A method for detecting genotoxic impurities in fosfomycin calcium

Abstract

This invention discloses a method for detecting genotoxic impurities in fosfomycin calcium. The method involves preparing a test solution and a reference solution using hydrochloric acid as a solvent, and then detecting the test solution and reference solution using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to obtain the content of genotoxic impurities in fosfomycin calcium. The genotoxic impurities are: 1,2-epoxypropyl diethyl phosphate, (2-propenyl)-diethyl phosphate, allyl diethyl phosphate, and propyl diethyl phosphate. This invention develops an analytical method capable of simultaneously detecting four diethyl phosphonate genotoxic impurities in fosfomycin calcium. This method exhibits good specificity, injection precision, linear range, limit of quantitation, limit of detection, accuracy, repeatability, intermediate precision, and robustness. It can be used for the detection and monitoring of diethyl phosphonate impurities in fosfomycin calcium raw materials, ensuring product quality and improving the safety of clinical medication.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for detecting genotoxic impurities in fosfomycin calcium. Background Technology

[0002] Calcium fosfomycin, chemical name: (-)-(1R,2S)-1,2-epoxypropylphosphonate calcium salt monohydrate, is a white crystalline powder. It is slightly soluble in water, practically insoluble in methanol, and insoluble in acetone or ether; its molecular formula is C3H5CaO4P·H2O, and its molecular weight is 194.14. Its structural formula is as follows:

[0003] .

[0004] Fosfomycin calcium is a broad-spectrum antibiotic active against Gram-positive bacteria, including multidrug-resistant (MDR) pathogens associated with life-threatening infections. Its mechanism of action involves inhibiting bacterial cell wall synthesis by inhibiting phosphoenolpyruvate transferase. This substance is available in various dosage forms, including tablets, capsules, and granules, and is widely used clinically, primarily for treating the following conditions: 1. Intestinal infections: bacterial enteritis, bacillary dysentery; 2. Urinary tract infections: cystitis, pyelonephritis, urethritis; 3. Dermatological and soft tissue infections: furuncles, anthrax, hidradenitis suppurativa, lymphadenitis, folliculitis; 4. Respiratory tract infections: nasopharyngitis, tonsillitis, tracheitis, early chronic bronchitis; 5. Ophthalmological infections: styes, dacryocystitis; 6. Gynecological infections: vaginitis, cervicitis.

[0005] As with any other active pharmaceutical ingredient, effective analytical methods must be established to quantitatively determine the content of the active pharmaceutical ingredient and its impurities in fosfomycin calcium raw material. In particular, it is necessary to detect genotoxic impurities with extremely low limit requirements to ensure that the drug reaches the necessary purity and improve the safety of clinical use.

[0006] Due to the small molecular size and high ionic polarity of fosfomycin calcium in solution, conventional reversed-phase or normal-phase liquid chromatography methods are not applicable, making its quantitative determination quite challenging. Furthermore, the lack of a chromophore in fosfomycin calcium molecules renders them undetectable using standard ultraviolet detectors, further increasing the difficulty of determination. The detection of genotoxic impurities such as diethyl phosphonate, which have extremely low limit requirements and similar structures, is even more challenging.

[0007] In the current industrial synthesis process of fosfomycin calcium, diethyl phosphonate impurities may be generated or degraded. The phosphonate structures in these impurities are classified as genotoxic warning structures. Because genotoxic impurities can induce gene mutations, causing chromosome breaks and rearrangements, even at extremely low concentrations, and possess potential carcinogenicity, they have received increasing attention in recent years. Research on genotoxic impurities has become a crucial aspect of ensuring drug quality.

[0008] To date, there are no reports on detection methods for genotoxic impurities such as diethyl phosphonate in fosfomycin calcium. Therefore, there is an urgent need to develop a highly specific and sensitive method for detecting diethyl phosphonate impurities to effectively control the quality of fosfomycin calcium and improve the safety of clinical use. Summary of the Invention

[0009] Therefore, embodiments of the present invention provide a method for detecting genotoxic impurities in fosfomycin calcium.

[0010] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0011] A method for detecting genotoxic impurities in fosfomycin calcium involves preparing a test solution and a reference solution using hydrochloric acid solution as a solvent, and then detecting the test solution and the reference solution using high performance liquid chromatography-tandem mass spectrometry to obtain the content of genotoxic impurities in fosfomycin calcium.

