Method for determining content of genotoxic impurities in sodium ibandronate

Abstract

The invention relates to the technical field of drug analysis and detection, and provides a method for determining the content of genotoxic impurities in sodium ibandronate, sodium ibandronate is subjected to extraction treatment to obtain a test solution, and the content of genotoxic impurities in the test solution is detected by gas chromatography; the extraction treatment specifically comprises the following steps: dissolving sodium ibandronate in water, adding inorganic salt and an organic solvent, standing for layering, and taking an organic phase; the genotoxic impurities comprise 1-bromopentane, dimethyl phosphite and trimethyl phosphate. Through the technical scheme, the problems of low detection sensitivity, poor applicability and poor specificity when the content of genotoxic impurities in sodium ibandronate is detected in related technologies are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and in particular to a method for determining the content of genotoxic impurities in ibandronate sodium. Background Art

[0002] Ibandronate is a commonly used anti-tumor drug and bone metabolism regulator, mainly used to treat a variety of diseases related to bone health, especially in the treatment of postmenopausal osteoporosis, bone pain caused by osteolytic bone metastasis of malignant tumors, and hypercalcemia. Impurity level is one of the important factors affecting the effectiveness and safety of drugs. Phosphoric acid and phosphorus oxychloride are used in the production process of ibandronate. The hydrolysis of the product will produce phosphorous acid. Since methanol is used for refining in the later stage of the process, if these two acids remain, their own acidity will have the characteristics of catalytic esterification reaction with methanol, and various methyl esters will be produced. Among them, organic phosphate compounds have carcinogenic warning structures and have the risk of damaging central nervous system and germ cells. Experimental research has determined that the residual amount of trimethyl phosphate and dimethyl phosphite is relatively large. Furthermore, 1-bromopentane is the starting material for preparing ibandronate sodium, which may remain in the finished product of ibandronate sodium. It is a small molecule halogenated aliphatic genotoxic impurity, which is the most common type of genotoxic impurities. Due to the strong reactivity of halogenated alkanes, it can directly react with biological macromolecules such as DNA, RNA and protein to undergo alkylation reactions, thereby causing DNA mutations, which may produce genotoxicity and carcinogenicity. Therefore, a method for detecting 1-bromopentane, trimethyl phosphate and dimethyl phosphite in ibandronate sodium is proposed, which is of great significance for the quality control of ibandronate sodium raw materials. Summary of the invention

[0003] The invention provides a method for determining the content of genotoxic impurities in ibandronate sodium, which solves the problems of low detection sensitivity, poor applicability and poor specificity when detecting the content of genotoxic impurities in ibandronate sodium in the related art.

[0004] The technical solution of the present invention is as follows: The present invention provides a method for determining the content of genotoxic impurities in ibandronate sodium. The ibandronate sodium is subjected to extraction treatment to obtain a test solution, and the content of genotoxic impurities in the test solution is detected by gas chromatography; The extraction treatment specifically comprises dissolving ibandronate sodium in water, adding an inorganic salt and an organic solvent, standing for stratification, and taking an organic phase; The genotoxic impurities include 1-bromopentane, dimethyl phosphite and trimethyl phosphate.

[0005] As a further technical solution, the inorganic salt is one of anhydrous sodium sulfate, sodium chloride, ammonium chloride, and ammonium sulfate; and the organic solvent is one of dichloromethane, toluene, n-hexane, and ethyl acetate.

[0006] As a further technical solution, the inorganic salt is anhydrous sodium sulfate or ammonium sulfate; and the organic solvent is dichloromethane or toluene.

[0007] As a further technical solution, in terms of g / mL, the mass volume ratio of ibandronate sodium to water is 1:10; In terms of g / mL, the mass volume ratio of the inorganic salt to water is 0.2 to 2:10; The volume ratio of the organic solvent to water is 3-10:10.

