Method for detecting main component zoledronic acid in zoledronic acid bulk drug and zoledronic acid preparation

Abstract

The invention discloses a method for detecting the main component zoledronic acid in zoledronic acid bulk drugs and preparations, which adopts high performance liquid chromatography for detection, and the chromatographic conditions of the liquid chromatography are as follows: a Kromasil 100-5-C18 chromatographic column is adopted, the mobile phase is acetonitrile: buffer solution = 4: 96 (v / v) for isocratic elution, the flow velocity is 0.8 mL / min, the column temperature is 20 DEG C, and the sample size is 10 [mu] L. The detection method provided by the invention can effectively separate the main components in the zoledronic acid bulk drug and the preparation, has good precision and linear relationship, is simple, convenient and rapid, can rapidly and accurately detect the main components in the zoledronic acid bulk drug and the preparation, and accords with related regulations.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting zoledronic acid, a main component of a zoledronic acid raw material drug and a preparation. Background Art

[0002] Zoledronic acid is a third-generation bisphosphonate drug with the chemical name 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-diphosphonic acid. The main pharmacological action of zoledronic acid is to inhibit bone resorption. It can inhibit osteoclast activity in vitro, induce osteoclast apoptosis, and block osteoclast resorption of mineralized bone and cartilage by binding to bone. Zoledronic acid can also inhibit the increase in osteoclast activity and the release of bone calcium induced by various stimulating factors produced by tumors. Zoledronic acid for injection is used in combination with standard anti-tumor drug treatment to treat bone damage in patients with bone metastasis of solid tumors and multiple myeloma patients, as well as the treatment of hypercalcemia caused by malignant tumors. In the existing zoledronic acid production process, some related substances may be introduced, which will affect the purity of the active ingredients of the drug and make it difficult to ensure the efficacy and safety of the drug. Therefore, it is urgent to develop a method for detecting zoledronic acid, the main ingredient in zoledronic acid raw materials and preparations, which is of great significance for the quality control of zoledronic acid. Summary of the invention

[0003] The present invention provides a detection method with excellent sensitivity, precision and linearity for the detection of zoledronic acid, a main component of zoledronic acid in zoledronic acid raw materials and preparations. The specific technical scheme is as follows:

[0004] A method for detecting zoledronic acid, a main component in a zoledronic acid bulk drug and a preparation, wherein the detection method is performed by high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: a Kromasil 100-5-C18 chromatographic column is used, acetonitrile: buffer = 4:96 v / v is used as a mobile phase for isocratic elution, wherein each 1L of the buffer contains 2 mL of triethylamine, and the pH is adjusted to 4.0 with phosphoric acid; the flow rate of the mobile phase is 0.6-1.0 mL / min, the column temperature is 20-30°C, the injection volume is 5-15 mL, and detection is performed by using an ultraviolet detector, and the detection wavelength is 210-220 nm.

[0005] The structure of the main component zoledronic acid is shown in Formula I:

[0006] (I).

[0007] In some embodiments, the detection limit of the main component is 75.15 ng / mL and the quantification limit is 250.50 ng / mL.

[0008] In some embodiments, the chromatographic column has a size of 250 mm×4.6 mm, 5 μm.

[0009] In some embodiments, the detection method satisfies at least one of the following conditions:

[0010] The flow rate of the mobile phase was 0.8 mL / min;

[0011] The column temperature was 25 °C;

[0012] The injection volume was 10 mL;

[0013] The detection wavelength was 215 nm.

[0014] Furthermore, in some embodiments, the chromatographic column Kromasil 100-5-C18 has 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 25°C, the injection volume is 10 μL, and the detection wavelength is 215 nm.

[0015] In some embodiments, the method for preparing the buffer solution includes: accurately pipetting triethylamine, placing it in a volumetric flask, adding ultrapure water to the scale, and adjusting the pH to 4.0 with phosphoric acid to use as a buffer solution.

[0016] In some embodiments, before the detection, the step 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.

