Method for detecting impurity A, impurity B, impurity C and impurity D in zoledronic acid bulk drug

Abstract

The invention belongs to the technical field of medicine analysis, and particularly relates to a method for detecting an impurity A, an impurity B, an impurity C and an impurity D. High performance liquid chromatography is adopted for detection, and chromatographic conditions are as follows: a Titank Phenyl-Hexyl chromatographic column is adopted as a chromatographic column; acetonitrile and a buffer solution with the ratio of 4: 96 v / v are used as a mobile phase for isocratic elution, and every 1 L of the buffer solution contains 8.00 g of sodium 1-octanesulfonate, 37.00 mg of EDTA-disodium, 10 mL of perchloric acid and 2 mL of phosphoric acid; the flow velocity of the mobile phase is 0.6-1.0 mL / min, the column temperature is 15-25 DEG C, the sample size is 5-15 mL, an ultraviolet detector is used for detection, and the detection wavelength is 210-220 nm; the method can detect impurities A, B, C and D in the zoledronic acid bulk drug, and can be used for quality control of the zoledronic acid bulk drug.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting impurities A, B, C and D in a zoledronic acid raw material drug. Background Art

[0002] Zoledronic acid is a third-generation bisphosphonate anti-osteoporosis drug, and its chemical name is 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-diphosphonic acid. Zoledronic acid is a new and highly effective bone resorption inhibitor API, which has obvious therapeutic and preventive effects in treating osteoporosis, increasing bone density and reducing the incidence of fractures. In the existing process of synthesizing zoledronic acid, related impurities including impurity A, impurity B, impurity C and impurity D may be produced, which may affect the purity of the chemical synthesis product and even exceed the limit specified in the quality standard, affecting its safety and effectiveness. Therefore, it is urgent to establish a detection method for impurities A, impurity B, impurity C and impurity D in zoledronic acid API with simple operation, good accuracy and high sensitivity. Summary of the invention

[0003] The present invention provides a detection method with good precision, linear relationship and accuracy for the detection of impurities A, B, C and D in zoledronic acid raw materials. The specific technical scheme is as follows:

[0004] A method for detecting impurities A, B, C and D in a zoledronic acid raw material drug, wherein the method adopts high performance liquid chromatography for detection, and the chromatographic conditions are as follows: a Titank Phenyl-Hexyl chromatographic column is adopted as a chromatographic column; acetonitrile: buffer = 4:96 v / v is used as a mobile phase for isocratic elution, wherein each 1L of the buffer contains 8.00 g of 1-octane sodium sulfonate, 37.00 mg of EDTA-disodium, 10 mL of perchloric acid and 2 mL of phosphoric acid; the flow rate of the mobile phase is 0.6-1.0 mL / min, the column temperature is 15-25°C, the injection volume is 5-15 mL, and detection is performed using an ultraviolet detector, and the detection wavelength is 210-220 nm;

[0005] The structures of impurity A, impurity B, impurity C and impurity D are shown in Formulas I, II, III and IV respectively:

[0006] (I) (II) (III) (IV).

[0007] In some embodiments, the chromatographic column has a size of 250 mm×4.6 mm×5 μm.

[0008] In some embodiments, the flow rate of the mobile phase is 0.8 mL / min.

[0009] In some embodiments, the column temperature of the chromatographic column is 18-22 °C, preferably 20 °C.

[0010] In some embodiments, the injection volume is 10 mL.

[0011] In some embodiments, the detection wavelength is 213-217 nm, preferably 215 nm.

[0012] In some embodiments, the method for preparing the buffer comprises:

[0013] Accurately weigh sodium 1-octane sulfonate and disodium EDTA, place them in a volumetric flask, then accurately pipette perchloric acid and phosphoric acid, add ultrapure water to dissolve and dilute to the scale, shake well, and use as a buffer solution.