[0012] The genotoxic impurities are: 1,2-epoxypropyl diethyl phosphate, (2-propenyl)-diethyl phosphate, propadienyl diethyl phosphate, and propyl diethyl phosphate.

[0013] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0014] C18 chromatographic column;

[0015] Gradient elution conditions for mobile phase: 0 min, 20% mobile phase B; 0-3 min, 20-50% mobile phase B; 3-7 min, 50-90% mobile phase B; 7-7.1 min, 90-20% mobile phase B; 7.1-10 min, 20% mobile phase B; wherein mobile phase A is 0.09-0.11% formic acid solution, and mobile phase B is acetonitrile.

[0016] This invention involves extensive research on liquid chromatography conditions, revealing that the elution program of the mobile phase significantly affects the retention time of target substances and the separation between chromatographic peaks. For example, when the initial proportion of mobile phase B is 30%, the peak shapes of 1,2-epoxypropyl diethyl phosphate, (2-propenyl)-diethyl phosphate, propadienyl diethyl phosphate, and propyl diethyl phosphate are all relatively good, but propadienyl diethyl phosphate and propyl diethyl phosphate do not achieve baseline separation. The study found that under the aforementioned liquid chromatography conditions, both the peak shapes of the four genotoxic impurities and the separation between each chromatographic peak can be considered simultaneously, effectively improving the accuracy of the detection method.

[0017] Further, the concentration of the hydrochloric acid solution is 0.05~0.2 mol / L. The present invention investigated the solvent; fosfomycin calcium has extremely low solubility in solvents such as water, methanol, and acetonitrile, exhibiting relatively good solubility only in acidic solutions. Further research revealed that, compared to other acids, such as formic acid and trifluoroacetic acid, a 0.05~0.2 mol / L hydrochloric acid solution not only meets the solubility requirements for fosfomycin calcium in the experiment but also effectively controls sample matrix interference, thereby ensuring the accuracy of the detection results. In some preferred embodiments, the concentration of the hydrochloric acid solution is 0.1 mol / L; under this concentration condition, it is more beneficial to improve the accuracy and sensitivity of the detection method.

[0018] Furthermore, the high-performance liquid chromatography (HPLC) conditions also include: a flow rate of 0.2–0.5 ml / min, a column temperature of 30–50 °C, and an injection volume of 5–10 μL. In some preferred embodiments, the flow rate is 0.25–0.35 ml / min, the column temperature is 38–42 °C, and the injection volume is 5 μL.

[0019] Furthermore, the chromatographic column was a Waters ACQUITY UPLC HSS T3 with dimensions of 2.1 mm × 100 mm and a diameter of 2.7 μm.

[0020] Furthermore, the mass spectrometry conditions are as follows:

[0021] Ionization method: Electrospray ionization source (ESI) + Multiple reaction monitoring (MRM); Ion Spray Voltage (IS): 4500V; Ion Source Temperature (TEM): 300℃; Nebulizer Gas (GS1): 15L / min; Heater Gas (GS2): 5L / min; Curtain Gas (CUR): 20psi; Collision Gas (CAD): 9L / min; Collision Cell Exit Potential (CXP): 10V; Entrance Potential (EP): 10V; Mass spectrometry acquisition parameters are as follows:

[0022] 1,2-Epoxypropyl-Diethyl Phosphate: Characteristic ion pair 195 / 167, declustering voltage 70V, collision energy 11eV;

[0023] (2-Propylene)-Diethyl phosphate: Characteristic ion pair 179 / 151, declustering voltage 66V, collision energy 15eV;

[0024] Diethyl allethyl phosphate: Characteristic ion pair 177 / 121, declustering voltage 71V, collision energy 19eV;

[0025] Diethyl propyl phosphate: Characteristic ion pair 181 / 153, declustering voltage 71V, collision energy 15eV.

[0026] Furthermore, the content of genotoxic impurities in fosfomycin calcium was calculated using the external standard method, according to the following formula (I):

[0027] (I)

[0028] In the formula, A 供 : The peak area of ​​characteristic ions of the target genotoxic impurities in the test solution;

[0029] m 对 Weigh the reference standard sample, in mg.