[0008] As a further technical solution, when the gas chromatography method is used for detection, the stationary liquid is 6% cyanopropylphenyl and 94% dimethylpolysiloxane in volume percentage.

[0009] As a further technical solution, when the gas chromatography method is used for detection, the initial column temperature is 70°C~90°C, maintained for 5 minutes, then increased to 220°C at a heating rate of 38~42°C / min, and maintained for 10 minutes; when the gas chromatography method is used for detection, the injection port temperature is 220°C and the detector temperature is 250°C; when the gas chromatography method is used for detection, the carrier gas used is nitrogen, the carrier gas flow rate is 1.9~2.1mL / min, and the split ratio is 8:1~12:1.

[0010] As a further technical solution, when the gas chromatography method is used for detection, the chromatographic column is a DB-624 capillary column.

[0011] As a further technical solution, the detection includes the following steps: using gas chromatography to test the reference solution and the test solution respectively, recording the chromatogram, and calculating the contents of 1-bromopentane, dimethyl phosphite and trimethyl phosphate by peak area using the external standard method.

[0012] As a further technical solution, the preparation method of the reference substance solution comprises the following steps: dissolving an inorganic salt in water, adding a reference substance stock solution, standing to separate the layers, and taking an organic phase to obtain a reference substance solution.

[0013] As a further technical solution, the preparation method of the reference substance stock solution comprises the following steps: dissolving 1-bromopentane, dimethyl phosphite and trimethyl phosphate in an organic solvent to obtain a reference substance stock solution.

[0014] The working principle and beneficial effects of the present invention are: In the present invention, ibandronate sodium is used to extract to obtain a test solution, and then gas chromatography is used to test the three genotoxic impurities of 1-bromopentane, dimethyl phosphite and trimethyl phosphate remaining in the test solution. The detection method has good specificity and high separation of the three analytes. When the interference of conventional solvents is excluded, blank solvents, other materials and sample solutions do not interfere with the determination at the retention time of 1-bromopentane, trimethyl phosphate and dimethyl phosphite.

[0015] The method of the present invention has high sensitivity, and the detection limits of 1-bromopentane, dimethyl phosphite, and trimethyl phosphate are 0.0011%, 0.0034%, and 0.0014%, respectively; when repeated injections are performed, the RSD values ​​of the peak areas of the quantitative limit concentration solutions are all <5%, which meets the requirements. In addition, the accuracy of the method of the present invention meets the requirements. It can be seen from the test results that the average recovery rate of the sample addition is within the range of 95% to 110%, and the RSD values ​​of the recovery rates at each concentration level are all <10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0017] Figure 1 The chromatogram of the test solution of Example 1; Figure 2 is the chromatogram of the reference substance solution of Example 1; Figure 3 Chromatogram of blank solvent for specificity test; Figure 4 Chromatogram of 1-bromopentane localization solution for specificity test; Figure 5 Chromatogram of trimethyl phosphate locating solution for specificity test; Figure 6 Chromatogram of dimethyl phosphite localization solution for specificity test; Figure 7 It is the linear regression curve diagram of 1-bromopentane; Figure 8 is the linear regression curve diagram of dimethyl phosphite; Fig. 9 is the linear regression curve diagram of trimethyl phosphate; Fig.10 Detection chromatogram for control group 1; Fig.11 Position the solution chromatogram for toluene. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] In the following examples, ibandronate sodium, batch number 230802, was homemade; Trimethyl phosphate, batch number 202209041, content 99.5%, sourced from Chengdu Aikoda Chemical Reagent Co., Ltd.; Dimethyl phosphite, batch number 12104154, content 98.7%, source Aladdin; Dichloromethane, batch number 2204011, sourced from Xilong Scientific Co., Ltd.; Intermediate I is 3-(N-methyl-N-n-pentylamino)propionitrile, batch number 230301, content of 99.67%, and is homemade.

