Method for analyzing related substances of zoledronic acid concentrated solution for injection

Abstract

The invention provides a method for analyzing related substances of a zoledronic acid concentrated solution for injection, and relates to the field of medicine analysis. According to the analysis method for the related substances of the zoledronic acid concentrated solution for injection, high performance liquid chromatography is adopted, and a chromatographic column adopts octadecylsilane chemically bonded silica as a filler; the flow velocity is 0.6 to 0.7 ml / min; the mobile phase is prepared from a phosphate buffer solution, ethanol and acetone in a volume ratio of (88-92): (4.5-5.5): (4.5-5.5). By adopting the detection method disclosed by the invention, the blank solvent and the auxiliary materials do not interfere with the peak appearance of impurities and main components, the impurity D is well separated from the main components, and the peak shape is good. Under various damage conditions, the solvent peak does not interfere with the determination of impurities and main peaks, the test solutions of the preparation and the raw materials are detected without impurities, the main peaks are pure peaks, and the sample is stable. In durability verification, all impurities and impurities adjacent to main components are well separated under different conditions. According to the method, the impurity D is effectively detected, the impurity D is better controlled, and the quality of the product can be fully reflected.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for analyzing related substances in a concentrated zoledronic acid solution for injection. Background Art

[0002] Zoledronic acid is a third-generation bisphosphonate inhibitor of osteoclasts developed and launched by Novartis. In March 2001, Novartis Pharma GmbH's zoledronic acid injection and zoledronic acid for injection were launched in the EU through the EMA route, with the trade name ZOMETA, and the specifications are 4mg and 4mg / 100ml. In March 2003, zoledronic acid concentrated solution for injection (specification is 4mg / 5ml) was launched in the EU. In August 2001, NOVARTIS's zoledronic acid injection (4mg / 100ml), zoledronic acid for injection (4mg) and zoledronic acid concentrated solution for injection (4mg / 5ml) were approved for marketing in the United States, all of which were listed as RLDs. They have all been withdrawn from the market (not for safety and effectiveness reasons). In 2005, zoledronic acid concentrated solution for injection (4mg / 5ml) was launched in Japan. In 2008, Novartis's zoledronic acid concentrated solution for injection (4mg / 5ml) was approved for import and launched in China.

[0003] The quality standard of zoledronic acid concentrated solution for injection is not included in the pharmacopoeia of any country. The import registration standard (standard number: JX20201000) and the YBH05372021 standard record the quality standard of zoledronic acid concentrated solution for injection, but there is no detection and analysis of impurity D (imidazole-1-acetic acid) in zoledronic acid concentrated solution for injection.

[0004] Combined with the impurity spectrum analysis of the raw materials of this product, the degradation impurity that may exist in the concentrated solution of zoledronic acid for injection is impurity D. It is of great significance to control impurity D and study it in method development. Research and development that can simultaneously detect the content of impurity D is of great significance and can fully reflect the quality of the product. Summary of the invention

[0005] In view of this, the present invention provides a method for analyzing related substances in a concentrated solution of zoledronic acid for injection.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The method for analyzing related substances of the concentrated solution of zoledronic acid for injection adopts high performance liquid chromatography, the chromatographic column: octadecylsilane bonded silica gel is used as a filler; the flow rate is 0.6-0.7ml / min; the mobile phase is prepared by phosphate buffer, ethanol and acetone in a volume ratio of 88-92:4.5-5.5:4.5-5.5.

[0008] Furthermore, the preparation method of the phosphate buffer comprises the following steps: taking anhydrous disodium hydrogen phosphate and tetrahexylammonium hydrogen sulfate, adding water to dissolve them, adding disodium ethylenediaminetetraacetate solution, and mixing them.

[0009] Furthermore, the mass volume ratio of the anhydrous disodium hydrogen phosphate, tetrahexyl ammonium hydrogen sulfate, and disodium ethylenediaminetetraacetate solution is 5.5-6g:4-5g:1.8-2.2ml, preferably 5.7g:4.5g:2ml.