[0017] Further, in some embodiments, the concentration of zoledronic acid in the bulk drug test solution is 1.0 mg / mL.

[0018] In some embodiments, before the detection, the step further 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 the preparation test solution.

[0019] Further, in some embodiments, the concentration of zoledronic acid in the test solution of the preparation is 1.0 mg / mL.

[0020] In some embodiments, before the detection, the method further includes preparing a zoledronic acid reference solution, and the preparation method of the zoledronic acid reference solution includes: taking a zoledronic acid reference, and diluting it with a mobile phase to prepare a zoledronic acid reference solution containing 1 mg of zoledronic acid per 1 mL.

[0021] On the other hand, the present invention also provides the application of the detection method in the quality control of zoledronic acid raw materials and preparations.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The detection method of the main component zoledronic acid in the zoledronic acid bulk drug and preparation provided by the present application realizes the qualitative and quantitative analysis of the main component in the zoledronic acid bulk drug preparation, and the method has strong specificity and low detection limit, and the detection limit of the main component is 75.15 ng / mL and the quantitative limit is 250.50 ng / mL. The detection requirements of the main component zoledronic acid in the zoledronic acid bulk drug preparation are met, and the main component linear relationship is good, and the correlation coefficient is greater than 0.99. The present invention adopts high performance liquid chromatography to detect the main component in the zoledronic acid bulk drug preparation, and the method is simple to operate, fast, and has good precision. The main component in the zoledronic acid bulk drug preparation can be detected quickly and accurately, and the whole process is reliable and controllable, suitable for practical application and promotion, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 This is a schematic diagram of the blank solution chromatogram in Example 1 of the present invention;

[0026] Figure 2 This is a schematic diagram of the chromatogram of the blank auxiliary material solution in Example 1 of the present invention;

[0027] Figure 3 This is a schematic diagram of the chromatogram of the test solution of the preparation in Example 1 of the present invention;

[0028] Figure 4 This is a schematic diagram of the chromatogram of the raw material drug test solution in Example 1 of the present invention;

[0029] Figure 5 This is a schematic diagram of the chromatogram of the zoledronic acid reference substance solution in Example 1 of the present invention;

[0030] Figure 6 Schematic diagram of the linear regression curve of the principal component of Example 3 of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] A method for detecting zoledronic acid, a main component in a zoledronic acid bulk drug and a preparation, wherein the detection method is performed by high performance liquid chromatography, and the main component structures are respectively shown in Formula I:

[0033] (I), the detection limit of the main component is 75.15 ng / mL and the quantification limit is 250.50 ng / mL.

[0034] As a preferred solution, the detection steps are as follows:

[0035] (1) Solution preparation: including blank solution, blank excipient solution, zoledronic acid reference substance stock solution, zoledronic acid reference substance solution, API test solution, and drug product test solution, including:

[0036] Buffer solution: accurately pipette 2 mL of triethylamine into a 1000 mL volumetric flask, add ultrapure water to the mark, and adjust the pH to 4.0 with phosphoric acid as the buffer solution;

[0037] Blank solution: acetonitrile-buffer as diluent, the acetonitrile-buffer (4:96, v / v);

[0038] 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 scale, 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 scale, mix well to prepare a blank excipient solution.

[0039] 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;

[0040] Zoledronic acid reference solution: accurately pipette 5 mL of zoledronic acid reference stock solution into a 10 mL volumetric flask, add diluent to dilute to the mark, shake well, and prepare a zoledronic acid reference solution with a concentration of 500 μg / mL;

[0041] API test solution: Accurately weigh 10.15 mg of zoledronic acid API, place in a 10 mL brown volumetric flask, dissolve in the diluent and dilute to the mark, shake well, and a 1.0 mg / mL zoledronic acid API test solution is prepared.

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

[0043] (2) The blank solution, blank excipient solution, zoledronic acid reference substance stock solution, zoledronic acid reference substance solution, test solution and test solution were detected 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 Kromasil 100-5-C18, 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 25 °C, the injection volume was 10 μL, and the detection wavelength was 215 nm.