[0014] In some embodiments, the detection limit of impurity A is 192.60 ng / mL, and the quantification limit is 629 ng / mL, the detection limit of impurity B is 454.50 ng / mL, and the quantification limit is 1515 ng / mL, the detection limit of impurity C is 126.00 ng / mL, and the quantification limit is 315 ng / mL, and the detection limit of impurity D is 150.00 ng / mL, and the quantification limit is 500 ng / mL.

[0015] In some embodiments, before the detection, the method further includes: preparing a bulk drug test solution, and the method for preparing the bulk drug test solution includes: diluting the zoledronic acid bulk drug with a mobile phase to obtain the bulk drug test solution.

[0016] Furthermore, the concentration of zoledronic acid in the bulk drug test solution is 0.5-1.5 mg / mL, preferably 1 mg / mL.

[0017] In some embodiments, before the detection, the method further comprises preparing a zoledronic acid reference solution and an impurity reference solution, wherein:

[0018] Zoledronic acid reference solution: Take zoledronic acid reference and dilute it with mobile phase to prepare a zoledronic acid reference solution containing 1 mg of zoledronic acid per 1 mL;

[0019] Impurity reference solution: Take impurity A reference, impurity B reference, impurity C reference and impurity D reference, and dilute them with mobile phase to prepare solutions with a concentration of 10~20μg for impurity A, impurity B, impurity C and impurity D.

[0020] In some embodiments, before the detection, the method further comprises preparing a zoledronic acid limit reference solution and a bulk drug limit spiked test solution, wherein:

[0021] Zoledronic acid limit reference solution: Take zoledronic acid reference and impurity A reference, impurity B reference, impurity C reference and impurity D reference, and dilute them with mobile phase to prepare a zoledronic acid limit reference solution with a zoledronic acid concentration of 1 mg / mL and impurity A, impurity B, impurity C and impurity D concentrations of 2 μg / mL;

[0022] API limit spiked test solution: Take zoledronic acid API and impurity A reference substance, impurity B reference substance, impurity C reference substance and impurity D reference substance, dilute with mobile phase to prepare API limit spiked test solution with a zoledronic acid concentration of 1 mg / mL and impurities A, impurity B, impurity C and impurity D concentrations of 2 μg / mL.

[0023] Furthermore, the chromatographic column is Titank Phenyl-Hexyl, with a specification of 250 mm×4.6 mm×5 μm, the mobile phase is acetonitrile: buffer = 4:96 (v / v) for isocratic elution, the flow rate is 0.8 mL / min, the column temperature is 20°C, the injection volume is 10 mL, and the detection wavelength is 215 nm.

[0024] In addition, the present invention also provides application of the detection method in quality control of zoledronic acid raw material medicine.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The detection method of impurities A, B, C and D in zoledronic acid raw materials provided in the present application realizes quantitative and qualitative analysis of impurities A, B, C and D in zoledronic acid raw materials. The method has strong specificity and low detection limit. The detection limit of impurity A is 192.60 ng / mL and the quantification limit is 629 ng / mL. The detection limit of impurity B is 454.50 ng / mL and the quantification limit is 1515 ng / mL. The detection limit of impurity C is 126.00 ng / mL and the quantification limit is 315 ng / mL. The detection limit of impurity D is 150.00 ng / mL and the quantification limit is 500 ng / mL. The detection requirements of impurities A, B, C and D in zoledronic acid raw materials are met, and the linear relationship of impurities A, B, C and D is good, and the correlation coefficients are all greater than 0.990. The invention adopts a high performance liquid chromatography method to detect impurities A, impurities B, impurities C and impurities D in a zoledronic acid raw material drug. The method is simple and fast to operate, has good precision, can quickly and accurately detect impurities A, impurities B, impurities C and impurities D in the zoledronic acid raw material drug, the whole process is reliable and controllable in operation, is suitable for practical application and promotion, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the technical description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 The system suitability chromatogram in Example 1 of the present invention; wherein A: blank solution; B: zoledronic acid limit reference solution; C: bulk drug test solution.

