CN113777180B

CN113777180B - Method for simultaneously detecting sitafloxacin and various isomer impurities thereof

Info

Publication number: CN113777180B
Application number: CN202010806358.9A
Authority: CN
Inventors: 曹龙祥; 郝妮媛; 李萍; 弓桦; 鲍鹏飞; 杨凯
Original assignee: Jumpcan Pharmaceutical Group Co ltd
Current assignee: Jumpcan Pharmaceutical Group Co ltd
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2023-05-02
Anticipated expiration: 2040-08-12
Also published as: CN113777180A

Abstract

The invention belongs to the technical field of analytical chemistry, and relates to a method for simultaneously detecting sitafloxacin and various isomer impurities thereof. The method utilizes high performance liquid chromatography to detect, and chromatographic conditions include: the chromatographic column adopts polysaccharide derivative coated reversed phase chiral chromatographic column; the mobile phase adopts a binary mobile phase system, the A phase is a triethylamine aqueous solution with the concentration of 0.45-0.55% v/v, and the B phase is acetonitrile; the elution mode adopts isocratic elution, and the volume ratio of the phase A to the phase B is 7:93-9:91. The method has the characteristics of both qualitative and quantitative determination, accurate and reliable result, good reproducibility, simple operation and the like, and can simultaneously and efficiently detect sitafloxacin and SRR, RSR and RSS optical isomers thereof, thereby realizing accurate monitoring of impurity spectrums and strict control of product quality.

Description

Method for simultaneously detecting sitafloxacin and various isomer impurities thereof

Technical Field

The invention belongs to the technical field of analytical chemistry, and relates to a method for simultaneously detecting sitafloxacin and various isomer impurities thereof.

Background

Sitafloxacin (Sitafloxacin/STFX/DU-6859 a) is a quinolone broad-spectrum antibacterial agent developed by Japanese first Co-product (Daiichi Sankyo), with CAS number 127254-12-0, chemical name 7- [ (7S) -7-amino-5-azaspiro [2.4] hept-5-yl ] -8-chloro-6-fluoro-1- [ (1R, 2S) -2-fluorocyclopropyl ] -1, 4-dihydro-4-oxoquinoline-3-carboxylic acid (structural formula shown below, containing 3 chiral centers, absolute configuration of SRS type, 7 optical isomers) and is often used clinically in the form of a hydrate (e.g., sesquihydrate) for treating serious bacterial infection or bacterial infection caused by drug resistant bacteria.

In chiral drugs, the presence of isomers (particularly optical isomers) in many cases affects the efficacy of drugs and may even cause toxic or side effects, and therefore it is very important to analyze and detect optical isomers in drugs having specific absolute configurations.

In terms of capillary electrophoresis, zhuang Xiaoqing et al used capillary electrophoresis based on beta-cyclodextrin and its derivatives to chiral resolve sitafloxacin and its three isomers (SSR/RRS/RSR) (Zhuang Xiaoqing, populus tomentosa, dibin et al, capillary electrophoresis chiral resolve sitafloxacin isomer [ J ], journal of drug analysis, 2012,32 (8): 1389-1393). Chinese patent application CN 107966489A discloses a method for simultaneously measuring sitafloxacin and five isomers thereof (RRS/SSR/RSR/SRR/SSS) by capillary electrophoresis. However, chiral resolution of sitafloxacin and isomers thereof by capillary electrophoresis has the disadvantages of complex operation, poor reproducibility, low sensitivity and the like.

In terms of high performance liquid chromatography, zhang Qiujia et al used HPLC method using D-phenylalanine/copper sulfate aqueous solution as mobile phase to determine two isomers (SSR/RRS) in sitafloxacin (Zhang Qiujia, chen Lei, de superb, HPLC method to determine sitafloxacin isomer [ J ], J. Chinese J. Antibiotics, 2012,37 (3): 237-239). In order to solve the problem of poor separation of sitafloxacin and its isomers, zhu Ziwei et al have adjusted D-phenylalanine to L-phenylalanine and determined three isomers (SSR/RRS/RSR) in sitafloxacin tablets by HPLC (Zhu Ziwei, MIGY, zhang Xiaoli et al, method study [ J ] of HPLC determination of sitafloxacin tablet isomers, J.China antibiotics 2015,40 (12): 944-947, 953). However, most of the existing HPLC detection methods focus only on two pairs (SSR/RRS and SRS/RSR) of the four pairs of enantiomers, while detection methods surrounding the other two pairs (RSS/SRR and RRR/SSS) are rarely reported.

In order to detect more isomer impurities in sitafloxacin, and also to more comprehensively monitor the product quality of sitafloxacin, and further ensure the medication safety of patients, development of a detection method for RSS/SRR type and/or RRR/SSS type isomers is needed.

