CN107345945B

CN107345945B - High performance liquid chromatography method for resolving glycopyrronium bromide enantiomer and checking impurities

Info

Publication number: CN107345945B
Application number: CN201610291461.8A
Authority: CN
Inventors: 于航; 杨晨
Original assignee: LIAONING PHARMA-UNION PHARMACEUTICAL Co Ltd
Current assignee: LIAONING PHARMA-UNION PHARMACEUTICAL Co Ltd
Priority date: 2016-05-05
Filing date: 2016-05-05
Publication date: 2019-12-17
Anticipated expiration: 2036-05-05
Also published as: CN107345945A

Abstract

The invention relates to a method for separating glycopyrronium bromide enantiomers by high performance liquid chromatography and checking enantiomer impurities. Separating the enantiomers of the glycopyrronium bromide by a high performance liquid chromatography, confirming the peak positions of the four enantiomers of the glycopyrronium bromide, and calculating the limits of the enantiomers in the glycopyrronium bromide according to a self-contrast method; the samples were prepared with n-hexane, and n-hexane-dehydrated ethanol-diethylamine (97: 3: 0.1) was used as the mobile phase. The method for separating the glycopyrronium bromide by adopting the high performance liquid chromatography has the advantages of good separation effect, low sample consumption, no need of reference substances for quantification, no pollution and low cost, and is suitable for the resolution of racemic glycopyrronium bromide.

Description

High performance liquid chromatography method for resolving glycopyrronium bromide enantiomer and checking impurities

Technical Field

The invention belongs to the technical field of medicines, and relates to a research on a method for resolving four chiral enantiomers of racemic muscarinic receptor antagonist glycopyrronium bromide by high performance liquid chromatography and an inspection method of enantiomer impurities thereof.

background

Glycopyrronium Bromide (Glycopyrronium Bromide) is a quaternary ammonium anticholinergic drug, the structure of the Glycopyrronium Bromide contains two chiral carbon atoms, two pairs of enantiomers (RS, SR, RR and SS) exist, namely four chiral enantiomers, products which are sold on the market at home and abroad are all mixtures of the enantiomers, namely a racemate, and the structure of the Glycopyrronium Bromide is shown as (I):

（）

The separation of chiral enantiomers, especially chiral drugs, has become one of the important subjects in the field of analytical chemistry in recent years, because the pharmacological action of a drug is realized by strict chiral recognition and matching with macromolecules in vivo, i.e. the chiral drug has significant stereoselectivity when acting with drug targets in a living body, so that in many cases, different enantiomers of the compound show great pharmacodynamic and pharmacokinetic differences in organisms, and the toxicological effects are different or even completely opposite. It was found that (3S, 2 'R) -glycopyrrolate has the worst cholinergic antagonism, whereas (3R, 2' S) -glycopyrrolate has the strongest cholinergic antagonism.

along with the understanding of the difference of biological activity between enantiomers of chiral drugs by people, the importance of developing single enantiomer drugs is gradually realized, the increasing importance of pharmaceutical companies on single enantiomer drugs in the world is reflected in the development of new drugs, the more important significance is to re-develop racemic drugs which are already on the market into single isomer drugs, because the new drugs are more time-saving and money-saving than the development of a brand new drug, and the single enantiomer drugs have the advantages of better possible curative effect, higher safety and less toxic and side effects, and the new drugs with the best curative effect and high safety are the targets of the efforts of the domestic and foreign pharmaceutical industries. Therefore, the prospect of developing single enantiomer medicaments for racemic medicaments through a chiral resolution way is very considerable.

The invention develops research aiming at chiral resolution of racemic drug glycopyrronium bromide, determines peak positions through 4 enantiomer monomers, performs content determination (when a certain monomer is used as a main drug) and enantiomer impurity content determination (when an enantiomer is an impurity), and has deep significance in developing new single-isomer drugs to determine the content and determining the content of the impurity (other three enantiomers).

Today, the safety and high efficiency of the research and development results become the primary focus of the pharmaceutical industry worldwide, and the separation and preparation technology of chiral chromatography plays an increasingly important role in the research and development of chiral drugs in recent years. Chiral drug quality control has attracted more and more attention and has become a hotspot in the field of drug research. The high performance liquid chromatography is one of the most commonly used chiral drug analysis methods, wherein the high performance liquid chromatography chiral stationary phase method is a classical, simple and rapid method, and the bonded cellulose chiral stationary phase has the characteristics of stable performance, good resolution effect and wide application range.

