Method for detecting phosphorous acid and phosphate ions in zoledronic acid raw material medicine and zoledronic acid preparation

Abstract

The invention relates to the technical field of drug analysis, and particularly discloses a method for detecting phosphorous acid and phosphate ions in zoledronic acid bulk drugs and preparations, related substances in the zoledronic acid bulk drugs and preparations are detected by using ion chromatography, and the chromatographic conditions of the ion chromatography are as follows: DionexIonPacTMAS18Analyl (4 * 250 mm) is used as a chromatographic column, DionexIonPacTMAG18Analyl (4 * 50 mm) is used as a guard column, the concentration of the phosphorous acid and phosphate ions in the zoledronic acid bulk drugs and preparations is 10-20%, and the concentration of the phosphorous acid and phosphate ions in the zoledronic acid bulk drugs and preparations is 10-20%. A 4.5 mmol / L sodium carbonate aqueous solution and a 1.4 mmol / L sodium bicarbonate aqueous solution are used as leacheate for isocratic elution, the flow rate is 1.0 mL / min, and the sample size is 10 microliters. The detection method provided by the invention can effectively separate phosphorous acid and phosphate ions in zoledronic acid bulk drugs and preparations, has the advantages of simplicity, rapidness, high precision and the like, can rapidly and accurately detect phosphorous acid and phosphate ions in zoledronic acid bulk drugs and preparations, 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 phosphorous acid and phosphate ions in a zoledronic acid raw material drug and a preparation. Background Art

[0002] Bisphosphonic acid compounds can be firmly adsorbed on the surface of hydroxyapatite, the main component of bone, inhibiting its formation and dissolution. Bisphosphonic acid can inhibit the calcification of soft tissue and the reabsorption of bone. Zoledronic acid (English name Zoledronic Acid), chemical name 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-diphosphonic acid, is a third-generation bisphosphonate drug, mainly used to treat malignant hypercalcemia and bone metastasis. Studies have found that in addition to inhibiting bone resorption, zoledronic acid also has direct and indirect anti-tumor activity, and can inhibit tumor angiogenesis by regulating the autocrine loop of VEGF-VEGFR2. In the process of producing ifosfamide raw materials by existing processes, related impurities such as phosphites and phosphates may be generated. The drug quality standards should strictly stipulate the limits of impurities for toxic impurities and toxic residual organic solvents.

[0003] Therefore, it is necessary to develop a method that can measure phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations, which is of great significance for the quality control of ifosfamide. Summary of the invention

[0004] Therefore, the present invention provides a method for detecting phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations, which has excellent sensitivity, precision and linearity and can meet the detection requirements of phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations. The technical scheme adopted is as follows:

[0005] A method for detecting phosphorous acid and phosphate ions in a zoledronic acid bulk drug and a preparation, wherein the phosphorous acid and phosphate ion structures are shown in Formula I and II, respectively:

[0006] (I) (II),

[0007] Ion chromatography was used to detect phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations, wherein the chromatographic conditions of the ion chromatography were: detection was performed using a conductivity detector and the chromatographic column was Dionex IonPac TM AS18Analytical, size: 4 mm × 250 mm, guard column: Dionex IonPac TMAG18Analytical, size 4 mm × 50 mm, isocratic elution was performed using 4.5 mmol / L sodium carbonate and 1.4 mmol / L sodium bicarbonate aqueous solution as eluent.

[0008] In some embodiments, the flow rate of the eluent is 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0009] In some embodiments, the injection volume is 8-12 μL, preferably 10 μL.

[0010] In some embodiments, the detection limit of phosphorous acid is 0.176 μg / mL, the quantification limit is 0.587 μg / mL, the detection limit of phosphate ion is 0.300 μg / mL, and the quantification limit is 1.000 μg / mL.

[0011] Furthermore, in some embodiments, before the ion chromatography is used for detection, the method further comprises: preparing a mixed reference solution, a bulk drug test solution, and a preparation test solution; wherein,

[0012] The preparation method of the mixed reference solution comprises: taking a phosphorous acid reference substance and a phosphate ion standard solution, adding water to dilute to prepare a solution containing 10 μg of phosphorous acid and 10 μg of phosphate ions per 1 mL;

[0013] The preparation method of the bulk drug test solution comprises: taking the bulk drug of zoledronic acid, diluting it with water to prepare a solution containing 1 mg of zoledronic acid per 1 mL;

[0014] The preparation method of the test solution of the preparation comprises: taking the zoledronic acid preparation, adding water to dilute it to prepare a solution containing 4 mg of zoledronic acid per 1 mL.