Disclosure of Invention

Problems to be solved by the invention

In order to make up the blank of the prior art and solve the problem that sitafloxacin and RSS type and SRR type isomer impurities thereof cannot be detected simultaneously and efficiently at present, the invention provides a method for simultaneously detecting sitafloxacin and various isomer (including RSS type and SRR type isomers) impurities thereof by adopting an HPLC method, so that sitafloxacin impurity mass spectrum is accurately monitored, and further product quality is strictly controlled.

Solution for solving the problem

The invention provides a method for simultaneously detecting sitafloxacin and various isomer impurities thereof, which is characterized in that the method utilizes high performance liquid chromatography to detect the sitafloxacin and various isomer impurities thereof, and chromatographic conditions of the high performance liquid chromatography comprise: the chromatographic column adopts polysaccharide derivative coated reversed phase chiral chromatographic column; the mobile phase adopts a binary mobile phase system, the A phase is a triethylamine aqueous solution with the concentration of 0.45-0.55% v/v, and the B phase is acetonitrile; the elution mode adopts isocratic elution, and the volume ratio of the phase A to the phase B is 7:93-9:91.

Specifically, in the above-described method, the isomers include RSS type isomers and SRR type isomers of sitafloxacin, and preferably include RSR type isomers.

Specifically, in the above method, the polysaccharide derivative coated reversed phase chiral column preferably has silica gel coated with cellulose-tris (4-chloro-3-methylphenyl carbamate) as a filler

OX-3R series analytical column.

Specifically, in the above method, the pH of the a phase is 8.5 to 9.0, preferably 8.8, more preferably adjusted by phosphoric acid.

Specifically, in the above method, the chromatographic conditions further include: column temperature: 25+/-2 ℃; a detector: UV detector or PDA detector; detection wavelength: 295+ -2 nm; flow rate: 0.7+/-0.1 ml/min; sample injection amount: 5.+ -. 2. Mu.l.

ADVANTAGEOUS EFFECTS OF INVENTION

Compared with the prior art, the method for simultaneously detecting sitafloxacin and various isomer impurities thereof has the characteristics of simultaneously taking qualitative and quantitative properties, accurate and reliable results, good repeatability, simple operation and the like, and can simultaneously and efficiently detect sitafloxacin and optical isomers thereof (SRR, RSR and RSS type), thereby realizing accurate monitoring of impurity spectrums and strict control of product quality.

Drawings

Fig. 1-4 show liquid chromatograms of the positioning solutions of each of the three isomers of sitafloxacin and SRR, RSR, RSS.

Fig. 5 shows a liquid chromatogram of a control solution containing sitafloxacin and three isomeric impurities.

Fig. 6 shows a liquid chromatogram of a sitafloxacin test solution.

Fig. 7 shows a liquid chromatogram of a loaded sample solution containing sitafloxacin and three isomeric impurities.

Fig. 8 shows a liquid chromatogram of a test solution of sitafloxacin sample (pilot and secondary test # 1).

Fig. 9 shows a liquid chromatogram of a test solution of sitafloxacin sample (pilot and secondary test # 2).

Fig. 10 shows a liquid chromatogram of a test solution of sitafloxacin sample (pilot and secondary test # 3).

Fig. 11 shows a liquid chromatogram of a test solution of sitafloxacin sample (pilot # 1).

Fig. 12 shows a liquid chromatogram of a test solution of sitafloxacin sample (pilot # 2).

Fig. 13 shows a liquid chromatogram of a test solution of sitafloxacin sample (pilot # 3).

Detailed Description

The term "sitafloxacin" as used herein refers to a compound having the CAS number 127254-12-0, chemical name 7- [ (7S) -7-amino-5-azaspiro [2.4] hept-5-yl ] -8-chloro-6-fluoro-1- [ (1 r, 2S) -2-fluorocyclopropyl ] -1, 4-dihydro-4-oxoquinoline-3-carboxylic acid or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof, unless otherwise specified.

The term "isomer" (or "isomer") refers to a compound having the same number and type of atoms and thus the same molecular weight, but a different spatial arrangement or configuration of atoms. The term "stereoisomer" refers to an isomer produced by the atoms in a molecule in a different spatial arrangement, and includes both "configurational isomer" and "conformational isomer". The term "configurational isomer" refers to isomers of atoms in a molecule that result from different spatial arrangements, and includes both "cis-trans isomers" and "optical isomers". The term "cis-trans isomer" refers to an isomer in which atoms (or groups) located on both sides of a double bond or ring system are located on the same side of the double bond or ring system as the result of the different positions relative to a reference plane, and in which atoms (or groups) are located on the opposite side of the double bond or ring system as the result of the trans-isomer, wherein "double bond" generally refers to a carbon-carbon double bond and also includes a carbon-nitrogen double bond and a nitrogen-nitrogen double bond. The term "optical isomer" (or "optical isomer", "optical body" or "optical isomer") refers to a stable isomer that has a vertical plane of asymmetry due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby enabling rotation of plane polarized light. Unless otherwise indicated, "isomer" as used in the present invention may refer to eight optical isomers including sitafloxacin (SRS type).