Disclosure of Invention

The invention provides a method for resolving glycopyrronium bromide enantiomers by high performance liquid chromatography, which is realized by confirming peak positions of four enantiomer monomers of glycopyrronium bromide and calculating the content of the enantiomers in the glycopyrronium bromide by a self-contrast method (without a contrast product), and mainly comprises the steps of sample preparation, liquid chromatography conditions and a quantitative calculation method; by examining chromatographic conditions, for example: the influence of chromatographic column packing, mobile phase proportion, detection wavelength, column temperature and the like on the separation of glycopyrronium bromide and isomers thereof comprehensively considers the peak shape and the peak position and selects the optimal separation condition. And respectively preparing solutions of glycopyrronium bromide monomers, confirming peak positions, and finally establishing a self-control method for quantifying and detecting enantiomer impurities in glycopyrronium bromide.

In the liquid phase condition, CHIRALCEL OZ-H chiral chromatographic columns provided by DAICEL (Daiillonite medicine chiral technology Co., Ltd.) are used, the chromatographic columns are suitable for being used in a normal-phase mobile phase, OZ is a polysaccharide derivative normal-phase coating type chiral chromatographic column, the surface of silica gel is coated with cellulose-tris (3-chloro-4-methylphenyl carbamate), OZ is a chiral filler which is actually used in the widest range at present, and is also a specified filler of USP L80, and excellent separation capability and tolerance of the OZ columns are reported in various documents; the ratio of n-hexane-absolute ethyl alcohol-diethylamine in the mobile phase is 97: 3: 0.1, and all reagents are required to be HPLC grade; detection wavelength: 220-240 nm; the temperature of the chromatographic column is 35-40 ℃,

In the invention, n-hexane is used for preparing glycopyrronium bromide and an enantiomer sample, the concentration of a sample is 0.5 mg/ml-2.0 mg/ml, 1% of self-contrast solution is prepared and is respectively injected, and the peak area of the enantiomer in the glycopyrronium bromide sample solution is not larger than the peak area of a main peak in the self-contrast solution (4 monomers have the same response value under the chromatographic condition, namely a correction factor meets 0.95-1.05).

the high performance liquid chromatography is used for detecting chiral drug isomers, and has the advantages of high efficiency, rapidness, simple and convenient operation, small sample consumption, low finished product, no need of reference substances, and satisfactory separation effect of glycopyrronium bromide and enantiomers. The mobile phase for high performance liquid chromatography resolution is easy to obtain, and has wide application prospect.

The features and advantages of the present invention will be apparent from the following examples and drawings.

Brief Description of Drawings

FIG. 1 is a high performance liquid chromatogram of (3R, 2' S) -type glycopyrronium bromide monomer

FIG. 2 is a high performance liquid chromatogram of (3R, 2' S) -type glycopyrronium bromide monomer

FIG. 3 is a high performance liquid chromatogram of (3R, 2' R) -type glycopyrronium bromide monomer

FIG. 4 is a high performance liquid chromatogram of (3R, 2' R) -type glycopyrronium bromide monomer

FIG. 5 is a high performance liquid chromatogram of (3S, 2' S) -type glycopyrronium bromide monomer

FIG. 6 is a high performance liquid chromatogram of (3S, 2' S) -type glycopyrronium bromide monomer

FIG. 7 is a high performance liquid chromatogram of (3S, 2' R) -type glycopyrronium bromide monomer

FIG. 8 is a high performance liquid chromatogram of (3S, 2' R) -type glycopyrronium bromide monomer

FIG. 9 is a mixed high performance liquid chromatogram of four monomers of glycopyrronium bromide

The specific implementation mode is as follows:

Example 1

The test instrument: shimadzu high performance liquid chromatograph (10 ADvp), Shimadzu ultraviolet detector (9A)

Chromatographic conditions are as follows: CHIRALCEL OZ-H chiral chromatography column (4.6 mm 250mm, 5 μm); mobile phase: n-hexane-absolute ethanol-diethylamine (97: 3: 0.1); detection wavelength: 230 nm; column temperature: 35 ℃; flow rate: 1.0 ml/min; sample introduction amount: 20 μ l.

