CN104407066B - A kind of retigabine has the analysis method of related substance - Google Patents
A kind of retigabine has the analysis method of related substance Download PDFInfo
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- CN104407066B CN104407066B CN201410626271.8A CN201410626271A CN104407066B CN 104407066 B CN104407066 B CN 104407066B CN 201410626271 A CN201410626271 A CN 201410626271A CN 104407066 B CN104407066 B CN 104407066B
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- acetonitrile
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- retigabine
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- triethylamine
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Abstract
The invention discloses a kind of retigabine and have the analysis method of related substance.This method uses high performance liquid chromatography, with octadecylsilane chemically bonded silica as chromatographic column filler, use UV-detector, select suitable organic facies and buffer solution for flowing phase, use gradient elution, efficiently solve the problem separating retigabine and having related substance.This method specificity is strong, reproducible, can guarantee that the controllability having related substance in retigabine.
Description
Technical field
The invention belongs to pharmaceutical technology field, relate to the analyzing detecting method of a kind of medicine, particularly retigabine is had
The method that related substance is measured.
Background technology
There is related substance closely related with drug quality, safety and curative effect, have the existence of related substance may reduce medicine
Curative effect, even cause toxic and side effects, therefore, it is necessary to controlled the kind of Drug-related by suitable determination method
Class and content, it is ensured that drug quality.
Retigabine is a kind of new antiepileptic medicine, chemical entitled N-(2-amino-4-(4-fluorine benzyl amino) phenyl) ammonia
Base Ethyl formate, reaches epilepsy effect by acting on neuron potassium-channel, and chemical structural formula is as follows:
Through retrieval, there is no the Patents for retigabine Related substance method or open source literature report at present.
Multiple retigabine can be had related substance to detect under same chromatographic condition by the present invention, has important to its quality control
Meaning.
Summary of the invention
It is an object of the invention to build a kind of retigabine and have the analysis method of related substance, the method answers specificity good,
Possible degradation impurity and possible residual intermediate effectively can be separated with main constituent, and highly sensitive, can be applicable to auspicious replacing
Add the quality control of shore.
The present inventor, for retigabine synthesis technique and degraded situation, identifies 6 impurity, the most succinylated
Thing, intermediate, 5-fluoro-2-nitroaniline, 5-fluoro-2-nitroaniline acylate, adjacent fluorine isomer and a fluorine isomer, chemistry knot
Structure formula is as follows:
Diacylate intermediate 5-fluoro-2-nitroaniline
Fluorine isomer between 5-fluoro-2-nitroaniline acylate neighbour's fluorine isomer
The present inventor is through screening conditions such as buffer solution composition, gradient elution program, and taking into account 6 has related substance
The needs separated, finally establish suitable Related substance method, are controlled by analytical cycle at 40min, and main constituent is with each
Have between related substance, be respectively arranged with between related substance and all can reach baseline separation, reached the purpose of foregoing invention.
The present invention is achieved through the following technical solutions:
A kind of retigabine has the analysis method of related substance, it is characterised in that used determination method is:
Use high performance liquid chromatography, with octadecylsilane chemically bonded silica as chromatographic column filler, use UV-detector,
Select suitable organic solvent and buffer solution for flowing phase, gradient elution；Described organic solvent is acetonitrile；Described buffer solution
For triethylamine aqueous solution, and triethylamine concentration is 0.05%-0.15% by volume, and with phosphorus acid for adjusting pH value to 2.0 ~ 3.0；Ladder
Degree elution program is 0～10min, and acetonitrile ratio is 27%, 10～20min, and acetonitrile ratio becomes 50% from 27%, 20～30min,
Acetonitrile ratio is 50%, 30～30.1min, and acetonitrile ratio becomes 27% from 50%, 30.1～40min, and acetonitrile ratio is 27%.
Preferably testing conditions: detection wavelength is 220nm；Flow velocity is 1.0ml/min；Sample size is 20 μ l；Flowing three second mutually
The concentration of amine aqueous solution is 0.1%, and pH value is 2.5；Sample preparation method is: weigh appropriate retigabine, adds a small amount of acetonitrile and makes molten
Solve, then with acetonitrile-0.1% triethylamine solution (with phosphorus acid for adjusting pH value to 2.5) (27:73) dilution, be configured to containing retigabine dense
Degree is the solution of 0.5mg/ml.
