CN103353491B - A method of with liquid chromatography for separating and determining alogliptin benzoate raw material and its preparation - Google Patents
A method of with liquid chromatography for separating and determining alogliptin benzoate raw material and its preparation Download PDFInfo
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- CN103353491B CN103353491B CN201310266993.2A CN201310266993A CN103353491B CN 103353491 B CN103353491 B CN 103353491B CN 201310266993 A CN201310266993 A CN 201310266993A CN 103353491 B CN103353491 B CN 103353491B
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Abstract
The invention belongs to analytical chemistry fields, the invention discloses a kind of methods with liquid chromatography for separating and determining alogliptin benzoate in relation to substance, this method is using octyl silane group silica gel as the chromatographic column of filler, using a certain proportion of inorganic acid salt buffer solution-organic phase as mobile phase, alogliptin benzoate and its content in relation to substance can be quantitative determined, to effectively control the quality of alogliptin benzoate and the formulation products containing alogliptin benzoate.The method of the present invention specificity is strong, and accuracy is high, easy to operate.
Description
Technical field
The invention belongs to analytical chemistry fields, and in particular to liquid chromatography for separating and determining alogliptin benzoate and its have
The method for closing substance.
Background technique
Alogliptin benzoate is serine protease DPP IV (DPP-IV) inhibitor, and internal pancreas can be maintained high
The level of blood glucose element sample peptide 1 (GLP-1) and glucose-dependent-insulinotropic polypeptide (GIP), promotes the secretion of insulin, thus
Hypoglycemic curative effect is played, treatment diabetes B is suitable for.Entitled 2- [[6- [(the 3R) -3-Amino-1- of alogliptin benzoate chemistry
piperidinyl]-3,4-dihydro-3-methyl-2,4-dioxo-1(2H)-pyrimidinyl]methyl]
Benzonitrile benzoate, molecular formula C25H27N5O4.Alogliptin benzoate structural formula is：
During synthesizing the compound, the intermediate for having several steps important may influence medicine due to removing not exclusively
The purity and quality of object, the related substance in these intermediate, that is, Control of drug quality, the conjunction for alogliptin benzoate
There are 6 at the related substance mainly controlled, is 1 6-Chloro-3-methyl-1H-pyrimidine-2 of intermediate, 4- respectively
Dione, intermediate 22-Bromomethyl-benzonitrile, 3 2- (6-Chloro-3-methyl-2,4- of intermediate
Dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl)-benzonitrile, 4 Piperidin-3- of intermediate
Ylamine acid amide, 5 2- of intermediate [6- (3-Amino-piperidin-1-yl) -3-methyl-2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-ylmethyl]-benzonitrile acid amide, 6 Benzoic of intermediate
Acid structural formula is respectively：
1 intermediate of intermediate, 2 intermediate 3
4 intermediate of intermediate, 5 intermediate 6
It is whether all to need in bulk pharmaceutical chemicals or preparation for the related substance introduced in synthesizing benzoic acids Egelieting
Quality control is carried out, therefore, alogliptin benzoate and its separation in relation to substance are realized, in alogliptin benzoate
The quality controlling party face of synthesis and production process has important practical significance.
Summary of the invention
The purpose of the present invention is to provide a kind of purity for analyzing alogliptin benzoate and it is separated in relation to substance
Method, to realize the separation and measurement of the associated substance of alogliptin benzoate, guarantee alogliptin benzoate and contain benzene
The quality of formic acid Egelieting preparation controls.
Purity of the present invention with liquid chromatography analysis alogliptin benzoate and it is separated in relation to substance
Method is the chromatographic column for using octyl silane group silica gel as filler, organic with a certain proportion of inorganic salt buffer solution-
It is mutually mobile phase.
Above-mentioned described chromatographic column is selected from Apollo-C using octyl silane group silica gel as filler8Or Ultimate-
C8。
Above-mentioned described organic phase is selected from following compound：Methanol, acetonitrile, propyl alcohol, isopropanol, tetrahydrofuran etc., preferably
For methanol.
