follows: chromatographic column: octadecyl bonded silica gel chromatographic column; mobile phase: taking 0.01mol/L sodium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; eluting; column temperature: 25-40 ℃; flow rate: 1.0ml/min; sample injection volume: 1-20 mul; a detector: UV detector: 210-230nm. The HPLC detection method has the advantages of good system applicability, detection limit, quantitative limit, durability, precision, accuracy and recovery rate, is favorable for quality control of the bromoanisole, and can better ensure the quality of the N-aryl pyridone medicines.
Description
Technical Field
The invention belongs to the field of medicine detection and analysis, and particularly relates to a high performance liquid chromatography method for detecting substances related to bromoanisole.
Background
The N-aryl pyridone is mainly a fibrosis inhibitor and is used for treating fibrosis, systemic sclerosis, sarcoidosis and allergic pneumonia. Para bromoanisole is an important starting material for synthesizing N-aryl pyridone medicines. Various reaction byproducts may be generated during the synthesis of p-bromoanisole. These reaction byproducts can participate in subsequent synthesis together with the p-bromoanisole, thereby generating new impurities, affecting the quality of N-aryl pyridone medicines and even affecting the medication safety of patients. Therefore, a detection method of related substances of the p-bromoanisole is established, and the impurity level of the p-bromoanisole is effectively controlled, so that the quality of the N-aryl pyridone medicines is ensured, and the method has very important significance.
The analytical methods of p-bromophenol and p-bromoanisole include capillary electrophoresis (reference [1] Wang Yufang. Capillary electrophoresis for detecting 2-aminophenol, p-bromophenol and Hg 2+ [ D ] Anqing, university of the teachers in natural water), surfactant-assisted-coagulation-floating dispersion liquid microextraction (SA-DLLME-SFO)/high performance liquid chromatography (reference [2] Guo Yayun, ding Yan, hu Xiaoya, etc.. Surfactant-assisted-coagulation-floating dispersion liquid microextraction/high performance liquid chromatography for measuring phenolic compounds [ J ] in environmental water samples, analytical test report, 2015 (09): 68-72), high Performance Liquid Chromatography (HPLC) (reference [3] Guan Gongfang. Bisphenol compound synthesis process research with specific structure, university of the college of general chemical industry, 2013), etc. In the HPLC method, the bromoanisole is only used as an internal standard for detection, and the sensitivity is not examined. Surfactant assisted-coagulation-floating dispersion liquid microextraction (SA-DLLME-SFO)/high performance liquid chromatography to measure p-bromophenol, the pretreatment is complex. The capillary electrophoresis method is used for measuring the p-bromophenol, the pretreatment is complex, and the sensitivity is not high. At present, no literature reports about analysis methods of p-bromophenol and o-bromoanisole in the p-bromoanisole exist, and no literature reports about analysis methods for detecting the o-bromoanisole.
Therefore, the quality of the p-bromophenol and the o-bromoanisole in the p-bromoanisole is better controlled for efficiently detecting the p-bromophenol and the o-bromoanisole in the p-bromoanisole. The invention provides a method for measuring p-bromophenol and o-bromoanisole in p-bromoanisole by high performance liquid chromatography with good separation effect, low detection limit and high sensitivity.
Disclosure of Invention
Aiming at the technical problems, the invention aims to establish an HPLC detection method for the substances related to the bromoanisole, which has good separation effect, low detection limit and high sensitivity.
The detection method of the invention comprises the following steps:
(1) Preparing a sample solution: dissolving p-bromoanisole with a solvent to obtain a sample solution;
(2) Preparing a reference substance solution: mixing p-bromophenol and o-bromoanisole with a solvent to obtain a reference substance solution;
(3) High performance liquid chromatograph detects: sample solution and reference substance solution are respectively injected, the content of p-bromophenol and o-bromoanisole in the p-bromoanisole is detected by HPLC method,
Wherein the HPLC chromatographic conditions are:
chromatographic column: octadecyl bonded silica gel chromatographic column;
mobile phase: taking 0.01mol/L sodium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; eluting;
column temperature: 25-40 ℃;
Flow rate: 1.0ml/min;
sample injection volume: 1-20 mu l
A detector: UV detector: 210-230nm.