[0015] Furthermore, in some embodiments, before the ion chromatography is used for detection, a blank excipient solution is prepared. The preparation method of the blank excipient solution comprises: taking 24 mg of sodium citrate and 220 mg of mannitol, adding 3 mL of water to dilute and dissolve to obtain a blank excipient solution.

[0016] Furthermore, in some embodiments, before the ion chromatography is used for detection, it also includes preparing a 100% spiked test solution of the preparation; the preparation method of the 100% spiked test solution of the preparation includes: taking the zoledronic acid preparation, the phosphorous acid reference substance and the phosphate ion standard solution, diluting them with water to prepare a solution containing 4 mg of zoledronic acid, 10 μg of phosphorous acid and 10 μg of phosphate ions per 1 mL.

[0017] In some embodiments, the ion chromatography column is Dionex IonPac TMAS18Analytical, size: 4 mm × 250 mm, guard column: Dionex IonPac TM AG18Analytical, with a specification of 4 mm×50 mm, used 4.5 mmol / L sodium carbonate and 1.4 mmol / L sodium bicarbonate aqueous solution as eluent for isocratic elution. The detector was a conductivity detector, the flow rate of the eluent was 1.0 mL / min, and the injection volume was 10 μL.

[0018] Compared with the prior art, the method for detecting phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations developed by the present invention has the following beneficial effects:

[0019] The detection method of phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations provided in the present application can realize the quantitative and qualitative analysis of phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations. The method has strong specificity and low detection limit. The detection limit of phosphorous acid in zoledronic acid is 0.176 μg / mL, the limit of quantification is 0.587 μg / mL, and the detection limit of phosphate ions is 0.300 μg / mL, and the limit of quantification is 1.000 μg / mL. It meets the detection requirements of phosphorous acid and phosphate ions in zoledronic acid raw materials, and phosphorous acid and phosphate ions have good linear relationship, good repeatability, high accuracy, and the correlation coefficient within the linear range of the standard curve is greater than 0.99. The invention adopts an ion chromatography detector to detect phosphorous acid and phosphate ions in a zoledronic acid raw material and a preparation. The method is simple and fast to operate, has good precision, can detect phosphorous acid and phosphate ions in a zoledronic acid raw material and a preparation quickly and accurately, 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

[0020] 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.

[0021] Figure 1 is the ion chromatogram of the blank solution in Example 1 of the present invention;

[0022] Figure 2 It is the ion chromatogram of the blank auxiliary material solution in Example 1 of the present invention;

[0023] Figure 3 is the ion chromatogram of the mixed reference solution in Example 1 of the present invention;

[0024] Figure 4 The ion chromatogram of the test solution of the preparation in Example 1 of the present invention;

[0025] Figure 5 Schematic diagram of the linear regression curve of phosphorous acid in Example 3 of the present invention;

[0026] Figure 6 Schematic diagram of the linear regression curve of phosphate ions in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] A method for detecting phosphorous acid and phosphate ions in a zoledronic acid raw material and preparation, wherein the detection method adopts ion chromatography for detection, and the phosphorous acid and phosphate ion structures are shown in Formula I and II, respectively:

[0029] (I) (II), the detection limit of phosphorous acid is 0.176 μg / mL, the quantification limit is 0.587 μg / mL, the detection limit of phosphate ion is 0.300 μg / mL, and the quantification limit is 1.000 μg / mL.

[0030] As a preferred solution, the detection steps are as follows:

[0031] (1) Solution preparation: including blank solution, mixed reference substance stock solution, mixed reference substance solution, blank excipient solution, test substance solution and 100% spiked test substance solution, of which:

[0032] Blank solution: ultrapure water (diluent);

[0033] Phosphorous acid reference substance stock solution: accurately weigh the phosphorous acid reference substance, place it in a 100 mL volumetric flask, dilute it to the mark with a diluent, and shake well to obtain the phosphorous acid reference substance stock solution;

[0034] Phosphate ion reference stock solution: Accurately pipette the phosphate ion standard solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare the phosphate ion reference stock solution.