[0020] Example 1 A method for determining the content of genotoxic impurities in ibandronate sodium. After ibandronate sodium is subjected to extraction treatment, a test solution is obtained, and the content of genotoxic impurities in the test solution is detected by gas chromatography; the genotoxic impurities include 1-bromopentane, dimethyl phosphite and trimethyl phosphate; the detection is as follows: Capillary column: DB-624, 30m×0.25mm×1.4µm; Chromatographic conditions: Based on volume percentage, a capillary column with 6% cyanopropylphenyl and 94% dimethylpolysiloxane as stationary liquid was used as the chromatographic column. The initial column temperature was 80°C, maintained for 5 minutes, then increased to 220°C at a heating rate of 40°C / min and maintained for 10 minutes; the injection port temperature was 220°C, the FID detector temperature was 250°C; the carrier gas was nitrogen, the split ratio was 10:1, and the carrier gas flow rate was 2mL / min; the injection volume was 1µL; The preparation method of the test solution comprises the following steps: taking 1.0 g of ibandronate sodium, accurately weighing it, placing it in a stoppered test tube, adding 10 mL of water to dissolve it, adding 1 g of anhydrous sodium sulfate and 10 mL of dichloromethane, shaking it well, standing it to separate into layers, taking the dichloromethane phase, and obtaining the test solution; The preparation method of the reference substance stock solution comprises the following steps: accurately weighing appropriate amounts of 1-bromopentane, dimethyl phosphite, and trimethyl phosphate, dissolving them with dichloromethane, and quantitatively diluting them to prepare a solution containing 25 µg of each per 1 mL.

[0021] The preparation method of the reference substance solution comprises the following steps: dissolving 1 g of anhydrous sodium sulfate in 10 mL of water, adding 10 mL of the reference substance stock solution, standing to separate layers, and taking the organic phase to obtain the reference substance solution; Determination: Take samples of the test solution and the reference solution respectively and record the chromatograms; calculate by peak area using the external standard method, and the content of 1-bromopentane, dimethyl phosphite and trimethyl phosphate shall not exceed 0.025%.

[0022] The chromatogram of the test solution is as follows Figure 1 The chromatogram of the reference solution is shown in Figure 2 shown.

[0023] The contents of 1-bromopentane, dimethyl phosphite and trimethyl phosphate were calculated by peak area according to the external standard method.

[0024] Calculation formula:

[0025] Where W 对 : The weight of 1-bromopentane, dimethyl phosphite and trimethyl phosphate reference substances, mg; P: Purity of 1-bromopentane, dimethyl phosphite, and trimethyl phosphate reference substances.

[0026] A 对 : Peak areas of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in the reference solution; V 对 : Dilution multiple of reference solution; A 样 : Peak areas of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in the test solution; W 样 : The weight of the test sample, mg; V 样 : dilution multiple of the test solution; In this batch of test samples, 1-bromopentane was not detected (i.e., <0.0011%), dimethyl phosphite was not detected (i.e., <0.0034%), and trimethyl phosphate was not detected (i.e., <0.0014%).

[0027] Example 2 A method for determining the content of genotoxic impurities in ibandronate sodium. After ibandronate sodium is subjected to extraction treatment, a test solution is obtained, and the content of genotoxic impurities in the test solution is detected by gas chromatography; the genotoxic impurities include 1-bromopentane, dimethyl phosphite and trimethyl phosphate; the detection is as follows: Capillary column: DB-624, 30m×0.25mm×1.4µm; Chromatographic conditions: Based on volume percentage, a capillary column with 6% cyanopropylphenyl and 94% dimethylpolysiloxane as stationary liquid was used as the chromatographic column. The initial column temperature was 70°C, maintained for 5 minutes, then increased to 220°C at a heating rate of 42°C / min and maintained for 10 minutes; the injection port temperature was 220°C, the FID detector temperature was 250°C; the carrier gas was nitrogen, the split ratio was 8:1, and the carrier gas flow rate was 1.9mL / min; the injection volume was 1µL; The preparation method of the test solution comprises the following steps: taking 1.0 g of ibandronate sodium, accurately weighing it, placing it in a stoppered test tube, adding 10 mL of water to dissolve it, adding 0.2 g of anhydrous sodium sulfate and 10 mL of dichloromethane, standing it to separate into layers, taking the dichloromethane phase, and obtaining the test solution; The preparation method of the reference substance stock solution comprises the following steps: accurately weighing appropriate amounts of 1-bromopentane, dimethyl phosphite, and trimethyl phosphate, dissolving them with dichloromethane, and quantitatively diluting them to prepare a solution containing 25 µg of each per 1 mL.

