CN112345668B - High performance liquid chromatography method for separating vildagliptin intermediate and R-type isomer - Google Patents
High performance liquid chromatography method for separating vildagliptin intermediate and R-type isomer Download PDFInfo
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- CN112345668B CN112345668B CN202011243419.1A CN202011243419A CN112345668B CN 112345668 B CN112345668 B CN 112345668B CN 202011243419 A CN202011243419 A CN 202011243419A CN 112345668 B CN112345668 B CN 112345668B
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Abstract
The invention discloses a high performance liquid chromatography method for separating a vildagliptin intermediate and an R-type isomer. The chromatographic conditions provided by the invention can effectively separate vildagliptin intermediate 5 from the R-type isomer of vildagliptin by using a conventional C18 chromatographic column without depending on an expensive chiral chromatographic column, and are obviously superior to the prior art.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a high performance liquid chromatography method for separating vildagliptin intermediate and R-type isomer.
Background
Diabetes is a chronic metabolic disease characterized by blood sugar rise due to insulin secretion and action defects, and oral hypoglycemic agents include insulin sensitizers (thiazolidinediones and biguanides), insulin secretion accelerators (nonsulfonylureas and sulfonylurea insulin secretion accelerators), alpha-glucosidase inhibitors and the like. In recent years, several main varieties of thiazolidinedione drugs, including troglitazone, rosiglitazone, pioglitazone and the like, find toxic and side effects such as liver injury, cardiovascular risk, cancer induction risk and the like in the clinical application process, and limit the application of the drugs. The novel hypoglycemic dipeptidyl peptidase-4 inhibitor drug has better safety and durability, and the total adverse reaction is similar to that of a placebo. Vildagliptin is an oral effective dipeptidyl peptidase-4 inhibitor, can effectively improve the blood sugar control of type 2 diabetes patients, and the pharmacological activity, the blood sugar reducing mechanism and the clinical curative effect of vildagliptin are proved by related tests, so that a new medicine selection is provided for the diabetes patients.
The chemical structure of vildagliptin is as follows.
A key intermediate in the vildagliptin synthesis process is an intermediate 5, and an R-type isomer impurity also exists in the intermediate 5. If the R-type isomer impurity is not completely removed, the purity of the final product vildagliptin is greatly influenced. Therefore, it is very necessary to monitor the content of the R isomer impurity in intermediate 5. The chemical structures of intermediate 5 and its R-isomer are as follows.
The separation difficulty of the intermediate 5 and the R-type isomer thereof is very large, and the prior art is assisted by a chiral chromatographic column (Chinese patent, publication No. CN 106568877A). However, chiral columns are expensive, several chiral columns are usually lost when a set of analytical methodologies is developed, and the analysis cost is high.
The addition of chiral additives in mobile phase is a method which makes it possible to separate chiral isomers, but the development of the method is extremely difficult, and not all chiral compounds can find suitable chiral additives.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a high performance liquid chromatography method for separating vildagliptin intermediate and R-type isomer.
The purpose of the invention is realized by the following technical scheme:
an HPLC method for separating vildagliptin intermediate and R-type isomer thereof comprises the following chromatographic conditions:
a chromatographic column: c18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase a phase: firstly, preparing a 5% acetonitrile water solution containing 20 mu M N-acetyl-S- (4-nitrophenyl) -L-cysteine, and then adjusting the pH value to 3.5 by using phosphoric acid, wherein the acetonitrile water solution is prepared immediately after use;
mobile phase B phase: 25% aqueous acetonitrile;
elution mode and ratio: the phase A and the phase B are eluted at equal degrees according to the volume ratio of 48: 52;
flow rate: 1.0 mL/min;
detection wavelength: 210 nm;
column temperature: 30 ℃;
the chemical structural formula of vildagliptin intermediate 5 and its R-isomer is as follows:
preferably, the sample size in the HPLC method is 10. mu.L.
Preferably, the C18 chromatography column is an Agilent Eclipse Plus C18(4.6 mm. times.250 mm, 5 μm).
Has the beneficial effects that:
the chromatographic conditions provided by the invention can effectively separate vildagliptin intermediate 5 from R-type isomer thereof by using a conventional C18 chromatographic column, and the expensive chiral chromatographic column is not required, so that the method is obviously superior to the prior art.
Drawings
FIG. 1 is a separation chromatogram of a vildagliptin intermediate 5 and an R-type isomer mixed reference substance under chromatographic conditions of the invention;
fig. 2 is a separation chromatogram of vildagliptin intermediate 5 and its R-isomer mixed reference substance comparing chromatographic conditions.
Detailed Description
The following examples are intended to embody the essential technical matters of the present invention, and the specific general details described in the following examples are not intended to limit the scope of the present invention.
Instrument and reagent
1. Instrument
Agilent 1260 liquid chromatograph (vacuum degasser, binary pump, autosampler), USA.
A Mettler toledo electronic balance (XS 105).
2. Reagent
The purity of the vildagliptin intermediate 5 reference substance and the purity of the vildagliptin intermediate 5R-type isomer reference substance are both more than or equal to 99%.
The chiral additive N-acetyl-S- (4-nitrophenyl) -L-cysteine was purchased from Jiuding chemistry and had a purity of greater than or equal to 98%.
