Method for detecting impurity B, impurity C and impurity D in zoledronic acid preparation

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting 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 the content of the impurity B, the impurity C and the impurity D in the zoledronic acid preparation, and is convenient for quality control of the zoledronic acid preparation.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting impurities B, C and D in a zoledronic acid preparation. 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, 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 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 B, impurity C and impurity D in zoledronic acid preparations that is simple to operate, accurate and sensitive. Summary of the invention

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

[0004] A method for detecting impurities B, C and D in a zoledronic acid preparation, wherein the chromatographic conditions are as follows: 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 8.00 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 at a detection wavelength of 210-220 nm;

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

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

[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 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.

[0013] In some embodiments, the preparation method of the buffer solution includes: accurately weighing sodium 1-octane sulfonate and disodium EDTA, placing them in a volumetric flask, then accurately pipetting perchloric acid and phosphoric acid, adding ultrapure water to dissolve and dilute to the scale, and shaking well to obtain a buffer solution.

[0014] Before the detection, the method also includes preparing a preparation test solution, and the preparation method of the preparation test solution includes: diluting the zoledronic acid preparation with a mobile phase to obtain a preparation test solution with a zoledronic acid concentration of 0.3-1.5 mg / mL.

[0015] Further, in some embodiments, the concentration of zoledronic acid in the test solution of the preparation is 0.5 mg / mL.

[0016] In some embodiments, before the test, the method further comprises preparing a blank excipient solution, an impurity reference solution, a zoledronic acid limit reference solution, and a formulation limit spiked test solution, wherein:

[0017] Blank excipient solution: Take appropriate amount of sodium citrate and mannitol, add into mobile phase to dissolve and dilute to prepare blank excipient solution;

[0018] Impurity reference solution: Take impurity B, impurity C and impurity D reference substances, add mobile phase to dilute, and prepare impurity reference solution with a concentration of 20 μg / mL for impurity B, impurity C and impurity D;

[0019] Zoledronic acid limit reference solution: Take zoledronic acid reference and impurity B, impurity C and impurity D reference, dilute with mobile phase, and prepare a zoledronic acid limit reference solution with a zoledronic acid concentration of 500 μg / mL and an impurity B, impurity C and impurity D concentration of 2 μg / mL;

[0020] Preparation limit spiked test solution: Take zoledronic acid preparation and impurity B, impurity C and impurity D reference substances, dilute with mobile phase to prepare a preparation limit spiked test solution with a zoledronic acid concentration of 500 μg / mL and an impurity B, impurity C and impurity D concentration of 2 μg / mL.

[0021] Furthermore, in some embodiments, 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.

[0022] On the other hand, the invention also provides application of the detection method in quality control of zoledronic acid preparations.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The detection method of impurities B, C and D in zoledronic acid preparations provided in the present application realizes quantitative and qualitative analysis of impurities B, C and D in zoledronic acid preparations. The method has strong specificity and low detection limit. 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 B, C and D in zoledronic acid preparations are met, and the linear relationship between impurities B, C and D is good, and the correlation coefficients are all greater than 0.99. The invention adopts a high performance liquid chromatography method to detect impurities B, impurity C and impurity D in a zoledronic acid preparation. The method is simple and fast to operate, has good precision, can quickly and accurately detect impurities B, impurity C and impurity D in the zoledronic acid preparation, and 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

[0025] 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.

[0026] Figure 1The specificity and system suitability chromatogram of Example 1 of the present invention, wherein: A: blank solution; B: blank excipient solution; C: zoledronic acid limit reference solution; D: preparation test solution; E: preparation limit spiked test solution.

[0027] Figure 2 Schematic diagram of the linear regression curve of impurity B in Example 3 of the present invention.

[0028] Figure 3 Schematic diagram of the linear regression curve of impurity C in Example 3 of the present invention.

[0029] Figure 4 Schematic diagram of the linear regression curve of impurity D in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] A method for detecting impurities B, C and D in a zoledronic acid preparation, wherein the detection method is carried out by high performance liquid chromatography, and the structures of the impurities B, C and D are shown in formulas I, II and III:

[0032] (I) (II) (III), 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, and the detection limit of the impurity D is 150.00 ng / mL, and the quantification limit is 500 ng / mL.