Preferably, the chromatographic column is YMC-Triart C, 250X 4.6mm X5 μm.
Preferably, the mobile phase elution mode is gradient elution.
Further preferably, the gradient elution procedure is:
| Time (minutes) | Acetonitrile (vol%) | 0.01Mol/L sodium dihydrogen phosphate solution (vol%) |
| 0 | 80 | 20 |
| 5 | 50 | 50 |
| 6 | 80 | 20 |
| 20 | 80 | 20 |
Preferably, the detection wavelength of the UV detector is 215nm.
Preferably, the column temperature is 40 ℃.
Preferably, the solvent is acetonitrile: water=80:20 (volume).
The invention provides a method for measuring related substances in p-bromoanisole for the first time, and the method can be used for rapidly and accurately detecting the contents of p-bromophenol and o-bromoanisole impurities in the p-bromoanisole. The HPLC detection method has the advantages of good system applicability, detection limit, quantitative limit, durability, precision, accuracy and recovery rate. The invention is beneficial to the quality control of the bromoanisole and the quality assurance of the N-aryl pyridone medicines.
Drawings
FIG. 1 is a specific chromatogram of a substance related to bromoanisole provided by the invention.
FIG. 2 is a solution chromatogram of the detection limit of p-bromophenol and o-bromoanisole provided by the invention.
FIG. 3 is a quantitative limiting solution chromatogram of p-bromophenol and o-bromoanisole provided by the invention.
FIG. 4 is a linear diagram of p-bromoanisole.
Fig. 5 is a linear diagram of o-bromoanisole.
FIG. 6 is a linear plot of p-bromophenol.
Detailed Description
The invention will be further described with reference to examples of the invention, which are given for illustrative purposes only and are not to be construed as limiting the invention.
No relevant literature report is found in the invention. By optimizing the mobile phase composition and ratio, the column Wen Dengse spectral conditions determine the optimal chromatographic conditions for detecting the relevant substances in p-bromoanisole. When the mobile phase is selected to be acetonitrile and water for isocratic detection, the peak has slight tailing, and when acetonitrile and water are used for gradient detection, the peak width is too wide, and the peak has slight tailing. When the aqueous phase was 0.05mol/L ammonium acetate (ph=5) or 0.5% acetic acid, both gradient and isocratic, baseline fluctuations, peak tailing, and less separation occurred. When the water phase is 0.01mol/L sodium dihydrogen phosphate aqueous solution isocratic detection, the baseline is stable, but the peak is trailing; when the aqueous phase is 0.01mol/L sodium dihydrogen phosphate aqueous solution gradient detection, the peak shape is basically symmetrical, the separation degree is higher, but the peak width is wider; when the column temperature is 40 ℃ and the aqueous phase is 0.01mol/L sodium dihydrogen phosphate aqueous solution gradient detection, the peak width is proper, the peak shape is basically symmetrical, and the separation degree is higher. By optimization, the final selected chromatographic conditions were as follows:
Chromatographic conditions:
Gradient elution procedure:
| Time (minutes) | Acetonitrile% | Water (0.01 mol/L sodium dihydrogen phosphate)% |
| 0 | 80 | 20 |
| 5 | 50 | 50 |
| 6 | 80 | 20 |
| 20 | 80 | 20 |
1. Detecting instrument and chromatographic conditions
Instrument: agilent high performance liquid chromatograph (HPLC 1260)
Chromatographic column: YMC-Triart C; (250X 4.6mm X5 μm)
2. Sample solution preparation: about 25mg of p-bromoanisole was weighed precisely, placed in a 50ml measuring flask, diluted to scale with solvent (acetonitrile: water=80:20), and shaken well.
Preparing a reference substance solution: respectively weighing 25mg of p-bromophenol and o-bromoanisole, precisely weighing, placing into a 100ml measuring flask, diluting to scale with solvent (acetonitrile: water=80:20), and shaking; 1ml of this solution was removed and placed in a 100ml measuring flask, diluted to scale with solvent (acetonitrile: water=80:20) and shaken well.
3. And (3) sample injection detection: and precisely measuring 20 mu l of each of the sample solution and the reference substance solution, and respectively injecting samples.