The invention specifically discloses a method for simultaneously detecting sitafloxacin and various isomer impurities thereof, which utilizes High Performance Liquid Chromatography (HPLC) to detect the sitafloxacin and various isomer impurities thereof, and the chromatographic conditions used comprise: the chromatographic column adopts polysaccharide derivative coated reversed phase chiral chromatographic column; the mobile phase adopts a binary mobile phase system, the A phase is a triethylamine aqueous solution with the concentration of 0.45-0.55% v/v, and the B phase is acetonitrile; the elution mode adopts isocratic elution, and the volume ratio of the phase A to the phase B is 7:93-9:91.

In one embodiment of the present invention, the isomers considered as impurities in the above-described process mainly include RSS-type isomers and SRR-type isomers of sitafloxacin.

In a preferred embodiment of the present invention, the isomers considered as impurities in the above process also include the RSR-type isomer of sitafloxacin.

In one embodiment of the present invention, the polysaccharide derivative coated reversed phase chiral chromatography column of the above method uses silica gel coated with cellulose-tris (4-chloro-3-methylphenyl carbamate) as a filler, and is suitable for use with a more polar mobile phase.

In a preferred embodiment of the present invention, the polysaccharide derivative coated reversed phase chiral chromatography column in the above method is

OX-3R series analytical column with packing particle size of 3 μm, column inner diameter of 4.6mm, column length of 50, 100, 150 or 250mm.

In one embodiment of the present invention, phase a in the above method is required to maintain a certain pH, which is usually 8.5 to 9.0, preferably 8.8, in order to facilitate separation and purification of the substance to be tested.

In a preferred embodiment of the present invention, the pH of the above-mentioned phase a is adjusted by phosphoric acid.

In one embodiment of the present invention, the chromatographic conditions in the above method further include column temperature, detector, detection wavelength, mobile phase flow rate, sample injection amount, and the like.

In a preferred embodiment of the present invention, the column temperature is 25.+ -. 2 ℃.

In a preferred embodiment of the invention, the detector is a UV detector or a PDA detector.

In a preferred embodiment of the present invention, the detection wavelength is 295.+ -.2 nm.

In a preferred embodiment of the present invention, the mobile phase flow rate is 0.7.+ -. 0.1ml/min.

In a preferred embodiment of the present invention, the amount of the above-mentioned sample is 5.+ -.2. Mu.l.

The technical scheme of the invention will be further described below with reference to specific examples. The instruments, medicines, reagents, etc. used in the following examples are all available by conventional commercial means unless otherwise specified.

Examples: detecting the content of sitafloxacin and isomer impurities thereof.

1. Instruments, reagents and medicines:

(1) Instrument:

waters e2695-2498-2998 high performance liquid chromatograph, manufactured by Waters corporation, U.S.A.;

XS205DU electronic balance, manufactured by Mertrex-tolidox, switzerland;

model FE 28 pH meter, manufactured by mertrele-tolidox, switzerland;

FGZ 200Z-type medicine illumination test box manufactured by Fuqi corporation of Beijing middle-family;

DHG-9030A type electrothermal constant temperature blast drying oven is manufactured by Shanghai Sorpe company;

DK-S24 type electric heating constant temperature water bath pot is produced by Shanghai Jing Hongzheng Co.

(2) Reagent:

phosphoric acid, analytically pure, lot number: AR 20180315, produced by the national drug group;

acetonitrile, chromatographic purity, lot number: JA077830, manufactured by merck, germany;

triethylamine, analytical grade, lot number: k1806002, produced by aladine company, shanghai;

methanol, chromatographic purity, lot number: i0989707903, manufactured by merck, germany;

ultrapure water is self-made.

(3) Control:

sitafloxacin sesquihydrate

436.84 g/mol), sitafloxacin RSR isomer sesquihydrate (/ -for the case of the salt>

436.84g/mol Sitafloxacin RSR isomer sesquihydrate

436.84g/mol Sitafloxacin RSR isomer sesquihydrate

436.84 g/mol), all manufactured by Suzhou Chu Kai pharmaceutical Co.