[0033] As a preferred solution, the detection steps are as follows:

[0034] (1) Solution preparation: including blank solution, blank excipient solution, zoledronic acid reference substance stock solution, impurity reference substance stock solution, impurity reference substance solution, zoledronic acid limit reference substance solution, preparation test substance solution, preparation limit spiked test substance solution, including:

[0035] Buffer solution: accurately weigh 8.00 g of sodium 1-octane sulfonate and 37.00 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, mix well, and use this as the buffer solution;

[0036] Blank solution: acetonitrile-buffer as diluent, the acetonitrile-buffer (4:96, v / v);

[0037] Blank excipient solution: accurately weigh 600 mg of sodium citrate and 5500 mg of mannitol, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to the mark, mix well, accurately pipette 5 mL of the above solution, place in a 20 mL volumetric flask, add diluent to dissolve and dilute to the mark, mix well, to prepare a blank excipient solution;

[0038] Zoledronic acid reference substance stock solution: accurately weigh 10 mg of zoledronic acid reference substance, place it in a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a 1 mg / mL zoledronic acid reference substance stock solution;

[0039] Impurity reference substance stock solution: accurately weigh 2 mg of each impurity reference substance, place it in a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare each impurity reference substance stock solution with a concentration of 200 μg / mL;

[0040] Impurity reference substance solution: 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 with a concentration of 20 μg / mL;

[0041] Zoledronic acid limit reference solution: accurately pipette 5 mL of zoledronic acid reference stock solution and 1 mL of each impurity reference solution into a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a zoledronic acid limit reference solution with 500 μg / mL zoledronic acid and 2 μg / mL of each impurity;

[0042] Preparation test sample stock solution: Take one tube of zoledronic acid lyophilized powder (4 mg based on anhydrous zoledronic acid), accurately add 4 mL of diluent, shake well, and prepare a preparation test sample stock solution with a concentration of 1.0 mg / mL.

[0043] Preparation test solution: accurately pipette 5 mL of the preparation test solution into a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a preparation test solution with a zoledronic acid concentration of about 500 μg / mL;

[0044] Preparation limit spiked test solution: accurately pipette 5 mL of preparation test stock solution and 1 mL of each impurity reference solution into a 10 mL volumetric flask, dilute to volume with diluent, shake well, and prepare a preparation limit spiked test solution with 500 μg / mL zoledronic acid and 2 μg / mL of each impurity;

[0045] (2) The blank solution, blank excipient solution, zoledronic acid reference stock solution, impurity reference stock solution, impurity reference solution, zoledronic acid limit reference solution, preparation test solution, and preparation limit spiked test solution were detected by high performance liquid chromatography, and 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.

[0046] 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.

[0047] Example 1: Solution preparation and specificity

[0048] (1) Buffer solution: Accurately weigh 8.00 g of sodium 1-octane sulfonate and 37.00 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, dissolve and dilute to volume with ultrapure water, and mix well to prepare the buffer solution.

[0049] (2) Blank solution: acetonitrile-buffer as diluent, the acetonitrile-buffer (v / v, 4:96);

[0050] (3) Blank excipient solution: Accurately weigh 600 mg of sodium citrate and 5500 mg of mannitol, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to the mark, mix well, accurately transfer 5 mL of the above solution, place in a 20 mL volumetric flask, add diluent to dissolve and dilute to the mark, mix well to prepare a blank excipient solution.

[0051] (4) Zoledronic acid reference substance stock solution: Accurately weigh 10 mg of zoledronic acid reference substance and place it in a 10 mL volumetric flask. Dilute to volume with diluent and shake well to prepare a 1 mg / mL zoledronic acid reference substance stock solution.

[0052] (5) 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 concentration of each impurity reference substance stock solution.

[0053] (6) Impurity reference solution: Accurately pipette 1 mL of each impurity reference stock solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare a 20 μg / mL impurity reference solution.