4. Detection condition verification
Preparing an impurity reference stock solution: taking 25mg of each of the o-bromoanisole reference substance and the p-bromophenol reference substance, precisely weighing, placing into a 20ml measuring flask, dissolving with a solvent (acetonitrile: water=80:20), diluting to scale, and shaking uniformly; precisely 5ml was measured, placed in a 50ml measuring flask, dissolved with solvent (acetonitrile: water=80:20) and diluted to scale, and shaken well.
(1) System applicability
Precisely weighing 25mg of p-bromoanisole, placing in 50ml, adding solvent (acetonitrile: water=80:20), dissolving and diluting to scale, and shaking; precisely measuring 0.5ml, placing in a 100ml measuring flask, diluting to scale with solvent, and shaking. The sample injection was repeated 6 times. The peak areas RSD of the 6 sample injections are respectively 0.18%, and the system applicability is good.
| Numbering device | 1 | 2 | 3 | 4 | 5 | 6 | Average peak area | RSD(％) |
| Peak area | 102.09 | 101.94 | 102.07 | 101.96 | 101.80 | 101.59 | 101.91 | 0.18 |
(2) Specialization of
Blank solution: solvent (acetonitrile: water=80:20)
Para-bromophenol positioning solution: proper amount of p-bromophenol is taken, precisely weighed, dissolved and diluted with a solvent (acetonitrile: water=80:20) to prepare a solution containing 2.5 μg of p-bromophenol per 1 ml.
O-bromoanisole positioning solution: proper amount of o-bromoanisole is taken, precisely weighed, dissolved and diluted by a solvent (acetonitrile: water=80:20) to prepare a solution with 2.5 mug of o-bromoanisole per 1 ml.
Sample solution: 25mg of p-bromoanisole is taken, precisely weighed, placed in a 50ml measuring flask, dissolved and diluted to a scale by adding a solvent (acetonitrile: water=80:20), and shaken well.
Control solution: 1ml of the impurity control stock solution is precisely measured, placed in a 50ml measuring flask, diluted to a scale with a solvent (acetonitrile: water=80:20), and shaken well.
Mixing solution: about 25mg of p-bromoanisole is taken, precisely weighed, 1ml of impurity reference substance stock solution is precisely added, the mixture is placed in a 50ml measuring flask, diluted to a scale with solvent (acetonitrile: water=80:20), and uniformly shaken.
20 Mu l of the solution is taken for sample injection, the blank solution has no interference to detection, the separation degree between the bromoanisole and adjacent impurities is 3.51, and the specificity is good. The detection map is shown in figure 1.
(3) Detection limit
P-bromophenol detection limit: the p-bromophenol control is weighed precisely, dissolved and diluted with a solvent (acetonitrile: water=80:20) to prepare a solution containing 0.053 μg of p-bromophenol per 1ml, which is used as a detection limit solution, and the detection limit is 0.053 μg/ml (signal to noise ratio is 3).
O-bromoanisole detection limit: the o-bromoanisole reference substance is taken as a proper amount, precisely weighed, dissolved and diluted by a solvent (acetonitrile: water=80:20) to prepare a solution containing 0.006 mug of o-bromoanisole per 1ml, and the detection limit is 0.006 mug/ml (signal to noise ratio is 3).
P-bromoanisole detection limit: the appropriate amount of the p-bromoanisole reference substance is taken, precisely weighed, dissolved and diluted by a solvent (acetonitrile: water=80:20) to prepare a solution containing 0.010 mug of p-bromoanisole per 1ml, and the detection limit is 0.010 mug/ml (signal to noise ratio is 3).
The detection limit diagram of the p-bromophenol and the o-bromoanisole is shown in figure 2.
(4) Quantitative limit
Para-bromophenol quantitative limit: proper amount of p-bromophenol is taken, precisely weighed, dissolved and diluted with a solvent (acetonitrile: water=80:20) to prepare a solution containing 0.11 μg of p-bromophenol per 1ml, which is used as a quantitative limit solution, and the quantitative limit is 0.11 μg/ml (signal to noise ratio is 10).
O-bromoanisole quantitative limit: proper amount of o-bromoanisole is precisely weighed, and a solvent (acetonitrile: water=80:20) is dissolved and diluted to prepare a solution containing 0.21 mug of o-bromoanisole per 1ml, wherein the solution is used as a quantitative limit solution, and the quantitative limit is 0.21 mug/ml (signal to noise ratio is 10).