[0029] Figure 2 Schematic diagram of the linear regression curve of impurity A in Example 3 of the present invention.

[0030] Figure 3 Schematic diagram of the linear regression curve of impurity B in Example 3 of the present invention.

[0031] Figure 4 Schematic diagram of the linear regression curve of impurity C in Example 3 of the present invention.

[0032] Figure 5 Schematic diagram of the linear regression curve of impurity D in Example 3 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The following will be combined with the drawings in 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 making creative work belong to the protection scope of the present invention.

[0034] A method for detecting impurities A, B, C and D in a zoledronic acid raw material drug, wherein the detection method is to detect by high performance liquid chromatography, and the structures of the impurities A, B, C and D are as shown in formulas I, II, III and IV:

[0035] (I) (II) (III) (IV), the detection limit of impurity A is 192.60 ng / mL, and the quantification limit is 629 ng / mL, the detection limit of impurity B is 454.50 ng / mL, and the quantification limit is 1515 ng / mL, the detection limit of impurity C is 126.00 ng / mL, and the quantification limit is 315 ng / mL, and the detection limit of impurity D is 150.00 ng / mL, and the quantification limit is 500 ng / mL.

[0036] As a preferred solution, the detection steps are as follows:

[0037] (1) Solution preparation: including blank solution, blank excipient solution, zoledronic acid reference solution, impurity reference stock solution, impurity reference solution, zoledronic acid limit reference solution, API test solution, API limit spiked test solution, including:

[0038] Buffer solution: accurately weigh 8 g of sodium 1-octane sulfonate and 37 mg of disodium EDTA, place them in a 1000 mL volumetric flask, then accurately pipette 10 mL of perchloric acid and 2 mL of phosphoric acid, add ultrapure water to dissolve and dilute to the mark, shake well, and use this as the buffer solution;

[0039] Blank solution: acetonitrile-buffer as diluent, the acetonitrile-buffer (4:96, v / v);

[0040] Zoledronic acid reference solution: accurately weigh 10 mg of zoledronic acid reference, place in a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a 1 mg / mL zoledronic acid reference solution;

[0041] Impurity reference substance stock solution: accurately weigh 2 mg of each impurity reference substance, place them in different 10 mL volumetric flasks, dilute to volume with diluent, shake well, and prepare each impurity reference substance stock solution with a concentration of 200 μg / mL;

[0042] Impurity reference substance solution-1: accurately pipette 1 mL of each impurity reference substance stock solution into a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare each impurity reference substance solution-1 with a concentration of 20 μg / mL; repeat the preparation to obtain each impurity reference substance solution-2;

[0043] Impurity reference solution-2: Accurately pipette 5 mL of each impurity reference solution-1 into a 10 mL volumetric flask, dilute with diluent and make up to the mark, shake well, and prepare each impurity reference solution-2 with a concentration of 10 μg / mL.

[0044] Zoledronic acid limit reference solution: accurately weigh 10 mg of zoledronic acid reference substance and place it in a 10 mL volumetric flask, then accurately pipette 1.5 mL of each impurity reference solution -1, dilute to volume with diluent, shake well, and prepare a zoledronic acid limit reference solution with 1 mg / mL zoledronic acid and 2 μg / mL of each impurity;

[0045] API test solution: accurately weigh 10 mg of zoledronic acid API, place in a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a 1 mg / mL API test solution;

[0046] API limit spiked test solution: accurately weigh 10 mg of zoledronic acid API and place it in a 10 mL volumetric flask, then accurately pipette 1.5 mL of each impurity reference solution -1, dilute to volume with diluent, shake well, and prepare a API limit spiked test solution with 1 mg / mL zoledronic acid and 2 μg / mL impurities;

[0047] (2) The blank solution, zoledronic acid reference solution, impurity reference stock solution, impurity reference solution, zoledronic acid limit reference solution, API test solution, and API limit spiked test solution were tested by high performance liquid chromatography, the chromatographic conditions of the liquid chromatography were as follows: detection was performed using an ultraviolet detector, the chromatographic column was Titank Phenyl-Hexyl, with a specification of 250 mm×4.6 mm×5 μm, the mobile phase was acetonitrile:buffer = 4:96 (v / v) for isocratic elution, the flow rate was 0.8 mL / min, the column temperature was 20°C, the injection volume was 10 mL, and the detection wavelength was 215 nm.