The Method validation of chromatographic process
1. there is related substance location test
Preparation retigabine sample solution, it is respectively arranged with related substance contrast solution, retigabine and to be respectively arranged with the mixing of related substance molten
Liquid, is injected separately into chromatograph of liquid, records chromatogram.The results are shown in Table 1, accompanying drawing 1 is shown in by collection of illustrative plates
Table 1 retigabine and each magazins' layout parametric results
Result shows: blank solvent peak is noiseless to measurement result, and retigabine is respectively arranged with between related substance and main constituent, respectively
Have between related substance and be attained by good separation；
2 detection limits, quantitative limit test
Signal to noise ratio method is used to measure retigabine and be respectively arranged with detection limit and the quantitative limit of related substance.Prepare auspicious for adding respectively
Shore and be respectively arranged with the stock solution of related substance, is diluted to finite concentration sample introduction, calculates the ratio (signal to noise ratio) of peak height and noise, its
Signal to noise ratio (S/N) is about the sample detection amount of 10 and is quantitative limit, and signal to noise ratio (S/N) is about the sample detection amount of 3 and is detection
Limit, the results are shown in Table 2
Table 2 quantitative limit and detection limit result of the test
Result shows: this method is high to the response being respectively arranged with related substance, can accurately control to be respectively arranged with the content of related substance；
3 linear tests
Precision weigh retigabine and 6 have related substance, add a small amount of acetonitrile and make dissolving, then with acetonitrile-0.1% triethylamine
Solution (with phosphorus acid for adjusting pH value to 2.5) (27:73) dilution, is configured to the solution of 7 parts of variable concentrations.Sample introduction record color respectively
Spectrogram, with concentration as abscissa, peak area is vertical coordinate, obtains retigabine and is respectively arranged with the equation of linear regression of related substance,
The results are shown in Table 3
Table 3 linear test result
Result shows: under this method, and retigabine and 6 have related substance in the range of finite concentration, all can present
Good is linear；
4 recovery tests
Precision weighs appropriate retigabine, adds a small amount of acetonitrile and makes dissolving, then (uses phosphoric acid with acetonitrile-0.1% triethylamine solution
Regulation pH value is to 2.5) (27:73) dilution, it is configured to containing the solution that retigabine concentration is 0.5mg/ml 9 parts, three parts is one group,
Related substance is had in right amount respectively so that being respectively arranged with the concentration of related substance in three groups of solution, to be respectively 0.5 μ g/ml(relative to each group of addition
The 50% of limit), 1.0 μ g/ml(are relative to the 100% of limit) with 1.5 μ g/ml(relative to the 150% of limit).Sample introduction also records chromatograph
Figure, calculates the response rate, the results are shown in Table 4
Table 4 recovery test result
Result shows: this method accuracy is good；
5 destructive testings
Precision weighs appropriate retigabine, breaks it respectively under the conditions of strong acid, highly basic, high temperature, illumination, oxidation etc.
Bad test, sample introduction also records chromatogram, the impurity situation that statistics is destroyed out, the results are shown in Table 5
Table 5 failure test impurity situation of change conclusive table
Conclusion: this method can be good at detecting the catabolite that retigabine destructive testing produces, material balance
Rate is all between 90%～110%, and degradation impurity meets the requirements with main peak separating degree, and main peak spectral purity the most all meets the requirements.Cause
This, can use above-mentioned chromatographic system to have related substance and catabolite thereof to measure in retigabine.
Accompanying drawing explanation
Accompanying drawing 1: retigabine has related substance location test contrast color spectrogram.
Specific embodiment
Following example are used for being further appreciated by the present invention, but are not limited to the scope of the present embodiment
Three batches of retigabine crude drug of embodiment have the mensuration of related substance
Precision weighs 3 crowdes of each 50mg of retigabine, is respectively placed in 100ml measuring bottle, adds a small amount of acetonitrile and makes dissolving, then uses second
Nitrile-0.1% triethylamine solution (with phosphorus acid for adjusting pH value to 2.5) (27:73) is diluted to scale, shakes up, obtains need testing solution.