[0054] (7) Zoledronic acid limit reference solution: Accurately pipette 5 mL of zoledronic acid reference stock solution and 1 mL of each impurity reference solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare a zoledronic acid limit reference solution with a zoledronic acid concentration of 500 μg / mL and each impurity concentration of 2 μg / mL;

[0055] (8) Preparation test sample stock solution: Take one tube of zoledronic acid lyophilized powder (containing 4 mg of anhydrous zoledronic acid), accurately add 4 mL of diluent, shake well, and prepare a preparation test sample stock solution with a concentration of 1.0 mg / mL.

[0056] (9) Preparation test solution: Accurately pipette 5 mL of the preparation test solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare a preparation test solution with a zoledronic acid concentration of 500 μg / mL.

[0057] (10) Limit-spiked test solution: Accurately pipette 5 mL of the test solution and 1 mL of each impurity reference solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare a limit-spiked test solution with a zoledronic acid concentration of 500 μg / mL and each impurity concentration of 2 μg / mL.

[0058] Liquid chromatography detection: The blank solution, blank excipient solution, zoledronic acid reference substance stock solution, impurity reference substance stock solution, impurity reference substance solution, zoledronic acid limit reference substance solution, preparation test solution, and preparation limit spiked test solution were detected by high performance liquid chromatography, and the chromatogram was recorded. The chromatogram is Figure 1 The above liquid chromatography conditions are:

[0059] 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.

[0060] from Figure 1It can be seen that the chromatographic peak of the impurity B reference substance appeared at the retention time of 3.155 min, the chromatographic peak of the impurity C reference substance appeared at the retention time of 17.329 min, and the chromatographic peak of the impurity D reference substance appeared at the retention time of 13.477 min, and the chromatographic peaks were well separated.

[0061] from Figure 1 It can be seen that the blank solution, blank excipient solution and preparation test solution have no interference with the detection of impurities B, impurity C and impurity D, indicating that the liquid chromatography method provided by the present invention has good specificity.

[0062] Example 2: Limit of Detection and Limit of Quantitation

[0063] 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 preparation 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.

[0064]

[0065] Table 1

[0066] 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.

[0067]

[0068] Table 2

[0069] Example 3: Linear Relationship

[0070] 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 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.

[0071] 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.

[0072]

[0073] Table 3

[0074] Combining Table 3 and Figure 2-Figure 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.

[0075] Example 4: Repeatability

[0076] The same batch of samples were taken, and the preparation limit spiked test solution was prepared in parallel. According to the detection method of impurities B, impurity C and impurity D in the zoledronic acid preparation described in Example 1, high performance liquid chromatography was used for detection. 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.

[0077]

[0078] Table 4

[0079] Example 5: Intermediate Precision

[0080] The preparation 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.

[0081]

[0082] Table 5

[0083] As can be seen from Table 5, the RSD of the peak area of ​​impurity B is 7.16%, the RSD of the peak area of ​​impurity C is 1.15%, and the RSD of the peak area of ​​impurity D is 2.00%. The results show that the liquid phase detection method for impurities B, impurity C and impurity D in the zoledronic acid preparation provided by the present invention has good intermediate precision.

[0084] Example 6: Accuracy

[0085] The accuracy test of impurity B, impurity C and impurity D is expressed as recovery rate (%), and three samples of the preparation quantitative limit concentration spiked recovery rate, the preparation 50% spiked recovery rate, the preparation 100% spiked recovery rate, and the preparation 150% spiked recovery rate were prepared in parallel as recovery test solutions. The recovery test 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 results are shown in Table 6.

[0086] The recovery rate was calculated according to the following formula: Recovery rate (%) = (measured amount - original amount) / theoretical addition amount × 100%

[0087]

[0088] Table 6

[0089] As can be seen from Table 6, at the quantitative limit level, the recovery rate of impurity B is in the range of 93.88% to 106.78%; at other levels, the recovery rate of impurity B is in the range of 80.65% to 103.92%; the average RSD of the four groups of spiked recoveries is 0.06%. At the quantitative limit level, the recovery rate of impurity C is in the range of 87.62% to 99.48%; at other levels, the recovery rate of impurity C is in the range of 93.43% to 100.08%; the average RSD of the four groups of spiked recoveries is 0.03%. At the quantitative limit level, the recovery rate of impurity D is in the range of 89.04% to 90.42%; at other levels, the recovery rate of impurity D is in the range of 102.25% to 110.02; the average RSD of the four groups of spiked recoveries is 0.02%. It shows that the detection method of impurities B, impurity C and impurity D in the zoledronic acid preparation provided by the present invention has good accuracy.

