Method and kit for detecting content of fosfomycin sodium for injection by using high performance liquid chromatography

Abstract

The invention relates to a method for detecting the content of fosfomycin sodium for injection by using high performance liquid chromatography. According to the method, an electrospray detector is used as a detector, a chromatographic column with octadecyl bonded silica gel as a filler and a formic acid aqueous solution as a mobile phase are adopted, and the content of fosfomycin is obtained by combining linear regression analysis and a specific calculation formula. The invention also relates to a kit comprising a chromatographic column and a mobile phase for use in the method of the invention. According to the method, the required fosfomycin content can be conveniently, quickly, accurately and efficiently measured, the operation is simple, the specificity, the reproducibility and the precision are good, the method is very suitable for detection of compatibility experiment samples of fosfomycin sodium for injection, and valuable and referable fosfomycin content data can be provided for clinical research of fosfomycin sodium related preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and relates to a drug content detection method and a kit, and in particular to a method and a kit for detecting the content of fosfomycin sodium for injection by using high performance liquid chromatography. Background Art

[0002] Fosfomycin has a small molecular weight and is a phosphoric acid derivative in structure, which has a different structure from other antibiotics. Fosfomycin itself is a free acid, and its pharmaceutical products mainly have two forms: fosfomycin calcium salt and fosfomycin sodium salt. Fosfomycin calcium salt is mainly made into oral preparations, while fosfomycin sodium salt is mainly made into injection preparations.

[0003] "Chinese Pharmacopoeia (Part II)" (2020 edition), "Japanese Pharmacopoeia" and "Korean Pharmacopoeia" all include fosfomycin sodium and fosfomycin sodium for injection, "European Pharmacopoeia" and "British Pharmacopoeia" include fosfomycin sodium, but the content determination methods included in the literature are all microbiological assays and titration methods, which cannot meet the detection requirements of high throughput, automation, simple operation, short detection cycle, good reproducibility, and high precision. Therefore, when those skilled in the art conduct clinical research on fosfomycin sodium related preparations, they cannot quickly, accurately and efficiently obtain reference fosfomycin content data, thereby increasing the economic cost and time cost of research and development.

[0004] In view of this, developing a convenient, fast, accurate and efficient method to detect the content of fosfomycin sodium for injection is of great significance for the clinical research of fosfomycin sodium related preparations. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for conveniently, quickly, accurately and efficiently using high performance liquid chromatography to detect the content of fosfomycin sodium for injection ((-)-(1R,2S)-1,2-epoxypropylphosphonic acid disodium salt, whose specific structural formula is shown in formula (I)).

[0006]

[0007] The purpose of the present invention is achieved through the following solutions:

[0008] A method for detecting the content of fosfomycin sodium for injection using high performance liquid chromatography, characterized in that the method comprises:

[0009] (1) Selection of chromatographic conditions

[0010] A chromatographic column with octadecyl bonded silica as a filler is used, and the column temperature of the chromatographic column is 25-35° C.; a formic acid aqueous solution is used as a mobile phase, wherein the formic acid content of the mobile phase is 0.18-0.3% based on the total mass of the formic acid aqueous solution, the mobile phase flow rate is 0.4-0.6 mL / min, and isocratic elution is performed;

[0011] (2) Solution preparation

[0012] Preparation of reference stock solution:

[0013] Prepare fosfomycin sodium reference substance, dissolve it in water, and dilute it with water to a fosfomycin concentration of 5 mg / mL;

[0014] Preparation of reference solution:

[0015] Pipet different amounts of reference substance stock solutions, place them in fixed volume containers, dilute with water to fixed volume, and obtain reference substance solutions with different fosfomycin concentrations;

[0016] Preparation of test solution:

[0017] Prepare the fosfomycin sodium injection test sample to be tested, dissolve it in water, and dilute it with water to a fosfomycin concentration of 0.45-0.55 mg / mL;

[0018] (3) Measurement method

[0019] Take the same volume of the test solution and the reference solution with different fosfomycin concentrations, inject them into the liquid chromatograph respectively, record the chromatogram according to the chromatographic conditions, perform linear regression analysis on the fosfomycin concentration of the reference solution and the measured fosfomycin peak area, and calculate the fosfomycin content according to the following formula:

[0020]

[0021] in:

[0022] A u is the peak area of ​​fosfomycin peak in the test solution;

[0023] C u is the theoretical concentration of fosfomycin in the test solution, in mg / mL;

[0024] The intercept is the intercept of the linear regression equation obtained for the reference solution;

[0025] The slope is the slope of the linear regression equation obtained for the reference solution.

