CN114113353B

CN114113353B - Method for separating and detecting ethyl acetate and/or related impurities

Info

Publication number: CN114113353B
Application number: CN202010896576.6A
Authority: CN
Inventors: 张�荣; 王俊入; 周春燕
Original assignee: Chongqing Huabangshengkai Pharm Co ltd
Current assignee: Chongqing Huabangshengkai Pharm Co ltd
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2023-05-02
Anticipated expiration: 2040-08-31
Also published as: CN114113353A

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for analyzing ethyl gative carboxylate and related impurities by utilizing a high performance liquid chromatography. The chromatographic column adopted by the method uses phenyl bonded silica gel as a filler, adopts buffer salt solution and organic solvent for gradient elution, and has the flow rate of 0.9-1.1 ml/min and the column temperature of 23-27 ℃. The analysis method provided by the invention can well separate and accurately quantify the impurities MOXH-SM1f and MOXH-SM1i which have the structure similar to that of the ethyl acetate of the gative carboxylic acid within 37 minutes, can effectively separate the impurities from other 7 known impurities possibly existing in the ethyl acetate of the gative carboxylic acid, and provides the separation and measurement problems of the known impurities in the ethyl acetate of the gative carboxylic acid which are not solved by the prior art.

Description

Method for separating and detecting ethyl acetate and/or related impurities

Technical Field

The invention relates to the technical field of medicine analysis, in particular to a method for analyzing ethyl gative carboxylate and related impurities by utilizing a high performance liquid chromatography.

Background

Moxifloxacin is a chemical with a molecular formula of C ₂₁ H ₂₄ FN ₃ O ₄ Fluoroquinolones as antibacterial agents. The DNA topoisomerase inhibitor can be used for treating social acquired pneumonia, chronic bronchitis acute episode, acute sinusitis, etc. caused by Staphylococcus aureus, bacillus influenza, pneumococcus, etc. Belongs to a fourth-generation quinolone antibacterial drug, and is a new-generation antibiotic with broad antibacterial spectrum. The product has strong antibacterial activity against common respiratory tract bacteria such as Streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and part of Staphylococcus aureus, and especially against Streptococcus pneumoniae. Clinically used for treating acute Dou Xianyan, acute episode of chronic bronchitis, community-acquired pneumonia, and skin infection and skin soft tissue infection without complications. The product has the characteristics of almost no photosensitive reaction, good tissue penetrating power, high concentration in lung tissues and good medicine for treating respiratory tract infection.

The ethyl acetate of the gatifloxacin is a key starting material of the moxifloxacin, and the quality control of the ethyl acetate of the gatifloxacin is beneficial to guaranteeing the quality of the moxifloxacin. According to the known impurity structural formula possibly contained in the finished product in the moxifloxacin hydrochloride EP10.3 pharmacopoeia quality standard, it is presumed that the following 2 impurities with a structure highly similar to that of ethyl acetate may exist in the raw material ethyl acetate:

at present, no related literature or data discloses a method for separating and detecting the two impurities.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a method for separating ethyl acetate and/or related impurities by an HPLC method, by which related substances can be efficiently separated and the separation time can be reduced.

In order to achieve the above purpose, the present invention provides the following solutions:

the method for separating the ethyl acetate of the gative carboxylic acid and/or related impurities by an HPLC method comprises the steps of taking phenyl bonding silica gel as a filler, and carrying out gradient elution by adopting a mobile phase A and a mobile phase B to separate the ethyl acetate of the gative carboxylic acid and/or related impurities; the related impurities are one or more of impurities MOXH-SM1a, MOXH-SM1b, MOXH-SM1e, MOXH-SM1m, MOXH-SM1g, MOXH-SM1h, MOXH-SM1j, MOXH-SM1f and MOXH-SM1i; the structural formula of the ethyl gative carboxylate and related impurities is specifically as follows:

the mobile phase A is buffer salt solution, and the mobile phase B is organic solvent;

further, the buffer salt solution is a mixed solution of potassium dihydrogen phosphate solution and triethylamine, and the pH value of the buffer salt solution is 2.8-3.2;

specifically, the pH of the buffer salt solution is 3.0;

specifically, the buffer salt solution uses phosphoric acid to adjust the pH;

further, the concentration of the potassium dihydrogen phosphate in the buffer salt solution is 0.005-0.05 mol/L, and the volume percentage of the triethylamine in the buffer salt solution is 0.05-5.0%.