[0048] As a preferred solution, a Titank Phenyl-Hexyl column was used with isocratic elution, a flow rate of 0.8 mL / min, and an injection volume of 10 μL.

[0049] Example 1: Solution preparation, specificity and system suitability

[0050] (1) Buffer solution: Accurately weigh 8.00 g of sodium 1-octane sulfonate and 37.00 mg of disodium EDTA, place in a 1000 mL volumetric flask, then accurately pipette 10 mL of perchloric acid and 2 mL of phosphoric acid, dissolve and dilute to volume with ultrapure water, and shake well to prepare the buffer solution.

[0051] (2) Blank solution: acetonitrile-buffer as diluent, the acetonitrile-buffer (v / v, 4:96);

[0052] (3) Zoledronic acid reference solution: Accurately weigh 10 mg of zoledronic acid reference, place in a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a 1 mg / mL zoledronic acid reference solution;

[0053] (4) Impurity reference substance stock solution: Accurately weigh 2 mg of each impurity reference substance and place it in a 10 mL volumetric flask. Dilute to volume with diluent and shake well to prepare a 200 μg / mL impurity reference substance stock solution; impurities refer to impurity A, impurity B, impurity C and impurity D.

[0054] (5) Impurity reference substance solution-1: Accurately pipette 1 mL of each impurity reference substance stock solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare each impurity reference substance solution-1 with a concentration of 20 μg / mL;

[0055] (6) Impurity reference solution-2: Accurately pipette 5 mL of each impurity reference solution-1 into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare each impurity reference solution-2 with a concentration of 10 μg / mL;

[0056] (7) Zoledronic acid limit reference solution: Accurately weigh 10 mg of zoledronic acid reference substance and place it in a 10 mL volumetric flask. Then accurately pipette 1.5 mL of each impurity reference solution -1, dilute to volume with diluent, and shake well to prepare a zoledronic acid limit reference solution with 1 mg / mL zoledronic acid and 2 μg / mL of each impurity.

[0057] (8) API test solution: Accurately weigh 10 mg of zoledronic acid API and place it in a 10 mL volumetric flask. Dilute to volume with diluent and shake well to prepare a 1 mg / mL API test solution.

[0058] (9) API limit spiked test solution: Accurately weigh 10 mg of zoledronic acid API and place it in a 10 mL volumetric flask. Accurately pipette 1 mL of each impurity reference solution and dilute to volume with diluent. Shake well to prepare API limit spiked test solution with 1 mg / mL zoledronic acid and 2 μg / mL of each impurity.

[0059] Liquid chromatography detection: The blank solution, zoledronic acid reference solution, impurity reference stock solution, impurity reference solution, zoledronic acid limit reference solution, bulk drug test solution, and bulk drug limit spiked test solution were detected by high performance liquid chromatography, and the chromatogram was recorded. The chromatogram is Figure 1 The liquid chromatography conditions in this embodiment are:

[0060] A Titank Phenyl-Hexyl (250×4.6 mm, 5 μm) chromatographic column was used with an isocratic elution of acetonitrile: buffer = 4:96 (v / v) with a flow rate of 0.8 mL / min, a column temperature of 20 °C, and an injection volume of 10 mL.

[0061] from Figure 1 It can be seen that the chromatographic peak of impurity A reference substance appeared at the retention time of 4.420 min, the chromatographic peak of impurity B reference substance appeared at the retention time of 3.208 min, the chromatographic peak of impurity C reference substance appeared at the retention time of 16.386 min, and the chromatographic peak of impurity D reference substance appeared at the retention time of 12.896 min, and the chromatographic peaks were well separated.

