CN109884201A - Determination of Impurities of Repaglinide in Repaglinide and Metformin Tablets by Detector Combined Method - Google Patents
Determination of Impurities of Repaglinide in Repaglinide and Metformin Tablets by Detector Combined Method Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 103
- 229960002354 repaglinide Drugs 0.000 title claims abstract description 80
- FAEKWTJYAYMJKF-QHCPKHFHSA-N GlucoNorm Chemical compound C1=C(C(O)=O)C(OCC)=CC(CC(=O)N[C@@H](CC(C)C)C=2C(=CC=CC=2)N2CCCCC2)=C1 FAEKWTJYAYMJKF-QHCPKHFHSA-N 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 44
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 229960003105 metformin Drugs 0.000 title claims abstract 6
- 239000012488 sample solution Substances 0.000 claims abstract description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 14
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 7
- 230000005284 excitation Effects 0.000 claims abstract description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000945 filler Substances 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
- 238000002347 injection Methods 0.000 claims abstract 3
- 239000007924 injection Substances 0.000 claims abstract 3
- 239000000243 solution Substances 0.000 claims description 27
- 239000013558 reference substance Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- 238000005259 measurement Methods 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000012088 reference solution Substances 0.000 claims 2
- 239000003814 drug Substances 0.000 abstract description 5
- 238000004587 chromatography analysis Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- OETHQSJEHLVLGH-UHFFFAOYSA-N metformin hydrochloride Chemical compound Cl.CN(C)C(=N)N=C(N)N OETHQSJEHLVLGH-UHFFFAOYSA-N 0.000 description 21
- 150000001875 compounds Chemical class 0.000 description 14
- 239000000843 powder Substances 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- FTCMVLQJMIXDSI-VWLOTQADSA-N ethyl 2-ethoxy-4-[2-[[(1s)-3-methyl-1-(2-piperidin-1-ylphenyl)butyl]amino]-2-oxoethyl]benzoate Chemical compound C1=C(OCC)C(C(=O)OCC)=CC=C1CC(=O)N[C@@H](CC(C)C)C1=CC=CC=C1N1CCCCC1 FTCMVLQJMIXDSI-VWLOTQADSA-N 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 229960004329 metformin hydrochloride Drugs 0.000 description 6
- -1 Metformin hydrochloride compound Chemical class 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 229940123208 Biguanide Drugs 0.000 description 4
- 150000004283 biguanides Chemical class 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
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- 239000002994 raw material Substances 0.000 description 4
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- 238000006731 degradation reaction Methods 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
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- 238000005303 weighing Methods 0.000 description 3
- RTGDFNSFWBGLEC-TVPGTPATSA-N 2-morpholin-4-ylethyl (z)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1h-2-benzofuran-5-yl)-4-methylhex-4-enoate Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(\C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-TVPGTPATSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003914 insulin secretion Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
- YREOLPGEVLLKMB-UHFFFAOYSA-N 3-methylpyridin-1-ium-2-amine bromide hydrate Chemical compound O.[Br-].Cc1ccc[nH+]c1N YREOLPGEVLLKMB-UHFFFAOYSA-N 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N Benzoic acid Natural products OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960004365 benzoic acid Drugs 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
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- 238000012937 correction Methods 0.000 description 1
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- 230000000593 degrading effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229960002163 hydrogen peroxide Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
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- 230000003834 intracellular effect Effects 0.000 description 1
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- 239000011812 mixed powder Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical group CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