[0030] P: Content of reference standard, expressed as percentage (%);

[0031] V 供 : Dilution factor of the test sample;

[0032] A 对 : The peak area of ​​characteristic ions of toxic impurities to the target gene in the reference solution;

[0033] m 供 The sample weight is expressed in mg.

[0034] V 对 : Dilution factor of reference standard.

[0035] The embodiments of the present invention have the following advantages:

[0036] This invention, through exploration of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection methods, has developed an analytical method capable of simultaneously detecting four diethyl phosphonate toxic impurities in fosfomycin calcium. This method exhibits good specificity, injection precision, linear range, limit of quantitation, limit of detection, accuracy, repeatability, intermediate precision, and robustness. It can be used for the detection and monitoring of diethyl phosphonate impurities in fosfomycin calcium raw materials, ensuring product quality and improving the safety of clinical medication. Attached Figure Description

[0037] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0038] Figure 1 is the MRM chromatogram of 1,2-epoxypropyl-phosphonic acid diethyl ester in the specific detection of this invention. A-blank solvent, B-reference solution, C-test solution, D-spiked test solution;

[0039] Figure 2 is the MRM chromatogram of (2-propenyl)-phosphonic acid diethyl ester in the specific detection of this invention. A-blank solvent, B-reference solution, C-test solution, D-spiked test solution;

[0040] Figure 3 is the MRM chromatogram of the specific detection of diethyl allenyl phosphate in this invention, where A is blank solvent, B is reference solution, C is test solution, and D is spiked test solution.

[0041] Figure 4 shows the MRM chromatogram of diethyl propyl phosphate in the specific detection of this invention, where A is blank solvent, B is reference solution, C is test solution, and D is spiked test solution.

[0042] Figure 5 Linear regression results of 1,2-epoxypropyl-phosphonic acid diethyl ester provided by the present invention;

[0043] Figure 6 The linear regression results of (2-propenyl)-phosphonate diethyl ester provided by this invention are shown in the figure.

[0044] Figure 7 Linear regression results for diethyl propadiene phosphate provided by this invention;

[0045] Figure 8 Linear regression results of diethyl propyl phosphate provided by this invention;

[0046] Figure 9 The overlapping MRM chromatogram of the reference solution provided in Comparative Example 1 of this invention;

[0047] Figure 10 This is an overlapping MRM chromatogram of the reference solution provided in Comparative Example 2 of the present invention. Detailed Implementation

[0048] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1 Detection Method

[0050] 1. Reagents and Materials

[0051] Formic acid: Grade AR, batch number A04222272, source QCS;

[0052] Acetonitrile: Grade HPLC, batch number 240808, source: Fisher;

[0053] Water: Grade distilled water, sourced from Watsons;

[0054] Hydrochloric acid: Grade AR, batch number 20250702, source: Huanghua Century Kebo Technology Development Co., Ltd.;

[0055] 1,2-Epoxypropylphosphonic acid diethyl ester reference standard: 100% purity, CAS No. 28423-96-3, batch No. 96659, source: QCS;

[0056] (2-Propylene)-phosphonate diethyl ester reference standard: content 95.43%, CAS No. 5954-65-4, batch No. DM25071236, source DMCHEM;

[0057] Diethyl allenyl phosphate reference standard: 100% purity, CAS No. 1609-72-9, batch No. 96658, source: QCS;

[0058] Diethyl propionate reference standard: purity 97%, CAS number 18812-51-6, batch number RH806014, source: Ron Reagents;

[0059] Fosfomycin calcium (raw material): batch numbers D50-240101, D50-240102, D50-240103, source: Chifeng Aike Pharmaceutical Technology Co., Ltd.

[0060] 2. Solution preparation

[0061] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0062] Test solution: Accurately weigh 100 mg of fosfomycin calcium, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0063] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonate diethyl ester reference standard, (2-propenyl)-phosphonate diethyl ester reference standard, allyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0064] Reference solution: Take 0.1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0065] 3. Instruments and Equipment

[0066] High performance liquid chromatography-mass spectrometry instrument: Model 5500+, serial number IE-5148, manufacturer AB.

