Method for detecting diastereoisomers in nenofloxacin malate sodium chloride injection

Abstract

The invention provides a method for detecting diastereoisomers in a nerofloxacin malate sodium chloride injection. The method comprises the following steps: separating optical isomers of the nerofloxacin malate sodium chloride injection by adopting a liquid chromatography; wherein the chromatographic condition of the liquid chromatography comprises a mobile phase, the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a trifluoroacetic acid solution, and the mobile phase B is methanol-tetrahydrofuran. According to the technical scheme, a trifluoroacetic acid solution is adopted as a mobile phase A, methanol-tetrahydrofuran is adopted as a mobile phase B, tetrahydrofuran is added in the mobile phase B, the peak pattern of a main peak is improved, the trailing factor is 1.06, the operation time can be shortened, and the peak pattern of the main peak is improved.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting diastereomers in nemofloxacin malate sodium chloride injection. Background Art

[0002] Ninofloxacin malate is clinically used to treat community-acquired pneumonia and is an anti-infective drug. There are two chiral carbons in the structure of ninoxacin and its salt ninoxacin malate, which can produce diastereoisomers. The isomers in the ninoxacin malate raw materials or preparations currently sold on the market are (3S, 5S) type, and the structural formula and nomenclature are as follows:

[0003] 7-[(3 S , 5 S )-3-amino-5-methyl-piperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid malate hemihydrate The structural formulas and names of other non-corresponding isomers are as follows:

[0004] 7-[(3S,5R)-3-Amino-5-methyl-piperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid or:

[0005] 7-[(3R,5S)-3-Amino-5-methyl-piperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid The diastereoisomers are process impurities and are generated in the substitution reaction. The conversion process is as follows:

[0006] The registration standard of the nemofloxacin malate sodium chloride injection of Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory includes the detection method of the above-mentioned non-corresponding isomers. The impurity is not controlled under the related substance inspection item, and only the separation degree of the impurity and nemofloxacin is required in the system suitability solution under the content determination item. However, this method has a long running time, a low column temperature, a high selectivity for the liquid chromatograph, and a poor peak shape of the main peak of the chromatogram. Summary of the invention

[0007] The main purpose of the invention is to provide a method for detecting diastereomers in nemofloxacin malate sodium chloride injection, aiming to solve the technical problem that the main peak shape of the chromatogram of the current detection method for impurities in nemofloxacin malate is poor.

[0008] To achieve the above object, the present invention provides a method for detecting diastereomers in nemofloxacin malate sodium chloride injection, which comprises the following steps: Liquid chromatography is used to separate the optical isomers of nemofloxacin malate sodium chloride injection; wherein the chromatographic conditions of the liquid chromatography include a mobile phase, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a trifluoroacetic acid solution, and the mobile phase B is methanol-tetrahydrofuran.

[0009] Optionally, the mobile phase A is a trifluoroacetic acid aqueous solution with a volume fraction of 0.1%.

[0010] Optionally, the mobile phase B is a methanol-tetrahydrofuran solvent with a volume ratio of 97.5:2.5.

[0011] Optionally, the volume ratio of the mobile phase A to the mobile phase B is 55:45.

[0012] Optionally, the chromatographic conditions further include a stationary phase, and the stationary phase is octadecylsilane bonded silica gel.

[0013] Optionally, the chromatographic conditions further include chromatographic column specifications, and the chromatographic column specifications are Welch Ultimate LP-C18 4.6*150mm, 5um.

[0014] Optionally, the chromatographic conditions further include a mobile phase flow rate, and the mobile phase flow rate is 1.0 mL per minute.

[0015] Optionally, the chromatographic conditions further include a chromatographic column temperature, and the chromatographic column temperature is 30°C.

[0016] Optionally, the chromatographic conditions also include a detection wavelength, and the detection wavelength is 297 nm.

[0017] Optionally, the chromatographic conditions further include an elution program, which is an isocratic elution with an injection volume of 20 μL and a running time of 15 min.

[0018] In the technical solution of the present invention, trifluoroacetic acid solution is used as mobile phase A, methanol-tetrahydrofuran is used as mobile phase B, tetrahydrofuran is added to mobile phase B to improve the peak shape of the main peak, the tailing factor is 1.06, the running time can be shortened, and the peak shape of the main peak is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the information shown in these drawings without paying creative work.

[0020] Figure 1 It is the liquid chromatogram of diastereoisomers in the traditional detection method; Figure 2 It is a liquid chromatogram of the test solution provided by the present invention.

[0021] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0022] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0023] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0024] The registration standard of nemonoxacin malate sodium chloride injection produced by Xinchang Pharmaceutical Factory of Zhejiang Medicine Co., Ltd. includes the detection method of the above-mentioned non-corresponding isomers. This impurity is not controlled under the related substance inspection item, and only the separation degree of this impurity and nemonoxacin is set in the system suitability solution under the content determination item.