[0026] In the method of the present invention, the detector used is an electrospray detector (CAD) and the nebulizer temperature is 35°C.

[0027] In addition, the present invention also relates to a kit comprising a chromatographic column and a mobile phase for the method of the present invention.

[0028] Compared with the prior art, the advantages and beneficial effects of the method of the present invention are:

[0029] The method of the present invention uses high performance liquid chromatography and an electrospray detector for detection, and is combined with linear regression analysis and a specific formula, is simple to operate, has good specificity, can conveniently, quickly, accurately and efficiently measure the required fosfomycin content, can meet the growing needs of high-standardization detection, such as high throughput, automation, short detection cycle, good reproducibility, high precision, etc., is very suitable for the detection of compatibility experimental samples of fosfomycin sodium for injection, and provides valuable and reference fosfomycin content data for clinical research on fosfomycin sodium related preparations, thereby saving the cost of preparation research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The HPLC chromatogram of the blank solution according to the chromatographic conditions of Example 1 is shown in FIG.

[0031] Figure 2 The HPLC chromatogram of the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1, wherein the concentration of fosfomycin in the reference solution is about 0.05 mg / mL.

[0032] Figure 3 The HPLC chromatogram of the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1, wherein the concentration of fosfomycin in the reference solution is about 0.25 mg / mL.

[0033] Figure 4 The HPLC chromatogram of the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1, wherein the concentration of fosfomycin in the reference solution is about 0.4 mg / mL.

[0034] Figure 5 The HPLC chromatogram of the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1, wherein the concentration of fosfomycin in the reference solution is about 0.5 mg / mL.

[0035] Figure 6 The HPLC chromatogram of the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1, wherein the concentration of fosfomycin in the reference solution is about 0.6 mg / mL.

[0036] Figure 7 The HPLC chromatogram of the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1, wherein the concentration of fosfomycin in the reference solution is about 0.75 mg / mL.

[0037] Figure 8The HPLC chromatogram of the fosfomycin sodium test solution according to the chromatographic conditions of Example 1 is shown.

[0038] Fig. 9 The figure is a linear relationship diagram between the fosfomycin concentration and the peak area of ​​the fosfomycin sodium reference solution according to the chromatographic conditions of Example 1.

[0039] Fig.10 The HPLC chromatogram of the blank auxiliary material solution is shown in the figure below: DETAILED DESCRIPTION

[0040] In the present invention, "reference substance" refers to the fosfomycin sodium working reference substance to be determined for the fosfomycin content, which is commercially available. Unless otherwise explicitly stated, "fosfomycin sodium working reference substance" and "fosfomycin sodium reference substance" are used synonymously.

[0041] In the present invention, the "test sample" refers to the fosfomycin sodium product for injection whose fosfomycin content is to be determined.

[0042] In the present invention, unless otherwise stated, all operations are carried out at room temperature and normal pressure.

[0043] In the present invention, "fosfomycin sodium for injection" refers to a fosfomycin sodium injection preparation product made of fosfomycin sodium and citric acid in a weight ratio of about 97:3.

[0044] In the present invention, the "compatibility experimental sample of fosfomycin sodium for injection" refers to a fosfomycin sodium injection preparation product made of fosfomycin sodium, citric acid and a solvent (eg, 5% glucose and sodium chloride).

[0045] All numerical values ​​used to express amounts, percentages or ratios in the specification and claims are to be understood as being modified in all cases by the term "about". In this regard, the term "about" as used herein may include a range of ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of the numerical value.

[0046] Unless otherwise expressly stated, the concentrations stated in the specification refer to mass concentrations.

[0047] Unless otherwise specified, the water described in the specification refers to ultrapure water, which can be homemade or commercially available.

[0048] Unless otherwise specified, in this manual, the “LOG(A u )" refers to the logarithm with base 10; "10^" refers to the power of 10, where "^" is the exponent symbol, which is used to represent a number (10 in this article) multiplied by itself a number of times (in this article, ) is the product of .