[0028] The preparation method of the reference substance solution comprises the following steps: dissolving 0.2 g of anhydrous sodium sulfate in 10 mL of water, adding 10 mL of the reference substance stock solution, standing to separate layers, and taking the organic phase to obtain the reference substance solution; Determination: Take samples of the test solution and the reference solution respectively and record the chromatograms.

[0029] Example 3 A method for determining the content of genotoxic impurities in ibandronate sodium. After ibandronate sodium is subjected to extraction treatment, a test solution is obtained, and the content of genotoxic impurities in the test solution is detected by gas chromatography; the genotoxic impurities include 1-bromopentane, dimethyl phosphite and trimethyl phosphate; the detection is as follows: Capillary column: DB-624, 30m×0.25mm×1.4µm; Chromatographic conditions: Based on volume percentage, a capillary column with 6% cyanopropylphenyl and 94% dimethylpolysiloxane as stationary liquid was used as the chromatographic column. The initial column temperature was 90°C, maintained for 5 minutes, then increased to 220°C at a heating rate of 38°C / min, and maintained for 10 minutes; the injection port temperature was 220°C, the FID detector temperature was 250°C; the carrier gas was nitrogen, the split ratio was 8:1, and the carrier gas flow rate was 2.1mL / min; the injection volume was 1µL; The preparation method of the test solution comprises the following steps: taking 1.0 g of ibandronate sodium, accurately weighing it, placing it in a stoppered test tube, adding 10 mL of water to dissolve it, adding 2 g of anhydrous sodium sulfate and 10 mL of dichloromethane, standing it to separate into layers, taking the dichloromethane phase, and obtaining the test solution; The preparation method of the reference substance stock solution comprises the following steps: accurately weighing appropriate amounts of 1-bromopentane, dimethyl phosphite, and trimethyl phosphate, dissolving them with dichloromethane, and quantitatively diluting them to prepare a solution containing 25 µg of each per 1 mL.

[0030] The preparation method of the reference substance solution comprises the following steps: dissolving 2 g of anhydrous sodium sulfate in 10 mL of water, adding 10 mL of the reference substance stock solution, standing to separate layers, and taking the organic phase to obtain the reference substance solution; Determination: Take samples of the test solution and the reference solution respectively and record the chromatograms.

[0031] Embodiments 4 to 7 The difference between Examples 4 to 7 and Example 1 is that in Examples 4 to 7, different inorganic salts are used in the preparation of the test solution, as shown in Table 1.

[0032] The recoveries of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in Example 1 and Examples 4 to 7 are shown in Table 1.

[0033] Table 1 Recovery rates of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in Example 1 and Examples 4 to 7

[0034] In Table 1, the results show that without adding inorganic salt, the recovery rates of the three impurities cannot meet the requirements. When the inorganic salt is anhydrous sodium sulfate or ammonium sulfate, the recovery rates of the three impurities can meet the requirements. When the inorganic salt is sodium chloride or ammonium chloride, the recovery rates of dimethyl phosphite and trimethyl phosphate are low and cannot meet the requirements.

[0035] Embodiment 8-10 The difference between Examples 8 to 10 and Example 1 is that in Examples 8 to 10, different organic solvents are used in the preparation of the test solution, as shown in Table 2.

