A method for determining related substances in cetirizine chloride tablets

Abstract

This invention provides a method for determining related substances in cetylpyridinium chloride lozenges. The method employs high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material; mobile phase A is 0.05% trifluoroacetic acid aqueous solution, and mobile phase B is acetonitrile, using linear gradient elution. The peak areas of each impurity in the test solution and the main peak area of ​​the control solution are measured, and the content of each impurity is calculated. This method has high specificity and good repeatability, ensuring the controllability of the process and degradation impurities in cetylpyridinium chloride lozenges.

Description

Technical Field

[0001] This invention belongs to the technical field of determining drug-related substances, and particularly relates to a method for determining the related substances of cetirizine chloride tablets. Background Technology

[0002] Cetylpyridinium chloride, as a quaternary ammonium salt and hexadecylpyridinium chloride, exerts its bactericidal effect by generating enzymes that inactivate energy, denaturing essential cellular proteins and disrupting cell membranes. In vitro tests have shown that this product has inhibitory and bactericidal effects on various oral pathogenic and non-pathogenic bacteria. This product can reduce or inhibit plaque formation, thus maintaining oral hygiene and eliminating bad breath.

[0003] Cetylpyridamole lozenges have a strong bactericidal effect against common oropharyngeal pathogens and enveloped viruses. While killing bacteria, they also inhibit the release of the inflammatory factor PGE2. They are effective in treating acute and subacute pharyngitis and gingivitis, relieving dry and sore throat. Furthermore, numerous studies have indicated that cetylpyridamole can reduce the viral load of SARS-CoV-2 in saliva, helping to reduce the spread of the virus.

[0004] Currently, no standards in various countries (the United States, the United Kingdom, the European Union, Japan, China, etc.) include methods for determining related substances in cetylpyridinium chloride lozenges, and a search of domestic and international literature has also failed to find such methods. Preliminary experimental studies have revealed that the related substances in cetylpyridinium chloride lozenges have increased compared to the raw material, necessitating the development of methods for detection and control. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for determining related substances in cetylpyridinium chloride lozenges. This method has good specificity and separation, can effectively separate possible degradation impurities and process impurities from the main components, and has high sensitivity, and can be applied to the quality control of cetylpyridinium chloride lozenges.

[0006] The method for detecting related substances in the raw material standard of cetylpyridinium chloride in the Chinese Pharmacopoeia is liquid chromatography gradient elution. However, due to interference from excipients in the formulation, this method is not suitable for the detection of related substances in cetylpyridinium chloride lozenges, and the detection conditions for related substances in lozenges need to be redeveloped.

[0007] This invention provides a method for determining related substances in cetirizine chloride tablets, comprising the following steps:

[0008] The test sample was dissolved in methanol by ultrasonication, centrifuged, and the supernatant was collected to obtain the test sample solution.

[0009] The test solution and the control solution were determined by high performance liquid chromatography.

[0010] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is 0.05% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is used.

[0011] Preferably, the method further includes preparing a control solution:

[0012] The test solution was diluted with methanol to obtain a solution containing 2.5 μg of cetylpyridinium chloride per 1 mL, which was used as a control solution.

[0013] Preferably, the gradient elution includes:

[0014] 0 min, Phase A: 72.5%–77.5%, Phase B: 27.5%–22.5%;

[0015] 50 min, Phase A: 40%, Phase B: 60%;

[0016] 61 min, Phase A: 40%, Phase B: 60%;

[0017] 75 min, Phase A: 17%, Phase B: 83%;

[0018] 90 min, Phase A: 17%, Phase B: 83%;

[0019] 95 min, Phase A: 10%, Phase B: 90%;

[0020] 105 min, Phase A: 10%, Phase B: 90%;

[0021] 106 min, Phase A: 75%, Phase B: 25%;

[0022] 116 min, Phase A: 75%, Phase B: 25%.

[0023] Preferably, the flow rate of the mobile phase is 0.9 mL to 1.1 mL per minute; the column temperature is 25 to 35 °C; and the detection wavelength is 259 nm.

[0024] Preferably, the system suitability solution used in the assay method is prepared according to the following method:

[0025] Take appropriate amounts of impurity I reference standard and cetylpyridinium chloride, place them in the same volumetric flask, dissolve and dilute with methanol to prepare a mixed solution containing 2.5 μg and 0.5 mg per ml, respectively.