[0010] Further, the disodium ethylenediaminetetraacetic acid solution: take disodium ethylenediaminetetraacetic acid dihydrate, add sodium hydroxide solution to dissolve it, add water to make up the volume, mix well, and obtain 0.3-0.4g of disodium ethylenediaminetetraacetic acid dihydrate per 100ml.

[0011] Furthermore, the pH value of the phosphate buffer is adjusted to 7.8-8.0 using a sodium hydroxide solution.

[0012] Furthermore, the sodium hydroxide solution is 1-2 mol / L.

[0013] Further, injection volume: 30-40 μl.

[0014] Further, the column temperature was 23-27°C.

[0015] Further, the detection wavelength is: 213-217nm.

[0016] Furthermore, the chromatographic column has a specification of 250 mm×4.6 mm, 5 μm or a chromatographic column with equivalent performance.

[0017] Further, test solution: accurately measure the test sample, place it in a volumetric flask, dilute it to the scale with the mobile phase, dilute it 5-10 times, shake it well, and use it as the test solution.

[0018] Further, the reference solution: take the impurity D reference, accurately weigh it, put it in a volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain the impurity D reference solution, containing 1.5-1.7 mg per 100 ml; take the zoledronic acid reference, accurately weigh it, put it in a volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain the zoledronic acid reference solution, containing 1.5-1.7 mg per 100 ml; accurately measure the impurity D reference solution and the zoledronic acid reference solution, respectively, and put them in the same volumetric flask, and the impurity D reference solution and the zoledronic acid reference solution are diluted 9-11 times by the mobile phase, shake well, and use them as the reference solution.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) By using the detection method of the present invention, the blank solvent and auxiliary materials do not interfere with the peaks of impurities and main components, and the impurity D is well separated from the main component, and the peak shape is good.

[0021] (2) Under all destruction conditions, the solvent peak did not interfere with the determination of impurities and the main peak. No impurities were detected in the test solutions of the preparations and raw materials. The main peaks were all pure peaks, and the samples were stable.

[0022] (3) In the durability verification, under different conditions, each impurity and the impurities adjacent to the main component were well separated.

[0023] (4) The method of the present invention can effectively detect impurity D, better control impurity D, and fully reflect the quality of the product. DETAILED DESCRIPTION

[0024] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0027] The Chinese name of impurity D is imidazole-1-acetic acid, with the structural formula:

[0028]

[0029] Example 1 - Analysis Method for Related Substances of Zoledronic Acid Concentrated Solution for Injection

[0030] Inspection method: High performance liquid chromatography

[0031] Chromatographic conditions:

[0032] Chromatographic column: Octadecylsilane bonded silica gel is used as filler ( C18 chromatographic column, 250 mm × 4.6 mm, 5 μm);

[0033] Detection wavelength: 215nm;

[0034] Column temperature: 25°C;

[0035] Injection volume: 40 μl;

[0036] Flow rate: 0.6ml / min;

[0037] Mobile phase: phosphate buffer (pH 7.9)-ethanol-acetone (90:5:5, v / v / v);

[0038] Among them, phosphate buffer (pH7.9): take 5.7g of anhydrous disodium hydrogen phosphate (equivalent to 14.38g of disodium hydrogen phosphate dodecahydrate), 4.5g of tetrahexylammonium hydrogen sulfate, add 900ml of water to dissolve, add 2ml of disodium ethylenediaminetetraacetic acid solution, mix well, adjust the pH value to 7.9 with 2mol / L sodium hydroxide solution, and mix well.

[0039] Disodium ethylenediaminetetraacetate solution: Take 0.365 g of disodium ethylenediaminetetraacetate dihydrate, add 5 ml of 2 mol / L sodium hydroxide solution to dissolve it, add water to 100 ml, mix well, and you have the solution.