[0062] from Figure 1 It can be seen that the blank solution and the API test solution have no interference with the detection of impurities A, B, C and D, indicating that the liquid chromatography method provided by the present invention has good specificity and system applicability.

[0063] Example 2: Limit of Detection and Limit of Quantitation

[0064] Detection limit and quantification limit: The impurity reference substance stock solutions prepared in Example 1 were quantitatively diluted step by step with diluent, and then detected by high performance liquid chromatography. The specific conditions of liquid chromatography were as described in Example 1. The spectrum was recorded. The detection limit and quantification limit of the raw material were obtained according to the signal-to-noise ratio of the detection limit being not less than 3:1 and the signal-to-noise ratio of the quantification limit being not less than 10:1. The results are shown in Table 1.

[0065]

[0066] Table 1

[0067] Six portions of quantitative limit solutions were prepared in parallel and detected by high performance liquid chromatography. The specific conditions of liquid chromatography were as described in Example 1. The spectra were recorded and the results are shown in Table 2, which shows that the quantitative limit determined in this application has excellent precision.

[0068]

[0069] Table 2

[0070] Example 3: Linear Relationship

[0071] The impurity reference solution with a concentration of 20 μg / mL prepared in Example 1 was diluted with a diluent to obtain a linear solution of impurity A with a concentration of 0.611 μg / mL to 6.114 μg / mL, a linear solution of impurity B with a concentration of 1.515 μg / mL to 9.100 μg / mL, a linear solution of impurity C with a concentration of 0.280 μg / mL to 11.200 μg / mL, and a linear solution of impurity D with a concentration of 0.571 μg / mL to 5.714 μg / mL.

[0072] The linear solution prepared above was detected by high performance liquid chromatography, and the specific conditions of liquid chromatography were as described in Example 1, and the spectrum was recorded. The standard curve was drawn with concentration C (μg / mL) as the abscissa and peak area A (mV) as the ordinate, and the regression equation was calculated. The results are shown in Table 3 below.

[0073]

[0074] Table 3

[0075] Combining Table 3 and Linear Figures 2 to 4 It can be seen that impurity B has a good linear relationship in the concentration range of 1.515 μg / mL~9.100 μg / mL, impurity C has a good linear relationship in the concentration range of 0.280 μg / mL~11.200 μg / mL, and impurity D has a good linear relationship in the concentration range of 0.571 μg / mL~5.714 μg / mL.

[0076] Example 4: Repeatability

[0077] Take the same batch of samples, prepare the bulk drug limit spiked test solution in parallel, and detect the impurities A, B, C and D in the zoledronic acid bulk drug according to the detection method of Example 1, using high performance liquid chromatography, the specific conditions of liquid chromatography are as described in Example 1, and the results are shown in Table 4. It can be seen from the results that the detection method provided in the present application has good repeatability.

[0078]

[0079] Table 4

[0080] Example 5: Intermediate Precision

[0081] The API limit spiked test solution prepared in Example 1 was taken and tested by high performance liquid chromatography. The specific conditions of liquid chromatography were as described in Example 1. The injection was repeated 6 times, with 10 μL injected each time. The test results are shown in Table 5.

[0082]

[0083] Table 5

[0084] As can be seen from Table 5, the RSD of the peak area of ​​impurity A is 0.92%, the RSD of the peak area of ​​impurity B is 3.42%, the RSD of the peak area of ​​impurity C is 0.44%, and the RSD of the peak area of ​​impurity D is 0.41%. The results show that the liquid phase detection method for impurities A, impurity B, impurity C and impurity D in the zoledronic acid bulk drug provided by the present invention has good intermediate precision.