[0026] Preferably, the sensitivity solution used in the determination method is prepared according to the following method:

[0027] Accurately measure an appropriate amount of the control solution and dilute it quantitatively with methanol to prepare a solution containing 0.25 μg per ml.

[0028] Preferably, the determination method uses methanol as a blank solution.

[0029] Preferably, the 1-tetradecylpyridine chloride in the test sample exhibits good linearity in the range of 0.47771 μg / ml to 3.5828 μg / ml;

[0030] Cetylpyridinium chloride exhibits good linearity in the range of 0.25106 μg / ml to 3.7659 μg / ml.

[0031] This invention provides a method for determining related substances in cetylpyridinium chloride lozenges. The method employs high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material; mobile phase A is 0.05% trifluoroacetic acid aqueous solution, and mobile phase B is acetonitrile, using linear gradient elution. The peak areas of each impurity in the test solution and the main peak area of ​​the control solution are measured, and the content of each impurity is calculated. This method has high specificity and good repeatability, ensuring the controllability of the process and degradation impurities in cetylpyridinium chloride lozenges. Attached Figure Description

[0032] Figure 1 The standard curve of cetylpyridinium chloride;

[0033] Figure 2 The standard curve for impurity I;

[0034] Figure 3 The spectrum of the blank solvent used in the method of the present invention;

[0035] Figure 4 The spectrum of the blank excipient solution used in the method of the present invention;

[0036] Figure 5 A chromatogram of a solution suitable for the system using the method of the present invention;

[0037] Figure 6 The spectrum of the solution obtained by the method of the present invention;

[0038] Figure 7 The chromatogram of the reference solution obtained by the method of the present invention;

[0039] Figure 8 The chromatogram of the test solution obtained by the method of the present invention;

[0040] Figure 9 The spectrum of the test solution spiked using the method of the present invention. Detailed Implementation

[0041] This invention provides a method for determining related substances in cetirizine chloride tablets, comprising the following steps:

[0042] The test sample was dissolved in methanol by ultrasonication, centrifuged, and the supernatant was collected to obtain the test sample solution.

[0043] The test solution and the control solution were determined by high performance liquid chromatography.

[0044] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is 0.05% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is used.

[0045] The related substances in the cetylpyridinium chloride tablets of this invention include impurity I and 1-octadecylpyridine chloride; impurity I is 1-tetradecylpyridine chloride.

[0046] Cetylpyridinium chloride is readily soluble in methanol. The sucrose content in cetylpyridinium chloride tablets is about 90%, and sucrose is insoluble in methanol. Therefore, methanol was chosen as the solvent, and the sample was extracted by centrifugation and filtration.

[0047] This invention involves dissolving the test sample in methanol using ultrasound, centrifuging, and collecting the supernatant to obtain the test solution. Specifically, take 5 tablets of this product (approximately equivalent to 10 mg of cetylpyridinium chloride), grind them into a fine powder, place them in a 20 ml volumetric flask, add an appropriate amount of methanol, dissolve by ultrasound, dilute to the mark with methanol, shake well, centrifuge, collect the supernatant, filter, and collect the filtrate as the test solution. The centrifugation speed is 5000 rpm, and the centrifugation time is 4–6 min. The supernatant is filtered through a 0.22 μm organic filter membrane.

[0048] After obtaining the test solution, the present invention uses high performance liquid chromatography to determine the test solution and the control solution;

[0049] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column (Waters SunFire C18, 4.6 mm × 250 mm, 5 μm or a column with equivalent performance); mobile phase A is 0.05% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is used.

[0050] In this invention, the flow rate of the mobile phase is 0.9 mL to 1.1 mL per minute; the column temperature is 25 to 35 °C; and the detection wavelength is 259 nm.

[0051] In this invention, the gradient elution includes:

[0052] 0 min, Phase A: 72.5%–77.5%, Phase B: 27.5%–22.5%;

[0053] 50 min, Phase A: 40%, Phase B: 60%;

[0054] 61 min, Phase A: 40%, Phase B: 60%;

[0055] 75 min, Phase A: 17%, Phase B: 83%;

[0056] 90 min, Phase A: 17%, Phase B: 83%;

[0057] 95 min, Phase A: 10%, Phase B: 90%;

[0058] 105 min, Phase A: 10%, Phase B: 90%;

[0059] 106 min, Phase A: 75%, Phase B: 25%;

[0060] 116 min, Phase A: 75%, Phase B: 25%.