Specifically, the mass concentration of the potassium dihydrogen phosphate in the buffer salt solution is 2.72g/L, and the volume percentage of triethylamine in the buffer salt solution is 0.3%;

further, the organic solvent is one or more of methanol, ethanol and acetonitrile;

specifically, the organic solvent is methanol;

further, the gradient elution is:

time/minute	Mobile phase a/%	Mobile phase B/%
			0	38-42	58-62
3	40	60
			20	30	70
23	20	80
			29	20	80
30	40	60
			37	40	60

；

Specifically, the gradient elution is:

time/minute	Mobile phase a/%	Mobile phase B/%
			0	40	60
3	40	60
			20	30	70
23	20	80
			29	20	80
30	40	60
			37	40	60

；

Further, the flow rate of the mobile phase in the method is 0.9-1.1 ml/min; in the method, the column temperature of the chromatographic column is 23-27 ℃;

specifically, the flow rate of the mobile phase in the method is 1.0ml/min; the column temperature of the chromatographic column in the method is 25 ℃.

It is a further object of the present invention to provide a method for identifying ethyl acetate and/or related impurities which is effective in identifying related substances and which reduces the identification time;

the method for identifying the ethyl acetate and/or related impurities by using the HPLC method comprises the steps of utilizing the method in the first aim to separate the ethyl acetate and related impurities, and introducing the ethyl acetate and related impurities into a detector for detection, wherein the detection wavelength of the detector is 210-340 nm; the related impurity is the related impurity in purpose one; comparing the detected chromatogram with a known reference chromatogram, and identifying whether the detected object contains the ethyl acetate and/or related impurities;

specifically, the detector detects a wavelength of 250nm.

It is a further object of the present invention to provide a method for determining ethyl acetate of a gative carboxylic acid and/or impurities MOXH-SM1f and/or impurities MOXH-SM1i by HPLC; the method can effectively measure the content of related substances and control the measurement time;

a method for measuring ethyl acetate and/or impurity MOXH-SM1f and/or impurity MOXH-SM1i by an HPLC method, wherein the ethyl acetate and/or impurity MOXH-SM1f and/or impurity MOXH-SM1i are separated by the method in the first aim, a chromatogram is obtained by detection in a detector with a detection wavelength of 250nm, and the content of the ethyl acetate and/or impurity MOXH-SM1f and/or impurity MOXH-SM1i is calculated according to the peak area;

specifically, the method comprises the following specific steps:

s1: reagent preparation

Dissolving a sample to be tested in a diluent to obtain a sample solution; dissolving a reference substance in a diluent to obtain a reference substance solution; placing the sample into a sample feeding disc with the temperature controlled between 2 and 8 ℃;

s2: separation

Separating the ethyl acetate and/or impurity MOXH-SM1f and/or impurity MOXH-SM1i using the method described in object one;

s3: authentication

Identifying whether the test object contains the ethyl acetate and/or the impurity MOXH-SM1f and/or the impurity MOXH-SM1i by using the method described in the second aim;

s4: content calculation

Measuring peak areas according to the obtained chromatograms, and calculating the contents of the ethyl acetate and/or the impurity MOXH-SM1f and/or the impurity MOXH-SM1i;

further, the diluent is water and acetonitrile;

specifically, the diluent is water and acetonitrile in a volume ratio of 1:1.

The invention has the beneficial effects that:

1. the analysis method provided by the invention can well separate and detect impurities MOXH-SM1f and MOXH-SM1i which have structures similar to that of the ethyl acetate of the gative carboxylic acid.

2. The separation degree between the main component of the analysis method and other known impurities possibly existing in the sample and impurities MOXH-SM1f and MOXH-SM1i is larger than 1.5, and the analysis method has the advantages of strong specificity, good durability, high sensitivity and good reproducibility.

3. The analysis method provided by the invention can well separate and accurately quantify the impurities MOXH-SM1f and MOXH-SM1i which have the structure similar to that of the ethyl acetate of the gative carboxylic acid within 37 minutes, can effectively separate the impurities from other 7 known impurities possibly existing in the ethyl acetate of the gative carboxylic acid, and provides the separation and measurement problems of the known impurities in the ethyl acetate of the gative carboxylic acid which are not solved by the prior art.