本发明属于药物分析领域，即通过一种高效液相色谱法串联两种检测器技术分离测定瑞格列奈二甲双胍片中瑞格列奈的5种杂质。色谱方法为：以辛烷基硅烷键合硅胶为填充剂；以磷酸二氢钾缓冲溶液(质量分数为0.1％～1％，pH值为2.0～8.0)‑乙腈为流动相；检测器为二极管阵列及荧光检测器串联，检测波长分别为210～310nm及激发波长200～300m、发射波长300～400nm；流速为0.5～2.0ml/min，进样体积为10～100μl，样品溶液浓度为0.01～1.0mg/ml。利用本发明方法可以准确定量计算瑞格列奈二甲双胍片中瑞格列奈的5种杂质，保证了瑞格列奈二甲双胍片的质量可控性。The invention belongs to the field of drug analysis, that is, five kinds of impurities in repaglinide in repaglinide metformin tablets are separated and determined by a high-performance liquid chromatography method with two detectors in series. The chromatography method is as follows: using octanylsilane-bonded silica gel as filler; using potassium dihydrogen phosphate buffer solution (mass fraction of 0.1% to 1%, pH value of 2.0 to 8.0)-acetonitrile as mobile phase; detector is a diode The array and the fluorescence detector are connected in series, the detection wavelength is 210-310nm, the excitation wavelength is 200-300m, and the emission wavelength is 300-400nm; the flow rate is 0.5-2.0ml/min, the injection volume is 10-100μl, and the sample solution concentration is 0.01- 1.0 mg/ml. The method of the invention can accurately and quantitatively calculate the five impurities of repaglinide in the repaglinide and metformin tablets, which ensures the quality controllability of the repaglinide and metformin tablets.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, and in particular to pass through high efficiency liquid chromatography for separating and determining compound Repaglinide
The method of Repaglinide impurity in diformin tablet.
Background technique
Repaglinide diformin tablet is Repaglinide and Metformin hydrochloride compound formulation, by Novo Nordisk Co., Ltd
(Novo Nordisk) is developed, and FDA approval is obtained on June 23rd, 2008, is listed in the U.S., trade nameIt does not list at home at present.The product is controlled for Therapeutic diet and motion exercise not can be effectively controlled high blood
The diabetes B (non-insulin-dependent) of sugar, wherein Repaglinide ingredient is Drugs Promoting Insulin Secretion, is methyl methylamine benzene
Formic acid (CMBA) derivative, by the ATP dependence K in conjunction with the specific receptor on beta Cell of islet film, promoting cell membrane+Channel is closed, and K is inhibited+From β cell drain, make membrane depolarization, so that open voltage relies on Ca2+Channel makes extracellular Ca2 +Into intracellular, promote the insulin secretion of storage.
Entitled S (+)-2- ethyoxyl-4- { 2- [(3- methyl-1-(2- (1- piperidyl) phenyl) butyl) of Repaglinide chemistry
Amino] -2- oxoethyl } benzoic acid, molecular formula C27H16N2O4, chemical structural formula r is as follows.
Repaglinide chemical structural formula
Starting material, intermediate, final product analog and the catabolite of Repaglinide in the synthesis process etc. may
As impurity, which is that diabetic takes conventional medicine for a long time, if the impurity content in product is higher, is easy to patient
Long term toxicity is generated, therefore strict control need to be carried out to the impurity in product.
Repaglinide raw material recorded in the world mainly existing National Pharmacopeia standard (European Pharmacopoeia, United States Pharmacopeia and
Chinese Pharmacopoeia), the structure and chemical formula information of the material impurities controlled are shown in Table 1.
The impurity structure and chemical formula of 1 Repaglinide of table
Impurity 1 and impurity 3 are the post-rift catabolite of Repaglinide amido bond, and impurity 2, impurity 4 are synthesising by-product,
Impurity 5 is that raw material brings impurity into, this 5 kinds of impurity will affect the product quality that the substance is prepared into after preparation, should carry out comprehensively
Control.Pharmacopoeia of each country is as follows to Repaglinide Control of Impurities situation: Chinese Pharmacopoeia version in 2015 is without known Control of Impurities, European medicine
In 9.2 editions standards of allusion quotation 5 kinds of known impurities are controlled with (wherein impurity E is optical isomer, has individual measuring method, not
Control the impurity C in USP standard), 3 kinds of known impurities of United States Pharmacopeia 42 editions control (wherein have 2 kinds of known impurities and Europe
Continent pharmacopeia is identical).Therefore, pharmacopoeia of each country cannot 5 kinds of impurity in control table 1 comprehensively.