Above-mentioned described method, the inorganic salt buffer solution-organic phase of mobile phase use gradient elution.
In above-mentioned described method, the inorganic salts for including in inorganic salt buffer solution are selected from phosphate, carbonate, citric acid
Salt, preferably phosphate.
The concentration for the inorganic salts for wherein including in inorganic salt buffer solution is 0.01~0.1mol/L, and preferred concentration is
0.02mol/L。
Method of separating and assaying of the present invention can be realized in accordance with the following methods：
1) it takes alogliptin benzoate or formulation samples containing alogliptin benzoate appropriate, is mixed with methanol or flowing
Sample is solved, the sample solution of every 1mL 0.1~1.5mg containing alogliptin benzoate is configured to.
2) setting flow rate of mobile phase is 0.5~1.5mL/min, and flow rate of mobile phase is preferably 1.0mL/min, Detection wavelength
For 200~250nm, best detection wavelength 215nm, column oven temperature is 10~40 DEG C, most preferably 20 DEG C of column oven temperature.
3) 10~50 μ L of sample solution 1) is taken, liquid chromatograph is injected, completes alogliptin benzoate and related substance
Separation determination.Wherein：
The model of high performance liquid chromatograph, has no special requirements, and the chromatograph that the present invention uses is Shimadzu：LC-20ATvp,
SPD-M20Avp
Chromatographic column：C8（Apollo, 250 × 4.6 mm, 5 μm）
Mobile phase：A：Potassium phosphate buffer（Weigh 2.72gKH2PO4, 0.94g sodium hexanesulfonate adds water 1000ml to make
Dissolution, KOH solution adjust pH to 5.5）；B：Methanol；Using gradient elution
Flow velocity：1.0mL/min
Detection wavelength：215nm
Column temperature：20℃
Sampling volume：10μL
The present invention uses C8(Apollo, 250 × 4.6 mm, 5 μm), can efficiently separate alogliptin benzoate and
It is in relation to substance.The present invention solves the problems, such as alogliptin benzoate and its separation determination in relation to substance, ensures that benzene
Formic acid Egelieting and its preparation it is quality controllable.
Detailed description of the invention
The high-efficient liquid phase chromatogram of Fig. 1 blank solvent
Fig. 2 alogliptin benzoate and its high-efficient liquid phase chromatogram in relation to substance
The high-efficient liquid phase chromatogram of Fig. 3 alogliptin benzoate raw material
The high-efficient liquid phase chromatogram of Fig. 4 alogliptin benzoate piece auxiliary material blank
The high-efficient liquid phase chromatogram of Fig. 5 alogliptin benzoate piece
Specific embodiment：
Following embodiment is not limited to the range of this implementation for further understanding the present invention.
Embodiment 1
Instrument and condition
High performance liquid chromatograph：Shimadzu：LC-20ATvp, SPD-M20Avp；
Chromatographic column：C8（Apollo, 250 × 4.6 mm, 5 μm）；
Mobile phase：A：Potassium phosphate buffer（Weigh 2.72gKH2PO4, 0.94g sodium hexanesulfonate adds water 1000ml to make
Dissolution, KOH solution adjust pH to 5.5）；B：Methanol；It is eluted by with Gradient
| T(min) | 0 | 5 | 15 | 25 | 30 | 40 | 50 |
| B% | 15 | 15 | 35 | 50 | 50 | 15 | 15 |
Flow velocity：1.0mL/min
Detection wavelength：215nm
Column temperature：20℃
Sampling volume：10μL
Experimental procedure
Alogliptin benzoate and its intermediate about 25mg are taken respectively, are set in 50mL measuring bottle, are added mobile phase（A：B=1：1）It is molten
Scale is solved and be diluted to, is shaken up, respectively takes and is configured to mixing sample solution in right amount.