Drawings

Fig. 1: HPLC chromatogram of the mixed solution;

fig. 2: detection limit HPLC chromatogram;

fig. 3: durability 1HPLC chromatogram;

fig. 4: durability 2HPLC chromatogram;

fig. 5: durability 3HPLC chromatogram;

fig. 6: durability 4HPLC chromatogram;

fig. 7: durability 5HPLC chromatogram;

fig. 8: durability 6HPLC chromatogram;

fig. 9: durability 7HPLC chromatogram;

fig. 10: durability 8HPLC chromatogram.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

Example 1

Taking a proper amount of the sample, precisely weighing, adding a diluent (water-acetonitrile, volume ratio is 1:1) for dissolving and quantitatively diluting to prepare a solution containing about 1mg in each 1ml, taking the solution as a sample solution, and immediately placing the sample solution into a sample injection tray with the temperature of 5 ℃ after preparation; precisely weighing 5.0ml, placing into a 50ml measuring flask, diluting to scale with diluent, shaking, precisely weighing 2.0ml, placing into a 100ml measuring flask, diluting to scale with diluent, shaking, and taking as control solution.

Chromatographic column (4.6mm.times.250 mm,5 μm) packed with phenyl-bonded silica gel; taking buffer salt (weighing 2.72g of monopotassium phosphate, adding 1000ml of water for dissolution, adding 3.0ml of triethylamine, adjusting pH to 3.0 with phosphoric acid) as a mobile phase A, taking methanol as a mobile phase B, performing linear gradient elution according to a table 1, and detecting the wavelength to be 250nm; the flow rate is 1.0ml per minute; the column temperature was 25 ℃.

TABLE 1 gradient elution table

Time [ (time ]Minute (min)	Mobile phase a (%)	Mobile phase B (%)
			0	40	60
3	40	60
			20	30	70
23	20	80
			29	20	80
30	40	60
			37	40	60

Accurately weighing a proper amount of a system applicability reference substance 1 (containing ethyl gative carboxylate MOXH-SM1, impurity MOXH-SM1i and impurity MOXH-SM1 f) of the ethyl gative carboxylate MOXH-SM1, accurately weighing, adding a diluent to dissolve and dilute the mixture to prepare a solution containing about 1mg of each 1ml, shaking the solution uniformly to serve as a system applicability solution, and immediately placing the solution into a sample injector with the temperature of 5 ℃ after preparation. Taking 10 μl, injecting into a liquid chromatograph, recording chromatogram, and sequentially making peaks according to impurities MOXH-SM1f, MOXH-SM1 and MOXH-SM1i, wherein the separation degree between the impurities MOXH-SM1f and the main component meets the requirement. Precisely measuring 10 μl of the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. And calculating the contents of the two impurities according to a main component comparison method added with the correction factors.

Example 2

Specialization of

Impurities that may be present in ethyl acetate of gative SM 1: the impurities MOXH-SM1a, MOXH-SM1b, MOXH-SM1e, MOXH-SM1m, MOXH-SM1g, MOXH-SM1h, MOXH-SM1j, MOXH-SM1f and MOXH-SM1i are 9 in total, and the impurities MOXH-SM1f and MOXH-SM1i are studied in the method.

Taking 10 μl of each of blank solution, impurity positioning solution, other impurity mixed solution, sample solution and mixed solution, sequentially feeding, recording chromatogram, and measuring the results shown in tables 2 and 3 and shown in fig. 1.

TABLE 2HPLC chromatogram integration results for the mixed solution

TABLE 3 determination results of specificity test

Conclusion: the main components of the blank solution and the sample solution do not interfere with the impurity MOXH-SM _1f And impurity MOXH-SM _1i Other known impurities and impurities MOXH-SM which may be present in the main component and the sample _1f And impurity MOXH-SM _1i The separation degree between the two is more than 1.5, and the method specificity meets the requirements.

Example 3

Detection limit

And taking the detection limit solution to continuously sample for 3 times, and calculating the ratio (signal to noise ratio) of the peak height of the main peak to the noise. The test results are shown in tables 4 and 5 and in FIG. 2.

TABLE 4 detection limit HPLC chromatogram integration results

TABLE 5 detection limit measurement results

Conclusion: the detection limit concentration of the impurity MOXH-SM1f is 0.074 mug/ml, the concentration in the test sample is 0.007%, and the signal to noise ratio average value is 14.2; the detection limit concentration of the impurity MOXH-SM1i is 0.065 mug/ml, the concentration in the test sample is 0.007%, and the signal to noise ratio average value is 10.9; the detection limit concentration of the main component SM1 is 0.064 mug/ml, the concentration of the main component SM1 in a test sample is 0.006%, and the signal to noise ratio average value is 8.4, which all meet the requirement of the detection limit.