[0067] Analytical balance (1 / 100,000): Model Securra-225D, serial number IE-5254, manufacturer Sartorius.

[0068] 4. Testing conditions

[0069] 4.1 Liquid Chromatography Conditions

[0070] Column: Waters ACQUITY UPLCHSS T3, 2.1mm*100mm, 2.7μm.

[0071] The gradient elution program is shown in Table 1 below. Mobile phase A: 0.1% formic acid solution; mobile phase B: acetonitrile. The 0.1% formic acid solution is prepared as follows: Take 1 ml of formic acid, place it in a 1000 ml volumetric flask, dilute to the mark with water, and mix well. Flow rate: 0.3 ml / min; injection volume: 5 μL; column temperature: 40℃.

[0072] Table 1

[0073]

[0074] 4.2 Mass Spectrometry Conditions

[0075] Ionization method: Electrospray ionization source (ESI) + Multiple reaction monitoring (MRM); Ion Spray Voltage (IS): 4500V; Ion Source Temperature (TEM): 300℃; Nebulizer Gas (GS1): 15L / min; Heater Gas (GS2): 5L / min; Curtain Gas (CUR): 20 psi; Collision Gas (CAD): 9 L / min; Collision Cell Exit Potential (CXP): 10V; Entrance Potential (EP): 10V; Mass spectrometry acquisition parameters for four diethyl phosphonate genotoxic impurities are shown in Table 2 below.

[0076] Table 2

[0077]

[0078] Quantitative analysis was performed using the external standard method. The content of genotoxic impurities in fosfomycin calcium was calculated according to the following formula (I):

[0079] (I)

[0080] In the formula, A 供 : The peak area of ​​characteristic ions of the target genotoxic impurities in the test solution;

[0081] m 对 Weigh the reference standard sample, in mg.

[0082] P: Content of reference standard, expressed as percentage (%);

[0083] V 供 : Dilution factor of the test sample;

[0084] A 对 : The peak area of ​​characteristic ions of toxic impurities to the target gene in the reference solution;

[0085] m 供 : Sample weight, mg;

[0086] V 对 : Dilution factor of reference standard.

[0087] Example 2: Validation of the Analytical Method

[0088] The method validation scheme was designed in accordance with the relevant requirements of the "Guiding Principles for Analytical Method Validation 9101 of the General Chapter of the Chinese Pharmacopoeia 2025 Edition". The method validation content includes specificity, limit of detection, linearity, accuracy, repeatability, intermediate precision, robustness, etc.

[0089] I. Exclusivity

[0090] (1) Solution preparation

[0091] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0092] Test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0093] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonate diethyl ester reference standard, (2-propenyl)-phosphonate diethyl ester reference standard, allyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0094] Reference solution: Take 0.1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0095] Spiked test solution: Weigh 100 mg of fosfomycin calcium (batch D50-240102) accurately, place it in a 10 ml volumetric flask, add 0.1 ml of reference stock solution, dilute to the mark with solvent, and shake well.

[0096] (2) Measurement

[0097] Take 5 μl each of blank solvent, reference solution, test solution, and spiked test solution, inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), and perform detection under the conditions described in Example 1. Record the chromatograms. The results are shown in Table 3 below.

[0098] Table 3

[0099]

[0100] The results show:

[0101] 1) In the blank solvent, there are no absorption peaks at the positions of the genotoxic impurities, indicating that the blank solvent has no effect on the experiment;

[0102] 2) No genotoxic impurities were detected in the test sample;

[0103] 3) The 1,2-epoxypropyl-phosphonic acid diethyl ester reference standard has an absorption peak at 5.01 min with a peak area of ​​108650; the (2-propenyl)-phosphonic acid diethyl ester reference standard has an absorption peak at 5.55 min with a peak area of ​​252787; the allyl phosphate diethyl ester reference standard has an absorption peak at 5.22 min with a peak area of ​​721851; and the propyl phosphate diethyl ester reference standard has an absorption peak at 5.94 min with a peak area of ​​390961.