[0040] Solution preparation:

[0041] Solvent: mobile phase;

[0042] Complete excipient solution: Take about 0.24g of sodium citrate and about 2.2g of mannitol, weigh accurately, place in a 50ml volumetric flask, add appropriate amount of water to dissolve and dilute to the scale, shake well, and use as the complete excipient solution.

[0043] Blank excipient solution: Accurately measure 2 ml of the total excipient solution, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and use it as the blank excipient solution.

[0044] Sensitivity solution: Take about 8 mg of zoledronic acid reference substance, accurately weigh it, place it in a 500ml volumetric flask, add water to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 20ml volumetric flask, dilute to the scale with mobile phase, shake well, accurately measure 1 ml again, place it in a 10ml volumetric flask, dilute to the scale with mobile phase, shake well, as the sensitivity solution.

[0045] Test solution: Accurately measure 2 ml of the product, place it in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and use it as the test solution.

[0046] Reference solution: Take about 8 mg of impurity D reference substance and zoledronic acid reference substance respectively, weigh accurately, place in 500 ml volumetric flasks respectively, add water to dissolve and dilute to the scale, shake well; accurately measure 1 ml of each, place in the same 50 ml volumetric flask, dilute to the scale with mobile phase, shake well, as reference solution.

[0047] System suitability requirements: In the sensitivity solution chromatogram, the signal-to-noise ratio of the zoledronic acid peak should be no less than 10.

[0048] Instrument and column balance:

[0049] Instrument balance: Without connecting the chromatographic column, flush the instrument with acetic acid-water (25:75, v / v) at a flow rate of 2 mL / min for about 1 hour, flush with water at a flow rate of 2 mL / min for about 2 hours, and then flush the instrument with the mobile phase at a flow rate of 2 mL / min for about 5 minutes. If the instrument is tested for other products or has not been used for a long time before testing the concentrated solution of zoledronic acid for injection, the instrument needs to be balanced.

[0050] Balance of the chromatographic column: Connect the chromatographic column, use the mobile phase at a flow rate of 0.6mL / min, take 40μl of this product and inject it directly 10 times until the chromatographic column balance test requirements are met. The running time for each injection is 3 minutes, and then use the mobile phase at a flow rate of 0.6ml / min to balance the chromatographic column. If the chromatographic column has not been used for a long time before the detection of the concentrated solution of zoledronic acid for injection or the chromatographic column is flushed and maintained during use, the chromatographic column needs to be balanced.

[0051] Column balance test: Accurately measure 40 μl of blank excipient solution and inject it into the liquid chromatograph at least twice, and record the chromatograms respectively. The retention times of the solvent peak and the blank excipient peak in the chromatogram should be repeatable.

[0052] Determination method: Accurately measure 40 μl of each of the solvent, blank excipient solution, sensitivity solution, reference solution, and test solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0053] Calculation formula:

[0054] Impurity content (%) = (A sample × W pairs × P pairs × test sample dilution factor) ÷ (A pairs × reference sample dilution factor × labeled amount) × 100%

[0055] Impurity content (%) = impurity D detected + other impurities detected

[0056] Where: Sample A - the peak area of ​​each impurity in the test solution;

[0057] A - peak area of ​​each component in the reference solution; W

[0058] Pair - weight of each reference substance, mg;

[0059] P - content / purity of each reference substance, %.

[0060] Specification: 5ml: 4mg

[0061] Result determination: If there are impurity peaks in the chromatogram recorded by the test solution (minus the chromatographic peaks at the corresponding positions of the blank excipient solution), the impurity D shall be calculated by the external standard method based on the peak area of ​​impurity D in the reference solution, and shall not exceed 0.2%; other impurities shall be calculated by the external standard method based on the peak area of ​​zoledronic acid in the reference solution, and shall not exceed 0.2%; the total amount of impurities shall not exceed 0.4%. Any chromatographic peak in the test solution that is smaller than the main peak area of ​​the sensitivity solution can be ignored (0.05%).