[0036] The recoveries of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in Example 1 and Examples 8 to 10 are shown in Table 2.

[0037] Table 2 Recovery rates of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in Example 1 and Example 8-10

[0038] In Table 2, the results show that when n-hexane and ethyl acetate are used as extraction solvents, the recovery rate cannot meet the requirements, and when toluene and dichloromethane are used as solvents, the recovery rate can meet the requirements, but toluene is more toxic, so dichloromethane is selected as the solvent.

[0039] Embodiment 11 The difference between this embodiment and embodiment 1 is that, in this embodiment, the mass of anhydrous sodium sulfate used in the preparation of the test solution is different, as shown in Table 3.

[0040] The recoveries of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in Examples 1 to 3 and Examples 5 and 11 are shown in Table 3.

[0041] Table 3 Recovery rates of 1-bromopentane, dimethyl phosphite and trimethyl phosphate in Examples 1 to 3 and Examples 5 and 11

[0042] In Table 3, the results show that when the inorganic salt is anhydrous sodium sulfate, the recovery rates of the three impurities can meet the requirements when the dosage is 1g or more.

[0043] Experimental Example 1: Verification of the test method The detection method of Example 1 was subjected to specificity test, solution stability test, linearity and range test, repeatability test, accuracy test, intermediate precision test and durability test.

[0044] 1. Specificity test Blank solvent: dichloromethane; blank solvent chromatogram is as follows Figure 3 shown.

[0045] N, N-dipentylmethylamine, intermediate I, methanol, ethanol, acetone, DMF, toluene, dimethylaminopropionitrile, 3-aminopropionitrile positioning solution: accurately weigh the above substances respectively, dissolve them in dichloromethane and quantitatively dilute them to make a solution containing 25μg of each per 1mL.

[0046] Benzene fixation solution: Accurately weigh benzene, dissolve it in dichloromethane and quantitatively dilute it to make a solution containing 25µg per 1mL, and shake well.

[0047] 1-Bromopentane fixation solution: Accurately weigh 1-bromopentane, dissolve it in dichloromethane and quantitatively dilute it to make a solution containing 25µg per 1mL, and shake well; the chromatogram of the 1-bromopentane fixation solution is as follows Figure 4 shown.

[0048] Trimethyl phosphate positioning solution: accurately weigh trimethyl phosphate, dissolve it in dichloromethane and quantitatively dilute it to make a solution containing 25µg per 1mL, and shake well; the chromatogram of the trimethyl phosphate positioning solution is as follows Figure 5 shown.

[0049] Dimethyl phosphite positioning solution: accurately weigh dimethyl phosphite, dissolve it in dichloromethane and quantitatively dilute it to make a solution containing 25µg per 1mL, shake well; the chromatogram of the dimethyl phosphite positioning solution is as follows Figure 6 shown.

[0050] Reference substance solution: Weigh 1-bromopentane, trimethyl phosphate, and dimethyl phosphite accurately, dissolve them in dichloromethane, and quantitatively dilute them to make solutions containing 25 µg each, as reference substance stock solutions. Weigh 1 g of anhydrous sodium sulfate, place it in a stoppered test tube, add 10 mL of water to dissolve it, then accurately add 10 mL of reference substance stock solution, shake, let stand to separate the layers, and take the dichloromethane layer.

[0051] Test solution: Take 1.0 g of ibandronate sodium, weigh it accurately, put it in a stoppered test tube, add 10 mL of water to dissolve it, add 1 g of anhydrous sodium sulfate to dissolve it, accurately add 10 mL of dichloromethane, shake well, let it stand to separate the layers, and take the dichloromethane layer.

[0052] The results were recorded in Table 4 according to the chromatographic conditions in Example 1.

[0053] Table 4 Specificity test results

[0054] 2 Solution stability test Blank solvent: dichloromethane.