2. Chromatographic conditions:

in this example sitafloxacin and its various isomer impurities were determined by HPLC using reversed phase chiral columns, the optimum chromatographic conditions being shown in table 1. Under this condition, the peak shape of each isomer is good, the retention time is reasonable, and the degree of separation between the peaks is also ideal.

TABLE 1

3. Preparing a solution:

3.1 preparation of a pre-prepared liquid:

the sitafloxacin reference substance and the three isomer (RSR, SRR and RSS) reference substances are respectively weighed about 1mg, precisely weighed, placed into four 10ml volumetric flasks, dissolved and diluted to scale by adding methanol, and uniformly shaken to obtain four pre-prepared solutions (the mass concentration of the four solutes is about 0.1mg/ml and recorded as 10% mass concentration).

3.2 preparation of positioning solution:

precisely weighing 1ml of each of the four pre-prepared solutions in item 3.1, placing into four 10ml volumetric flasks, adding methanol for dilution to scale, and shaking uniformly to obtain four positioning solutions (the mass concentration of the four solutes is about 0.01mg/ml, and recorded as 1% mass concentration).

3.3 preparation of control solution:

precisely weighing 1ml of each of the four pre-prepared solutions in item 3.1, placing into a 10ml volumetric flask, adding methanol to dilute to scale, and shaking to obtain reference solution (the mass concentration of each of the four solutes is about 0.01mg/ml, and recorded as 1% mass concentration).

3.4 preparation of sample solution:

about 10mg of sitafloxacin sample (sample to be measured) is weighed, precisely weighed, placed in a 10ml volumetric flask, dissolved by adding methanol, diluted to a scale, and shaken uniformly to obtain sample solution.

3.5 preparation of sample solution:

and (3) weighing about 10mg of sitafloxacin to be tested (to-be-tested sample), precisely weighing, placing into a 10ml volumetric flask, adding a proper amount of methanol for dissolution, precisely weighing 0.5ml of each of the three isomer pre-prepared solutions in the 3.1 item, adding into the volumetric flask, adding methanol for dilution to a scale, and shaking uniformly to obtain a sample-adding to-be-tested solution.

4. And (3) measuring:

4.1 measurement method:

precisely measuring 5 μl of the sample solution in item 3, injecting into a liquid chromatograph, detecting according to the chromatographic conditions in item 2, recording corresponding chromatograms, and calculating the content of each component.

4.2 detection criteria:

impurity content limits are controlled with reference to import drug registration standards, and isomer impurity content limits are as follows: the content of the RSR/SRR/RSS type isomer is not more than 0.5%, and the sum of the contents of the three isomers is not more than 1.0%.

5. And (3) methodological verification:

5.1 specificity:

precisely weighing 5 μl of each of the four positioning solutions, the control solution, the sample solution and the sample-adding sample solution in example 3.1, injecting into a high performance liquid chromatograph, detecting according to the chromatographic conditions in item 2, and recording the chromatograms, wherein the results are shown in fig. 1 to 7 and table 2.

TABLE 2

As can be seen by comparing fig. 1 to 5, in the HPLC spectra (fig. 1 to 4) of the four positioning solutions, the retention times of sitafloxacin, SRR type isomer, RSR type isomer and RSS type isomer were 15.588min, 22.114min, 32.081min and 41.721min, respectively; based on the above, chromatographic peaks corresponding to sitafloxacin and SRR, RSR, RSS isomer thereof can be clearly identified in the HPLC spectrogram (figure 5) of the reference solution, the retention time of the four chromatographic peaks is sequentially increased, the minimum separation degree between the components is 5.681, and the requirements are met.

However, no chromatographic peaks for the above three isomers could be observed in the HPLC profile of the test solution (fig. 6), indicating that the batch of sitafloxacin test contained almost no impurity of SRR, RSR, RSS type. To further examine the feasibility of the method, the same chromatographic conditions were used to determine the sample-loaded sample solution, and the chromatographic peak of the isomer impurity was observed in the HPLC spectrum (fig. 7), and the same ideal degree of separation was found between the chromatographic peak and the main peak of sitafloxacin.

In addition, because the detection method of the invention utilizes a triethylamine-acetonitrile binary mobile phase system, the blank solvent does not interfere with the detection of a main peak (while when the detection is carried out by a method of Zhang Qiujia et al, the used derivatization agent leads to easy drift of a baseline, the time required for balancing is longer, and some instruments have the problem of larger baseline noise); at the same time, other types of impurities do not interfere with the detection of the target isomer chromatographic peak.

The results show that the detection method disclosed by the invention can be used for simultaneously, efficiently and accurately detecting the content of SRR/RSR/RSS type isomer in the sitafloxacin sample, has higher feasibility and is suitable for popularization and application.