[0062] (1) Test according to the method of Example 1

[0063] Determination method:

[0064] Solvent: Mobile phase

[0065] Impurity D reference solution: Take about 1 mg of each impurity D reference, accurately weigh, place in a 10 ml volumetric flask, add mobile phase to dilute to the scale, shake well; accurately measure 1 ml, place in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, as the impurity D positioning solution.

[0066] Test solution: Take 2 ml of concentrated zoledronic acid solution for injection, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0067] Blank excipient solution: Take 2 ml of the complete excipient solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0068] Results and Conclusions:

[0069] Table 1 Optimization of relevant material conditions - Positioning test results

[0070]

[0071] Conclusion: Under this chromatographic condition, blank solvent and excipients do not interfere with the peaks of impurities and main components, and impurity D is well separated from the main component with a good peak shape.

[0072] (2) Forced degradation

[0073] Table 2: Validation of relevant material methods - Destructive test - Summary of results

[0074]

[0075]

[0076] Conclusion: Under various destruction conditions, the solvent peak does not interfere with the determination of impurities and main peaks, no impurities are detected in the test solutions of preparations and raw materials, the main peaks are all pure peaks, and the samples are stable.

[0077] (3) Elimination of interference from related substances and auxiliary materials

[0078] The blank auxiliary material of this product can elute a chromatographic peak under the chromatographic conditions of the relevant substances, and needs to be deducted: the method developed in this embodiment deducts the auxiliary material peak of the test solution by setting a blank auxiliary material solution prepared simultaneously.

[0079] (4) Determination of system suitability

[0080] System suitability requirements: In the sensitivity solution chromatogram, the signal-to-noise ratio of the zoledronic acid peak should be no less than 10.

[0081] (5) Durability verification

[0082] In the verification of the durability of the detection method of the present invention, under different conditions, each impurity and the impurities adjacent to the main component were well separated.

[0083] The method validation includes: specificity, linearity and range, limit of quantification and limit of detection, accuracy test, repeatability test, intermediate precision test, durability and solution stability test. The validation shows that this method is suitable for the detection of related substances in concentrated solution of zoledronic acid for injection.

[0084] 5.1 Exclusivity

[0085] 5.1.1 Acceptability criteria

[0086] (1) Positioning solution: Blank solvent should not interfere with the determination; blank excipients should not interfere with the determination.

[0087] (2) Forced degradation test: Under all conditions, the auxiliary material peak and solvent peak do not interfere with the determination of impurities and main peak, the separation degree between the main peak and adjacent impurities should meet the requirements, the main peak is a pure peak, and the material balance should be between 90% and 110%.

[0088] 5.1.2 Results Summary

[0089] (1) Positioning solution: Blank solvent and excipient solution did not interfere with the determination; the signal-to-noise ratio of the sensitivity solution was 39.93 and not less than 10, which met the requirements; impurity D and zoledronic acid in the reference solution elute in sequence, with a separation degree of 39.12 greater than 1.5, and the specificity test met the requirements.

[0090] (2) Forced degradation test: Under various destructive conditions, the solvent peak did not interfere with the determination of impurities and main peaks. No impurities were detected in the test solutions of the preparations and raw materials. The main peaks were all pure peaks, and the material balance was between 90% and 110%.

[0091] 5.2 Linear range

[0092] 5.2.1 Acceptability criteria

[0093] The concentration of the linear solution is used as the abscissa and the peak area is used as the ordinate. The least square method is used for linear regression to calculate the correlation coefficient. Within the limit range of 0.01% to 0.6%, the linear correlation coefficient r is not less than 0.990, and the ratio of the absolute value of the Y-axis intercept to the 100% theoretical concentration response value should not be greater than 25%;

[0094] 5.2.2 Results Summary

[0095] The solvent does not interfere with the determination. The linear correlation coefficient of zoledronic acid impurity D is 0.99999, which is greater than 0.990. The ratio of the absolute value of the Y-axis intercept to the 100% theoretical concentration response value is 1.27%, which is less than 25%, and meets the requirements. The linear correlation coefficient of zoledronic acid is 0.99743, which is greater than 0.990. The ratio of the absolute value of the Y-axis intercept to the 100% theoretical concentration response value is 10.88%, which is less than 25%, and the linear test meets the requirements.