[0035] Mixed reference substance stock solution: accurately weigh the phosphorous acid reference substance, place it in a 10 mL volumetric flask, dilute it to the mark with a diluent, and shake well; accurately pipette the above solution and phosphate ion standard solution separately, place them in the same 20 mL volumetric flask, dilute it to the mark with a diluent, and shake well to prepare the phosphorous acid and phosphate ion mixed reference substance stock solution;

[0036] Mixed reference substance solution: accurately pipette the mixed reference substance stock solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to obtain a phosphorous acid and phosphate ion mixed reference substance solution;

[0037] Blank excipient solution: accurately weigh appropriate amounts of sodium citrate and mannitol, add 3 mL of ultrapure water to dissolve, and shake well to prepare a blank excipient solution;

[0038] API test solution: accurately weigh zoledronic acid API (batch number: 20221001), place in a 20 mL volumetric flask, dilute to volume with diluent, and shake well to prepare API test solution;

[0039] Preparation test solution: accurately weigh the zoledronic acid preparation (batch number: 210215AJ), place it in a 10 mL volumetric flask, dilute it to the mark with a diluent, and shake well to prepare the preparation test solution; the zoledronic acid preparation described in this example is a zoledronic acid lyophilized powder injection for injection.

[0040] Preparation 100% spiked test solution: Accurately weigh the zoledronic acid preparation (batch number: 210215AJ), place it in a 10 mL volumetric flask, accurately add the mixed reference stock solution, dilute to the scale with diluent, and shake well to prepare the preparation 100% spiked test solution of phosphorous acid, phosphate ion and zoledronic acid.

[0041] (2) The blank solution, mixed reference solution, blank excipient solution, test solution and 100% spiked test solution were tested by ion chromatography. The chromatographic conditions of the ion chromatography were as follows: a conductivity detector was used for detection and the chromatographic column was Dionex IonPac TM AS18Analytical, size: 4 mm × 250 mm, guard column: Dionex IonPac TM AG18Analytical, size 4 mm × 50 mm, isocratic elution with 4.5 mmol / L sodium carbonate and 1.4 mmol / L sodium bicarbonate aqueous solution as eluent. As a preferred solution, the flow rate is 1.0 mL / min and the injection volume is 10 μL.

[0042] In the examples of the present application, the concentration of the phosphate ion standard solution is 1 mg / ml.

[0043] Example 1: Preparation and detection of solution

[0044] (1) Blank solution: ultrapure water (diluent).

[0045] (2) Phosphorous acid reference stock solution: Accurately weigh 10 mg of phosphorous acid reference solution and place it in a 100 mL volumetric flask. Dilute to volume with diluent and shake well to obtain a phosphorous acid concentration of 100 μg / mL.

[0046] (3) Phosphate ion reference stock solution: Accurately pipette 1 mL of phosphate ion standard solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to obtain a phosphate ion concentration of 100 μg / mL.

[0047] (4) Mixed reference stock solution: Accurately weigh 10 mg of phosphorous acid reference substance and place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well; accurately pipette 2 mL of the above solution and phosphate ion standard solution respectively, place them in the same 20 mL volumetric flask, dilute to the mark with diluent, and shake well to prepare a mixed reference stock solution with phosphorous acid and phosphate ion concentrations of 100 μg / mL each.

[0048] (5) Mixed reference solution: Accurately pipette 1 mL of the mixed reference stock solution into a 10 mL volumetric flask, dilute to volume with diluent, and shake well to prepare a mixed reference solution with a phosphorous acid and phosphate ion concentration of 10 μg / mL.

[0049] (6) Blank excipient solution: Accurately weigh 24 mg of sodium citrate and 220 mg of mannitol, add 3 mL of ultrapure water to dissolve, and shake well to prepare a blank excipient solution.

[0050] (7) API test solution: Accurately weigh 20 mg of zoledronic acid API and place it in a 20-mL volumetric flask. Dilute to volume with diluent and shake well to obtain a 1 mg / mL API test solution.

[0051] (8) Preparation test solution: Accurately weigh 40 mg of zoledronic acid preparation as anhydrous zoledronic acid, place it in a 10-mL volumetric flask, dilute to volume with diluent, and shake well to obtain a preparation test solution with a zoledronic acid concentration of 4 mg / mL.