In addition, compound Repaglinide diformin tablet currently without can standards of pharmacopoeia method for reference, and due to compound
Contain larger amount of Metformin hydrochloride ingredient (mass ratio of Metformin hydrochloride and Repaglinide is 500:1) in preparation,
To the detection of Repaglinide and its impurity there are severe jamming under the conditions of diode array detector, Repaglinide in each pharmacopeia
Standard method (being all made of diode array detector) the i.e. prior art is not particularly suited for the miscellaneous of the Repaglinide in the Compound Tablet
Quality Control system.
Patent application CN101929987B is only the detection of one of Repaglinide Material synthesis process intermediate, in this
Mesosome is identical as EP impurity C.EP impurity C belongs to degradation impurity, should be controlled during the storage of finished product." HPLC method is surveyed
Determine the related substance of Repaglinide in Repaglinide diformin tablet piece " (University Of Shenyang's journal, 2016) using European Pharmacopoeia
Chromatographic condition in 7.0 version methods only will test device and be changed to fluorescence detector, control three kinds of impurity in European Pharmacopoeia
(A, B,D)." the related substance of Repaglinide in the principal component own control hair measurement compound preparation of correction up factorization method " (drug
Analysis magazine, 2015), it is consistent using chromatographic condition and EP 7.0, Metformin hydrochloride is reduced by optimization sample solvent and is interfered,
And the measurement concentration method of sample to be tested is improved to improve sensitivity.Though this method uses acetonitrile as Solvents Solvent, hydrochloric acid two
First biguanides still has certain solubility in acetonitrile, cannot exclude its response in chromatography completely.By map in text as it can be seen that yin
Property sample solution and impurity A described in the text (i.e. impurity 1 in the present invention) to go out peak position very close, make under method specificity
Drop；In addition, the slice weight due to sample to be tested is larger, if wanting to be formulated into the sample to be tested concentration in text, need with minimal amount of molten
Liquid dissolves a large amount of solid powders, this will affect the veracity and precision of the method.
The present invention is using two kinds of detector combinations, to 5 kinds of impurity of Repaglinide in compound Repaglinide diformin tablet
It is controlled comprehensively.Compared with method in above-mentioned patent and document, this law advantage is: (1) can exclude hydrochloric acid two in compound preparation
The interference that first biguanides detects impurity 1 (Metformin hydrochloride is in fluorescence detection wherein without response)；(2) by using specific chromatography
Column realizes the separation of impurity 5 Yu Repaglinide, is controlled together, increases safety (3) fluorescence detector of sample to auspicious
Ge Lienai and its sensitivity with higher of impurity 1,2,4,5, therefore sample to be tested concentration in testing sample solution can be prepared
In reduced levels, enhance the operability of sample preparation, enhances the veracity and precision of measuring method.(4) same measured
Under concentration, the response in fluorescence detector of impurity 3 is lower, and response is relatively in diode array detector for this impurity
Height is detected in diode array detector using series diode array detector method and is controlled impurity 3.
In conclusion the method in the present invention highlights the novelty of the combination of two kinds of detectors and chromatographic column optimization, compared with
On the basis of easy sample preparation process, lower detectable concentration, it can control more comprehensively in compound preparation in lower specification component
Impurity level, enhance the safety of sample.Party's forensic science simplicity, operability and specificity are stronger, with higher
Sensitivity, accuracy and precision
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of high performance liquid chromatographies point by two kinds of detectors of connecting
Method from Repaglinide impurity in measurement compound Repaglinide diformin tablet can be to multiple with overcome the deficiencies in the prior art
The separation determination of the progress related impurities of Repaglinide in square Repaglinide diformin tablet.
The present invention is realized by following technological means.
Detector is combined the impurity of Repaglinide in method measurement Repaglinide diformin tablet, carries out in the following ways:
Using high performance liquid chromatography, chromatographic condition includes: using octyl silane group silica gel as filler；With di(2-ethylhexyl)phosphate
Hydrogen potassium buffer solution and acetonitrile are mobile phase, are separated using gradient elution；Detector is diode array and fluorescence detector string
Connection, diode array detector Detection wavelength are 210~310nm, 200~300m of excitation wavelength, the transmitted wave of fluorescence detector
Long 300~400nm；0.5~2.0ml/min of flow velocity, 10~100 μ l of sampling volume, column temperature are 20~50 DEG C.