[0104] 4) The spiked test solution of 1,2-epoxypropyl-phosphonic acid diethyl ester showed a peak at 5.02 min with a peak area of ​​130082, and the recovery rate of the spiked test solution peak area compared with the reference standard peak area was 119.73%. The spiked test solution of (2-propenyl)-phosphonic acid diethyl ester showed a peak at 5.55 min with a peak area of ​​287661, and the recovery rate of the spiked test solution peak area compared with the reference standard peak area was 113.80%. The spiked diethyl propadiene phosphate solution showed a peak at 5.23 min with a peak area of ​​768,428, and the recovery rate of the spiked test sample peak area compared with the reference standard peak area was 106.45%. The spiked diethyl propadiene phosphate solution showed a peak at 5.94 min with a peak area of ​​419,341, and the recovery rate of the spiked test sample peak area compared with the reference standard peak area was 107.26%, both within the range of 80%-120%.

[0105] The above demonstrates that this method has good specificity.

[0106] II. Sample injection precision

[0107] (1) Solution preparation

[0108] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0109] Test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0110] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonate diethyl ester reference standard, (2-propenyl)-phosphonate diethyl ester reference standard, allyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0111] Reference solution: Take 0.1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0112] (2) Measurement

[0113] Take 5 μl of the reference solution and inject it into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument. Perform six consecutive injections and analyze the sample under the conditions described in Example 1. Record the chromatogram. The injection precision results are shown in Table 4 below.

[0114] Table 4

[0115]

[0116] The results showed that the retention time RSDs of 1,2-epoxypropyl-phosphonate diethyl ester, (2-propenyl)-phosphonate diethyl ester, propadienyl phosphate diethyl ester, and 1,2-epoxypropyl-phosphonate diethyl ester were all less than 2%, and the peak area RSDs were all less than 10%, indicating good injection precision.

[0117] III. Linear

[0118] (1) Solution preparation

[0119] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0120] Test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0121] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonate diethyl ester reference standard, (2-propenyl)-phosphonate diethyl ester reference standard, allyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0122] Linear stock solution: Accurately transfer 1 ml of the reference stock solution into a 10 ml volumetric flask, add solvent to make up to volume, and mix well.

[0123] Linear solution 1: Accurately transfer 0.1 ml of the linear stock solution into a 10 ml volumetric flask, add solvent to make up to volume, and mix well.

[0124] Linear solution 2: Accurately transfer 0.5 ml of the linear stock solution into a 10 ml volumetric flask, add solvent to make up to volume, and mix well.

[0125] Linear solution 3: Accurately transfer 1 ml of the linear stock solution into a 10 ml volumetric flask, add solvent to make up to volume, and mix well.

[0126] Linear solution 4: Accurately transfer 1.5 ml of the linear stock solution into a 10 ml volumetric flask, add solvent to make up to volume, and mix well.

[0127] Linear solution 5: Accurately transfer 2.0 ml of the linear stock solution into a 10 ml volumetric flask, add solvent to make up to volume, and mix well.

[0128] (2) Measurement

[0129] Accurately measure 5 μl of each of the above linear solutions, inject them into a high-performance liquid chromatography-mass spectrometry instrument, and perform detection according to the conditions in Example 1, and record the chromatogram.

[0130] Linear regression was performed with concentration (ng / ml) as the x-axis and peak area as the y-axis to calculate the linear equation. The results are shown in Table 5 below.

[0131] Table 5

[0132]

[0133] The results show that all linear correlation coefficients (r) are greater than 0.99, indicating a good linear relationship.

[0134] IV. Limit of Quantification

[0135] Take 1.5 μl of the linear solution and inject it into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument. Perform six consecutive injections and detect the chromatograms according to the conditions in Example 1. The results are shown in Table 6 below.

[0136] Table 6

[0137]

[0138] The results showed that the average S / N of each genotoxic impurity was greater than 10, and the peak area RSD was less than 10% after six consecutive injections of the limit of quantitation solution, indicating that each genotoxic impurity could be quantitatively detected.

[0139] V. Detection Limit

[0140] (1) Solution preparation

[0141] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution (i.e., linear solution 1), place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0142] (2) Measurement

[0143] Take 5 μl of each of the three limit-of-detection solutions, inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument, and perform detection according to the conditions in Example 1. Record the chromatograms. The results are shown in Table 7 below.

[0144] Table 7

[0145]

[0146] The results showed that all toxic impurities could be effectively detected.