[0049] On the one hand, the present invention provides a method for detecting the content of fosfomycin sodium for injection using high performance liquid chromatography, characterized in that the method comprises:

[0050] (1) Selection of chromatographic conditions

[0051] A chromatographic column with octadecyl bonded silica as a filler is used, and the column temperature of the chromatographic column is 25-35° C.; a formic acid aqueous solution is used as a mobile phase, wherein the formic acid content of the mobile phase is 0.18-0.3% based on the total mass of the formic acid aqueous solution, the mobile phase flow rate is 0.4-0.6 mL / min, and isocratic elution is performed;

[0052] (2) Solution preparation

[0053] Preparation of reference stock solution:

[0054] Prepare fosfomycin sodium reference substance, dissolve it in water, and dilute it with water to a fosfomycin concentration of 5 mg / mL;

[0055] Preparation of reference solution:

[0056] Pipet different amounts of reference substance stock solutions, place them in fixed volume containers, dilute with water to fixed volume, and obtain reference substance solutions with different fosfomycin concentrations;

[0057] Preparation of test solution:

[0058] Prepare the fosfomycin sodium injection test sample to be tested, dissolve it in water, and dilute it with water to a fosfomycin concentration of 0.45-0.55 mg / mL;

[0059] (3) Measurement method

[0060] Take the same volume of the test solution and the reference solution with different fosfomycin concentrations, inject them into the liquid chromatograph respectively, record the chromatogram according to the chromatographic conditions, perform linear regression analysis on the fosfomycin concentration of the reference solution and the measured fosfomycin peak area, and calculate the fosfomycin content according to the following formula:

[0061]

[0062] in:

[0063] A u is the peak area of ​​fosfomycin peak in the test solution;

[0064] C u is the theoretical concentration of fosfomycin in the test solution, in mg / mL;

[0065] The intercept is the intercept of the linear regression equation obtained for the reference solution;

[0066] The slope is the slope of the linear regression equation obtained for the reference solution.

[0067] In a preferred embodiment of the invention, the detector used is an electrospray detector (CAD) with a nebulizer temperature of 35°C.

[0068] In a preferred embodiment of the present invention, the chromatographic column with octadecyl bonded silica as filler is selected from Waters T3 (150*4.6mm, 3μm) (Waters, USA) or chromatographic column with equivalent performance.

[0069] In the present invention, the chromatographic column with equivalent performance refers to a chromatographic column that is comparable to the target chromatographic column (e.g., Waters The selectivity of the chromatographic columns is consistent, which is manifested in the consistent order of the elution of the chromatographic peaks, and the retention values ​​of the solutes and the separation between the chromatographic peaks are as consistent as possible. The selection of chromatographic columns with equivalent performance can be achieved by methods known to those skilled in the art, such as the Snyder / Dolan method, the Euerby method, the Hoogmartens method, and the United States Pharmacopoeia method.

[0070] In the present invention, isocratic elution refers to an elution method in which the composition ratio and flow rate of the mobile phase remain constant during the analysis cycle of the sample components during the high performance liquid chromatography operation.

[0071] In the present invention, linear regression analysis refers to univariate linear regression analysis, which is a statistical analysis method that uses regression analysis in mathematical statistics to determine the quantitative relationship between two variables (an independent variable x and a dependent variable y) that are interdependent. The result is represented by a linear regression equation y=kx+b and an r value, where x is the concentration of fosfomycin in the reference solution, y is the peak area of ​​fosfomycin measured at a specific fosfomycin concentration, k is the slope of the linear regression equation, b is the intercept of the linear regression equation, and r is the slope of the linear regression equation. 2 =Be the correlation coefficient.The whole variation of the correlation coefficient reaction dependent variable fosfomycin peak area can be explained by the ratio of independent variable fosfomycin concentration through regression relationship, show the linear relationship between the degree of fit between experimental data and fitting function and independent variable fosfomycin concentration and dependent variable fosfomycin peak area, the r value is closer to 1, the degree of fit is higher, and the linear relationship is better.According to the difference of chromatographic conditions, the fosfomycin concentration of reference substance solution is different from the linear regression analysis result of measured fosfomycin peak area.Specific results are shown in the embodiment part below.