[0096] Table 3 Linearity test results

[0097]

[0098] 5.3 Limit of quantification and detection limit

[0099] 5.3.1 Acceptability criteria

[0100] In the quantitative limit solution, the signal-to-noise ratio of each peak should be no less than 10, the RSD of the retention time of each peak should be no more than 1.0%, the RSD of the peak area ≤10%, and the quantitative limit should be less than the reporting limit (0.05%); the signal-to-noise ratio of each peak in the detection limit solution should be no less than 3.

[0101] 5.3.2 Results Summary

[0102] The blank solvent does not interfere with the determination; the signal-to-noise ratio of the sensitivity solution is 19.10, not less than 10, which meets the requirements; the signal-to-noise ratio of the detection limit solution of zoledronic acid impurity D is 3.41, not less than 3, which meets the requirements; the minimum signal-to-noise ratio of the 6 injections with the quantitative limit is 22.36, all of which are not less than 10, the RSD of the retention time of the 6 injections is 0.22%, less than 1.0%, and the RSD of the peak area is 3.43%, less than 10%, all of which meet the requirements; the signal-to-noise ratio of the detection limit solution of zoledronic acid is 6.02, not less than 3, which meets the requirements; the minimum signal-to-noise ratio of the 6 injections with the quantitative limit is 13.91, all of which are not less than 10, the RSD of the retention time of the 6 injections is 0.16%, less than 1.0%, and the RSD of the peak area is 3.35%, less than 10%, and the quantitative limit and detection limit tests meet the requirements.

[0103] Table 4 Results of limit of quantitation and limit of detection tests

[0104]

[0105] 5.4 Accuracy

[0106] 5.4.1 Acceptability criteria

[0107] Nine samples of the test solution were added for recovery, and the average recovery rates of impurity D were in the range of 80% to 120%, with RSDs of ≤10%.

[0108] 5.4.2 Results Summary

[0109] Neither blank solvent nor auxiliary material solution interferes with the determination; the signal-to-noise ratio of the sensitivity solution is 24.16, not less than 10, which meets the requirements; the average recovery rate of impurity D in 9 sample recovery test solutions is 91.9%, between 80% and 120%, and the RSD is 1.17%, less than 10%; the accuracy is good.

[0110] 5.5 Durability

[0111] 5.5.1 Acceptability criteria

[0112] The S / N of the main peak of the sensitivity solution under each condition should be no less than 10:1, and the blank auxiliary material solution should not interfere with the detection of impurity D and the main component.

[0113] Add the sample to the test solution and calculate according to the external standard method: if the detected amount is less than the reporting limit, no comparison will be made;

[0114] Limit <0.5%: change value ≤20%;

[0115] 0.5%≤Limit<5%:Change value≤10%;

[0116] Limit ≥5%: Change value ≤5%.

[0117] 5.5.2 Results Summary

[0118] Blank solvents and excipients do not interfere with the determination; under various chromatographic conditions, the minimum signal-to-noise ratio of the sensitivity solution is 14.36 and not less than 10, which meets the requirements; under various conditions such as durability wavelength change of ±2nm, flow rate change of 0.1ml / min, column temperature change of ±2℃, fine-tuning of mobile phase A reagent, and different chromatographic columns, the maximum detection amount of impurity D is 0.215%, and the maximum rate of change is 11.35%, both of which are no more than 20%, indicating good durability.

[0119] 5.6 Repeatability

[0120] 5.6.1 Acceptability criteria

[0121] 6 samples:

[0122] If the detected amount is less than the reporting limit (0.05%), it will not be compared;

[0123] Detection amount <0.1%: RSD≤30%;

[0124] 0.1%≤Detection amount<0.2%:RSD≤20%;

[0125] 0.2%≤Detection amount<0.5%:RSD≤10%;

[0126] 0.5%≤Detection amount<5%:RSD≤5.0%;

[0127] 5%≤Detection amount:RSD≤2.5%.