[0061] The determination method provided by the present invention also includes the preparation of a control solution, the preparation of impurity I reference stock solution I, the preparation of impurity I reference stock solution II, the preparation of a system suitability solution, and the preparation of a sensitivity solution.

[0062] The control solution described in this invention is prepared by the following method:

[0063] The test solution was diluted with methanol to obtain a solution containing 2.5 μg of cetylpyridinium chloride per 1 mL, which was used as a control solution.

[0064] Specifically, the control solution is a 2.5 μg / mL solution obtained by diluting the test sample solution 500 μg / mL 200 times.

[0065] Preparation of impurity I reference standard stock solution I: Weigh approximately 25 mg of impurity I reference standard accurately, place it in a 25 ml volumetric flask, dissolve and dilute with methanol to the mark, and shake well to obtain the solution.

[0066] Preparation of Impurity I Reference Stock Solution II: Accurately measure 1.0 ml of Impurity I Reference Stock Solution I and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with methanol to prepare a solution containing approximately 0.05 mg of Impurity I per ml. Shake well to obtain the solution.

[0067] System suitability solution: Weigh approximately 10 mg of cetylpyridinium chloride reference standard accurately and place it in a 20 ml volumetric flask. Add 1.0 ml of impurity I reference standard stock solution II, then dissolve and dilute with methanol to the mark to prepare a mixed solution containing 2.5 μg of impurity I and 0.5 mg of cetylpyridinium chloride per ml. Shake well to obtain the solution.

[0068] Sensitivity solution: Accurately measure 1.0 ml of the control solution, place it in a 10 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0069] Preparation of blank solution: methanol.

[0070] Final determination method: Octadecylsilane-bonded silica gel was used as the stationary phase (Waters SunFire C18, 4.6 mm × 250 mm, 5 μm or equivalent column); 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B. A linear gradient elution was performed according to the table below, with an initial ratio of mobile phase A to mobile phase B (72.5–77.5: 27.5–22.5); the flow rate was 0.9–1.1 ml / min; the detection wavelength was 259 nm; the column temperature was 25–35 °C; and the injection volume was 10 μl. The peak areas of each impurity in the test solution and the main peak area of ​​the control solution were measured, and the content of each impurity was calculated.

[0071] To further illustrate the present invention, the following detailed description of a method for determining related substances in cetylpyridinium chloride tablets provided by the present invention is provided in conjunction with embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0072] Example 1

[0073] To verify the effectiveness of the method for determining substances according to this invention, the inventors conducted numerous experiments and scientifically validated the final determined method. The validation experiments are as follows:

[0074] (1) Preparation of diluent: Methanol is used as diluent.

[0075] (2) Preparation of the test solution: Take 5 tablets of this product, grind them into a fine powder, accurately weigh (approximately equivalent to 10 mg of cetylpyridinium chloride), place them in a 20 ml volumetric flask, add an appropriate amount of methanol, sonicate to dissolve and dilute to the mark, shake well, centrifuge (5000 rpm, 5 min), take the supernatant and filter it through a 0.22 μm organic filter membrane, and take the filtrate as the test solution. The chromatogram of the test solution is shown in [reference needed]. Figure 8 .

[0076] (3) Preparation of the reference solution: Accurately measure 0.5 ml of the test solution and place it in a 100 ml volumetric flask. Dilute quantitatively to the mark with methanol and mix well. The chromatogram of the reference solution is shown in [reference image]. Figure 7 .

[0077] (4) Preparation of impurity I reference standard stock solution I: Weigh approximately 25 mg of impurity I reference standard accurately, place it in a 25 ml volumetric flask, dissolve and dilute with methanol to the mark, shake well, and the solution is ready.

[0078] (5) Preparation of impurity I reference stock solution II: Accurately measure 1.0 ml of impurity I reference stock solution I, place it in a 20 ml volumetric flask, add methanol to dissolve and dilute to the mark to prepare a solution containing about 0.05 mg of impurity I per ml, shake well, and the solution is ready.