Example 4

Durability to chromatographic conditions

Taking mixed solution under the special item, respectively using normal chromatographic conditions, testing different column temperatures, column flow rates, mobile phase B-phase methanol initial proportion and mobile phase A-phase buffer salt pH value of a predetermined test, respectively testing after an instrument system is stable, recording the separation degree between peaks, and inspecting the relative retention time and normalized content change condition of known impurities. The test results are shown in tables 2, 6-14, FIG. 1, and FIGS. 3-10 below.

TABLE 6 durability 1HPLC chromatogram integration results

TABLE 7 durability 2HPLC chromatogram integration results

Table 8 durability 3HPLC chromatogram integration results

Table 9 durability 4HPLC chromatogram integration results

Table 10 durability 5HPLC chromatogram integration results

Table 11 durability 6HPLC chromatogram integration results

Table 12 durability 7HPLC chromatogram integration results

TABLE 13 durability 8HPLC chromatogram integration results

TABLE 14 determination of durability test for chromatographic condition change

Conclusion: when the chromatographic conditions slightly fluctuate, the separation degree between the impurity MOXH-SM1f and the main peak is more than 1.5, and the separation degree between the impurity MOXH-SM1i and the adjacent components is more than 1.4, so that the method has better durability.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

The method for separating the ethyl acetate of the gative carboxylic acid and related impurities by an HPLC method is characterized in that a chromatographic column adopted by the method takes phenyl bonding silica gel as a filler, and a mobile phase A and a mobile phase B are adopted for gradient elution to separate the ethyl acetate of the gative carboxylic acid and related impurities; the related impurities are impurities MOXH-SM1f and impurities MOXH-SM1i; the structural formula of the ethyl gative carboxylate and related impurities is specifically as follows:

；

the mobile phase A is buffer salt solution, and the mobile phase B is organic solvent; the buffer salt solution is a mixed solution of potassium dihydrogen phosphate solution and triethylamine, and the pH value of the buffer salt solution is 2.8-3.2; the mass concentration of the potassium dihydrogen phosphate in the buffer salt solution is 0.005-0.05 mol/L, and the volume percentage of the triethylamine in the buffer salt solution is 0.05-5.0%; the organic solvent is methanol; the gradient elution specifically comprises the following steps:

time/minute Mobile phase a/% Mobile phase B/% 0 38-42 58-62 3 40 60 20 30 70 23 20 80 29 20 80 30 40 60 37 40 60

。
2. The method according to claim 1, wherein the flow rate of the mobile phase in the method is 0.9 to 1.1ml/min; the temperature of the chromatographic column in the method is 23-27 ℃.
A method for identifying ethyl acetate and related impurities by an HPLC method, which is characterized in that the ethyl acetate and related impurities are separated by the method in claim 1 and are detected by a detector with the detection wavelength of 210-340 nm; the related impurity is the related impurity in claim 1; comparing the detected chromatogram with the chromatogram of the known reference substance, and identifying whether the detected substance contains the ethyl acetate and related impurities.
A method for determining ethyl acetate and impurities MOXH-SM1f and MOXH-SM1i by an hplc method, characterized in that ethyl acetate and impurities MOXH-SM1f and MOXH-SM1i are separated by the method according to claim 1, detected in a detector with a detection wavelength of 250nm to obtain a chromatogram, and the contents of ethyl acetate and impurities MOXH-SM1f and impurities MOXH-SM1i are calculated according to peak areas.
5. The method according to claim 4, characterized in that the method comprises the following specific steps:

s1: reagent preparation

Dissolving a sample to be tested in a diluent to obtain a sample solution; dissolving a reference substance in a diluent to obtain a reference substance solution; placing the prepared reagent into a sample injection disc with the temperature controlled between 2 and 8 ℃;

s2: separation

Separating the ethyl acetate and impurities MOXH-SM1f and MOXH-SM1i using the process of claim 1;

s3: authentication

Identifying whether the test object contains the ethyl acetate and impurities MOXH-SM1f and MOXH-SM1i by the method of claim 3;

s4: content calculation

From the obtained chromatogram, the peak area was measured, and the contents of the ethyl acetate and the impurities MOXH-SM1f and MOXH-SM1i were calculated.
6. The method of claim 5, wherein the diluent is water or acetonitrile.