5.2 stability of solution:

preparing a sample solution, a sample 1% self-control solution and a system applicability solution respectively, inspecting the solution stability after being placed for different time, and making corresponding requirements by referring to verification and confirmation of an analysis method in GMP quality control as follows:

(1) In the sample solution, the RSD of the main peak area and the main peak content is less than 2%, and the RSD of the main peak retention time is less than or equal to 1%; RSD of isomer content (calculated as peak area normalization) should be in agreement: impurity less than or equal to 0.1%, RSD less than or equal to 30%; 0.1 to 0.2 percent of impurity, and RSD is less than or equal to 20 percent; 0.2 to 0.5 percent of impurity, and less than or equal to 10 percent of RSD; 0.5 to 1.0 percent of impurity, and less than or equal to 5 percent of RSD; impurities are more than or equal to 1.0%, and RSD is less than or equal to 2%;

(2) In the 1% self-control solution of the test sample, the RSD of the area of the main peak is less than or equal to 5%;

(3) In the system applicability solution, the RSD of the main peak area is less than 2%, and the RSD of the isomer peak area is less than or equal to 5%.

5.2.1 examination of test solutions:

taking about 10mg of sitafloxacin to be tested, precisely weighing, placing into a 10ml volumetric flask, adding methanol for dissolving, diluting to scale, and shaking uniformly to obtain a test solution.

Under the condition of room temperature (20+/-10 ℃), 5 mu l of each sample solution is precisely measured at 0h, 3h, 6h, 9h, 12h, 15h, 18h and 24h, and the sample solutions are injected into a liquid chromatograph, detected according to the chromatographic condition in item 2, and chromatograms are recorded, and the results are shown in Table 3.

TABLE 3 Table 3

As shown in Table 3, after the sample solution was left at room temperature for 24 hours, the RSD of the main peak area was 0.18%, the RSD of the main peak content was 0, both were less than 2%, and the RSD of the main peak retention time was 0.08%, less than 1%, both satisfying the requirements, indicating that the sample solution was stable at room temperature for 24 hours.

5.2.2 investigation of test 1% self-control solution:

precisely measuring 1ml of the sample solution in the example 5.2.1, placing in a 100ml volumetric flask, adding methanol to dilute to scale, and shaking uniformly to obtain the 1% self-control solution of the sample.

Under the condition of room temperature (20+/-5 ℃), 5 mu l of each 1% self-control solution is precisely measured at 0h, 3h, 6h, 9h, 12h, 15h, 18h and 24h, the solution is injected into a liquid chromatograph, detection is carried out according to the chromatographic condition in item 2, and a chromatogram is recorded, and the results are shown in Table 4.

TABLE 4 Table 4

As can be seen from Table 4, after the 1% self-control solution of the test sample was left at room temperature for 24 hours, the RSD of the main peak area was 0.50% and less than 5%, which is satisfactory, indicating that the 1% self-control solution of the test sample was stable at room temperature for 24 hours.

5.2.3 investigation of System applicability solution:

about 2mg of each of the three isomers (SRR/RSR/RSS) was taken, precisely weighed, placed in different 20ml volumetric flasks, dissolved in methanol and diluted to the scale, and shaken well to obtain three isomer stock solutions. Taking about 10mg of sitafloxacin to be tested, precisely weighing, placing into a 10ml volumetric flask, adding a proper amount of methanol for dissolution, precisely measuring 0.5ml of each of the three isomer stock solutions, placing into the volumetric flask, adding methanol for dilution to a scale, and shaking uniformly to obtain a system applicability solution.

5. Mu.l of each of the system-suitable solutions was precisely measured at room temperature (20.+ -. 5 ℃ C.) for 0h, 3h, 6h, 9h, 12h, 15h, 18h and 24h, and the measured solutions were injected into a liquid chromatograph, followed by detection under the chromatographic conditions in item 2, and the chromatograms were recorded, and the results are shown in Table 5.

TABLE 5

As can be seen from Table 5, the RSD of the main peak area was 0.39% or less than 2%; the RSD of the SRR type isomer peak area was 0.50%, the RSD of the RSR type isomer peak area was 0.48%, and the RSD of the RSS type isomer peak area was 0.26%, both less than 5%, indicating that the system applicability solution was stable within 24h at room temperature.

5.3 sensitivity:

about 2mg of sitafloxacin and about 2mg of each of three isomers (SRR/RSR/RSS) are respectively taken, precisely weighed, placed in different 20ml volumetric flasks, dissolved by adding methanol, diluted to a scale and uniformly shaken to obtain stock solution. And respectively precisely measuring each appropriate amount of the four stock solutions, placing the four stock solutions into the same volumetric flask, and adding methanol to dilute the four stock solutions into a mixed reference substance solution with a certain concentration. Gradually diluting the mixed reference substance solution, and recording the signal to noise ratio (S/N) of the corresponding chromatogram.