[0079] (6) Preparation of the spiked test solution: Take 5 tablets of this product, grind them into powder, accurately weigh them, place them in a 20 ml volumetric flask, accurately add 1.0 ml of impurity I reference standard stock solution, dissolve and dilute to the mark with methanol, shake well, centrifuge (5000 rpm, 5 min), take the supernatant and filter it through a 0.22 μm organic filter membrane, the filtrate is used as the spiked test solution. The chromatogram of the 100% spiked test solution is shown in [reference needed]. Figure 9 .

[0080] (7) System Suitability Solution: Accurately weigh approximately 10 mg of cetylpyridinium chloride reference standard and place it in a 20 ml volumetric flask. Add 1.0 ml of Impurity I reference standard stock solution II, then dissolve and dilute to the mark with methanol to prepare a mixed solution containing 2.5 μg of Impurity I and 0.5 mg of cetylpyridinium chloride per ml. Shake well to obtain the solution. The chromatogram of the system suitability solution is shown below. Figure 5 .

[0081] (8) Sensitivity solution: Accurately measure 1.0 ml of the control solution and place it in a 10 ml volumetric flask. Dilute to the mark with methanol and mix well. See the chromatogram of the sensitivity solution below. Figure 6 .

[0082] (9) Preparation of blank excipient solution: Take a blank excipient solution (a mixed powder of sucrose, povidone K30, hydroxypropyl cellulose, carbomer, menthol, and magnesium stearate prepared according to the formulation) and proceed with the same procedure as the test solution. The chromatogram of the blank excipient solution is shown in [reference needed]. Figure 4 .

[0083] (9) Preparation of blank solution: methanol. The chromatogram of the blank solution is shown below. Figure 3 .

[0084] (10) Final determination method: Octadecylsilane bonded silica gel was used as the packing material (Waters SunFire C18, 4.6 mm × 250 mm, 5 μm or equivalent column); 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B. The initial ratio of mobile phase A to mobile phase B (72.5~77.5: 27.5~22.5) was used for linear gradient elution according to the table below; the flow rate was 0.9 ml to 1.1 ml per minute; the detection wavelength was 259 nm; the column temperature was 25℃~35℃; and the injection volume was 10 μl.

[0085]

[0086] System suitability test

[0087] Take the control solution, prepare five parallel samples according to the relevant substances determination method, inject them into the chromatograph, record the chromatograms, and examine the system suitability. The experimental results are shown in the table below:

[0088] Table 1 System Applicability Results

[0089] serial number 1 2 3 4 5 average RSD% Reference peak area 17.86 17.85 17.35 17.74 17.75 17.71 1.18

[0090] The system suitability test showed that the RSD% of the peak area of ​​the control main peak was less than 10.0%, which met the requirements. The test results indicate that the system suitability of this determination method is good.

[0091] Specificity test

[0092] Blank solvent, blank excipient solution, and test solution were prepared according to the above method, and the relevant substances were determined according to the method for determining cetirizine chloride tablets. The test results are shown in the table below:

[0093] Table 2 Results of specificity test

[0094]

[0095] The experimental results show that the blank solvent and blank excipients do not interfere with the determination of related substances, and the method for determining related substances has good specificity.

[0096] Linear range

[0097] Accurately weigh appropriate amounts of cetylpyridinium chloride and impurity I to prepare 50 ml of mixed reference stock solutions with concentrations of 10 μg / ml and 20 μg / ml, respectively. Dilute with methanol to prepare linear solutions of different concentrations, with impurity I and cetylpyridinium chloride at 0.5% as 100% concentration, within the range of the limit of quantitation to 150% limit. Measure the peak area according to the relevant substances determination method. Perform linear regression analysis with peak area as the abscissa (X) and concentration as the ordinate (Y). The results are shown in the table below:

[0098] Table 3. Preparation of linear solutions of cetylpyridinium chloride.