[0072] In a preferred embodiment of the present invention, the column temperature of the chromatographic column is 25°C, 30°C or 35°C.

[0073] In a preferred embodiment of the present invention, an aqueous formic acid solution is used as the mobile phase, wherein the formic acid content of the mobile phase is 0.18-0.3%, particularly 0.24%, based on the total mass of the aqueous formic acid solution. Further preferably, the flow rate of the mobile phase is 0.45-0.55 mL / min, such as 0.45 mL / min, 0.5 mL / min or 0.55 mL / min. Preferably, the injection volume of the mobile phase is 10-40 μL, preferably 15-30 μL, particularly 20 μL.

[0074] In the present invention, the formic acid used to prepare the mobile phase is chromatographic grade formic acid, especially liquid chromatography-mass spectrometry (LC / MS) grade formic acid, with a purity of >99%. Commercially available products include LC / MS grade, Pierce TM LC / MS grade, etc. The preparation of a solution having a specific concentration (eg, mass concentration or volume concentration) for use as a mobile phase is well known to those skilled in the art.

[0075] In a preferred embodiment, the formic acid used in the mobile phase is LC / MS grade with a purity of 99.6%.

[0076] Optionally, in the above-mentioned method of the present invention, the formic acid used to prepare the mobile phase can be dried before use by methods known to those skilled in the art, including but not limited to inorganic desiccants (such as anhydrous copper sulfate, anhydrous magnesium sulfate), molecular sieves, vacuum low-temperature distillation, etc.

[0077] In a preferred embodiment of the present invention, the concentration of fosfomycin in the fosfomycin sodium test solution is 0.45-0.55 mg / mL, preferably 0.5 mg / mL.

[0078] In a preferred embodiment of the present invention, the concentration of fosfomycin in the fosfomycin sodium reference solution is 0.01-0.85 mg / mL, preferably 0.02-0.80 mg / mL, and particularly 0.05-0.75 mg / mL.

[0079] In the present invention, the constant volume container used to prepare the reference substance solution is a conventional constant volume container known to those skilled in the art, such as a volumetric flask. Preferably, the volume of the constant volume container is 10-50 mL, preferably 20 mL.

[0080] In a preferred embodiment of the present invention, the injection volume of the fosfomycin sodium test solution and the reference solution is 5-40 μL, preferably 10-30 μL, more preferably 15-25 μL, especially 20 μL.

[0081] In a specific embodiment of the present invention, the content of fosfomycin obtained by the method is in the range of 90%-110%. The content of fosfomycin is in the range specified in the relevant standards of fosfomycin sodium for injection in the 17th edition of the Japanese Pharmacopoeia.

[0082] In the method of the present invention, a blank excipient solution is also used as a negative control solution for verifying the specificity of the method. The blank excipient solution is prepared by dissolving a certain amount of anhydrous citric acid in water and diluting with water to a desired concentration. Preferably, the concentration of citric acid in the blank excipient solution is 20-40 μg / mL, preferably 25-35 μg / mL, and particularly 30 μg / mL.

[0083] It should be noted that, since the structures of fosfomycin and fosfomycin sodium are known and have a constant molar mass difference, that is, the characterization parameters of the two (such as content or concentration, etc.) can be easily converted into each other by calculation methods well known to those skilled in the art, therefore, the above-mentioned method of the present invention can be used to determine both the content of fosfomycin and the content of fosfomycin sodium.

[0084] In another aspect, the present invention also provides a kit comprising a chromatographic column and a mobile phase for use in the method of the present invention.

[0085] In a preferred embodiment of the present invention, in the kit, the chromatographic column is a chromatographic column with octadecyl bonded silica gel as a filler, and the mobile phase is a formic acid aqueous solution, wherein the formic acid content of the mobile phase is 0.24% based on the total mass of the formic acid aqueous solution.

[0086] In the present invention, the kit can be used in a conventional manner known to those skilled in the art, and it can be used to test any fosfomycin sodium injection sample to be tested for fosfomycin or fosfomycin sodium content, including but not limited to the fosfomycin sodium reference substance, test substance and other commercially available fosfomycin sodium injection preparation products used in the method described in the first aspect of the present invention.