When the signal-to-noise ratio is 10, the concentration at this time is calculated as the quantitative limit concentration, and 6 parts of the concentration are diluted by the same method, the RSD of the measured peak area is less than 10%, and the RSD of the retention time is less than 1%. When the signal-to-noise ratio is 3, the concentration at the moment is calculated, namely the detection limit concentration. The above requirements are formulated with reference to the validation and validation of analytical methods in GMP quality control.

5. Mu.l of each of the above solutions of different concentrations was measured precisely, and the measured solution was injected into a liquid chromatograph, followed by measurement under the chromatographic conditions described in item 2, and the chromatograms were recorded, and the results are shown in Table 6.

TABLE 6

For sitafloxacin, the limit of detection was 0.11ng and the limit of quantification was 0.28ng. The RSD of the peak area of the 6 quantitative limited samples is 8.89% and is less than 10%; the RSD of the retention time is 0.10%, less than 1%.

For the SRR type isomer, the limit of detection was 0.20ng and the limit of quantification was 0.60ng. The RSD of the peak area of the 6 quantitative limit samples is 2.40% and less than 10%; the RSD of the retention time was 0.05%, less than 1.0%.

For the RSR type isomer, the limit of detection was 0.26ng and the limit of quantification was 0.79ng. The RSD of the peak area of the 6 quantitative limited samples is 9.24% and less than 10%; the RSD of the retention time was 0.06%, less than 1.0%.

For the RSS type isomer, the limit of detection was 0.34ng and the limit of quantification was 0.95ng. The RSD of the peak area of the 6 quantitative limit samples is 5.68% and less than 10%; the RSD of the retention time was 0.08%, less than 1.0%.

The above results indicate that the sensitivity of the method is good.

5.4 linear range:

taking the concentration of the 1% self-control solution (about 10 mug/ml) of the sample in the 5.2 item as 100%, the linear relationship of 7 to 9 concentration points of sitafloxacin and various isomers thereof in the 1% self-control solution of the sample in the range of quantitative limit concentration to 150% concentration was examined. Linear regression is performed with the concentration on the abscissa and the peak area on the ordinate, the correlation coefficient r should not be less than 0.998, and the intercept should not be greater than 25%. The above requirements are formulated with reference to the validation and validation of analytical methods in GMP quality control.

About 2mg of sitafloxacin and about 2mg of each of three isomers (SRR/RSR/RSS) are respectively taken, precisely weighed, placed in different 20ml volumetric flasks, dissolved by adding methanol, diluted to a scale and uniformly shaken to obtain stock solution. Then respectively measuring 20 mu l, 50 mu l, 0.1ml, 0.2ml, 0.3ml, 0.5ml, 0.8ml, 1ml and 1.5ml of each stock solution precisely, placing the stock solutions into different 10ml volumetric flasks, adding methanol to dilute to a scale, and shaking uniformly to obtain a linear solution. The linear solution preparation method is shown in Table 7, and the results are shown in Table 8.

TABLE 7

TABLE 8

The SRR type isomer has good linear relation in the concentration range of 2% -150%, namely 0.2-15 mug/ml, meets the test requirement, the linear equation is y= 35980.330x-3796.515, r=1.000, is larger than 0.998, and the intercept is 1.05% and smaller than 25%.

The RSR isomer has good linear relation in the concentration range of 2% -150%, namely 0.2-15 mug/ml, meets the test requirement, the linear equation is y= 32850.340x-4541.668, r=0.999, is larger than 0.998, and the intercept is 1.30% and smaller than 25%.

The RSS isomer has good linear relation in the concentration range of 2% -150%, namely 0.2-15 mug/ml, meets the test requirement, and has the linear equation of y= 35083.380x-3362.124, r=1.000, more than 0.998, intercept of 0.85% and less than 25%.

Sitafloxacin has good linear relation in the concentration range of 2% -150%, namely the concentration range of 0.2-15 mug/ml, meets the test requirement, and has a linear equation of y= 35290.740x-1102.266, r=0.999, more than 0.998, an intercept of 0.27% and less than 25%.

In addition, the correction factors for SRR, RSR and RSS type isomers were 0.98, 1.07 and 1.01, respectively.

5.5 recovery rate:

about 2mg of each of three isomer (SRR/RSR/RSS) controls is respectively taken, precisely weighed, placed in different 20ml volumetric flasks, dissolved and diluted to scale by adding methanol, and uniformly shaken to obtain stock solution. Precisely measuring 0.5ml of stock solutions of the three isomers respectively, placing the stock solutions into a 10ml volumetric flask, adding methanol for dilution to a scale, and shaking uniformly to obtain a mixed control solution.