[0099] serial number Dilution method Relative to the limit concentration level Cetylpyridamole concentration (μg / ml) 1# Linear stock solution 2.5ml → 100ml 10％ 0.25 2# Linear stock solution 5.0 ml → 100 ml 20％ 0.50 3# Linear stock solution 2.5ml → 20ml 50％ 1.25 4# Linear stock solution 5.0 ml → 20 ml 100％ 2.5 5# Linear stock solution 7.5ml → 20ml 150％ 3.75

[0100] Table 4. Preparation of linear solution concentration for impurity I

[0101]

[0102]

[0103] Table 5. Results of linearity test for cetylpyridinium chloride

[0104]

[0105] Table 6 Results of linearity test for impurity I

[0106]

[0107] The standard curve of cetylpyridinium chloride is shown below. Figure 1The standard curve for impurity I is shown below. Figure 2 The experimental results showed that impurity I and cetylpyridinium chloride exhibited good linearity in the concentration ranges of 0.47771 μg / ml to 3.5828 μg / ml and 0.25106 μg / ml to 3.7659 μg / ml, respectively.

[0108] Limit of quantitation and limit of detection

[0109] Take stock solutions of reference standards (impurity I 1000 μg / ml, cetylpyridinium chloride 500 μg / ml, 1-octadecylpyridine chloride 500 μg / ml), and further dilute with methanol to prepare solutions of 0.48 μg / ml, 0.24 μg / ml, and 0.24 μg / ml. Inject these solutions to determine the limits of quantitation (LOQ) and detection (LOD). The concentration with a signal-to-noise ratio of 10:1 is the LQ, and the LQ concentrations are confirmed as impurity I 0.48 μg / ml, cetylpyridinium chloride 0.24 μg / ml, and 1-octadecylpyridine chloride 0.24 μg / ml. The concentration with a signal-to-noise ratio of 3:1 is the LOD, and the LOD concentrations are confirmed as impurity I 0.14 μg / ml, cetylpyridinium chloride 0.07 μg / ml, and 1-octadecylpyridine chloride 0.07 μg / ml. Then, prepare six solutions with LQ concentrations and determine the peak area of ​​the six solutions according to the relevant substances determination method to determine the precision of the LQ.

[0110] Table 7 Limit of Quantification and Limit of Detection

[0111]

[0112] Acceptable standards: The signal-to-noise ratio for the limit of quantitation shall not be less than 10:1, and the signal-to-noise ratio for the limit of detection shall not be less than 3:1.

[0113] Table 8 Precision of Quantitation Limit

[0114]

[0115] The results showed that the limit of quantitation (LOQ) concentrations for cetylpyridinium chloride were 0.24 μg / ml, equivalent to 10% of the limit concentration; the LQ concentration for impurity I was 0.48 μg / ml, equivalent to 19% of the limit concentration; and the LQ concentration for 1-octadecylpyridine chloride was 0.24 μg / ml, equivalent to 10% of the limit concentration. The limit of detection (LOD) concentrations for cetylpyridinium chloride were 0.07 μg / ml, equivalent to 3% of the limit concentration; the LOD concentration for impurity I was 0.14 μg / ml, equivalent to 6% of the limit concentration; and the LOD concentration for 1-octadecylpyridine chloride was 0.07 μg / ml, equivalent to 3% of the limit concentration. The RSDs of the peak areas for cetylpyridinium chloride, impurity I, and 1-octadecylpyridine chloride in the six parallel needle LOD solutions were 4.41%, 2.52%, and 8.91%, respectively, with signal-to-noise ratios greater than 10. These results indicate good precision in the LOD measurements.

[0116] Repeatability

[0117] Accurately weigh approximately 25 mg of impurity I reference standard, dissolve and dilute to the mark with methanol in a 25 ml volumetric flask, and shake well. Accurately measure 5.0 ml of this solution and place it in a 100 ml volumetric flask. Dilute to the mark with methanol solution and shake well to obtain a stock solution of impurity I reference standard (concentration 50 μg / ml). Take 5 tablets of this product, grind them into powder, accurately weigh them, place them in a 20 ml volumetric flask, accurately add 1.0 ml of impurity I reference standard stock solution, dissolve and dilute to the mark with methanol, shake well, centrifuge (5000 rpm, 5 min), and filter the supernatant through a 0.22 μm organic filter membrane. Use the filtrate as the spiked test solution. Prepare 6 parallel solutions. Accurately measure 0.5 ml of each spiked test solution and place it in a 100 ml volumetric flask. Dilute to the mark with methanol and shake well to obtain the spiked control solution.