Precision measures need testing solution 1ml, puts in 100ml measuring bottle, with acetonitrile-0.1% triethylamine solution (with phosphorus acid for adjusting pH value extremely
2.5) (27:73) is diluted to scale, shakes up, and obtains contrast solution；
Chromatographic condition: using Shimadzu high performance liquid chromatograph, UV-detector, chromatographic column filler is octadecylsilane key
Close silica gel (model is 150mm × 4.6mm × 5 m)；With 0.1% triethylamine solution (with phosphorus acid for adjusting pH value to 2.5) for flowing phase
A, with acetonitrile as Mobile phase B, gradient elution program is 0～10min, and acetonitrile ratio is 27%, 10～20min, acetonitrile ratio from
27% becomes 50%, 20～30min, acetonitrile ratio is 50%, 30～30.1min, and acetonitrile ratio becomes 27% from 50%, 30.1～
40min, acetonitrile ratio is 27%；Detection wavelength is 220nm；Flow velocity is 1.0ml/min；
Precision measures contrast solution and each 20 μ l of need testing solution, is injected separately in chromatograph of liquid, and sample introduction also records color
Spectrogram, uses the Self-control method of the correction up factor to calculate the content being respectively arranged with related substance, the results are shown in Table 6
6 three batches of retigabine crude drug of table have the measurement result of related substance
Claims (3)
1. a retigabine has the analysis method of related substance, it is characterised in that:
Use high performance liquid chromatography, with octadecylsilane chemically bonded silica as chromatographic column filler, use UV-detector, select
Suitable organic solvent and buffer solution are flowing phase, gradient elution；
Described organic solvent is acetonitrile, and described buffer solution is triethylamine aqueous solution, and triethylamine concentration is by volume
0.05%-0.15%, and with phosphorus acid for adjusting pH value to 2.0～3.0, gradient elution program is 0～10min, and acetonitrile ratio is
27%, 10～20min, acetonitrile ratio becomes 50% from 27%, and 20～30min, acetonitrile ratio is 50%, 30～30.1min, second
Nitrile ratio becomes 27% from 50%, and 30.1～40min, acetonitrile ratio is 27%；
Described retigabine has related substance to be following 6 kinds:
Diacylate；
Intermediate；
5-fluoro-2-nitroaniline；
5-fluoro-2-nitroaniline acylate；
Adjacent fluorine isomer；
Between fluorine isomer.
2. Related substance method as claimed in claim 1, it is characterised in that detection wavelength is 210nm～240nm；Flow velocity
It is 0.8～1.3ml/min；Sample size is 5～50 μ l；In buffer solution, triethylamine concentration is 0.1%, and pH value is 2.5.
3. Related substance method as claimed in claim 1, it is characterised in that sample preparation method is: weigh the most auspicious replacing
Add shore, add a small amount of acetonitrile and make dissolving, then dilute with the mixed solution of acetonitrile and 0.1% triethylamine, in this mixed solution, triethylamine
The pH value of solution phosphoric acid regulates to 2.5, and acetonitrile is 27:73 with the ratio of triethylamine solution, is configured to containing retigabine concentration
Solution for 0.5mg/ml.
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Non-Patent Citations (5)
| Title |
|---|
| HPLC测定瑞替加滨的含量;黄超等;《中国现代应用药学》;20121031;第29卷(第10期);950-952 * |
| Identification and characterization of four process-related impurities in retigabine;Xiangjun Wang等;《Journal of Pharmaceutical and Biomedical Analysis》;20120821;第71卷;148-151 * |
| Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine;Michal Douša等;《Journal of Pharmaceutical and Biomedical Analysis》;20140203;第94卷;71-76 * |
| 新型抗癫痫药瑞替加滨的遗传毒性杂质的控制;刁荣蓉等;《首都医药》;20140930;79-80 * |
| 色谱及其联用技术在药物杂质分析中的应用;杜薇等;《海峡药学》;20131231;第25卷(第5期);1-5 * |
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