Olopatadine hydrochloride oral solution related substance detection method

Abstract

The invention provides a method for detecting related substances of an olopatadine hydrochloride oral solution, which comprises the following steps: preparing a solvent, an auxiliary contrast solution, a test solution and the like, and carrying out separation detection by adopting a gradient elution procedure and a special chromatographic column, and chromatographic conditions comprise that a mobile phase A is a mixed solution of a supramolecular complex-phosphate buffer solution and acetonitrile in a specific ratio; the mobile phase B is phosphate buffer solution-acetonitrile; a zirconium-doped octyl silane bonded silica gel chromatographic column with the surface zirconium oxide loading capacity of 2.8-3.5 wt% and the pore diameter of 7-9 nm is used, and gradient elution is carried out. The method can be used for detecting related substances including impurities A, B, C, E and the like, the separation degree of each impurity is remarkably improved, the separation degree of a main component and each degraded impurity meets the requirements, and the method is good in system applicability and system precision, high in specificity, good in linear relation, high in accuracy and suitable for large-scale popularization and application, and the quantitation limit and the detection limit reach the standard. The related substances in the olopatadine hydrochloride oral solution can be accurately and reliably detected.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting related substances in olopatadine hydrochloride oral solution. Background Art

[0002] Olopatadine hydrochloride oral solution is a drug commonly used to treat allergic diseases. Its main active ingredient, olopatadine hydrochloride, effectively inhibits the release of allergic mediators such as histamine, thereby alleviating allergic symptoms. Due to its ease of administration and rapid onset of action, this oral solution has gained widespread clinical favor and is widely used to alleviate the symptoms of diseases such as allergic rhinitis and chronic urticaria, achieving excellent therapeutic results for numerous patients.

[0003] In drug quality control, the detection of related substances in olopatadine hydrochloride oral solution is a key step in ensuring drug safety and efficacy. However, existing detection technologies have many shortcomings in detecting its impurities. Existing detection methods are ineffective in separating impurities. Due to the wide variety of impurities in olopatadine hydrochloride oral solution, and the fact that some impurities have similar structures to the main components, traditional analytical methods have difficulty in effectively separating these impurities. For example, when using conventional chromatographic columns and mobile phase systems, impurity peaks easily overlap with the main peak, resulting in inaccurate quantitative and qualitative analysis of impurities and the inability to accurately determine the impurity content, which in turn affects the accurate assessment of drug quality. On the other hand, existing mobile phase systems, such as single phosphate buffer or acetonitrile systems, have poor stability and are easily affected by pH fluctuations or temperature changes, resulting in retention time drift and reduced resolution. In addition, some methods lack sensitivity and are unable to accurately detect trace degradation products. In addition, existing methods do not adequately assess excipient interference. Excipient components in oral solutions may mask impurity peaks or generate false-positive signals, making it impossible to accurately distinguish olopatadine hydrochloride from its impurities, leading to deviations in test results. At the same time, most methods do not provide correction factors for key impurities and rely solely on external standard quantification, making it difficult to eliminate errors caused by matrix effects. Summary of the Invention

[0004] In view of this, the present invention proposes a method for detecting related substances in olopatadine hydrochloride oral solution to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows:

[0006] A method for detecting related substances in olopatadine hydrochloride oral solution comprises the following steps:

[0007] S1 prepares solvent, excipient control solution, test solution, control solution, sensitivity solution, and system suitability solution;

[0008] S2. injecting each solution into a high performance liquid chromatograph for detection;

[0009] S3. analyzing the test results;

[0010] The chromatographic conditions for the detection by the high performance liquid chromatography method include:

[0011] (1) Mobile phase A: a mixture of supramolecular complex-phosphate buffer and acetonitrile with a pH of 3.8-4.0 and a volume ratio of (75-85):(15-25);

[0012] (2) Mobile phase B: phosphate buffer-acetonitrile with a volume ratio of (40-50):(50-70);

[0013] (3) Chromatographic column: A zirconium-doped octylsilane bonded silica gel chromatographic column (Zr-C8 column) was used, with a surface zirconium oxide loading of 2.8-3.5 wt% and a pore size distribution of 7-9 nm;

[0014] (4) The elution procedure is gradient elution, and the gradient elution procedure is specifically as follows:

[0015]

[0016] Furthermore, the related substances of the Olopatadine Hydrochloride Oral Solution are specifically as follows:

[0017] Impurity A of formula (I) is [(E)-3-(dimethylamino)propylidene]-6-11-dihydrodibenzo[b,e]oxepin-2-acetic acid, the molecular formula of which is C 21 H 23 NO3, molecular weight 337.41;

[0018] Impurity B of formula (II) is 11-[(Z)-3-(dimethylamino)propylidene]-6,11-dihydrodibenzo[b,e]oxepin-2-acetic acid N-oxide, with the molecular formula C 21 H 23 NO4, molecular weight 353.41;

[0019] Impurity C of formula (III) is 11-[(Z)-3-(dimethylamino)propylidene]-6,11-dihydrodibenzo[b,e]oxepane-2-carbaldehyde, with the molecular formula C 20 H 21 NO2, molecular weight 307.39;

[0020] The impurity E of formula (IV) is 2-(11-oxo-6,11-dihydrodibenzo[b,e]oxepan-2-yl)acetic acid, the molecular formula of which is C 16 H 12 O4, molecular weight 268.26;

[0021]

[0022] Furthermore, the solvent is a phosphate buffer, specifically, 13-14g of potassium dihydrogen phosphate is added to 950-1050ml of water, then 0.9-1.1ml of triethylamine is added, the pH value is adjusted to 3.7-3.9 with phosphoric acid, and then mixed with acetonitrile in a volume ratio of 68:32-72:28.

[0023] Furthermore, 1.9-2.1 g of the excipient control solution was accurately weighed and placed in a 9.5-10.5 ml volumetric flask, diluted to the mark with the solvent and shaken well.

[0024] Furthermore, the test solution is prepared by accurately weighing 1.9-2.1 mg of olopatadine hydrochloride into a 9.5-10.5 ml volumetric flask, diluting to the mark with the solvent and shaking well; the control solution is prepared by accurately weighing the olopatadine hydrochloride reference substance, dissolving it in the solvent and quantitatively diluting it to prepare a solution containing 0.9-1.1 μg per 1 ml.

[0025] Furthermore, the sensitivity solution is prepared by precisely measuring 0.9-1.1 ml of the reference solution into a 9.5-10.5 ml volumetric flask, diluting to the mark with the solvent and shaking well.

[0026] Furthermore, the system suitability solution is prepared by dissolving olopatadine hydrochloride, impurity A, impurity B, impurity C, and impurity E reference substances in the solvent and quantitatively diluting the solution to prepare a mixed solution containing 0.19-0.21 mg of olopatadine hydrochloride and 0.9-1.1 μg of each impurity per ml.

[0027] Furthermore, the mobile phase A is prepared by first dissolving hydroxypropyl-β-cyclodextrin in a phosphate buffer solution with a pH of 3.8-4.0, controlling the dissolution temperature at 40±2°C, and ultrasonically degassing for 10-15 minutes before mixing with acetonitrile to obtain mobile phase A.

[0028] Furthermore, the zirconium-doped octylsilane bonded silica gel chromatography column specifically comprises the following steps:

[0029] (1) Silica gel pretreatment: 3-5 μm spherical silica gel was removed by stirring under reflux at 80-100°C for 6-8 hours with a mixture of concentrated hydrochloric acid and methanol in a volume ratio of 1:3-1:5, then washed with water until neutral and dried at 120-150°C;

[0030] (2) Preparation of zirconium source solution: dissolve zirconium oxychloride in anhydrous ethanol to make a 0.05-0.15 mol / L solution, add glacial acetic acid at a molar ratio of 1:1-3:1 to zirconium source material, and stir well;

[0031] (3) Zirconium doping treatment: pre-treated silica gel was added with zirconium source solution at a ratio of 1:5-1:10 (g / mL), stirred in an oil bath at 60-80°C under nitrogen protection for 12-24 hours, filtered, washed with alcohol, and dried in a vacuum at 60-80°C for 8-12 hours;

[0032] (4) Octylsilane bonding reaction: zirconium-doped silica gel is added with anhydrous toluene, and octyltrichlorosilane is slowly added dropwise at a ratio of 1.5:1-3:1, and refluxed at 110-130°C under nitrogen protection and stirred for 18-36 hours, cooled, filtered, and washed to obtain bonded silica gel;

[0033] (5) End-capping treatment: bonded silica gel is dispersed in anhydrous toluene, with a mass volume ratio of bonded silica gel to anhydrous toluene of 1:5-1:10 (g / mL), and trimethylsilyl chloride is added, with a molar ratio of 2:1-4:1 to the amount of silanol groups remaining on the surface of the silica gel. Stir at 80-100°C under nitrogen protection for 6-12 hours, filter, wash with alcohol water, and vacuum dry at 60-80°C for 12-24 hours;

[0034] (6) Chromatographic column filling: The dry silica gel was loaded into a 150 mm × 4.6 mm, 5 μm column tube using a homogenization method, with ultrasonic assistance, and the head was sealed after filling to obtain a zirconium-doped octylsilane bonded silica gel chromatographic column.