[0052] (9) 100% spiked test solution of preparation: Accurately weigh 40 mg of zoledronic acid preparation test sample and place it in a 10 mL volumetric flask. Accurately pipette the mixed reference stock solution into the same volumetric flask, dilute to volume with diluent, and shake well to prepare a 100% spiked test solution with a phosphorous acid and phosphate ion concentration of 10 μg / mL and a zoledronic acid concentration of 4 mg / mL.

[0053] Ion chromatography detection: The blank solution, mixed reference solution, blank excipient solution, API test solution, preparation test solution and preparation 100% spiked test solution were detected using an ion chromatography detector, and the chromatograms were recorded. The chromatograms are as follows: Figure 1 The above ion chromatography conditions are:

[0054] Using Dionex IonPac TM AS18 Analytical (4 mm × 250 mm) ion chromatography column, Dionex IonPac TM The AG18Analytical (4 mm × 50 mm) guard column was used, and 4.5 mmol / L sodium carbonate and 1.4 mmol / L sodium bicarbonate aqueous solution were used as eluents for isocratic elution. The detector was a conductivity detector, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0055] from Figure 3 It can be seen that the chromatographic peak of the phosphorous acid reference substance appeared at the retention time of 10.810 min, and the chromatographic peak of the phosphate ion reference substance appeared at the retention time of 13.587 min, and the chromatographic peaks were well separated.

[0056] from Figures 1 to 4 It can be seen that the blank solution, blank auxiliary material solution and zoledronic acid test sample have no interference with the detection of phosphorous acid and phosphate ions, indicating that the ion chromatography 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 reference substance stock solution prepared in Example 1 was quantitatively diluted step by step with a diluent, and then detected by ion chromatography. The specific conditions of ion chromatography were as described in Example 1. The spectrum was recorded. The detection limit and quantification limit of the raw material and the preparation were obtained with a signal-to-noise ratio of not less than 3:1 for the detection limit and a noise ratio of not less than 10:1 for the quantification limit. The results are shown in Table 1.

[0059]

[0060] Table 1

[0061] Six quantitative limit solutions were prepared in parallel and detected by ion chromatography. The specific conditions of ion 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 mixed reference substance stock solution of Example 1 was diluted stepwise to obtain linear solutions with concentrations of 2 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL, respectively.

[0066] The linear solution prepared above was detected by ion chromatography. The specific conditions of ion 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 Figure 5 , Figure 6 It can be seen that phosphorous acid and phosphate ions have good linear relationships in the concentration range of 2 μg / mL ~20 μg / mL.

[0070] Example 4: Intermediate Precision

[0071] The mixed reference solution prepared in Example 1 was taken and tested by ion chromatography. The specific conditions of ion 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 4.

[0072]

[0073] Table 4

[0074] As can be seen from Table 4, the RSD of the phosphorous acid peak area is 1.3%, and the RSD of the phosphate ion peak area is 2.0%. The results show that the detection method of phosphorous acid and phosphate ions in the zoledronic acid bulk drug and preparation provided by the present invention has good precision.

[0075] Example 5: Repeatability

[0076] The same batch of samples were taken, and 100% of the test solution was spiked in parallel. The detection method of phosphorous acid and phosphate ions described in Example 1 was used for detection by ion chromatography-conductivity detector. The specific conditions of ion chromatography were as described in Example 1, and the results are shown in Table 5. It can be seen from the results that the detection method provided by the present application has good repeatability.

[0077]

[0078] Table 5

[0079] Example 5: Accuracy

[0080] The accuracy test of phosphorous acid and phosphate ions is expressed as recovery (%). Three portions of 50% spiked test solution, 100% spiked test solution, and 150% spiked test solution of the preparation are prepared in parallel as recovery test solutions. The recovery test solution prepared above is tested by ion chromatography. The specific conditions of ion chromatography are as described in Example 1. The spectrum is 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 addition amount × 100%

[0082]

[0083] Table 6

[0084] As can be seen from Table 6, the recovery rate of phosphorous acid is in the range of 93.87% to 100.44%, the average recovery rate is 97.58%, and the recovery rate RSD is 2.5%; the recovery rate of phosphate ions is in the range of 98.21% to 104.39%, the average recovery rate is 99.80%, and the recovery rate RSD is 1.9%, indicating that the detection method of phosphorous acid and phosphate ions in the zoledronic acid bulk drug and preparation provided by the present invention has good accuracy.