Blank reagent solution and mixing sample solution are taken respectively, carry out efficient liquid phase chromatographic analysis, record by above-mentioned condition
Chromatogram, the result is shown in Figure 1, Fig. 2.
The chromatographic peak that retention time is 28.494min in Fig. 2 is the chromatographic peak of alogliptin benzoate, remaining chromatographic peak is
The chromatographic peak of each intermediate of alogliptin benzoate, as seen from the figure, alogliptin benzoate can reach with in-between body
Baseline separation meets the requirement of Chinese Pharmacopoeia.
Embodiment 2
Instrument and condition
High performance liquid chromatograph：Shimadzu：LC-20ATvp, SPD-M20Avp；
Chromatographic column：C8（Apollo, 250 × 4.6 mm, 5 μm）；
Mobile phase：A：Potassium phosphate buffer（Weigh 2.72gKH2PO4, 0.94g sodium hexanesulfonate adds water 1000ml to make
Dissolution, KOH solution adjust pH to 5.5）；B：Methanol；It is eluted by with Gradient
| T(min) | 0 | 5 | 15 | 25 | 30 | 40 | 50 |
| B% | 15 | 15 | 35 | 50 | 50 | 15 | 15 |
Flow velocity：1.0mL/min
Detection wavelength：215nm
Column temperature：20℃
Sampling volume：10μL
Experimental procedure
Alogliptin benzoate raw material about 25mg is taken, sets in 50mL measuring bottle, adds mobile phase（A：B=1：1）It dissolves and is diluted to
Scale shakes up, as test solution.
Test solution is taken, efficient liquid phase chromatographic analysis is carried out by above-mentioned condition, records chromatogram, as a result see Fig. 3.
The chromatographic peak that retention time is 28.452min in Fig. 3 is the chromatographic peak of alogliptin benzoate, can be demonstrate,proved by figure
Bright, the chemical purity of alogliptin benzoate reaches the requirement of bulk pharmaceutical chemicals, and this law can be used for the quality of alogliptin benzoate
Monitoring.
Embodiment 3
Instrument and condition
High performance liquid chromatograph：Shimadzu：LC-20ATvp, SPD-M20Avp；
Chromatographic column：C8(Apollo, 250*4.6mm, 5 μm)；
Mobile phase：A：Potassium phosphate buffer（Weigh 2.72gKH2PO4, 0.94g sodium hexanesulfonate adds water 1000ml to make
Dissolution, KOH solution adjust pH to 5.5）；B：Methanol；It is eluted by with Gradient
| T(min) | 0 | 5 | 15 | 25 | 30 | 40 | 50 |
| B% | 15 | 15 | 35 | 50 | 50 | 15 | 15 |
Flow velocity：1.0mL/min
Detection wavelength：215nm
Column temperature：20℃
Sampling volume：10μL
Experimental procedure
It takes alogliptin benzoate piece appropriate, is approximately equivalent to alogliptin benzoate 12.5mg, set in 25mL measuring bottle, add stream
Dynamic phase（A：B=1：1）Ultrasonic treatment dissolution, and use mobile phase（A：B=1：1）It is diluted to scale, is shaken up, filters, subsequent filtrate is taken to make
For test solution.
Test solution is taken, carries out efficient liquid phase chromatographic analysis by above-mentioned condition, records chromatogram, and carry out blank with method
Auxiliary material test, is as a result shown in Fig. 4, Fig. 5.
Fig. 4 proves that blank auxiliary does not interfere the measurement of this product, and Fig. 5 proves that this law can be used for alogliptin benzoate piece
Quality-monitoring.The chromatographic peak of 28.384min is the chromatographic peak of alogliptin benzoate.
Show from Fig. 1-Fig. 5：Method of the invention can clearly separate alogliptin benzoate with wherein mesosome,
And can accurately carry out detecting quantitatively, to calculate the content of alogliptin benzoate, to effectively control benzoic acid A Gelie
The product quality in spit of fland.