Quantitative limit of p-bromoanisole: proper amount of p-bromoanisole is taken, precisely weighed, dissolved and diluted by a solvent (acetonitrile: water=80:20) to prepare a solution containing 0.040 mug of p-bromoanisole per 1ml, and the quantitative limit is 0.040 mug/ml (signal-to-noise ratio is 10).
The quantitative limit diagram of the p-bromophenol and the o-bromoanisole is shown in figure 3.
(5) Linearity of
Para bromoanisole linearity: taking a proper amount of p-bromoanisole reference substances, respectively quantitatively diluting to prepare 5.00μg/ml、3.75μg/mll、3.00μg/ml、2.50μg/ml、2.00μg/ml、1.50μg/ml、1.00μg/ml、0.50μg/ml、0.25μg/ml、0.20μg/ml、0.04μg/ml,, and precisely measuring 20 mu l of sample injection. A linear regression curve was established with the concentration on the abscissa and the peak area on the ordinate, with the linear regression equation y= 34.4121x-0.3767 and r 2 =0.9994.
O-bromoanisole linearity: taking a proper amount of o-bromoanisole reference substances, respectively quantitatively diluting to prepare 5.00μg/ml、3.75μg/mll、3.00μg/ml、2.50μg/ml、2.00μg/ml、1.50μg/ml、1.00μg/ml、0.50μg/ml、0.25μg/ml、0.20μg/ml,, and precisely measuring 20 mu l of sample injection. A linear regression curve was established with the concentration on the abscissa and the peak area on the ordinate, with the linear regression equation y= 55.2522x-0.7624 and r 2 = 0.9992.
Linearity of p-bromophenol: taking a proper amount of p-bromophenol reference substances, respectively quantitatively diluting to prepare 5.00μg/ml、3.75μg/mll、3.00μg/ml、2.50μg/ml、2.00μg/ml、1.50μg/ml、1.00μg/ml、0.50μg/ml、0.25μg/ml、0.20μg/ml、0.11μg/ml,, and precisely measuring 20 mu l of each sample. Establishing a linear regression curve with the concentration as an abscissa and the peak area as an ordinate, wherein a linear regression equation is y= 35.2327x-0.8721; r2=0.9998.
(6) Precision of
About 25mg of p-bromoanisole is taken, precisely weighed, 1ml of impurity reference substance stock solution is precisely added, the mixture is placed in a 50ml measuring flask, diluted to a scale with solvent (acetonitrile: water=80:20), and uniformly shaken. 6 parts were prepared in parallel. 20 μl of each sample was measured precisely, and the chromatogram was recorded. The RSD of the 6-time measurement results of the p-bromophenol is 0.73%; the RSD of the o-bromoanisole 6 times measurement result is 0.46%.
At different times, another operator takes about 25mg of p-bromoanisole, precisely measures, precisely adds 1ml of impurity reference substance stock solution, places the stock solution into a 50ml measuring flask, dilutes the stock solution to scale with solvent (acetonitrile: water=80:20), and shakes the stock solution uniformly. 6 parts were prepared in parallel. 20 μl of each sample was measured precisely, and the chromatogram was recorded. The RSD of 6 times of measurement results of the p-bromophenol is 0.18%, and the RSD of 12 times of measurement results of the p-bromophenol is 0.65%; the RSD of the o-bromoanisole 6 times is 0.16%, and the RSD of the o-bromoanisole 12 times is 0.71%.
(7) Accuracy of
About 25mg of p-bromoanisole is taken, precisely weighed, 0.3ml, 1ml and 1.2ml of impurity reference substance stock solution are respectively precisely added, the stock solution is respectively placed in 50ml measuring flasks, diluted to scale with solvent (acetonitrile: water=80:20), and uniformly shaken. 3 parts were prepared in parallel, 9 parts in total. 20 μl of each sample was measured precisely, and the chromatogram was recorded. Recovery was calculated as (measured amount-sample amount)/addition amount×100%.
The recovery rate RSD of the p-bromophenol for 9 times is 0.43%; the recovery rate RSD of the o-bromoanisole for 9 times is 2.32 percent.