[0087] It should be noted that in this specification, various features, parameters, conditions, other elements and combinations thereof described for the method for detecting the content of fosfomycin sodium for injection using high performance liquid chromatography are applicable to the kit of the present invention.

[0088] The method of the present invention will be further described and illustrated in more detail below in conjunction with specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0089] The reagents or instruments used are as follows:

[0090] Water Homemade ultrapure water

[0091] Formic acid Thermo Fisher Scientific Inc.,

[0092] LC / MS grade product, purity 99.6%, model

[0093] A11750

[0094] Fosfomycin Sodium Reference Material Guangdong Liwei Pharmaceutical Co., Ltd.

[0095] Fosfomycin Sodium for Injection Test Sample Sinopharm Guorui Pharmaceutical Co., Ltd.

[0096] High performance liquid chromatograph Thermo Fisher Scientific Inc., model

[0097] Ultimate 3000

[0098] Ultrapure water machine Sichuan Water Dilution Treatment Equipment Co., Ltd.

[0099] 1 / 100,000 balance SHIMADZU, model AP135W

[0100] One-millionth balance Sartorius, Germany, model

[0101] MSA6-6S-OCE-DM

[0102] Electronic balance Shenzhen APT Electronic Technology Co., Ltd., model

[0103] APTP456A

[0104] 1. Chromatographic conditions

[0105] Example 1

[0106] Instrument: High performance liquid chromatography-electrospray detector (purchased from Thermo Fisher Scientific, model: Ultimate 3000)

[0107] Chromatographic column: Waters T3 (150*4.6mm, 3μm)

[0108] Column temperature: 30°C;

[0109] Mobile phase: aqueous formic acid solution with a formic acid content of 0.24%, isocratic elution

[0110] Flow rate: 0.5 mL / min;

[0111] Injection volume: 20 μL.

[0112] The results are as follows Figures 1 to 10 shown.

[0113] Example 2

[0114] The chromatographic conditions were the same as those in Example 1, except that the column temperature of the chromatographic column was 25°C.

[0115] Example 3

[0116] The chromatographic conditions were the same as those in Example 1, except that the column temperature of the chromatographic column was 35°C.

[0117] Example 4

[0118] The chromatographic conditions were the same as those in Example 1, except that the flow rate of the mobile phase was 0.45 mL / min.

[0119] Example 5

[0120] The chromatographic conditions were the same as those in Example 1, except that the flow rate of the mobile phase was 0.55 mL / min.

[0121] 2. Sample solution preparation

[0122] 2.1 Reference substance stock solution

[0123] Accurately weigh an appropriate amount of fosfomycin sodium reference substance (batch number S20230901), dissolve it in water, and dilute it with water to a fosfomycin concentration of about 5 mg / mL, and shake well.

[0124] 2.2 Reference solution

[0125] Accurately pipette 0.2mL, 1mL, 1.6mL, 2mL, 2.4mL and 3mL of the reference substance stock solution into 20mL volumetric flasks respectively, dilute to the scale with water, shake well to obtain a reference substance solution with a fosfomycin concentration range of about 0.05-0.75mg / mL.

[0126] 2.3 Test solution

[0127] Accurately weigh an appropriate amount of fosfomycin sodium injection test sample (batch number 2304001), dissolve it in water, and dilute it with water to a fosfomycin concentration of about 0.5 mg / mL, and shake well.

[0128] 3. Determination of Fosfomycin Content

[0129] Accurately measure 20 μL of the test solution prepared as above and the reference solution with different fosfomycin concentrations, inject them into the liquid chromatograph respectively, and record the chromatograms according to the chromatographic conditions of Examples 1-5, respectively, wherein the chromatograms of the reference solution (fosfomycin concentration is about 0.05 mg / mL), the reference solution (fosfomycin concentration is about 0.25 mg / mL), the reference solution (fosfomycin concentration is about 0.4 mg / mL), the reference solution (fosfomycin concentration is about 0.5 mg / mL), the reference solution (fosfomycin concentration is about 0.6 mg / mL), the reference solution (fosfomycin concentration is about 0.75 mg / mL) and the test solution of Example 1 are as follows: Figure 2 , 3 , 4, 5, 6, 7 and Figure 8 Then, the fosfomycin concentration of the reference solution of Example 1 and the measured fosfomycin peak area were subjected to linear regression analysis to obtain a linear regression equation, and the fosfomycin content was obtained according to the following calculation formula:

[0130]

[0131] in:

[0132] A u is the peak area of ​​fosfomycin peak in the test solution;

[0133] C u is the theoretical concentration of fosfomycin in the test solution, in mg / mL;

[0134] The intercept is the intercept of the linear regression equation obtained for the reference solution;

[0135] The slope is the slope of the linear regression equation obtained for the reference solution.