Claims (7)
1. a kind of method of the liquid chromatography for separating and determining alogliptin benzoate in relation to substance, it is characterised in that：Octyl silicon
Alkane bonded silica gel is the chromatographic column of filler, and using phosphate buffer solution-methanol as mobile phase, gradient is,
The related substance of the alogliptin benzoate of method separation determination is alogliptin benzoate reaction intermediate, and structural formula is such as
Under：
。
2. method of separating and assaying according to claim 1, chromatographic column is selected from the chromatography that brand is Ultimate or Apollo
Column.
3. the method for separating and assaying according to claim 1, oneself containing 0.94mg/ml in the phosphate buffer solution
Alkyl sulfonic acid sodium.
4. method of separating and assaying according to claim 1, phosphate is potassium dihydrogen phosphate.
5. method of separating and assaying according to claim 1, the concentration of the phosphate buffer solution is 0.02mol/L,
The pH value of buffer is preferably 5.5.
6. the method for separating and assaying according to claim 1, which is characterized in that including following steps：
1）Take alogliptin benzoate or formulation samples containing alogliptin benzoate appropriate, respectively with methanol or flowing phased soln
Sample is configured to sample solution of every 1mL containing 0.1~1.5mg of alogliptin benzoate and its intermediate；
2）Setting flow rate of mobile phase is 0.5~1.5mL/min, and Detection wavelength is 200~250nm, and column oven temperature is 10~40
DEG C, gradient is
；
3）Take 1）10~50 μ L of sample solution, inject liquid chromatograph, complete alogliptin benzoate and its related substance
Separation determination.
7. method of separating and assaying according to claim 6, step 2）The flow rate of mobile phase is 1.0mL/min；Detection
Wavelength is 215 nm.
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| CN104749269B (en) * | 2013-12-31 | 2018-12-28 | 中美华世通生物医药科技（武汉）有限公司 | A method of enantiomter impurity in Egelieting bulk pharmaceutical chemicals and preparation is measured using HPLC |
| CN104316606B (en) * | 2014-09-24 | 2020-07-07 | 万特制药(海南)有限公司 | Method for separating and measuring vildagliptin related substances by liquid chromatography |
| CN105527348B (en) * | 2014-09-28 | 2018-07-24 | 中美华世通生物医药科技（武汉）有限公司 | Separation analysis alogliptin benzoate preparation and its method in relation to substance |
| CN104569172A (en) * | 2014-11-05 | 2015-04-29 | 广东东阳光药业有限公司 | Method for detecting dissolution rate of alogliptin benzoate tablets by using liquid chromatography |
| CN105738517B (en) * | 2016-02-24 | 2016-11-09 | 中山万汉医药科技有限公司 | A kind of measure the method having related substance in bent Ge Lieting piece |
| CN105699547B (en) * | 2016-04-22 | 2017-03-29 | 中山万远新药研发有限公司 | It is a kind of determine succinum love song Ge Lieting raw materials in about material method |
| CN109580835A (en) * | 2018-12-31 | 2019-04-05 | 辰欣药业股份有限公司 | A method of with the related substance of liquid chromatography for separating and determining Egelieting |
| CN109668988B (en) * | 2019-02-27 | 2021-07-02 | 浙江华贝药业有限责任公司 | Method for analyzing and determining 2- (dibromomethyl) -4-fluorobenzonitrile in trelagliptin succinate |
| CN111537622B (en) * | 2019-11-29 | 2021-11-26 | 杭州华东医药集团新药研究院有限公司 | Method for detecting impurities of duloxetine and salts thereof |
| CN112480074A (en) * | 2020-12-08 | 2021-03-12 | 湖南千金湘江药业股份有限公司 | 3, 4-dihydropyrimidine benzonitrile derivative, and preparation method and application thereof |
| CN115656389B (en) * | 2022-11-17 | 2024-02-06 | 重庆科瑞南海制药有限责任公司 | Method for measuring content of R-3-aminopiperidine dihydrochloride in alogliptin benzoate by using LC-MS |
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