[0085] Example 6: Accuracy

[0086] The accuracy test of impurity A, impurity B, impurity C and impurity D is expressed in recovery (%), and three samples of the spiked recovery rate of the limit of quantitative concentration of the raw material drug, the spiked recovery rate of 50% of the raw material drug, the spiked recovery rate of 100% of the raw material drug, and the spiked recovery rate of 150% of the raw material drug are prepared in parallel as recovery test solutions. The recovery test solution prepared above was detected by high performance liquid chromatography. The specific conditions of liquid chromatography are as described in Example 1, and the spectrum is recorded. The results are shown in Table 6.

[0087] The recovery rate was calculated according to the following formula: Recovery rate (%) = (measured amount - original amount) / theoretical added amount × 100%.

[0088]

[0089]

[0090] Table 6

[0091] As can be seen from Table 6, at the quantitative limit level, the recovery rate of impurity A is 83.42%~115.20%, and the RSD average value of four groups of spiked recoveries is 0.03%. At the quantitative limit level, the recovery rate of impurity B is 76.62%~86.94%, and the RSD average value of four groups of spiked recoveries is 0.03%. At the quantitative limit level, the recovery rate of impurity C is 71.20%~110.39%, and the RSD average value of four groups of spiked recoveries is 0.03%. At the quantitative limit level, the recovery rate of impurity D is 80.45%~117.43%, and the RSD average value of four groups of spiked recoveries is 0.02%. It shows that the detection method of impurities A, impurity B, impurity C and impurity D in the zoledronic acid bulk drug provided by the present invention has good accuracy.

[0092] Example 7: Solution Stability

[0093] The zoledronic acid limit reference solution, the bulk drug test solution and the bulk drug limit spiked test solution prepared in Example 1 were placed at room temperature for 0 h, 2 h, 6 h, 10 h, and 12 h, and then detected by high performance liquid chromatography. The specific conditions of liquid chromatography are as described in Example 1. The spectrum was recorded, the peak area was examined, and the RSD was calculated. The results are shown in Table 7.

[0094]

[0095]

[0096] Table 7

[0097] As can be seen from Table 7, the zoledronic acid limit reference solution, the API test solution, and the API limit spiked test solution were all stable within 12 h.

[0098] Example 8: Durability

[0099] The zoledronic acid limit reference solution, the bulk drug test solution and the bulk drug limit spiked test solution prepared in Example 1 were tested by high performance liquid chromatography under different conditions. The specific conditions of liquid chromatography are as described in Example 1. The spectra were recorded, the peak areas were investigated, and the content RSD was calculated. The results are shown in Table 8.

[0100]

[0101]

[0102]

[0103]

[0104] Table 8

[0105] As can be seen from Table 8, the RSD of the impurity A content of the zoledronic acid limit reference solution determined under different conditions is 0.27%, the RSD of the impurity B content is 0.13%, the RSD of the impurity C content is 0.13%, and the RSD of the impurity D content is 0.18%; the RSD of the impurity A content in the bulk drug test solution is 0.18%, and the RSD of the impurity B content is 0.31%; the RSD of the impurity A content of the bulk drug limit spiked test solution is 0.13%, the RSD of the impurity B content is 1.42%, the RSD of the impurity C content is 0.13%, and the RSD of the impurity D content is 0.12%. The results show that the liquid phase detection method for impurities A, impurity B, impurity C and impurity D in the zoledronic acid bulk drug provided by the present invention has good durability.

[0106] Example 9: Sample determination

[0107] According to the detection method of impurities A, B, C and D in zoledronic acid bulk drug provided in Example 1, the contents of impurities A, B, C and D in 3 batches of zoledronic acid bulk drug samples were detected. The results are shown in Table 9:

[0108]

[0109] Table 9

[0110] It can be seen from Table 9 that the content of impurity A in the three batches of zoledronic acid raw materials is in the range of 0.05%~0.06%, the content of impurity B is in the range of 0.02%~0.03%, and impurity C and impurity D are not detected, all of which meet the limit requirements (<0.3%).