System suitability solution is prepared: Repaglinide and each impurity mixed solution, the mixing using organic reagent and water are molten
Liquid is prepared, and wherein Repaglinide concentration is 1~100 μ g/ml, and impurity reference substance concentration is respectively 0.02~2 μ g/ml.
Impurity reference substance solution: each impurity reference substance solution is all made of organic reagent and the mixed solution of water is prepared, concentration
It is 0.02~2 μ g/ml.
Sample solution prepare: the mixed solution of sample solution organic reagent and water prepare, concentration be containing Repaglinide 1~
100μg /ml。
Measurement: above-mentioned solution is injected separately into high performance liquid chromatograph, records chromatogram.
In the method, potassium dihydrogen phosphate buffer solution mass fraction 0.1%~1% in mobile phase, pH value 2.0~8.0；
Potassium dihydrogen phosphate buffer solution and acetonitrile initial volume ratio are 90:10~70:30, and final mobile phase ratio is 10:90~30:
70。
In the method, Detection wavelength is preferably that diode array detector Detection wavelength is 240nm, fluorescence detector
Excitation wavelength be 244nm, launch wavelength 348nm.
In the method, column temperature is 40 DEG C.
In the method, flow velocity is preferably 1.0ml/min.
In the method, organic solvent described in the mixed solution of organic solvent and water is ethyl alcohol, the body of ethyl alcohol and water
Product compares 1:1.
In the method, system suitability solution contains 10 μ g/ml of Repaglinide, each 1.0 μ g/ml of impurity reference substance 1~5；
Each impurity reference substance solution distinguishes impure 1~5 reference substance, 1.0 μ g/ml；Sample solution 10 μ g/ml containing Repaglinide.
In the method, the impurity 1~5 of Repaglinide is shown in Table 1 in Repaglinide diformin tablet.
In the method, with fluorescence detector checked for impurities 1,2,4,5, with diode array detector checked for impurities 3.
Beneficial effects of the present invention: establishing the method that two kinds of detectors are used in series detection, and it is auspicious can to control simultaneously compound
In Ge Lienai diformin tablet 5 kinds of Repaglinides impurity content (including in European Pharmacopoeia Repaglinide raw material standard require control
1 kind of not identical impurity in the 4 kinds of impurity and United States Pharmacopeia Repaglinide raw material standard of system).This law can realize Repaglinide and two
First biguanides is kept completely separate with each impurity of Repaglinide, between melbine, Repaglinide and other impurities and each impurity peaks
Separating degree is all larger than 1.5,.Under the conditions of this law, the concentration and peak area of each impurity peaks are at good linear relationship, and each impurity exists
Sensitivity in corresponding detector is higher, and Repaglinide, impurity 1, impurity 2, impurity 3, impurity 4, the detection limit of impurity 5 are respectively
0.06ng,0.07 ng,0.05ng,0.2ng,0.1ng,0.06ng.Method of the invention can be accurately to compound Repaglinide diformazan
The impurity of Repaglinide carries out quantitative analysis in biguanides piece, to ensure that the quality controllable of compound Repaglinide diformin tablet
Property.
Detailed description of the invention
1 Repaglinide impurity of Fig. 1 embodiment, 1 map.
1 Repaglinide impurity of Fig. 2 embodiment, 2 map.
1 Repaglinide impurity of Fig. 3 embodiment, 3 map.
1 Repaglinide impurity of Fig. 4 embodiment, 4 map.
1 Repaglinide impurity of Fig. 5 embodiment, 5 map.
1 system suitability solution map of Fig. 6 embodiment.
1 negative sample solution map of Fig. 7 embodiment.
1 test solution map of Fig. 8 embodiment.
Wherein: A is fluorescence detector map, and B is diode array detector map.