The acetonitrile is chromatographically pure, the water is ultrapure water, and the percentage concentration of the acetonitrile water solution is the volume percentage concentration of the acetonitrile.
Second, method and results
1. Chromatographic conditions
A chromatographic column: agilent Eclipse Plus C18(4.6 mm. times.250 mm, 5 μm);
mobile phase a phase: firstly, preparing a 5% acetonitrile water solution containing 20 mu M N-acetyl-S- (4-nitrophenyl) -L-cysteine, and then adjusting the pH value to 3.5 by using phosphoric acid, wherein the acetonitrile water solution is prepared immediately after use;
mobile phase B phase: 25% acetonitrile in water;
elution mode and ratio: the phase A and the phase B are eluted at equal degrees according to the volume ratio of 48: 52;
flow rate: 1.0 mL/min;
detection wavelength: 210 nm;
column temperature: 30 ℃;
sample introduction amount: 10 μ L.
2. Solution preparation
Mixing the reference solution: and mixing a proper amount of mobile phase A and B according to a volume ratio of 48:52 to obtain a solvent, and preparing a mixed reference solution containing 0.5mg/mL vildagliptin intermediate 5 reference substance and 0.5mg/mL vildagliptin intermediate 5R-type isomer reference substance.
Vildagliptin intermediate 5 control solution: and mixing a proper amount of the mobile phase A and the mobile phase B according to the volume ratio of 48:52 to serve as a solvent, and preparing a vildagliptin intermediate 5 reference solution containing 0.1mg/mL vildagliptin intermediate 5 reference.
Vildagliptin intermediate 5R isomer control solution: and mixing a proper amount of the mobile phase A and the mobile phase B according to the volume ratio of 48:52 to obtain a vildagliptin intermediate 5R-type isomer reference solution containing 0.1mg/mL vildagliptin intermediate 5R-type isomer reference.
3. Analysis of sample introduction
Respectively and precisely measuring 10 mu L of mixed reference substance solution, vildagliptin intermediate 5 reference substance solution and vildagliptin intermediate 5R-type isomer reference substance solution, injecting into a liquid chromatograph, analyzing according to the chromatographic conditions, and recording a chromatogram. As a result, as shown in fig. 1, under the chromatographic conditions, vildagliptin intermediate 5 and its R-isomer can be separated at baseline with a resolution of more than 2.0.
In order to embody the separation effect of chiral additive N-acetyl-S- (4-nitrophenyl) -L-cysteine on vildagliptin intermediate 5 and R-type isomer thereof, a comparison test is set, and the only difference of the comparison test is that the mobile phase A phase in chromatographic conditions does not contain N-acetyl-S- (4-nitrophenyl) -L-cysteine, and the others are completely consistent. As a result, as shown in fig. 2, vildagliptin intermediate 5 co-eluted with its R-isomer under the chromatographic conditions, and could not be separated.
The test results show that the vildagliptin intermediate 5 and the R-type isomer thereof can be effectively separated by using the conventional C18 chromatographic column under the chromatographic conditions provided by the invention, and the expensive chiral chromatographic column is not required, so that the method is obviously superior to the prior art.
The above-described embodiments are intended to embody the essential technical content of the present invention, and the scope of the present invention should not be limited by the specific general details described in the above-described embodiments.
Claims (2)
1. A high performance liquid chromatography method for separating vildagliptin intermediate and R-type isomer is disclosed, wherein the intermediate is an intermediate 5 with the following chemical structure, the R-type isomer has the following chemical structure, and the chromatographic conditions are as follows:
a chromatographic column: agilent Eclipse Plus C18, specification of 4.6mm × 250mm, 5 μm;
mobile phase a phase: firstly, preparing a 5% acetonitrile water solution containing 20 mu M N-acetyl-S- (4-nitrophenyl) -L-cysteine, and then adjusting the pH value to 3.5 by using phosphoric acid, wherein the acetonitrile water solution is prepared immediately after use;
mobile phase B phase: 25% acetonitrile in water;
elution mode and ratio: the phase A and the phase B are eluted at equal degrees according to the volume ratio of 48: 52;
flow rate: 1.0 mL/min;
detection wavelength: 210 nm;
column temperature: 30 ℃;
the chemical structural formula of vildagliptin intermediate 5 and its R-isomer is as follows:
2. the high performance liquid chromatography method of claim 1, wherein: the amount of sample was 10. mu.L.
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| HU231050B1 (en) * | 2010-08-19 | 2020-02-28 | Egis Gyógyszergyár Nyrt. | Process for the preparation of a pharmaceutical active ingredient |
| CN103804266B (en) * | 2014-02-21 | 2016-06-08 | 张家港威胜生物医药有限公司 | A kind of synthetic method of vildagliptin intermediate |
| CN106338564B (en) * | 2016-09-27 | 2018-10-12 | 海口南陆医药科技股份有限公司 | A method of for detecting enantiomter in vildagliptin intermediate |
| CN106568877A (en) * | 2016-11-09 | 2017-04-19 | 河北科技大学 | Analysis method for Vildagliptin intermediate-5 enantiomer detection |
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