[0128] 5.6.2 Results Summary

[0129] Neither blank solvent nor excipient solution interferes with the determination; the repeatability of the reference substance was sampled continuously for 5 times, and the RSDs of the peak areas of zoledronic acid and zoledronic acid impurity D were 1.01% and 1.27%, respectively, both less than 2.0%, which met the requirements; the signal-to-noise ratio of the sensitivity solution was 24.91, not less than 10, which met the requirements; the average value of the detection amount of impurity D in 6 repeatability sample solutions was 0.192%, and the RSD was 1.13%, less than 20%, with good repeatability.

[0130] 5.7 Intermediate precision

[0131] 5.7.1 Acceptability criteria

[0132] 12 samples: Detection amount < reporting limit, no comparison; Detection amount < 0.1%: RSD ≤ 40%;

[0133] 5.7.2 Results Summary

[0134] Neither blank solvent nor auxiliary material solution interferes with the determination; in the intermediate precision, the reference substance was sampled continuously for 5 times, and the RSDs of the peak areas of zoledronic acid and zoledronic acid impurity D were 1.96% and 0.33%, respectively, both less than 2.0%, which met the requirements; the signal-to-noise ratio of the sensitivity solution was 26.40, not less than 10, which met the requirements; the average value of the detection amount of impurity D in 6 samples of the intermediate precision test solution was 0.194%, and the RSD was 0.39%, which was less than 20%, intermediate precision test.

[0135] Comparative Example 1 - YBH05372021 Standard Zoledronic Acid Concentrated Solution for Injection Related Substances Method Repeat

[0136] Chromatographic conditions

[0137] Chromatographic column: C18250mm×4.6mm, 5μm; detection

[0138] Wavelength: 215nm;

[0139] Column temperature: 30°C;

[0140] Injection volume: 40 μl;

[0141] Flow rate: 1.2ml / min

[0142] Mobile phase: sodium hydrogen phosphate mixed solution (take 14.38g of sodium hydrogen phosphate dodecahydrate and 4.5g of tetrahexyl ammonium hydrogen sulfate, add 900ml of water to dissolve, shake well, and obtain) - acetonitrile - disodium ethylenediaminetetraacetic acid solution (900:100:2, v / v / v) (adjust the pH value to 7.9 with 2mol / L sodium hydroxide solution)

[0143] Note: Disodium ethylenediaminetetraacetate solution: Take 0.365g of disodium ethylenediaminetetraacetate, place it in a 100ml volumetric flask, add 5ml of 2mol / L sodium hydroxide solution to dissolve it, dilute it to the scale with water, shake well, and you have it.

[0144] Determination method:

[0145] Solvent: Mobile phase

[0146] Impurity D localization solution: Take about 1 mg of each impurity D reference substance, accurately weigh, place in a 10 ml volumetric flask, add mobile phase to dilute to the scale, and shake well; accurately measure 0.5 ml, place in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, as the impurity D localization solution.

[0147] Test solution: Take 2 ml of concentrated zoledronic acid solution for injection, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0148] Results and Conclusions:

[0149] Table 5. Results of repeated-positioning tests on relevant substance conditions of YBH05372021 standard

[0150]

[0151] Conclusion: Under the chromatographic conditions of the YBH05372021 standard related substances, impurity D eluted earlier and the blank solvent interfered with the detection of impurity D.

[0152] Comparative Example 2

[0153] The main difference from Example 1 is that the flow rate is 1.2 ml / min.

[0154] Results and Conclusions:

[0155] Table 6 Optimization of relevant material conditions - Positioning test results

[0156]

[0157] Conclusion: The blank solvent elutes at the main peak position and still interferes with the main component.