Specific embodiment
Following embodiment is described further the content of present invention, does not limit the present invention.
Repaglinide used in following instance and its impurity reference substance 1,2,3,4 are all from Chinese food drug assay research
Institute, impurity 5 are purchased from Beijing Pu Xi Science and Technology Ltd., and Repaglinide diformin tablet is self-control (lot number 201410701), phosphoric acid
Potassium dihydrogen is that analysis is pure, and acetonitrile and ethyl alcohol are chromatographically pure.
Embodiment 1
Instrument: Agilent1260 type high performance liquid chromatograph (G1329B autosampler, G1316A thermostatted column compartment,
G1311C quaternary pump, G4212B diode array detector, G1321B fluorescence detector).
Chromatographic column: AgelaVENUSIL ASB C18 chromatographic column (4.6mm × 250mm, 5 μm).
Mobile phase: with 0.4% potassium dihydrogen phosphate buffer solution of mass fraction (using phosphorus acid for adjusting pH value 3.2) for mobile phase A,
Acetonitrile is Mobile phase B, is separated using gradient elution.Gradient elution is carried out according to table 2.
2 gradient elution table (volume ratio) of table/%
Detector: diode array and fluorescence detector series connection, Detection wavelength is respectively 240nm and excitation wavelength 244nm,
Launch wavelength 348nm.
Flow velocity: 1.0ml/min, sampling volume: 50 μ l, column temperature: 40 DEG C.
Sample solution prepares dilution: alcohol-water (volume ratio 1:1).
Impurity positions solution: taking 1,2,3,4,5 reference substance of impurity each appropriate, dissolve and diluted with dilution respectively and be made often
The solution of impure 1.0 μ g in 1 ml, shake up to get.
System suitability solution: taking Repaglinide and 1,2,3,4,5 reference substance of impurity each appropriate, accurately weighed, with dilution
Liquid, which is dissolved and diluted, is made in every 1ml the mixed solution containing 10 μ g of Repaglinide and the equal 1.0 μ g of each impurity, shake up to get.
The preparation of test solution: Repaglinide diformin tablet 10 are taken, finely ground to take piece powder appropriate at powder, precision claims
It is fixed, the solution that the 10 μ g containing Repaglinide in every 1ml is made is dissolved and diluted with dilution, is shaken up, filters, takes subsequent filtrate to obtain the final product.
Negative sample solution is prepared: the mixed powder for taking Metformin hydrochloride and each auxiliary material to mix with prescription ratio is appropriate, accurate
It is weighed, the solution that hydrochloric melbine 5mg in every 1ml is made is dissolved and diluted with dilution, is shaken up, and is filtered, is taken subsequent filtrate
To obtain the final product.
Measurement: taking impurity positioning solution and system suitability solution to be injected separately into liquid chromatograph, record chromatogram, according to
The appearance time (see FIG. 1 to FIG. 5) of impurity positioning each impurity peaks of solution positions the impurity in system suitability solution.System
Unite in applicability chromatography map, impurity 1 in fluorescence detector, impurity 2, Repaglinide, impurity 5, impurity 4 appearance time successively
For 2.156,5.485,17.665,18.466,24.699min (impurity 3 responds lower in fluorescence detector), between adjacent peak
Separating degree is followed successively by 10.289,35.921,2.513,19.438；Impurity 1 in diode array detector, impurity 2, impurity 3,
Repaglinide, impurity 5, impurity 4 appearance time be followed successively by 2.148,5.473,9.724,17.646,18.448,24.680
Min, separating degree is followed successively by 10.801,15.416,30.622,2.677,20.565 between adjacent peak, meets the requirements (see figure
6).Negative sample solution and test solution is taken to be injected separately into liquid chromatograph, negative sample solution is in main peak and impurity peak position
It sets noiseless (see Fig. 7), does not there are impurity peaks to detect (see Fig. 8) in test solution.