[0055] Test solution: Take 1.0 g of ibandronate sodium, weigh it accurately, put it in a stoppered test tube, add 10 mL of water to dissolve it, add 1 g of anhydrous sodium sulfate to dissolve it, accurately add 10 mL of dichloromethane, shake well, let it stand to separate the layers, and take the dichloromethane layer.

[0056] Spiked test solution: Take 1.0 g of ibandronate sodium, weigh it accurately, put it in a stoppered test tube, add 10 mL of water to dissolve it, add 1 g of anhydrous sodium sulfate to dissolve it, accurately add 10 mL of reference substance stock solution (preparation method is the same as under "1 Specificity test"), shake well, let it stand to separate the layers, and take the dichloromethane layer.

[0057] Reference solution: Preparation method is the same as “1 Specificity test”.

[0058] The results were recorded in Table 5 according to the chromatographic conditions in Example 1.

[0059] Table 5 Solution stability test results

[0060] 3 Linearity and range tests Weigh 1-bromopentane, trimethyl phosphate, and dimethyl phosphite reference substances respectively, add dichloromethane to dissolve and gradually dilute to prepare a series of reference substance mixed solutions with concentrations of 10%, 25%, 50%, 75%, 100%, 150% and 200% of the quantitative limit of each impurity, and the limit concentration. Weigh 1g of anhydrous sodium sulfate, place it in a stopper test tube, add 10mL of water to dissolve it, accurately add 10mL of the series of reference substance mixed solutions, shake it well, let it stand to stratify, take the dichloromethane layer, measure it according to the chromatographic conditions in Example 1, draw a linear graph with concentration as the horizontal coordinate and peak area as the vertical coordinate, calculate the regression equation and correlation coefficient r, and calculate the ratio of the impurity y-axis intercept to the 100% response value. The results are shown in Table 6.

[0061] The linear regression curve of 1-bromopentane is as follows Figure 7 As shown, the linear regression curve of dimethyl phosphite is as follows Figure 8 As shown, the linear regression curve of trimethyl phosphate is as follows Fig. 9 shown.

[0062] Table 6 Linearity test results

[0063] 4 Repeatability test 4.1 Solution preparation Blank solvent: dichloromethane.

[0064] Reference solution: Preparation method is the same as “1 Specificity test”.

[0065] Test solution: Preparation method is the same as “1 Specificity test”.

[0066] Spiked test solution: Take 1.0g of the product, weigh accurately, place in a stoppered test tube, add 10mL of water to dissolve, add 1g of anhydrous sodium sulfate to dissolve, accurately add 10mL of reference solution, shake well, let stand to separate, and take the dichloromethane layer. Prepare 6 copies in parallel.

[0067] 4.2 Acceptance criteria In the 6 spiked test solutions, the recovery rate of each component peak was RSD≤10%.

[0068] 4.3 Measurement and results The chromatographic conditions in Example 1 were used to measure and record the chromatogram. The recovery and RSD value of each peak were calculated by the external standard method. The results are recorded in Table 7.

[0069] Table 7 Repeatability test results

[0070] 5 Accuracy test 5.1 Solution preparation Reference substance stock solution: Preparation method is the same as “1 Specificity test”.

[0071] 50% reference stock solution: Accurately measure 1 mL each of 1-bromopentane reference stock solution, trimethyl phosphate reference stock solution, and dimethyl phosphite reference stock solution, place them in the same 100 mL volumetric flask, dilute to the scale with dichloromethane, and shake well.

[0072] 100% reference stock solution: Accurately measure 2 mL each of 1-bromopentane reference stock solution, trimethyl phosphate reference stock solution, and dimethyl phosphite reference stock solution, place them in the same 100 mL volumetric flask, dilute to the scale with dichloromethane, and shake well.

[0073] 150% reference stock solution: Accurately measure 3 mL each of 1-bromopentane reference stock solution, trimethyl phosphate reference stock solution, and dimethyl phosphite reference stock solution, place them in the same 100 mL volumetric flask, dilute to the scale with dichloromethane, and shake well.