[0085] Example 6: Solution Stability

[0086] The mixed reference solution prepared in Example 1 was taken as a stability solution, and after being placed at room temperature for 0 h, 2 h, 5 h, 20 h, 22.5 h, and 27 h, it was detected by ion chromatography-conductivity detector method. The specific conditions of liquid chromatography were as described in Example 1. The spectrum was recorded, its peak area was examined, and its RSD was calculated. The results are shown in Table 7;

[0087] The 100% spiked test solution of Example 4 was placed at room temperature for 0 h, 2 h, 5 h, 20 h, 22.5 h, and 27 h, and then detected by ion chromatography-conductivity detector method. 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;

[0088] Table 7

[0089] It can be seen from Table 7 that the mixed reference solution of phosphorous acid and phosphate ion is stable within 27 h, and the 100% spiked test solution of the preparation is stable within 27 h.

[0090] Example 7: Actual sample measurement

[0091] According to the detection method of phosphorous acid and phosphate ions in the zoledronic acid bulk drug and preparation provided in Example 1, the ion chromatography-conductivity detector method was used for detection, the peak area was investigated, and the RSD was calculated. The results are shown in Table 8:

[0092] Table 8

[0093] From the chromatograms of the five consecutive injections of the mixed reference solution in Table 8, phosphorous acid and phosphate ions were not detected in the three batches of samples of the API and the preparation; the content of phosphorous acid and phosphate ions in the API and the preparation did not exceed the limit of 0.25%, and met the limit requirements.

[0094] 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 phosphorous acid and phosphate ions in zoledronic acid bulk drugs and preparations, characterized in that: Ion chromatography was used to detect phosphorous acid and phosphate ions in zoledronic acid raw materials and preparations, wherein the chromatographic conditions of the ion chromatography were: detection was performed using a conductivity detector and the chromatographic column was Dionex IonPac TM AS18Analytical, size: 4 mm × 250 mm, guard column: Dionex IonPac TM AG18Analytical, size 4 mm × 50 mm, isocratic elution was performed using 4.5 mmol / L sodium carbonate and 1.4 mmol / L sodium bicarbonate aqueous solution as eluent.

2. The detection method according to claim 1, characterized in that The flow rate of the eluent is 0.8-1.2 mL / min.

3. The detection method according to claim 2, characterized in that The flow rate of the eluent was 1.0 mL / min.

4. The detection method according to claim 1, characterized in that The injection volume was 8~12 μL.

5. The detection method according to claim 4, characterized in that The injection volume was 10 μL.

6. The detection method according to any one of claims 1 to 5, characterized in that The detection limit of the phosphorous acid is 0.176 μg / mL, and the quantification limit is 0.587 μg / mL; the detection limit of the phosphate ion is 0.300 μg / mL, and the quantification limit is 1.000 μg / mL.

7. The detection method according to claim 1, characterized in that: Before using ion chromatography for detection, the method also includes: preparing a mixed reference solution, a raw material test solution, and a preparation test solution; wherein, The preparation method of the mixed reference solution comprises: taking a phosphorous acid reference substance and a phosphate ion standard solution, adding water to dilute to prepare a solution containing 10 μg of phosphorous acid and 10 μg of phosphate ions per 1 mL; The preparation method of the bulk drug test solution comprises: taking the bulk drug of zoledronic acid, diluting it with water to prepare a solution containing 1 mg of zoledronic acid per 1 mL; The preparation method of the test solution of the preparation comprises: taking the zoledronic acid preparation, adding water to dilute it to prepare a solution containing 4 mg of zoledronic acid per 1 mL.

8. The detection method according to claim 1, characterized in that Before the ion chromatography is used for detection, a blank auxiliary material solution is also prepared. The preparation method of the blank auxiliary material solution includes: taking 24 mg of sodium citrate and 220 mg of mannitol, adding 3 mL of water to dilute and dissolve to obtain a blank auxiliary material solution.

9. The detection method according to claim 1, characterized in that Before the ion chromatography is used for detection, the method also includes preparing a 100% spiked test solution of the preparation; the preparation method of the 100% spiked test solution of the preparation includes: taking the zoledronic acid preparation, the phosphorous acid reference substance and the phosphate ion standard solution, diluting them with water to prepare a solution containing 4 mg of zoledronic acid, 10 μg of phosphorous acid and 10 μg of phosphate ion 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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