[0044] As a preferred solution, a Kromasil 100-5-C18 column was used with isocratic elution, a flow rate of 0.8 mL / min, and an injection volume of 10 μL.

[0045] Example 1: Preparation and detection of solution

[0046] (1) Buffer solution: Accurately pipette 2 mL of triethylamine into a 1000 mL volumetric flask, add ultrapure water to the mark, and adjust the pH to 4.0 with phosphoric acid to prepare the buffer solution.

[0047] (2) Blank solution: acetonitrile-buffer as diluent, the acetonitrile-buffer (4:96, v / v);

[0048] (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 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;

[0049] (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.

[0050] (5) Zoledronic acid reference solution: Accurately pipette 5 mL of zoledronic acid reference stock solution into a 10 mL volumetric flask, add diluent to dilute to the mark, and shake well to prepare a zoledronic acid reference solution with a concentration of 500 μg / mL.

[0051] (6) API test solution: Accurately weigh 10 mg of zoledronic acid API and place it in a 10 mL volumetric flask. Dissolve it in the diluent and make up to the mark. Shake well to prepare a zoledronic acid API test solution with a concentration of about 1.0 mg / mL.

[0052] (9) Preparation test 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 solution with a zoledronic acid concentration of about 1.0 mg / mL.

[0053] Liquid chromatography detection: The blank solution, blank excipient solution, zoledronic acid reference solution, raw material test solution, and preparation test solution are detected by high performance liquid chromatography, and the chromatogram is recorded. Figure 1 The above liquid chromatography conditions are:

[0054] A Kromasil 100-5-C18 (250×4.6 mm, 5 μm) 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 25 °C, and an injection volume of 10 μL.

[0055] from Figure 5 It can be seen that the chromatographic peak of the main component zoledronic acid reference substance appeared at the retention time of 3.518 min.

[0056] from Figures 1 to 5 It can be seen that the blank solution, blank auxiliary material solution and preparation test solution have no interference with the detection of the main component, indicating that the liquid chromatography method provided by the present invention has good specificity.

[0057] Example 2: Limit of Detection and Limit of Quantitation

[0058] Detection limit and quantification limit: The zoledronic acid reference solution prepared in Example 1 was quantitatively diluted step by step with a 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 main component zoledronic acid 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.

[0059]

[0060] Table 1

[0061] 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.

[0062]

[0063] Table 2

[0064] Example 3: Linear Relationship

[0065] The 500 μg / mL zoledronic acid reference solution prepared in Example 1 was diluted with a diluent to obtain linear solutions with concentrations of 100.2 μg / mL, 250.5 μg / mL, 501 μg / mL, 751.5 μg / mL, and 1002 μg / mL, respectively.

[0066] 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.

[0067]

[0068] Table 3

[0069] Combining Table 3 and Linear Figure 6 It can be seen that the main component has a good linear relationship in the concentration range of 100.2 μg / mL~1002 μg / mL.

[0070] Example 4: Repeatability

[0071] The same batch of samples were taken, and the preparation test solution was prepared in parallel. According to the detection method of the zoledronic acid raw material and the main component zoledronic acid in the preparation described in Example 1, high performance liquid chromatography was used for detection. The specific conditions of liquid chromatography were 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.

[0072]

[0073] Table 4

[0074] Example 5: Intermediate Precision

[0075] The preparation test solution prepared in Example 1 was taken, and the preparation limit spiked test solution prepared above was 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.

[0076]

[0077] Table 5

[0078] As can be seen from Table 5, the RSD of the peak area of ​​the main component is 0.30%, and the results show that the liquid phase detection method for the main component in the zoledronic acid bulk drug and preparation provided by the present invention has good intermediate precision.

[0079] Example 6: Accuracy

[0080] The accuracy test of the main component is expressed as recovery (%), and three samples of the quantitative limit concentration spiked recovery rate, the preparation 50% spiked recovery rate sample, the preparation 100% spiked recovery rate sample, and the preparation 150% spiked recovery rate sample were prepared in parallel as recovery test solutions. The recovery test solutions prepared above were detected by high performance liquid chromatography. The specific conditions of liquid chromatography were as described in Example 1, and the spectra were recorded. The results are shown in Table 6.