The test of 2 specificity of embodiment
The present invention has carried out shakedown Degrading experiment to compound Repaglinide diformin tablet, with strong illumination, high temperature,
Acid, basic hydrolysis and the method for oxidation are destroyed, to study the separating degree of degradation impurity and main peak, and in Diode Array Detector
The peak purity of main peak, the specificity to method of proof are verified in device.Take compound Repaglinide diformin tablet 20, it is finely ground at
Powder weighs piece powder respectively and is formulated as follows solution.
A. sample solution: it is appropriate (being approximately equivalent to 0.5mg Repaglinide) to weigh piece powder, sets in 50ml measuring bottle, adds dilution
It dissolves Repaglinide and to scale, shakes up, filter, take subsequent filtrate as sample solution.
B. acid destroys sample solution: weighing piece powder in right amount (the about 1mg containing Repaglinide), sets in 100ml measuring bottle, is added
4mol/L hydrochloric acid solution 2ml, sealing, is placed at room temperature for 30min, adjusts pH value to neutrality with 4mol/L sodium hydroxide solution, use is dilute
It releases liquid and is diluted to scale, shake up, filter, subsequent filtrate is taken to destroy sample solution as acid.
C. alkali destroys sample solution: weighing piece powder in right amount (the about 1mg containing Repaglinide), sets in 100ml measuring bottle, is added
4mol/L sodium hydroxide solution 2ml, sealing, is placed at room temperature for 30min, adjusts pH value to neutrality with 4mol/L hydrochloric acid solution, use is dilute
It releases liquid and is diluted to scale, shake up, filter, subsequent filtrate is taken to destroy sample solution as alkali.
D. Oxidative demage sample solution: it is appropriate (the about 1mg containing Repaglinide) to weigh piece powder, sets in 100ml measuring bottle, addition
6% hydrogenperoxide steam generator 5ml, is placed at room temperature for 30min, sets 80 DEG C of water-baths and places 10min, taking-up is put to room temperature, dilute with dilution
It releases to scale, shakes up, filter, take subsequent filtrate as Oxidative demage sample solution.
E. high temperature sample solution: it is appropriate (the about 1mg containing Repaglinide) to weigh piece powder, sets in 100ml measuring bottle, adds dilution
Ultrasound 2min makes to dissolve liquid in right amount, and sealing sets and places 60h at 60 DEG C, then sets in 80 DEG C of water-baths and place 3h, takes out, lets cool to room
Temperature is shaken up with diluted to scale, and filtration takes subsequent filtrate as high temperature sample solution.
F. strong illumination destroys sample solution: weighing piece powder in right amount (the about 1mg containing Repaglinide), sets in 100ml measuring bottle, add
Dilution makes to dissolve in right amount, sets and places 72h under the strong light of 4500lx, takes out, lets cool to room temperature, with diluted to scale, shake
Even, filtration takes subsequent filtrate to destroy sample solution as strong illumination.
Precision measures above-mentioned each 50 μ l of solution and injects liquid chromatograph, is analyzed with this method chromatographic condition, is as a result seen
Table 3.
3 failure test result of table
The results show that this product is relatively stable under the conditions of high temperature and strong illumination, there is not degradation in two kinds of detectors
Peak is detected, and is destroyed in sample solution in oxidation and acid and is detected more impurity；Under this chromatographic condition, sample is by strength
After destruction, main peak purity is 990 or more, and each to destroy sample solution material balance, main peak is accorded with front and back impurity peaks separating degree
It closes and requires.
The detection limit test of embodiment 3
Take Repaglinide and its impurity 1,2,3,4,5 each appropriate, it is accurately weighed, it is dissolved and is diluted to a series of with dilution
The solution of concentration, sample introduction measurement take the concentration point of signal-to-noise ratio >=3 for detection limit, as a result are as follows:
Repaglinide, impurity 1, impurity 2, impurity 3, impurity 4, the detection limit of impurity 5 are respectively 0.06ng, 0.07ng, 0.05
Ng, 0.2ng, 0.1ng, 0.06ng, each defects inspecting limit is in 0.05% concentration of principal component hereinafter, meet determination of foreign matter sensitivity
It is required that.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (7)
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