[0025] The specific chromatographic conditions and typical spectra are shown in the following table and Figure 1 As shown: Chromatographic conditions: octadecylsilane bonded silica gel as filler; 0.1% trifluoroacetic acid solution as mobile phase A, methanol as mobile phase B, gradient elution according to the table below; flow rate 1.0 mL per minute; column temperature 10°C; detection wavelength 297 nm; injection volume 20 μL.

[0026]

[0027] However, this method has a long running time, low column temperature, high selectivity for liquid chromatography, and poor main peak shape of the chromatogram.

[0028] In view of this, the present invention proposes a method for detecting diastereomers in nemofloxacin malate sodium chloride injection, which uses liquid chromatography to separate the optical isomers of nemofloxacin malate sodium chloride injection; wherein the chromatographic conditions of the liquid chromatography are as follows: Use octadecylsilane bonded silica gel as filler (Welch Ultimate LP-C18 4.6mm150mm, 5m or equivalent chromatographic column); 0.1% trifluoroacetic acid solution as mobile phase A, methanol-tetrahydrofuran (volume ratio 97.5:2.5) as mobile phase B, mobile phase A-mobile phase B (volume ratio 55:45) isocratic elution; flow rate is 1.0mL per minute; column temperature is 30℃; detection wavelength is 297nm; injection volume is 20μL; running time is 15min.

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention. Example 1

[0030] Determination of Optical Isomers of Nemofloxacin Malate 1. Experimental Materials High performance liquid chromatography (instrument brand: Agilent model: 1260); Column: Welch Ultimate LP-C18 4.6mm150mm; (3S, 5S)-Nenoxacin malate standard, (3S, 5R)-Nenoxacin malate standard, and (3R, 5S)-Nenoxacin malate standard were prepared by Xinchang Pharmaceutical Factory of Zhejiang Medicine Co., Ltd.; methanol was chromatographic grade, water was deionized water, and the other reagents were of analytical grade.

[0031] 2. Test methods and results 1. Solution preparation Resolution determination solution: Take appropriate amounts of nemonoxacin malate reference substance and impurity B reference substance, accurately weigh them, dissolve them in solvent and dilute them to prepare a mixed solution containing approximately 0.02 mg of nemonoxacin and 0.4 μg of impurity B per 1 ml.

[0032] Test solution: Take 1.0 ml of nemonoxacin malate sodium chloride injection, place it in a 100 ml volumetric flask, dilute to the scale with mobile phase, and shake well.

[0033] 2. Chromatographic conditions Column: Welch Ultimate LP-C18 4.6mm150mm; Mobile phase A is a trifluoroacetic acid aqueous solution with a volume fraction of 0.1%; mobile phase B is methanol-tetrahydrofuran (volume ratio 97.5:2.5); the volume ratio of mobile phase A to mobile phase B is 55:45.

[0034] Elution procedure: isocratic elution; Flow rate: 1.0 mL / min; Column temperature: 30°C; Detection wavelength: 297nm.

[0035] Injection volume: 20 μL.

[0036] 3. Separation degree investigation See also Figure 2 In the separation determination solution chromatogram, the separation degree of nemonoxacin peak and impurity B peak was 2.1.

[0037] In summary, this scheme uses trifluoroacetic acid solution as mobile phase A and methanol-tetrahydrofuran as mobile phase B. The addition of tetrahydrofuran to mobile phase B improves the peak shape of the main peak, with a tailing factor of 1.06, which can shorten the running time and improve the peak shape of the main peak. After mixing mobile phase A and mobile phase B, isocratic elution is performed, and the running time is shortened from 82 minutes to 15 minutes; the column temperature is increased from 10°C to 30°C, reducing the limitations of instrument selection; and the simultaneous control of impurities under the main component content determination system also reduces R&D costs.

[0038] The technical features of the above embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0039] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for detecting diastereomers in nemofloxacin malate sodium chloride injection, characterized in that: The following steps are involved: Liquid chromatography is used to separate the optical isomers of nemofloxacin malate sodium chloride injection; wherein the chromatographic conditions of the liquid chromatography include a mobile phase, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a trifluoroacetic acid solution, and the mobile phase B is methanol-tetrahydrofuran.

2. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The mobile phase A is a trifluoroacetic acid aqueous solution with a volume fraction of 0.1%.

3. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The mobile phase B is a methanol-tetrahydrofuran solvent with a volume ratio of 97.5:2.

5.

4. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is 55:

45.

5. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The chromatographic conditions also include a stationary phase, and the stationary phase is octadecylsilane bonded silica gel.

6. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The chromatographic conditions also include chromatographic column specifications, which are Welch Ultimate LP-C184.6*150mm, 5um.

7. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The chromatographic conditions also include a mobile phase flow rate, which is 1.0 mL per minute.

8. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The chromatographic conditions also include a chromatographic column temperature, which is 30°C.

9. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The chromatographic conditions also include a detection wavelength, which is 297 nm.

10. The method for detecting diastereomers in nemofloxacin malate sodium chloride injection according to claim 1, characterized in that: The chromatographic conditions also include an elution program, which is an isocratic elution with an injection volume of 20 μL and a running time of 15 min.

Images