[0081] The recovery rate was calculated according to the following formula: Recovery rate (%) = (measured amount - original amount) / theoretical added amount × 100%;

[0082]

[0083] Table 6

[0084] As can be seen from Table 6, the recovery rate of the main component zoledronic acid is in the range of 105.12% to 111.40%; the average RSD of the three groups of spiked recoveries is 0.001%, indicating that the main component of zoledronic acid provided by the present invention, the detection method of zoledronic acid in the preparation has good accuracy.

[0085] Example 7: Solution Stability

[0086] The zoledronic acid reference solution and the preparation 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.

[0087]

[0088] Table 7

[0089] As can be seen from Table 7, the zoledronic acid reference solution and the test solution of the preparation were stable within 12 h.

[0090] Example 8: Durability

[0091] The test solution of the zoledronic acid preparation prepared in Example 1 was taken and detected by high performance liquid chromatography under different conditions. The specific conditions of liquid chromatography were as described in Example 1. The spectrum was recorded, the peak area was examined, and the content RSD was calculated. The results are shown in Table 8.

[0092]

[0093] Table 8

[0094] As can be seen from Table 8, the RSD of the main component content of the zoledronic acid preparation test solution measured under different conditions is 0.11%, and the results show that the liquid phase detection method of the main component in the zoledronic acid preparation provided by the present invention has good durability.

[0095] Example 9: Sample determination

[0096] According to the detection method of the main component in the zoledronic acid bulk drug and preparation provided in Example 1, the contents of the main components in three batches of zoledronic acid bulk drug and preparation samples were detected respectively. The results are shown in Table 9.

[0097]

[0098] Table 9

[0099] It can be seen from Table 9 that the content of the main component in the three batches of zoledronic acid raw materials and preparations is 104.71%~106.78%.

[0100] 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 zoledronic acid, a main component of zoledronic acid in a zoledronic acid bulk drug or preparation, characterized in that: The detection method is to use high performance liquid chromatography for detection, wherein the chromatographic conditions of the high performance liquid chromatography are: using a Kromasil100-5-C18 chromatographic column, using acetonitrile: buffer = 4:96 v / v as the mobile phase for isocratic elution, wherein each 1L of the buffer contains 2 mL of triethylamine, and the pH is adjusted to 4.0 with phosphoric acid; the flow rate of the mobile phase is 0.6-1.0 mL / min, the column temperature is 20-30°C, the injection volume is 5-15 mL, and an ultraviolet detector is used for detection, and the detection wavelength is 210-220 nm.

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 was 25 °C; The injection volume was 10 mL; The detection wavelength was 215 nm.

4. The detection method according to claim 1, characterized in that: The preparation method of the buffer solution comprises: Accurately pipette triethylamine into a volumetric flask, add ultrapure water to the mark, and adjust the pH to 4.0 with phosphoric acid as a buffer.

5. The detection method according to claims 1 to 4, characterized in that The detection limit of the main component zoledronic acid is 75.15 ng·mL -1 , the limit of quantification was 250.50 ng·mL -1 .

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

7. The detection method according to claim 6, characterized in that The concentration of zoledronic acid in the bulk drug test solution was 1.0 mg / mL.

8. The detection method according to claim 1, characterized in that: Before the detection, the method also includes preparing a preparation test solution. The preparation method of the preparation test solution includes: diluting the zoledronic acid preparation with a mobile phase to obtain the preparation test solution.

9. The detection method according to claim 1, characterized in that Before the detection, the method also includes preparing a zoledronic acid reference solution. The preparation method of the zoledronic acid reference solution includes: taking a zoledronic acid reference, diluting it with a mobile phase to prepare a zoledronic acid reference solution containing 1 mg of zoledronic acid per 1 mL.

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

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