[0035] Furthermore, during the gradient elution process, the flow rate is 1.0±0.02 ml / min; the column temperature is 30±1° C.; the detection wavelength is 299±2 nm; and the injection volume is 28-32 μl.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) High separation efficiency: The use of a zirconium-doped octylsilane bonded silica gel column combined with a gradient elution procedure achieves baseline separation of olopatadine hydrochloride and key degradation products such as impurities A, B, C, and E, effectively solving the problem of overlapping adjacent impurity peaks and improving detection accuracy.

[0038] (2) High sensitivity and wide linear range: By optimizing the mobile phase composition, hydroxypropyl-β-cyclodextrin-modified phosphate buffer, and gradient elution conditions, the detection limit and quantification limit are reduced. This allows the detection of extremely trace impurities, effectively avoiding the potential impact of low-level impurities on drug quality and meeting the strict requirements for impurity quantification.

[0039] (3) Strong specificity: It can effectively eliminate the interference of blank solvents, excipients and complex matrices, and achieve efficient separation of main components and impurities under various degradation conditions, meeting the requirements of quality control and stability research.

[0040] The method of the present invention has high sensitivity, strong specificity, a wide linear range and high precision, significantly improving the reliability and efficiency of the detection of related substances in olopatadine hydrochloride oral solution, providing strong technical support for drug quality control, and meeting the strict requirements of pharmacopoeias and regulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the linear equation diagram of impurity A;

[0042] Figure 2 is the linear equation diagram of olopatadine hydrochloride;

[0043] Figure 3 is the linear equation diagram of impurity B;

[0044] Figure 4 is the linear equation diagram of impurity C;

[0045] Figure 5 is the linear equation diagram of impurity E. DETAILED DESCRIPTION

[0046] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0047] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0048] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0049] Example 1 - System suitability and system precision test

[0050] 1. Solution preparation

[0051] Solvent: Phosphate buffer (take 13.6g of potassium dihydrogen phosphate, add 1000ml of water, add 1ml of triethylamine, and adjust the pH to 3.8 with phosphoric acid) - acetonitrile (70:30)

[0052] Test solution: Accurately weigh the product (2 mg of Olopatadine hydrochloride) into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0053] Reference solution: Accurately measure the olopatadine hydrochloride reference substance and quantitatively dilute it with solvent to prepare a solution containing 1 μg per 1 ml.

[0054] 2. Operational Procedure: A zirconium-doped octylsilane bonded silica gel column (Zr-C8 column) was used, with a surface zirconium oxide loading of 3.0 wt% and a pore size distribution of 8 nm. 30 μl of the control solution was precisely measured and injected into the liquid chromatograph for six consecutive injections. The peak area and retention time of the olopatadine hydrochloride peak were recorded for each injection.

[0055] 3. Results and Conclusions

[0056] Table 1:

[0057]

[0058] Conclusion: After 6 consecutive injections of the control solution, the RSD of the peak area of ​​olopatadine hydrochloride was 3.3%, less than 10.0%; the RSD of the retention time was 0.1%, less than 1.0%. The system suitability and system precision were good and met the requirements of the analytical method.

[0059] Example 2 - Specificity

[0060] 1. Solution preparation:

[0061] Solvent: phosphate buffer (take 13.6 g of potassium dihydrogen phosphate, add 1000 ml of water, add 1 ml of triethylamine, and adjust the pH to 3.8 with phosphoric acid)-acetonitrile (70:30).

[0062] Excipient control solution: Accurately weigh about 2 g of blank excipient solution (excluding olopatadine hydrochloride), place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0063] Test solution: Accurately weigh an appropriate amount of this product (approximately equivalent to 2 mg of olopatadine hydrochloride), place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0064] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent to make a solution containing approximately 1 μg per 1 ml.

[0065] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0066] Reference substance stock solution: Take appropriate amount of impurity A, impurity B, impurity C, and impurity E reference substances, weigh them accurately, place them in different volumetric flasks, add appropriate amount of solvent to dissolve and quantitatively dilute to make solutions containing approximately 200μg, 200μg, 200μg, and 200μg of each in each ml.