The sample preparation process is as follows:

The concentration of four monomer solutions of glycopyrronium bromide is respectively prepared to be 0.15mg/ml, samples are respectively injected, 2 needles are continuously fed into each sample, the peak position is determined, and a comparison response value is calculated (the response values of the four monomers are nearly consistent, the correction factor is 0.95-1.05, no correction is needed), the results are shown in figures 1 and 2 as (3R,2 'S) -type glycopyrronium bromide monomer high-efficiency liquid chromatogram maps, figures 3 and 4 as (3R, 2' R) -type glycopyrronium bromide monomer high-efficiency liquid chromatogram maps, figures 5 and 6 as (3S, 2 'S) -type glycopyrronium bromide monomer high-efficiency liquid chromatogram maps, and figures 7 and 8 as (3S, 2' R) -type glycopyrronium bromide monomer high-efficiency liquid chromatogram maps.

Example 2

The chromatographic conditions were the same as in example 1. Preparing a mixed solution of four monomers (the concentration of the monomers is 0.15 mg/ml), observing the separation degrees of the four monomers, wherein the separation degrees are all more than 2.0, and meeting the detection requirement, and the result is shown in a monomer mixed high performance liquid chromatogram map in figure 9.

Conclusion

The patent establishes a method for separating glycopyrronium bromide enantiomers by high performance liquid chromatography and checking enantiomer impurities. The method has the advantages of simplicity, high separation efficiency, high separation speed, small sample volume, no need of reference substances and low analysis cost. Generally, only some common reagents are needed, all reagents are not used in large quantities, and the beneficial effects are mainly shown in that:

The separation efficiency is high, the chiral separation of four monomers is realized, the separation time is short, the separation is realized within 10 minutes, and the requirement of impurity inspection is met.

The sample consumption is small, and the sample required by each sample introduction is only a few microliters

A self-comparison method is adopted for impurity quantification, a comparison product (external standard) is not needed, and cost is saved.

The method has the advantages of high detection sensitivity, good durability, wide application of chromatographic columns and reagents used under chromatographic conditions, and good guiding significance for isomer separation.

Claims

1. A high performance liquid chromatography method for resolving glycopyrronium bromide enantiomer and checking impurities is characterized in that the method mainly comprises the steps of sample preparation, investigation and analysis of high performance liquid chromatography conditions; separating glycopyrronium bromide and enantiomers thereof by high performance liquid chromatography, and confirming peak positions of four enantiomer monomers of the glycopyrronium bromide; preparing each sample by using n-hexane, and using the n-hexane-absolute ethyl alcohol-diethylamine as a mobile phase, wherein the mobile phase is prepared by the following steps: n-hexane-absolute ethyl alcohol-diethylamine 97: 3: 0.1, all reagents are required to be HPLC grade; the chromatographic conditions are as follows: CHIRALCEL OZ-H chiral column length: 150 mm-250 mm; inner diameter: 2.1-4.6 mm; the particle size of the filler is as follows: 5 μm.

2. The high performance liquid chromatography method for resolving glycopyrronium bromide enantiomers and checking impurities as claimed in claim 1, which is characterized in that: the high performance liquid chromatograph system is washed through the normal phase, must not contain moisture in any module part, and the detection wavelength: 220-240 nm, the temperature of the chromatographic column is 35-40 ℃, and the flow rate is 0.9-1.1 ml/min.

3. The high performance liquid chromatography method for resolving glycopyrronium bromide enantiomer and checking impurities as claimed in claim 1, wherein n-hexane is used for preparing racemic glycopyrronium bromide analysis sample and four enantiomer monomer samples, wherein n-hexane is required to be HPLC grade.

4. The high performance liquid chromatography method for resolving glycopyrronium bromide enantiomers and checking impurities as claimed in claim 1, which is characterized in that: four enantiomer monomer samples are respectively injected to obtain four enantiomers with peak positions as follows: RS, RR, SS, SR.

5. The high performance liquid chromatography method for resolving glycopyrronium bromide enantiomers and checking impurities as claimed in claim 1, which is characterized in that: the method for calculating the enantiomer of glycopyrronium bromide comprises the steps of respectively preparing a glycopyrronium bromide test solution and a 1% self-contrast solution, and respectively injecting samples, wherein the peak area of enantiomer impurities in the glycopyrronium bromide test solution is not larger than the peak area of a main peak in the self-contrast solution.

6. The high performance liquid chromatography method for resolving glycopyrronium bromide enantiomers and checking impurities as claimed in claim 1, which is characterized in that: the method can be used for separating glycopyrronium bromide and isomers, and simultaneously, the content of the enantiomer can be calculated by adopting a self-contrast method.

7. The high performance liquid chromatography method for resolving glycopyrronium bromide enantiomers and checking impurities as claimed in claim 1, which is characterized in that: when glycopyrrolate and the isomers are used in combination with other drugs, the content of glycopyrrolate and the isomers in the mixed solution can be checked by adopting the method.