Taking about 10mg of sitafloxacin sample, precisely weighing, placing in a 10ml volumetric flask, adding methanol for dissolving and diluting to scale, and shaking uniformly to obtain background solution.

And (3) arranging a plurality of test groups in parallel, wherein each group takes about 10mg of sitafloxacin test sample, accurately weighing the sitafloxacin test sample, placing the sitafloxacin test sample in a 10ml volumetric flask, adding a proper amount of methanol for dissolution, adding 0.4 ml, 0.5ml or 0.6ml of one of the stock solutions of the three isomers into each group respectively (the relative concentration is 80%, 100% and 120% of the limit of the stock solutions respectively), adding methanol for dilution to a scale, and shaking uniformly to obtain the recovery rate test sample solution.

Each of 5 μl of the above recovery test solution was precisely measured, and the measured solutions were injected into a liquid chromatograph, followed by detection according to the chromatographic conditions in item 2, recording the chromatograms, and calculating the recovery and RSD according to the external standard method (refer to the verification and confirmation of analytical methods in the chinese pharmacopoeia and GMP quality control, and the corresponding requirements were formulated as follows: recovery in the range of 90% -108%, RSD less than 5%), and the results are shown in table 9.

TABLE 9

The recovery rates of SRR, RSR and RSS type isomer are 101.41%, 104.16% and 96.30% respectively, 90% -108% respectively, and RSD is 1.99%, 1.31% and 2.36% respectively, and less than 5% respectively. The recovery rate of each isomer accords with the verification requirement, which shows that the detection method has good accuracy.

5.6 durability:

after appropriate adjustment of individual parameters in chromatographic conditions (such as flow rate, column temperature, binary mobile phase ratio, detection wavelength, etc.), the method is shown to have good durability if the measurement results are not substantially significantly affected.

With reference to the verification and validation of analytical methods in the chinese pharmacopoeia and GMP quality control, specific requirements are formulated as follows:

(1) In the system applicability solution, the separation degree between chromatographic peaks of each component is not less than 1.5;

(2) In the sensitivity solution, the signal to noise ratio of the main peak is not lower than 10;

(3) In the sample solution, if isomer peaks exist, the content of each isomer is calculated according to a self-comparison method, wherein the RSD of the isomer content meets the requirements (the impurity is less than or equal to 0.1 percent, the RSD is less than or equal to 40 percent, the impurity is 0.1 to 0.2 percent, the RSD is less than or equal to 30 percent, the impurity is 0.2 to 0.5 percent, the RSD is less than or equal to 15 percent, the impurity is 0.5 to 1.0 percent, the RSD is less than or equal to 7.5 percent, the impurity is more than or equal to 1.0 percent and the RSD is less than or equal to 4 percent).

The parameter adjustment conditions for each durability test are shown in table 10.

Table 10

Parameters (parameters)	Basic value	Adjustment range
			Detection wavelength	295nm	±2nm
Binary flow phase ratio	8:92	±1％
			Flow rate	0.7ml/min	±0.1ml/min
Column temperature	25℃	±2℃
			Sample injection volume	5μl	±2μl

About 2mg of each of the three isomers (SRR/RSR/RSS) was taken, precisely weighed, placed in different 20ml volumetric flasks, dissolved in methanol and diluted to the scale, and shaken well to obtain three isomer stock solutions.

Taking about 10mg of sitafloxacin to be tested, precisely weighing, placing into a 10ml volumetric flask, adding a proper amount of methanol for dissolution, precisely measuring 0.5ml of each of the three isomer stock solutions, placing into the volumetric flask, adding methanol for dilution to a scale, and shaking uniformly to obtain a system applicability solution.

Taking about 10mg of sitafloxacin to be tested, precisely weighing, placing into a 10ml volumetric flask, adding methanol for dissolving, diluting to scale, and shaking uniformly to obtain a test solution. Precisely measuring 1ml of the sample solution, placing in a 100ml volumetric flask, adding methanol to dilute to scale, and shaking to obtain 1% self-control solution of the sample.

Precisely measuring 0.5ml of 1% self-control solution of the sample, placing the sample in a 10ml volumetric flask, adding methanol to dilute the sample to a scale, and shaking the sample uniformly to obtain a sensitivity solution.

Taking about 10mg of sitafloxacin to be tested, precisely weighing, placing into a 10ml volumetric flask, adding a proper amount of methanol for dissolution, precisely measuring 0.5ml of each of the three isomer stock solutions, placing into the volumetric flask, adding methanol for dilution to a scale, and shaking uniformly to obtain a sample adding to be tested solution. Precisely measuring 1ml of the sample solution, placing in a 100ml volumetric flask, diluting to scale with methanol, and shaking to obtain 1% self-control solution of the sample.