[0067] Reference substance stock solution 1: Accurately measure 2 ml of each reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0068] Spiked test solution: Accurately weigh an appropriate amount of the product (olopatadine hydrochloride 4 mg), place it in a 20 ml volumetric flask, add 11.0 ml of the reference substance stock solution, dilute to the scale with solvent, and shake well.

[0069] Impurity A, Impurity B, Impurity C, Impurity E location solution: Accurately measure 1 ml of each reference substance stock solution and place it in a 200 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0070] System suitability solution: Take 10 mg of olopatadine hydrochloride reference substance and place it in a 50 ml volumetric flask. Add 0.25 ml each of the impurity stock solutions of Impurity A, Impurity B, Impurity C, and Impurity E. Dissolve in solvent and quantitatively dilute to make a mixed solution containing 0.2 mg of olopatadine hydrochloride and approximately 1 μg of each impurity per 1 ml.

[0071] 2. Strong degradation test solution: prepare undamaged, acid-damaged, alkaline-damaged, oxidatively-damaged, high-temperature-damaged, and light-damaged API solutions, test sample solutions, excipient control solutions, and corresponding control solutions.

[0072] Table 2:

[0073]

[0074]

[0075]

[0076] Acid-base destruction blank solution: Measure 2 ml of 1 mol / L hydrochloric acid solution, place it in a 20 ml volumetric flask, add 2 ml of 1 mol / L sodium hydroxide solution, dilute to the scale with solvent, shake well, and obtain.

[0077] Oxidative destruction blank solution: Measure 2 ml of 3% hydrogen peroxide solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0078] 3. Operation process: Accurately measure 30 μl of each of the above solutions, inject them into a liquid chromatograph (the instrument and chromatographic column are the same as those in Example 1), and record the chromatogram.

[0079] 4. Results and Conclusions

[0080] Table 3:

[0081]

[0082]

[0083] Table 4: Strong degradation test

[0084]

[0085] Table 5: Conservation of mass:

[0086] condition content% Total impurities% Mass conservation% Undamaged API solution 100.00 Not detected 100.00 Acid destruction of API solutions 100.93 0.01 100.94 Alkali destruction of API solution 100.39 Not detected 100.39 Oxidative damage to API solutions 99.34 0.2 99.54 High temperature destroys the API solution 101.82 0.02 101.84 Light damage to API solutions 99.93 0.4 100.33 Undestroyed test solution 100.00 Not detected 100.00 Acid destruction test solution 100.04 0.03 100.07 Alkali destruction test solution 100.07 0.02 100.09 Oxidative destruction of test solution 95.31 4.2 99.51 High temperature destroys the test solution 99.73 Not detected 99.73 Light destruction of test solution 99.53 1.0 100.53

[0087] Conclusion: 1) Blank solvent and excipient control solution showed no interference under all test conditions;

[0088] 2) Under each degradation condition, the blank solution and excipient control solution also showed no interference.

[0089] 3) In the sensitivity solution chromatogram, the signal-to-noise ratio of the olopatadine hydrochloride peak was 22.

[0090] 4) In the system suitability solution chromatogram, impurity A, olopatadine hydrochloride, impurity B, impurity C, and impurity E appeared in sequence. The resolutions between the olopatadine hydrochloride peak, the impurity B peak, and the impurity C peak were 9.8, 4.7, and 3.8, respectively, all meeting the requirements.

[0091] 5) In the strong degradation test, the mass was conserved under all degradation conditions, and the mass conservation range was 90%-110%.

[0092] This indicates that the analytical method has good specificity, is not interfered by other peaks, and can effectively detect related substances in olopatadine hydrochloride oral solution.

[0093] Example 3 - Limit of Quantitation Test

[0094] 1. Solution preparation: Accurately prepare standard solutions of impurity A, olopatadine hydrochloride, impurity B, impurity C, and impurity E, and gradually dilute them to different concentrations.

[0095] 2. Operation process: A zirconium-doped octylsilane bonded silica gel chromatography column (Zr-C8 column) with a surface zirconium oxide loading of 3.0 wt% and a pore size distribution of 8 nm was used. 30 μl of each of the above solutions was accurately measured and injected into a liquid chromatograph. The sample was injected continuously for 6 times and the chromatogram was recorded.