[0118] According to the relevant substance determination method, determine the peak area of ​​each impurity in the spiked test solution and the peak area of ​​the main peak in the spiked control solution, calculate the content of each impurity, calculate the impurity content of 6 spiked test samples, and calculate RSD (n=6).

[0119] Table 9 Repeatability

[0120]

[0121] Six samples were measured in parallel. The RSDs of impurity I, other individual impurities, and total impurities were 2.15%, 5.14%, and 1.37%, respectively, all less than 10%. The experimental results show that the method for determining related substances in cetirizine tablets has good repeatability.

[0122] intermediate precision

[0123] Different operators (B) prepared the solutions at different times according to the "repeatability" test. Following the relevant substance determination method, the peak areas of each impurity in the spiked test solution and the main peak area of ​​the spiked control solution were measured. The content of each impurity was calculated, and the impurity content of the six spiked test samples was calculated, along with the RSD (n=6). Finally, combining the six repeatability data (measured by operator A), the RSD of the 12 data points was calculated.

[0124] Table 10 Intermediate Precision

[0125]

[0126] The RSDs of impurity I, other individual impurities, and total impurities in six samples, determined by different operators at different times, were 4.13%, 3.90%, and 3.57%, respectively, all less than 15%, meeting the requirements. The RSDs of impurity I, other individual impurities, and total impurities in 12 sample solutions were 7.10%, 4.64%, and 6.32%, respectively, all less than 20%. The test results indicate that this method has good intermediate precision in determining related substances in cetylpyridinium chloride tablets.

[0127] Accuracy (Recovery Rate)

[0128] Impurity I reference standard stock solution: Weigh approximately 25 mg of impurity I reference standard accurately, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, accurately measure 5.0 ml, place it in a 100 ml volumetric flask, dilute to the mark with methanol solution, shake well, and the solution is obtained (concentration is 50 μg / ml).

[0129] 50% Spiked Test Solution: Take 5 tablets of this product (equivalent to 10 mg of cetylpyridinium chloride), grind them into powder, accurately weigh them, place them in a 20 ml volumetric flask, accurately add 0.5 ml of impurity I reference stock solution, dissolve and dilute to the mark with methanol, shake well, centrifuge (5000 rpm, 5 min), take the supernatant and filter it through a 0.22 μm organic filter membrane. The filtrate is used as the 50% spiked test solution. Prepare 3 parallel solutions.

[0130] 100% Spiked Test Solution: Take 5 tablets of this product (equivalent to 10 mg of cetylpyridinium chloride), grind them into powder, accurately weigh them, place them in a 20 ml volumetric flask, accurately add 1.0 ml of impurity I reference stock solution, dissolve and dilute to the mark with methanol, shake well, centrifuge (5000 rpm, 5 min), take the supernatant and filter it through a 0.22 μm organic filter membrane. The filtrate is used as the 100% spiked test solution. Prepare 3 parallel solutions.

[0131] 150% Spiked Test Solution: Take 5 tablets of this product (equivalent to 10 mg of cetylpyridinium chloride), grind them into powder, accurately weigh them, place them in a 20 ml volumetric flask, accurately add 1.5 ml of impurity I reference stock solution, dissolve and dilute to the mark with methanol, shake well, centrifuge (5000 rpm, 5 min), take the supernatant and filter it through a 0.22 μm organic filter membrane. The filtrate is used as the 150% spiked test solution. Prepare 3 parallel solutions.

[0132] Spiked control solution: Accurately measure 0.5 ml of each spiked test solution and place them in a 100 ml volumetric flask. Dilute to the mark with methanol and shake well to prepare the spiked control solution.

[0133] Prepare three aliquots of the spiked test solution at each concentration, with impurity concentrations of 0.25%, 0.5%, and 0.75% (equivalent to 50%, 100%, and 150% of the limit concentration), respectively. Determine the peak area of ​​each impurity in the spiked test solution and the peak area of ​​the main peak in the spiked control solution according to the relevant substances determination method. Calculate the content of each impurity and the sample recovery rate.