[0074] Reference solution: Preparation method is the same as “1 Specificity test”.

[0075] 50% spiked test solution: Take 1.0g of the product, weigh accurately, place in a stoppered test tube, add 10mL of water to dissolve, add 1g of anhydrous sodium sulfate to dissolve, accurately add 10mL of 50% reference stock solution, shake well, let stand to separate, and take the dichloromethane layer. Prepare 3 copies in parallel.

[0076] 100% spiked test solution: Take 1.0g of this product, weigh accurately, place in a stoppered test tube, add 10mL of water to dissolve, add 1g of anhydrous sodium sulfate to dissolve, accurately add 10mL of 100% reference substance stock solution, shake well, let stand to separate, and take the dichloromethane layer. Prepare 3 copies in parallel.

[0077] 150% spiked test solution: Take 1.0g of this product, weigh accurately, place in a stoppered test tube, add 10mL of water to dissolve, add 1g of anhydrous sodium sulfate to dissolve, accurately add 10mL of 150% reference substance stock solution, shake well, let stand to separate, and take the dichloromethane layer. Prepare 3 copies in parallel.

[0078] 5.2 Acceptance criteria In the 9 test solutions, the average recovery rate of each peak should be between 90% and 110%, and the RSD should be ≤10%.

[0079] 5.3 Measurement and results The chromatographic conditions in Example 1 were used to determine the recovery and RSD of each impurity. The results are recorded in Table 8.

[0080] Table 8 Accuracy test results

[0081] 6 Intermediate precision test The same batch of samples were taken and measured by different analysts on different dates using different equipment and different chromatographic columns. The experiment was carried out according to the requirements under "4 Repeatability Test". The RSD of the recovery rate of each impurity in the 12 solutions prepared by the two analysts was calculated and the results are recorded in Table 9.

[0082] Table 9 Intermediate precision test results

[0083] 7 Durability test 7.1 Robust Chromatographic Conditions The chromatographic conditions are shown in Table 10.

[0084] Table 10 Durability Chromatographic Conditions

[0085] 7.2 Solution preparation Blank solvent: dichloromethane.

[0086] Reference substance solution: Weigh 1-bromopentane, trimethyl phosphate, and dimethyl phosphite accurately, dissolve them in dichloromethane, and quantitatively dilute them to make a solution containing 25µg of each per 1mL as the reference substance stock solution. Weigh 1g of anhydrous sodium sulfate, place it in a stoppered test tube, add 10mL of water to dissolve it, then accurately add 10mL of the reference substance stock solution, shake it, let it stand to separate the layers, and take the dichloromethane layer.

[0087] Spiked test solution: Take 1.0 g of the product, weigh accurately, place in a stoppered test tube, add 10 mL of water to dissolve, add 1 g of anhydrous sodium sulfate to dissolve, accurately add 10 mL of reference substance stock solution, shake well, let stand to separate, and take the dichloromethane layer.

[0088] 7.3 Acceptance criteria Under various durability change test conditions, (1) The blank solvent should be free of interference; (2) In the spectrum of the reference solution, 1-bromopentane, dimethyl phosphite and trimethyl phosphate appear in sequence. The separation between the peaks should be no less than 1.5, and the RSD of the peak area of ​​each impurity peak should be ≤10%.

[0089] (3) Under each durability change condition, the RSD of the recoveries of each peak in the spiked test solution is ≤10%.

[0090] 7.4 Measurement and results After the chromatographic system is stable, under each durability test condition, the reference solution is accurately measured and continuously sampled 3 times, and then the test solution is accurately measured and injected into the gas chromatograph, and the chromatogram is recorded. The results are recorded in Table 13.

[0091] Table 11 Durability test results

[0092] Experimental Example 2: Changing the temperature program Experimental group 1: Chromatographic conditions were the same as in Example 1; Control group 1: The only difference from Example 1 is that the chromatographic column is as follows: CP-Volamine 30×0.32 mm Agilent.