[0096] 3. Results and Conclusions

[0097] Table 6:

[0098]

[0099]

[0100] Conclusion: The signal-to-noise ratio of impurity A was 17, the RSD of the peak area was 9.9%, and the limit of quantification was 0.0953 μg / ml, equivalent to 0.05% of the test solution; the signal-to-noise ratio of impurity B was 12, the RSD of the peak area was 7.8%, and the limit of quantification was 0.1026 μg / ml, equivalent to 0.05% of the test solution; the signal-to-noise ratio of impurity B was 12, the RSD of the peak area was 8.5%, and the limit of quantification was 0.1032 μg / ml, equivalent to 0.05% of the test solution; the signal-to-noise ratio of impurity C was 12, the RSD of the peak area was 6.7%, and the limit of quantification was 0.0232 μg / ml, equivalent to 0.01% of the test solution; the signal-to-noise ratio of impurity E was 15, the RSD of the peak area was 8.2%, and the limit of quantification was 0.1045 μg / ml, equivalent to 0.05% of the test solution.

[0101] Example 4 - Detection Limit Test

[0102] 1. Solution preparation: Accurately prepare the standard solution of the substance to be measured, gradually dilute it to a certain concentration and inject it. Calculate its signal-to-noise ratio. Generally, the sample concentration with a signal-to-noise ratio (S / N) ≥ 3 is the detection limit concentration, and its ratio to the theoretical sample concentration is the detection limit (requires 3 measurements).

[0103] 2. Operation process: Use a zirconium-doped octylsilane bonded silica gel chromatography column (Zr-C8 column) with a surface zirconium oxide loading of 3.0 wt% and a pore size distribution of 8 nm. Accurately measure 30 μl of each of the above solutions and inject them into the liquid chromatograph. Continuously inject the sample three times and record the chromatogram.

[0104] 3 Results and Conclusions

[0105] Table 7:

[0106]

[0107]

[0108] Conclusion: The signal-to-noise ratio of impurity A is 7, and the detection limit concentration is 0.0477μg / ml, equivalent to 0.02% of the test solution; the signal-to-noise ratio of olopatadine hydrochloride is 5, and the detection limit concentration is 0.0513μg / ml, equivalent to 0.03% of the test solution; the signal-to-noise ratio of impurity B is 5, and the detection limit concentration is 0.0516μg / ml, equivalent to 0.03% of the test solution; the signal-to-noise ratio of impurity C is 4, and the detection limit concentration is 0.0116μg / ml, equivalent to 0.01% of the test solution; the signal-to-noise ratio of impurity E is 6, and the detection limit concentration is 0.0523μg / ml, equivalent to 0.03% of the test solution.

[0109] Example 5 - Linearity and range testing

[0110] 1. Solution preparation: Solvent: Same as Example 1.

[0111] Reference substance stock solution: same as Example 2.

[0112] Linear stock solution: Accurately measure 2 ml of each reference stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0113] 1) Quantitative limit solution.

[0114] 2) 50% linear solution: Accurately measure 0.5 ml of the linear stock solution and place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0115] 3) 80% linear solution: Accurately measure 0.8 ml of the linear stock solution and place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0116] 4) 100% linear solution: Accurately measure 1 ml of the linear stock solution and place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0117] 5) 150% linear solution: Accurately measure 1.5 ml of the linear stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0118] 6) 200% linear solution: Accurately measure 2 ml of the linear stock solution and place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0119] 2. Operation: A zirconium-doped octylsilane bonded silica gel column (Zr-C8 column) with a surface zirconium oxide loading of 3.0 wt% and a pore size distribution of 8 nm was used. Accurately measure 30 μl of each of the above solutions and inject them into a liquid chromatograph, recording the chromatogram.

[0120] 3. Results and Conclusions

[0121] Table 8:

[0122]

[0123] Table 9:

[0124]

[0125] Table 10:

[0126]

[0127] Table 11:

[0128]

[0129]

[0130] Table 12:

[0131]

[0132] Table 13:

[0133] name Linear equations Slope Correction Factor Impurity A y=18.6077x+0.4078 18.6077 0.7 Olopatadine hydrochloride y=13.1257x+0.0619 13.1257 1.0 Impurity B y=14.0379x-0.2655 14.0379 0.9 Impurity C y=66.0930x+0.0969 66.0930 0.2 Impurity E y=22.4668x+0.5637 22.4668 0.6

[0134] Conclusion: The concentration range of impurity A is 0.0953ug / ml~1.9050ug / ml, the linear equation is y=18.6077x+0.4078, and the correlation coefficient r is 0.9996; the concentration range of olopatadine hydrochloride is 0.1026ug / ml~2.0513ug / ml, the linear equation is y=13.1257x+0.0619, and the correlation coefficient r is 0.9998; the concentration range of impurity B is 0.1032ug / ml~2.0635ug / ml , the linear equation is y=14.0379x-0.2655, and the correlation coefficient r is 0.9989; the impurity C concentration range is 0.0232ug / ml~1.8562ug / ml, the linear equation is y=66.0930x+0.0969, and the correlation coefficient r is 0.9999; the impurity E concentration range is 0.1045ug / ml~2.0895ug / ml, the linear equation is y=22.4668x+0.5637, and the correlation coefficient r is 0.9992.