[0111] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for detecting impurities A, B, C and D in a zoledronic acid bulk drug, characterized in that: The detection method is to use high performance liquid chromatography for detection, and the chromatographic conditions are: a Titank Phenyl-Hexyl chromatographic column is used as a chromatographic column; acetonitrile: buffer = 4:96 v / v is used as a mobile phase for isocratic elution, wherein each 1L of the buffer contains 8.00g of 1-octane sodium sulfonate, 37.00mg of EDTA-disodium, 10mL of perchloric acid and 2mL of phosphoric acid; the flow rate of the mobile phase is 0.6-1.0mL / min, the column temperature is 15-25°C, the injection volume is 5-15mL, and an ultraviolet detector is used for detection, and the detection wavelength is 210-220nm; The structures of impurity A, impurity B, impurity C and impurity D are shown in Formulas I, II, III and IV respectively: (I), (II), (III), (Ⅳ).

2. The detection method according to claim 1, characterized in that: The specifications of the chromatographic column are 250 mm×4.6 mm×5 μm.

3. The detection method according to claim 1, characterized in that: At least one of the following conditions must be met: The flow rate of the mobile phase was 0.8 mL / min; The column temperature of the chromatographic column is 18~22℃; The injection volume was 10 mL; The detection wavelength is 213~217 nm.

4. The detection method according to claim 1, characterized in that: The preparation method of the buffer solution comprises: Accurately weigh sodium 1-octane sulfonate and disodium EDTA, place them in a volumetric flask, then accurately pipette perchloric acid and phosphoric acid, add ultrapure water to dissolve and dilute to the scale, shake well, and use as a buffer solution.

5. The detection method according to any one of claims 1 to 4, characterized in that: The detection limit of the impurity A is 192.60 ng / mL, and the quantification limit is 629 ng / mL. The detection limit of the impurity B is 454.50 ng / mL, and the quantification limit is 1515 ng / mL. The detection limit of the impurity C is 126.00 ng / mL, and the quantification limit is 315 ng / mL. The detection limit of the impurity D is 150.00 ng / mL, and the quantification limit is 500 ng / mL.

6. The detection method according to claim 1, characterized in that: Before the detection, the method also includes preparing a bulk drug test solution, and the preparation method of the bulk drug test solution includes: diluting the zoledronic acid bulk drug with a mobile phase to obtain a bulk drug test solution with a concentration of 0.5-1.5 mg / mL.

7. The detection method according to claim 6, characterized in that: The concentration of zoledronic acid in the bulk drug test solution is 1 mg / mL.

8. The detection method according to claim 1, characterized in that: Before the test, the method further includes preparing a zoledronic acid reference solution and an impurity reference solution, wherein: Zoledronic acid reference solution: Take zoledronic acid reference and dilute it with mobile phase to prepare a zoledronic acid reference solution containing 1 mg of zoledronic acid per 1 mL; Impurity reference solution: Take impurity A reference, impurity B reference, impurity C reference and impurity D reference, and dilute them with mobile phase to prepare solutions with a concentration of 10~20μg for impurity A, impurity B, impurity C and impurity D.

9. The detection method according to claim 1, characterized in that: Before testing, the method also includes preparing a zoledronic acid limit reference solution and a bulk drug limit spiked test solution, wherein: Zoledronic acid limit reference solution: Take zoledronic acid reference and impurity A reference, impurity B reference, impurity C reference and impurity D reference, and dilute them with mobile phase to prepare a zoledronic acid limit reference solution with a zoledronic acid concentration of 1 mg / mL and impurity A, impurity B, impurity C and impurity D concentrations of 2 μg / mL; API limit spiked test solution: Take zoledronic acid API and impurity A reference substance, impurity B reference substance, impurity C reference substance and impurity D reference substance, dilute with mobile phase to prepare API limit spiked test solution with a zoledronic acid concentration of 1 mg / mL and impurities A, impurity B, impurity C and impurity D concentrations of 2 μg / mL.

10. Use of the detection method according to any one of claims 1 to 9 in the quality control of zoledronic acid bulk drug.

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