[0147] VI. Accuracy and Repeatability

[0148] (1) Solution preparation

[0149] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0150] Test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0151] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonate diethyl ester reference standard, (2-propenyl)-phosphonate diethyl ester reference standard, allyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0152] Reference solution: Take 0.1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0153] 100% spiked test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, add 100 μl of the reference stock solution, dilute to the mark with solvent, and shake well. Prepare 6 parallel solutions.

[0154] 50% spiked test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, add 50 μl of the reference stock solution, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0155] 150% spiked test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, add 150 μl of the reference stock solution, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0156] (2) Measurement

[0157] Accurately measure 5 μl each of the test solution, reference solution, 50% spiked test solution, 100% spiked test solution, and 150% spiked test solution, and inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument. Perform detection under the conditions described in Example 1 and record the chromatograms. The results are shown in Table 8 below.

[0158] Table 8

[0159]

[0160]

[0161] The results showed that the recoveries of all toxic impurities in the 6 100% spiked test solutions were between 80% and 120%, and the RSDs were all less than 10%, indicating good repeatability results. The recoveries of all toxic impurities in the 12 spiked test solutions were between 80% and 120%, and the RSDs were all less than 10%, indicating good accuracy results.

[0162] 7. Intermediate precision (different personnel, different times)

[0163] (1) Solution preparation

[0164] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0165] Test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0166] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonate diethyl ester reference standard, (2-propenyl)-phosphonate diethyl ester reference standard, allyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0167] Reference solution: Take 0.1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0168] 100% spiked test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, add 100 μl of the reference stock solution, dilute to the mark with solvent, and shake well. Prepare 12 parallel solutions.

[0169] (2) Measurement

[0170] Accurately measure 5 μl each of the test solution, reference solution, and 100% spiked test solution, inject them into the high-performance liquid chromatograph, and perform detection according to the conditions in Example 1. Record the chromatograms. The results are shown in Table 9 below.

[0171] Table 9

[0172]

[0173]

[0174] The results showed that the RSD of the recovery rate of each toxic impurity in the 12 100% test solutions was less than 10%, indicating that the intermediate precision experiment results were good.

[0175] 8. Durability

[0176] (1) Solution preparation

[0177] Solvent / Blank solvent: 0.1 mol / L hydrochloric acid solution.

[0178] Test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0179] Reference stock solutions: Accurately weigh appropriate amounts of 1,2-epoxypropyl-phosphonic acid diethyl ester reference standard, (2-propenyl)-phosphonic acid diethyl ester reference standard, propadienyl phosphate diethyl ester reference standard, and propyl phosphate diethyl ester reference standard, dissolve and dilute with solvent to prepare a solution containing 375 ng of each of the above four reference standards per 1 ml.

[0180] Reference solution: Take 0.1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0181] 100% spiked test solution: Accurately weigh 100 mg of fosfomycin calcium (batch D50-240102), place it in a 10 ml volumetric flask, add 100 μl of the reference stock solution, dilute to the mark with solvent, and shake well. Prepare 12 parallel solutions.

[0182] (2) Measurement

[0183] Accurately measure 5 μl each of the test solution, reference solution, and 100% spiked test solution, and inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument. Perform detection according to the conditions in Example 1 and record the chromatograms. The results are shown in Table 10 below.

[0184] Table 10

[0185]

[0186] Conclusion: With varying chromatographic conditions, the RSD of the recoveries of each genotoxic impurity was less than 10% under all conditions, indicating good robustness test results.

[0187] IX. Sample Testing

[0188] The method of Example 1 was used to test the fosfomycin calcium raw material, and the results are shown in Table 11 below.

[0189] Table 11

[0190]

[0191] The results showed that no toxic impurities were detected in any of the three batches of test samples, meeting the requirements.

[0192] Comparative Example 1

[0193] The only difference between this comparative example and Example 1 is the gradient elution program in the chromatographic conditions. The mobile phase gradient elution program of this comparative example is shown in Table 12 below.

[0194] Table 12

[0195]

[0196] The reference solution was tested under the chromatographic conditions of Comparative Example 1, and the overlapping chromatograms were recorded. (See figure) Figure 9 The results showed that baseline separation was not achieved among the four genotoxic impurities.