[0135] Example 6 - Accuracy

[0136] 1. Solution preparation:

[0137] Solvent: phosphate buffer (take 13.6 g of potassium dihydrogen phosphate, add 1000 ml of water, add 1 ml of triethylamine, and adjust the pH to 3.8 with phosphoric acid)-acetonitrile (70:30).

[0138] Excipient control solution: Accurately weigh about 4 g of blank excipient solution (excluding olopatadine hydrochloride), place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0139] Test sample stock solution: Accurately weigh an appropriate amount of this product (approximately equivalent to 80 mg of olopatadine hydrochloride), place it in a 200 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0140] Test solution: Accurately measure 10 ml of the test sample stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0141] Control solution: Accurately measure an appropriate amount of background test solution and quantitatively dilute it with solvent to make a solution containing approximately 1 μg per 1 ml.

[0142] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0143] Reference substance stock solution: Take appropriate amount of impurity A, impurity B, impurity C, and impurity E reference substances, weigh them accurately, place them in different volumetric flasks, add appropriate amount of solvent to dissolve and quantitatively dilute to make solutions containing approximately 200μg, 200μg, 200μg, and 200μg of each in each ml.

[0144] System suitability solution: Take approximately 10 mg of olopatadine hydrochloride, accurately weigh it, and place it in a 50 ml volumetric flask. Add 0.25 ml each of the reference substance stock solutions of Impurity A, Impurity B, Impurity C, and Impurity E. Dissolve it in solvent and dilute to the mark. Shake well.

[0145] Accuracy stock solution: Accurately measure 2 ml of each reference stock solution, place it in the same 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0146] 50% accuracy solution: Accurately measure 10 ml of the test sample stock solution into a 20 ml volumetric flask, add 0.5 ml of the accuracy stock solution, dilute to the mark with solvent, and shake well. Prepare three replicates.

[0147] 50% control solution: Accurately measure an appropriate amount of 50% accuracy solution and quantitatively dilute with solvent to produce a solution containing approximately 1 μg per 1 ml. Prepare 3 aliquots.

[0148] 100% accuracy solution: Accurately measure 10 ml of the test sample stock solution into a 20 ml volumetric flask, add 1 ml of the accuracy stock solution, dilute to the mark with solvent, and shake well. Prepare three replicates.

[0149] 100% control solution: Accurately measure an appropriate amount of 100% accuracy solution and quantitatively dilute with solvent to produce a solution containing approximately 1 μg per 1 ml. Prepare three aliquots.

[0150] 150% accuracy solution: Accurately measure 10 ml of the test sample stock solution into a 20 ml volumetric flask. Add 1.5 ml of each accuracy stock solution, dilute to the mark with solvent, and shake well. Prepare three replicates.

[0151] 150% control solution: Accurately measure an appropriate amount of 150% accuracy solution and quantitatively dilute with solvent to produce a solution containing approximately 1 μg per 1 ml. Prepare three aliquots.

[0152] 2. Operation process: Use a zirconium-doped octylsilane bonded silica gel chromatography column (Zr-C8 column) with a surface zirconium oxide loading of 3.0 wt% and a pore size distribution of 8 nm. Accurately measure 30 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0153] 3. Results and Conclusions

[0154] Table 14:

[0155]

[0156] Table 15:

[0157]

[0158]

[0159] Conclusion: The average recovery rate of impurity A at each concentration ranged from 93.41% to 98.89%; the average recovery rate of impurity B at each concentration ranged from 97.52% to 103.35%; the average recovery rate of impurity C at each concentration ranged from 93.49% to 96.03%; the average recovery rate of impurity E at each concentration ranged from 97.44% to 102.65%; the RSDs of the recoveries of impurity A, impurity B, impurity C, and impurity E were 5.0%, 4.4%, 2.9%, and 3.8%, respectively.