[0136] Under the chromatographic conditions of Example 1, the peak area measurement results of the fosfomycin sodium reference solution and the test solution at specific fosfomycin concentrations are shown in Table 1.

[0137] Table 1 Peak area determination results of fosfomycin sodium reference solution and test solution at specific fosfomycin concentrations

[0138]

[0139] The linear relationship between the fosfomycin concentration and the peak area of ​​the fosfomycin sodium reference solution is shown in the figure below: Fig. 9 As shown. The linear regression equation obtained is y=0.7677x+1.3975, r=0.9998, where x is the concentration of fosfomycin in the reference solution and y is the peak area of ​​fosfomycin. The results show that fosfomycin has a good linear relationship in the concentration range of 0.05-0.75 mg / mL.

[0140] When the theoretical fosfomycin concentration of the fosfomycin sodium test solution is about 0.5 mg / mL and the fosfomycin peak area is 14.9521, it can be calculated by the above calculation formula that the fosfomycin content in the fosfomycin sodium test solution is 94.03%.

[0141] The linear regression analysis results of the reference substance and the fosfomycin content of the test substance obtained under different chromatographic conditions (chromatographic conditions of Examples 1-5) are shown in Table 2 below.

[0142] Table 2 Linear regression analysis of reference substances and fosfomycin content of test substances obtained under different chromatographic conditions

[0143]

[0144] *k is the slope of the linear regression equation obtained for the reference solution.

[0145] *b is the intercept of the linear regression equation obtained for the reference solution.

[0146] It can be seen from the above results that the content of fosfomycin obtained by the method of the present invention under different chromatographic conditions is within the range of 90%-110%.

[0147] 4. Validation of the method of the present invention

[0148] 4.1 Preparation of relevant solutions

[0149] (1) Blank solution

[0150] Homemade ultrapure water was used as the blank solution.

[0151] (2) Blank excipient solution

[0152] Accurately weigh an appropriate amount of anhydrous citric acid, dissolve it in water, and dilute it with water to a citric acid concentration of about 30 μg / mL, and shake well.

[0153] 4.2 Verification Method

[0154] (1) System adaptability

[0155] The blank solution and the reference solution (fosfomycin concentration of about 0.05, 0.25, 0.4, 0.5, 0.6, 0.75 mg / mL) prepared as above were respectively injected into the liquid chromatograph, and the chromatogram was recorded according to the chromatographic conditions of Example 1. Figure 1 As shown, there is no interfering peak in the blank solution.

[0156] (2) Exclusivity

[0157] The blank excipient solution, the reference solution (fosfomycin concentration is about 0.5 mg / mL) and the test solution prepared as above were respectively injected into the liquid chromatograph, and the chromatogram was recorded according to the chromatographic conditions of Example 1. Fig.10 As shown, there is no interfering peak in the blank excipient solution.

[0158] (3) Repeatability

[0159] Accurately weigh 6 parts of the same batch of fosfomycin sodium injection test sample (batch number 2304001), dissolve in water, and dilute with water to a fosfomycin concentration of about 0.5 mg / mL, shake well, and obtain 6 parts of fosfomycin sodium test solution. The peak areas are measured respectively according to the chromatographic conditions of Example 1. After calculation, the relative standard deviation (RSD) of the fosfomycin content is 1.62%, indicating that the method of the present invention has good repeatability.

[0160] (4) Precision

[0161] Another experimenter used another device with exactly the same configuration to prepare 6 portions of the test solution in parallel according to repeatability. The chromatogram was recorded and the peak area was determined according to the chromatographic conditions of Example 1. After calculation, the relative standard deviation (RSD) of the fosfomycin content of 6 portions of repeatability and 6 portions of precision was 1.38%, indicating that the method of the present invention has good precision.