[0158] Comparative Example 3

[0159] The main difference from Example 1 is that the flow rate is 0.8 ml / min.

[0160] Results and Conclusions:

[0161] Table 7 Positioning test results

[0162]

[0163] Conclusion: Under this chromatographic condition, the blank excipient interferes with the detection of impurity D.

[0164] Comparative Example 4

[0165] The main difference from Example 1 is that the mobile phase is prepared from phosphate buffer, ethanol and acetone in a volume ratio of 90:6:4. The results show that impurity D peaks earlier and the auxiliary material peak interferes with the detection of impurity D.

[0166] Comparative Example 5

[0167] The main difference from Example 1 is that the mobile phase is prepared from phosphate buffer, methanol and acetonitrile in a volume ratio of 90:6:4. The results show that the separation degree between the impurity D peak and the auxiliary material peak is less than 1.5, which interferes with the detection.

[0168] Comparative Example 6

[0169] The main difference from Example 1 is that the pH value of the phosphate buffer is 7.7. Phosphate buffer: Take 5.7 g of anhydrous disodium hydrogen phosphate and 4.5 g of tetrahexyl ammonium hydrogen sulfate, add 900 ml of water to dissolve, add 2 ml of disodium ethylenediaminetetraacetic acid solution, mix well, and adjust the pH value to 7.7 with 2 mol / L sodium hydroxide solution.

[0170] The results showed that the blank solvent eluted at the main peak position, interfering with the main component.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing related substances in a concentrated solution of zoledronic acid for injection, characterized in that: The invention adopts a high performance liquid chromatography method, wherein the chromatographic column uses octadecylsilane bonded silica gel as a filler; the mobile phase is prepared from a phosphate buffer, ethanol and acetone in a volume ratio of 88-92:4.5-5.5:4.5-5.5; the preparation method of the phosphate buffer comprises the following steps: taking anhydrous disodium hydrogen phosphate and tetrahexylammonium hydrogen sulfate, adding water to dissolve them, adding disodium ethylenediaminetetraacetate solution and mixing; the flow rate is 0.6-0.7 ml / min.

2. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The mass volume ratio of the anhydrous disodium hydrogen phosphate, tetrahexyl ammonium hydrogen sulfate and disodium ethylenediaminetetraacetate solution is 5.5-6g:4-5g:1.8-2.2ml.

3. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 2, characterized in that: The disodium ethylenediaminetetraacetic acid solution is prepared by taking disodium ethylenediaminetetraacetic acid dihydrate, adding sodium hydroxide solution to dissolve it, adding water to make up the volume, and mixing well to obtain 0.3-0.4 g of disodium ethylenediaminetetraacetic acid dihydrate per 100 ml.

4. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The pH value of the phosphate buffer is adjusted to 7.8-8.0 with sodium hydroxide solution.

5. The method for analyzing related substances of the concentrated solution of zoledronic acid for injection according to claim 3 or 4, characterized in that: The sodium hydroxide solution is 1-2 mol / L.

6. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: Injection volume: 30-40 μl.

7. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The column temperature was 23-27°C.

8. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The specifications of the chromatographic column are 250 mm × 4.6 mm, 5 μm or a chromatographic column with equivalent performance.

9. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: Test solution: Accurately measure the test sample, place it in a volumetric flask, dilute it to the scale with mobile phase, dilute it 5-10 times, shake it well, and use it as the test solution.

10. The method for analyzing related substances of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: Reference solution: take impurity D reference, accurately weigh, place in a volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain impurity D reference solution, containing 1.5-1.7 mg per 100 ml; take zoledronic acid reference, accurately weigh, place in a volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain zoledronic acid reference solution, containing 1.5-1.7 mg per 100 ml; accurately measure impurity D reference solution and zoledronic acid reference solution respectively, place in the same volumetric flask, and both impurity D reference solution and zoledronic acid reference solution are diluted 9-11 times by the mobile phase, shake well, and use as reference solution; the impurity D is imidazole-1-acetic acid.

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