[0160] Example 7 - Solution Stability

[0161] 1. Solution Preparation

[0162] Solvent: phosphate buffer (take 13.6 g of potassium dihydrogen phosphate, add 1000 ml of water, add 1 ml of triethylamine, and adjust the pH to 3.8 with phosphoric acid)-acetonitrile (70:30).

[0163] Reference substance stock solution: Take appropriate amount of impurity A, impurity B, impurity C, and impurity E reference substances, weigh them accurately, place them in different volumetric flasks, add appropriate amount of solvent to dissolve and quantitatively dilute to make solutions containing approximately 200μg, 200μg, 200μg, and 200μg of each in each ml.

[0164] Reference substance stock solution 1: Accurately measure 2 ml of each reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0165] Spiked test solution: Accurately weigh an appropriate amount of the product (olopatadine hydrochloride 4 mg), place it in a 20 ml volumetric flask, add 1.0 ml of the reference stock solution, dilute to the scale with solvent, and shake well.

[0166] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent to make a solution containing approximately 1 μg per 1 ml.

[0167] 2. Operation process

[0168] The control solution and the spiked test solution were placed at room temperature. 30 μl of each solution was accurately measured at 0 h and at different time points, injected into the liquid chromatograph, and the chromatogram was recorded.

[0169] 3. Results and Conclusions

[0170] Table 15:

[0171]

[0172] Table 16:

[0173]

[0174] Conclusion: Under room temperature conditions, the reference solution is stable within 86.5 hours; the test solution is stable within 86.5 hours; no new degradation impurities are produced in both the reference solution and the test solution.

[0175] Example 8 - Filter membrane adsorption

[0176] 1. Solution Preparation

[0177] Reference substance stock solution: Take appropriate amount of impurity A, impurity B, impurity C, and impurity E reference substances, weigh them accurately, place them in different volumetric flasks, add appropriate amount of solvent to dissolve and quantitatively dilute to make solutions containing approximately 200μg, 200μg, 200μg, and 200μg of each in each ml.

[0178] Reference substance stock solution 1: Accurately measure 2 ml of each reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0179] Spiked test solution: Accurately weigh an appropriate amount of the product (approximately equivalent to 4 mg of olopatadine hydrochloride) and place it in a 20 ml volumetric flask. Add 1.0 ml of the reference stock solution 1, dilute to the mark with solvent, shake well, and use this as the unfiltered solution. Filter and discard the initial filtrate (0, 2, 4, 6 ml), and take the subsequent filtrate.

[0180] 2. Operation process: Use a zirconium-doped octylsilane bonded silica gel chromatography column (Zr-C8 column) with a surface zirconium oxide loading of 3.0 wt% and a pore size distribution of 8 nm. Accurately measure 30 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0181] 3. Results and Conclusions

[0182] Table 17:

[0183]

[0184] Conclusion: PTFE filter membrane has no adsorption.

[0185] 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 scope of protection of the present invention.

Claims

1. A method for detecting related substances in olopatadine hydrochloride oral solution, characterized in that: The following steps are involved: S1 prepares solvent, excipient control solution, test solution, control solution, sensitivity solution, and system suitability solution; S2. injecting each solution into a high performance liquid chromatograph for detection; S3. Analyze the test results; The chromatographic conditions for the detection by the high performance liquid chromatography method include: (1) Mobile phase A: a mixture of supramolecular complex-phosphate buffer and acetonitrile with a pH of 3.8-4.0 and a volume ratio of (75-85):(15-25); (2) Mobile phase B: phosphate buffer-acetonitrile with a volume ratio of (40-50):(50-70); (3) Chromatographic column: A zirconium-doped octylsilane bonded silica gel chromatographic column with a surface zirconium oxide loading of 2.8-3.5 wt% and a pore size distribution of 7-9 nm; (4) The elution procedure is gradient elution, and the gradient elution procedure is specifically as follows:

2. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: The related substances of the olopatadine hydrochloride oral solution are specifically as follows: Impurity A of formula (I) is [(E)-3-(dimethylamino)propylidene]-6-11-dihydrodibenzo[b,e]oxepin-2-acetic acid, with the molecular formula C 21 H 23 NO3, molecular weight 337.41; Impurity B of formula (II) is 11-[(Z)-3-(dimethylamino)propylidene]-6,11-dihydrodibenzo[b,e]oxepin-2-acetic acid N-oxide, with the molecular formula C 21 H 23 NO4, molecular weight 353.41; Impurity C of formula (III) is 11-[(Z)-3-(dimethylamino)propylidene]-6,11-dihydrodibenzo[b,e]oxepane-2-carbaldehyde, with the molecular formula C 20 H 21 NO2, molecular weight 307.39; The impurity E of formula (IV) is 2-(11-oxo-6,11-dihydrodibenzo[b,e]oxepan-2-yl)acetic acid, the molecular formula of which is C 16 H 12 O4, molecular weight 268.26; 3. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: The solvent is a phosphate buffer solution, specifically, 13-14 g of potassium dihydrogen phosphate is added to 950-1050 ml of water, 0.9-1.1 ml of triethylamine is added, the pH value is adjusted to 3.7-3.9 with phosphoric acid, and the solution is mixed with acetonitrile in a volume ratio of 68:32-72:

28.

4. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: For the excipient control solution, accurately weigh 1.9-2.1 g of blank excipient and place it in a 9.5-10.5 ml volumetric flask, dilute to the mark with the solvent and shake well.

5. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: The test solution is prepared by accurately weighing 1.9-2.1 mg of olopatadine hydrochloride into a 9.5-10.5 ml volumetric flask, diluting to the mark with the solvent and shaking well; the control solution is prepared by accurately weighing the olopatadine hydrochloride reference substance, dissolving it in the solvent and quantitatively diluting it to prepare a solution containing 0.9-1.1 μg per 1 ml.

6. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: The sensitivity solution is prepared by precisely measuring 0.9-1.1 ml of the reference solution and placing it in a 9.5-10.5 ml volumetric flask, diluting it to the mark with the solvent and shaking it well.

7. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, characterized in that: The system suitability solution is prepared by dissolving olopatadine hydrochloride, impurity A, impurity B, impurity C, and impurity E reference substances in the solvent and quantitatively diluting the solution to prepare a mixed solution containing 0.19-0.21 mg of olopatadine hydrochloride and 0.9-1.1 μg of each impurity per ml.

8. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: The mobile phase A is prepared by first dissolving hydroxypropyl-β-cyclodextrin in a phosphate buffer solution with a pH of 3.8-4.0, controlling the dissolution temperature at 40±2° C., and ultrasonically degassing for 10-15 minutes before mixing with acetonitrile to obtain the mobile phase A.

9. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: The specific steps of the zirconium-doped octylsilane bonded silica gel chromatographic column are as follows: (1) Silica gel pretreatment: 3-5 μm spherical silica gel was removed by stirring under reflux at 80-100°C for 6-8 hours with a mixture of concentrated hydrochloric acid and methanol in a volume ratio of 1:3-1:5, then washed with water until neutral and dried at 120-150°C; (2) Preparation of zirconium source solution: dissolve zirconium oxychloride in anhydrous ethanol to make a 0.05-0.15 mol / L solution, add glacial acetic acid at a molar ratio of 1:1-3:1 to zirconium source material, and stir well; (3) Zirconium doping treatment: pre-treated silica gel was added with zirconium source solution at a ratio of 1:5-1:10 (g / mL), stirred in an oil bath at 60-80°C under nitrogen protection for 12-24 hours, filtered, washed with alcohol, and dried in a vacuum at 60-80°C for 8-12 hours; (4) Octylsilane bonding reaction: zirconium-doped silica gel is added with anhydrous toluene, and octyltrichlorosilane is slowly added dropwise at a ratio of 1.5:1-3:1, and refluxed at 110-130°C under nitrogen protection and stirred for 18-36 hours, cooled, filtered, and washed to obtain bonded silica gel; (5) End-capping treatment: bonded silica gel is dispersed in anhydrous toluene, with a mass volume ratio of bonded silica gel to anhydrous toluene of 1:5-1:10 (g / mL), and trimethylsilyl chloride is added, with a molar ratio of 2:1-4:1 to the amount of silanol groups remaining on the surface of the silica gel. Stir at 80-100°C under nitrogen protection for 6-12 hours, filter, wash with alcohol water, and vacuum dry at 60-80°C for 12-24 hours; (6) Chromatographic column filling: The dry silica gel was loaded into a 150 mm × 4.6 mm, 5 μm column tube using a homogenization method, with ultrasonic assistance, and the head was sealed after filling to obtain a zirconium-doped octylsilane bonded silica gel chromatographic column.

10. The method for detecting related substances in olopatadine hydrochloride oral solution according to claim 1, wherein: During the gradient elution process, the flow rate was 1.0±0.02 ml / min; the column temperature was 30±1° C.; the detection wavelength was 299±2 nm; and the injection volume was 28-32 μl.

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