[0090] Example 7: Solution Stability

[0091] The zoledronic acid limit reference solution, the preparation test solution and the preparation limit spiked test solution prepared in Example 1 were placed at room temperature for 0 h, 1 h, 3 h, 7 h, and 12 h, 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 peak area was examined, and the RSD was calculated. The results are shown in Table 7.

[0092]

[0093] Table 7

[0094] As can be seen from Table 7, the zoledronic acid limit reference solution, formulation test solution and formulation limit spiked test solution were all stable within 12 h.

[0095] Example 8: Durability

[0096] The zoledronic acid limit reference solution, the preparation test solution and the preparation limit spiked test solution prepared in Example 1 were tested by high performance liquid chromatography under different conditions. The specific conditions of liquid chromatography were as described in Example 1. The spectra were recorded, the peak areas were examined, and the content RSD was calculated. The results are shown in Table 8.

[0097]

[0098]

[0099] Table 8

[0100] As can be seen from Table 8, the RSD of the impurity B content of the zoledronic acid limit reference solution measured under different conditions is 0.16%, the RSD of the impurity C content is 0.12%, and the RSD of the impurity D content is 0.11%; the RSD of the impurity B content of the preparation limit spiked test solution is 0.23%, the RSD of the impurity C content is 0.24%, and the RSD of the impurity D content is 0.11%. The results show that the liquid phase detection method for impurities B, impurity C and impurity D in the zoledronic acid preparation provided by the present invention has good durability.

[0101] Example 9: Sample determination

[0102] According to the detection method of impurities B, C and D in zoledronic acid preparations provided in Example 1, the contents of impurities B, C and D in 3 batches of zoledronic acid preparation samples were detected. The results are shown in Table 9:

[0103]

[0104] Table 9

[0105] As can be seen from Table 8, impurity B, impurity C and impurity D were not detected in the three batches of zoledronic acid preparations and met the limit requirements (<0.5%).

[0106] 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 B, C and D in a zoledronic acid preparation, 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 8.00 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 an ultraviolet detector is used for detection, and the detection wavelength is 210-220 nm; The structures of impurity B, impurity C and impurity D are shown in Formula I, II and III respectively: (I), (II), (III).

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 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 preparation test solution, and the preparation method of the preparation test solution includes: diluting the zoledronic acid preparation with a mobile phase to obtain a preparation test solution with a zoledronic acid concentration of 0.3-1.5 mg / mL.

7. The detection method according to claim 6, characterized in that: The concentration of zoledronic acid in the test solution of the preparation is 0.5 mg / mL.

8. The detection method according to claim 1, characterized in that: Before the test, the method also includes preparing a blank excipient solution, an impurity reference solution, a zoledronic acid limit reference solution, and a formulation limit spiked test solution, wherein: Blank excipient solution: Take appropriate amount of sodium citrate and mannitol, add into mobile phase to dissolve and dilute to prepare blank excipient solution; Impurity reference solution: Take impurity B, impurity C and impurity D reference substances, add mobile phase to dilute, and prepare impurity reference solution with a concentration of 20 μg / mL for impurity B, impurity C and impurity D; Zoledronic acid limit reference solution: Take zoledronic acid reference and impurity B, impurity C and impurity D reference, dilute with mobile phase, and prepare a zoledronic acid limit reference solution with a zoledronic acid concentration of 500 μg / mL and an impurity B, impurity C and impurity D concentration of 2 μg / mL; Preparation limit spiked test solution: Take zoledronic acid preparation and impurity B, impurity C and impurity D reference substances, dilute with mobile phase to prepare a preparation limit spiked test solution with a zoledronic acid concentration of 500 μg / mL and an impurity B, impurity C and impurity D concentration of 2 μg / mL.

9. The detection method according to claim 1, characterized in that: 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.

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

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