And precisely measuring 5 mu l of each of the system applicability solution, the sensitivity solution, the sample-adding test solution and the 1% self-control solution, injecting into a liquid chromatograph, detecting according to the durability chromatographic conditions, and recording chromatograms, wherein the results are shown in tables 11-15.

TABLE 11

When the sample injection volume changes by +/-2 mu l, the separation degree of a main peak and an adjacent isomer chromatographic peak in the system applicability solution is more than 1.5; in the sensitivity solution, the signal to noise ratio of the main peak is more than 10; in the sample solution, the RSD of the contents of the SRR, the RSR and the RSS type isomers meets the requirements, so that the detection method has good durability in the aspect of sample injection volume.

Table 12

When the detection wavelength changes by +/-2 nm, the separation degree of a main peak and an adjacent isomer chromatographic peak in the system applicability solution is more than 1.5; in the sensitivity solution, the signal to noise ratio of the main peak is more than 10; in the sample solution, the RSD with the contents of the SRR, the RSR and the RSS type isomers meets the requirements, so that the detection method has good durability in the aspect of detection wavelength.

TABLE 13

When the flow rate of the mobile phase changes by +/-0.1 ml/min, the separation degree of a main peak and an adjacent isomer chromatographic peak in the system applicability solution is more than 1.5; in the sensitivity solution, the signal to noise ratio of the main peak is more than 10; in the sample solution, the RSD with the contents of the SRR, the RSR and the RSS type isomers meets the requirements, so that the detection method has good durability in terms of flow rate.

TABLE 14

When the column temperature changes by +/-2 ℃, the separation degree of a main peak and an adjacent isomer chromatographic peak in a system applicability solution is more than 1.5; in the sensitivity solution, the signal to noise ratio of the main peak is more than 10; in the sample solution, the RSD with the contents of SRR, RSR and RSS type isomers meets the requirements, so that the detection method has good durability in the aspect of column temperature.

TABLE 15

When the binary mobile phase ratio (V _{Phase A} :V _{Phase B} ) When the variation is +/-1%, the separation degree of a main peak and an adjacent isomer chromatographic peak in the system applicability solution is more than 1.5; in the sensitivity solution, the signal to noise ratio of the main peak is more than 10; in the sample solution, the RSD of the contents of the SRR, the RSR and the RSS type isomers meets the requirements, which shows that the detection method has good durability in the aspect of binary mobile phase proportion.

6. Determination of sitafloxacin and its various isomer impurities in different batches of test samples:

small pilot samples 3 (lot #1, small pilot #2, small pilot # 3) and pilot samples 3 (lot #1, pilot #2, pilot # 3) of sitafloxacin were prepared in parallel according to the method of item 3.1, and the content (%) of each isomer impurity in the test sample was examined as measured according to the chromatographic condition of item 2, and the results are shown in fig. 8 to 13.

As can be seen from fig. 8 to 13, the presence of the three isomer impurities of sitafloxacin was not detected in the sample solutions of the 6 batches.

Claims

1. A method for simultaneously detecting sitafloxacin and various isomer impurities thereof is characterized in that,

the method utilizes high performance liquid chromatography to detect sitafloxacin and various isomer impurities thereof, and chromatographic conditions of the high performance liquid chromatography comprise:

the chromatographic column takes silica gel with the surface coated with cellulose-tris (4-chloro-3-methylphenyl carbamate) as a filler;

the mobile phase adopts a binary mobile phase system, the A phase is a triethylamine aqueous solution with the concentration of 0.45-0.55% v/v, and the B phase is acetonitrile;

the elution mode adopts isocratic elution, and the volume ratio of the phase A to the phase B is 7:93-9:91;

the isomers include RSS type isomers, SRR type isomers and RSR type isomers of sitafloxacin.

2. The method of claim 1, wherein the step of determining the position of the substrate comprises,

the chromatographic column is CHIRALCEL ^® OX-3R series analytical column.

3. The method of claim 1, wherein the step of determining the position of the substrate comprises,

the pH value of the phase A is 8.5-9.0.

4. The method of claim 3, wherein the step of,

the pH value of the phase A is 8.8.

5. The method according to claim 3 or 4, wherein,

the pH value of the A phase is regulated by phosphoric acid.

6. The method of claim 1, wherein the step of determining the position of the substrate comprises,

the chromatographic conditions further include: column temperature: 25+/-2 ℃; a detector: UV detector or PDA detector; detection wavelength: 295±2nm; flow rate: 0.7+/-0.1 ml/min; sample injection amount: 5.+ -. 2. Mu.l.