[0134] Table 11 Accuracy Results

[0135]

[0136] The recovery rates of this method for high, medium and low concentrations were all between 80% and 115%, with an average recovery rate of 94.7% and an RSD of less than 15%, all meeting the requirements. The experimental results show that this method has high accuracy and good recovery rate in the determination of related substances in cetylpyridinium chloride tablets.

[0137] Durability test

[0138] System suitability solution, sensitivity solution, control solution, and spiked test solution were prepared, and robustness tests were conducted on column temperature, flow rate, initial mobile phase ratio, and different chromatographic columns according to the relevant substances determination method. The test results are shown in the table below:

[0139] Table 12 Durability Test Results

[0140]

[0141] Conclusion: Under all experimental conditions, the resolution between impurity I and cetylpyridinium chloride peaks in the system suitability solutions was greater than 1.5. In the sensitivity solutions, the signal-to-noise ratio of the cetylpyridinium chloride peak was greater than 10. The number of impurities in the spiked test solutions was consistent. The RSDs of impurity I, other individual impurities, and total impurities were 3.37%, 5.26%, and 3.03%, respectively, all less than 20%, meeting the requirements. The experimental results indicate that this method has good robustness in determining related substances in cetylpyridinium chloride tablets.

[0142] Three batches of cetylpyridinium chloride tablets were taken, and test solutions were prepared according to the method. The relevant substances were determined by liquid chromatography. The impurities detected were: impurity I, unknown impurity and 1-octadecylpyridine chloride; impurity I was 1-tetradecylpyridine chloride.

[0143] As shown in the above embodiments, this invention provides a method for determining related substances in cetylpyridinium chloride lozenges. This method employs high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material; 0.05% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, using linear gradient elution; the peak areas of each impurity in the test solution and the main peak area of ​​the control solution are measured, and the content of each impurity is calculated. This method has high specificity and good repeatability, ensuring the controllability of the process and degradation impurities in cetylpyridinium chloride lozenges.

[0144] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining related substances in cetirizine chloride tablets, comprising the following steps: The test sample was dissolved in methanol by ultrasonication, centrifuged, and the supernatant was collected to obtain the test sample solution. The test solution and the control solution were determined by high performance liquid chromatography. The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is 0.05% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is used.

2. The determination method according to claim 1, characterized in that, This also includes the preparation of a control solution: The test solution was diluted with methanol to obtain a solution containing 2.5 μg of cetylpyridinium chloride per 1 mL, which was used as a control solution.

3. The determination method according to claim 1, characterized in that, The gradient elution includes: 0 min, Phase A: 72.5%–77.5%, Phase B: 27.5%–22.5%; 50 min, Phase A: 40%, Phase B: 60%; 61 min, Phase A: 40%, Phase B: 60%; 75 min, Phase A: 17%, Phase B: 83%; 90 min, Phase A: 17%, Phase B: 83%; 95 min, Phase A: 10%, Phase B: 90%; 105 min, Phase A: 10%, Phase B: 90%; 106 min, Phase A: 75%, Phase B: 25%; 116 min, Phase A: 75%, Phase B: 25%.

4. The determination method according to claim 1, characterized in that, The flow rate of the mobile phase was 0.9 mL to 1.1 mL per minute; the column temperature was 25 to 35 °C; and the detection wavelength was 259 nm.

5. The determination method according to claim 1, characterized in that, The system suitability solution used in the assay method is prepared according to the following method: Take appropriate amounts of impurity I reference standard and cetylpyridinium chloride, place them in the same volumetric flask, dissolve and dilute with methanol to prepare a mixed solution containing 2.5 μg and 0.5 mg per ml, respectively.

6. The determination method according to claim 2, characterized in that, The sensitivity solution used in the assay method is prepared according to the following method: Accurately measure an appropriate amount of the control solution and dilute it quantitatively with methanol to prepare a solution containing 0.25 μg per ml.

7. The determination method according to claim 1, characterized in that, The assay method used methanol as a blank solution.

8. The determination method according to claim 1, characterized in that, The 1-tetradecylpyridine chloride in the test sample showed good linearity in the range of 0.47771 μg / ml to 3.5828 μg / ml; Cetylpyridinium chloride exhibits good linearity in the range of 0.25106 μg / ml to 3.7659 μg / ml.

Images