[0093] Detection chromatogram Figures 10-11 As shown in the figure, it can be seen that Fig.11 Chromatogram of toluene (residual solvent of ibandronate sodium) solution Fig.10 In control group 1, dimethyl phosphite peaked at the same position, interfering with the determination of dimethyl phosphite.

[0094] This indicates that the specific chromatographic column in the chromatographic conditions of the present invention improves the detection accuracy of genotoxic impurities in ibandronate sodium.

[0095] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the content of genotoxic impurities in ibandronate sodium, characterized in that: After the ibandronate sodium is subjected to extraction treatment, a test solution is obtained, and the content of genotoxic impurities in the test solution is detected by gas chromatography; The extraction treatment specifically comprises dissolving ibandronate sodium in water, adding an inorganic salt and an organic solvent, standing for stratification, and taking an organic phase; The genotoxic impurities include 1-bromopentane, dimethyl phosphite and trimethyl phosphate.

2. A method for determining the content of genotoxic impurities in ibandronate sodium according to claim 1, characterized in that: The inorganic salt is one of anhydrous sodium sulfate, sodium chloride, ammonium chloride and ammonium sulfate; the organic solvent is one of dichloromethane, toluene, n-hexane and ethyl acetate.

3. A method for determining the content of genotoxic impurities in ibandronate sodium according to claim 2, characterized in that: The inorganic salt is anhydrous sodium sulfate or ammonium sulfate; the organic solvent is dichloromethane or toluene.

4. The method for determining the content of genotoxic impurities in ibandronate sodium according to claim 1, characterized in that: In terms of g / mL, the mass volume ratio of ibandronate sodium to water is 1:10; In terms of g / mL, the mass volume ratio of the inorganic salt to water is 0.2 to 2:10; The volume ratio of the organic solvent to water is 3-10:

10.

5. The method for determining the content of genotoxic impurities in ibandronate sodium according to claim 1, characterized in that: When the gas chromatography method is used for detection, the fixed liquid is 6% cyanopropylphenyl and 94% dimethylpolysiloxane in terms of volume percentage.

6. The method for determining the content of genotoxic impurities in ibandronate sodium according to claim 1, characterized in that: When the gas chromatography method is used for detection, the initial column temperature is 70°C~90°C, maintained for 5 minutes, then increased to 220°C at a heating rate of 38~42°C / min, and maintained for 10 minutes; when the gas chromatography method is used for detection, the injection port temperature is 220°C and the detector temperature is 250°C; when the gas chromatography method is used for detection, the carrier gas used is nitrogen, the carrier gas flow rate is 1.9~2.1mL / min, and the split ratio is 8:1~12:

1.

7. The method for determining the content of genotoxic impurities in ibandronate sodium according to claim 1, characterized in that: When the gas chromatography method is used for detection, the chromatographic column is a DB-624 capillary column.

8. The method for determining the content of genotoxic impurities in ibandronate sodium according to claim 1, characterized in that: The detection comprises the following steps: using gas chromatography to test the reference solution and the test solution respectively, recording the chromatogram, and calculating the contents of 1-bromopentane, dimethyl phosphite and trimethyl phosphate by peak area according to the external standard method.

9. The method for determining the content of genotoxic impurities in ibandronate sodium according to claim 8, characterized in that: The preparation method of the reference substance solution comprises the following steps: dissolving an inorganic salt in water, adding a reference substance stock solution, standing to separate layers, and taking an organic phase to obtain a reference substance solution.

10. A method for determining the content of genotoxic impurities in ibandronate sodium according to claim 9, characterized in that: The preparation method of the reference substance stock solution comprises the following steps: dissolving 1-bromopentane, dimethyl phosphite and trimethyl phosphate in an organic solvent to obtain the reference substance stock solution.

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