[0162] Surprisingly, from the above results, it can be seen that the method of the present invention is capable of conveniently, quickly, accurately and efficiently measuring the required fosfomycin content by high performance liquid chromatography and electrospray ionization detector detection, combined with linear regression analysis and a specific formula. The method is simple to operate, has good specificity, reproducibility and precision, is very suitable for the detection of compatibility experimental samples of fosfomycin sodium for injection, and can provide valuable and reference fosfomycin content data for clinical research on fosfomycin sodium related preparations, which is of great significance for the development of fosfomycin sodium related preparations.

[0163] (5) Comparison with the test results of pharmacopoeia methods

[0164] The microbiological assay (titer method) was used to test the same batch of samples (batch number 2304001), and the product report result (average value) was 100.0%; the method of the present invention was used to test 6 samples of the same batch, and the average repeatability result was 96.31%, and the relative deviation was 1.88%. The results show that the results of the method of the present invention are reliable.

Claims

1. A method for detecting the content of fosfomycin sodium for injection using high performance liquid chromatography, characterized in that: The method comprises: (1) Selection of chromatographic conditions A chromatographic column with octadecyl bonded silica as a filler is used, and the column temperature of the chromatographic column is 25-35° C.; a formic acid aqueous solution is used as a mobile phase, wherein the formic acid content of the mobile phase is 0.18-0.3% based on the total mass of the formic acid aqueous solution, the mobile phase flow rate is 0.4-0.6 mL / min, and isocratic elution is performed; (2) Solution preparation Preparation of reference stock solution: Prepare fosfomycin sodium reference substance, dissolve it in water, and dilute it with water to a fosfomycin concentration of 5 mg / mL; Preparation of reference solution: Pipet different amounts of reference substance stock solutions, place them in fixed volume containers, dilute with water to fixed volume, and obtain reference substance solutions with different fosfomycin concentrations; Preparation of test solution: Prepare the fosfomycin sodium injection test sample to be tested, dissolve it in water, and dilute it with water to a fosfomycin concentration of 0.45-0.55 mg / mL; (3) Measurement method Take the same volume of the test solution and the reference solution with different fosfomycin concentrations, inject them into the liquid chromatograph respectively, record the chromatogram according to the chromatographic conditions, perform linear regression analysis on the fosfomycin concentration of the reference solution and the measured fosfomycin peak area, and calculate the fosfomycin content according to the following formula: in: A u is the peak area of ​​fosfomycin peak in the test solution; C u is the theoretical concentration of fosfomycin in the test solution, in mg / mL; The intercept is the intercept of the linear regression equation obtained for the reference solution; The slope is the slope of the linear regression equation obtained for the reference solution.

2. The method according to claim 1, characterized in that The detector used in the method is an electrospray detector.

3. The method according to claim 1, characterized in that The chromatographic column with octadecyl bonded silica as filler is selected from Waters T3 or equivalent column.

4. The method according to claim 1, characterized in that: The concentration of fosfomycin in the fosfomycin sodium reference solution is 0.01-0.85 mg / mL, preferably 0.02-0.80 mg / mL, and particularly 0.05-0.75 mg / mL.

5. The method according to claim 1, characterized in that The formic acid content of the mobile phase is 0.24% based on the total mass of the formic acid aqueous solution.

6. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.45-0.55 mL / min, such as 0.45 mL / min, 0.5 mL / min or 0.55 mL / min.

7. The method according to claim 1, characterized in that The concentration of fosfomycin in the fosfomycin sodium test solution is 0.5 mg / mL; preferably, the injection volume of the fosfomycin sodium test solution and the reference solution is 5-40 μL, preferably 10-30 μL, and more preferably 15-25 μL.

8. The method according to any one of claims 1 to 7, characterized in that A blank excipient solution is also used as a negative control solution to verify the specificity of the method, wherein the concentration of citric acid in the blank excipient solution is 20-40 μg / mL, preferably 25-35 μg / mL, and particularly 30 μg / mL.

9. A kit comprising a chromatography column and a mobile phase for use in the method according to any one of claims 1 to 8.

10. The kit according to claim 9, wherein the chromatographic column is a chromatographic column using octadecyl bonded silica gel as a filler, and the mobile phase is a formic acid aqueous solution, wherein the formic acid content of the mobile phase is 0.24% based on the total mass of the formic acid aqueous solution.

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