(8) Solution stability
About 25mg of p-bromoanisole is taken, precisely weighed, placed in a 50ml measuring flask, diluted to a scale with a solvent (acetonitrile: water=80:20) and shaken well; accurately adding 1ml of impurity reference stock solution, placing into a 50ml measuring flask, diluting to scale with solvent (acetonitrile: water=80:20), and shaking; 20 μl of sample is measured and injected precisely for 0 day, 1 day and 3 days respectively, and the chromatogram is recorded. The RSD of the measuring result of the p-bromoanisole sample solution within 3 days is 1.01%; the RSD of the p-bromophenol control solution is 1.02% within 3 days; the RSD of the o-bromoanisole reference substance solution is 1.23% in 3 days, and the solution has good stability in 3 days.
(9) Durability of
About 25mg of p-bromoanisole is taken, precisely weighed, 1ml of impurity reference substance stock solution is precisely added, the mixture is placed in a 50ml measuring flask, diluted to a scale by a solvent (acetonitrile: water=80:20), and uniformly shaken to serve as a durability solution, and the durability condition of the method under the conditions of changing the column temperature to 25-35 ℃ and the flow rate to 0.9-1.1 ml/min is examined respectively. 20 μl of each sample was measured precisely, and the chromatogram was recorded. The RSD of the p-bromophenol determination was 7.38%; the RSD of the o-bromoanisole measurement result is 6.50%. The durability is good.
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Claims (5)
1. The HPLC detection method for the content of related substances in the p-bromoanisole is characterized by comprising the following steps:
(1) Preparing a sample solution: dissolving p-bromoanisole with a solvent to obtain a sample solution;
(2) Preparing a reference substance solution: mixing p-bromophenol and o-bromoanisole with a solvent to obtain a reference substance solution;
(3) High performance liquid chromatograph detects: sample solution and reference substance solution are respectively injected, the content of p-bromophenol and o-bromoanisole in the p-bromoanisole is detected by HPLC method,
Wherein the HPLC chromatographic conditions are:
chromatographic column: octadecyl bonded silica gel chromatographic column;
mobile phase: taking 0.01mol/L sodium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; gradient elution is carried out;
column temperature: 25-40 ℃;
Flow rate: 1.0ml/min;
Sample injection volume: 1-20 mul;
A detector: UV detector: 210-230nm;
The gradient elution procedure was:
。
2. The HPLC detection method for the content of related substances in the p-bromoanisole as in claim 1, wherein: the column was YMC-Triart C, 250X 4.6mm X5. Mu.m.
3. The HPLC detection method for the content of related substances in the p-bromoanisole as in claim 1, wherein: the detection wavelength of the UV detector is 215nm.
4. The HPLC detection method for the content of related substances in the p-bromoanisole as in claim 1, wherein: the column temperature was 40 ℃.
5. The HPLC detection method for the content of related substances in the p-bromoanisole as in claim 1, wherein: the solvent is acetonitrile: water=80:20.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2005259A1 (en) * | 1970-02-05 | 1971-08-19 | Chemische Fabrik Kalk GmbH 5000 Köln Kalk | P-bromophenol prepn using brominated sol-vent |
| US4447660A (en) * | 1980-06-03 | 1984-05-08 | Pcuk Produits Chimiques Ugine Kuhlmann | Process for the isomerization of ortho-and para-bromophenols or the ethers thereof into meta-bromophenols and corresponding ethers |
| GB8729738D0 (en) * | 1986-12-23 | 1988-02-03 | Bromine Compounds Ltd | Process for selective para-bromination of phenol & its derivatives |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6982356B2 (en) * | 2003-09-17 | 2006-01-03 | General Electric Company | Method for preparation of para-brominated hydroxyaromatic compounds |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2005259A1 (en) * | 1970-02-05 | 1971-08-19 | Chemische Fabrik Kalk GmbH 5000 Köln Kalk | P-bromophenol prepn using brominated sol-vent |
| US4447660A (en) * | 1980-06-03 | 1984-05-08 | Pcuk Produits Chimiques Ugine Kuhlmann | Process for the isomerization of ortho-and para-bromophenols or the ethers thereof into meta-bromophenols and corresponding ethers |
| GB8729738D0 (en) * | 1986-12-23 | 1988-02-03 | Bromine Compounds Ltd | Process for selective para-bromination of phenol & its derivatives |
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