[0197] Comparative Example 2

[0198] The only difference between this comparative example and Example 1 is the gradient elution program in the chromatographic conditions. The gradient elution program of this comparative example is shown in Table 13 below.

[0199] Table 13

[0200]

[0201] The reference solution was tested under the chromatographic conditions of Comparative Example 2, and the overlapping chromatograms were recorded. (See figure) Figure 10 The results showed that baseline separation was not achieved among the four genotoxic impurities.

[0202] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting genotoxic impurities in fosfomycin calcium, characterized in that, Test solutions and reference solutions were prepared using hydrochloric acid solution as solvent, and the test solutions and reference solutions were detected by high performance liquid chromatography-tandem mass spectrometry to obtain the content of genotoxic impurities in fosfomycin calcium. The genotoxic impurities are: 1,2-epoxypropyl diethyl phosphate, (2-propenyl)-diethyl phosphate, propadienyl diethyl phosphate, and propyl diethyl phosphate. The high-performance liquid chromatography (HPLC) conditions are as follows: C18 chromatographic column; Gradient elution conditions for mobile phase: 0 min, 20% mobile phase B; 0-3 min, 20-50% mobile phase B; 3-7 min, 50-90% mobile phase B; 7-7.1 min, 90-20% mobile phase B; 7.1-10 min, 20% mobile phase B; wherein mobile phase A is 0.09-0.11% formic acid solution, and mobile phase B is acetonitrile.

2. The method for detecting genotoxic impurities in fosfomycin calcium according to claim 1, characterized in that, The concentration of the hydrochloric acid solution is 0.05~0.2 mol / L.

3. The method for detecting genotoxic impurities in fosfomycin calcium according to claim 2, characterized in that, The concentration of the hydrochloric acid solution is 0.1 mol / L.

4. The method for detecting genotoxic impurities in fosfomycin calcium according to claim 1, characterized in that, The high-performance liquid chromatography conditions also include: a flow rate of 0.2~0.5 ml / min, a column temperature of 30~50℃, and an injection volume of 5~10 μL.

5. The method for detecting genotoxic impurities in fosfomycin calcium according to claim 4, characterized in that, The flow rate was 0.25~0.35 ml / min, the column temperature was 38~42℃, and the injection volume was 5 μL.

6. The method for detecting genotoxic impurities in fosfomycin calcium according to claim 1, characterized in that, The chromatographic column was a Waters ACQUITY UPLC HSS T3, with dimensions of 2.1 mm × 100 mm and a diameter of 2.7 μm.

7. The method for detecting genotoxic impurities in fosfomycin calcium according to any one of claims 1-6, characterized in that, Mass spectrometry conditions are: Ionization method: Electrospray ion source ESI + Multiple reaction monitoring; Electrospray voltage: 4500 V; Ion source temperature: 300 °C; Nebulizer gas: 15 L / min; Auxiliary heater gas: 5 L / min; Gas curtain gas: 20 psi; Collision gas: 9 L / min; Collision cell exit voltage: 10 V; Entrance voltage: 10 V; Mass spectrometry acquisition parameters as follows: 1,2-Epoxypropyl-Diethyl Phosphate: Characteristic ion pair 195 / 167, declustering voltage 70V, collision energy 11eV; (2-Propylene)-Diethyl phosphate: Characteristic ion pair 179 / 151, declustering voltage 66V, collision energy 15eV; Diethyl allethyl phosphate: Characteristic ion pair 177 / 121, declustering voltage 71V, collision energy 19eV; Diethyl propyl phosphate: Characteristic ion pair 181 / 153, declustering voltage 71V, collision energy 15eV.

8. The method for detecting genotoxic impurities in fosfomycin calcium according to claim 1, characterized in that, The content of genotoxic impurities in fosfomycin calcium was calculated using the external standard method, according to the following formula (I): （I） In the formula, A 供 : peak area of characteristic ion of target genotoxic impurity in test solution; m 对 : control sample, amount in mg; P: Content of reference standard, expressed as percentage (%); V 供 : dilution factor of the test sample; A 对 : peak area of characteristic ion of target genotoxic impurity in control solution; m 供 : sample weight, in mg; V 对 : dilution factor of control

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