Method for detecting related substances in cetylpyridinium chloride lozenge

Abstract

The invention belongs to the technical field of medical analysis, and particularly relates to a method for detecting related substances in cetylpyridinium chloride buccal tablets. The method for detecting related substances in cetylpyridinium chloride lozenges fills the blank of methods for detecting related impurities in cetylpyridinium chloride lozenges; the method is high in repeatability, good in linear relation, high in precision and stable and reliable in result, quality control over the cetylpyridinium chloride buccal tablets is achieved, and an effective guarantee is provided for the quality of the cetylpyridinium chloride buccal tablets.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis, and in particular relates to a method for detecting related substances of cetylpyridinium chloride lozenges. Background Art

[0002] Cetylpyridinium chloride is a cationic quaternary ammonium compound. As a surfactant, it inhibits and kills bacteria mainly by reducing surface tension. Its structure is shown below:

[0003]

[0004] Cetylpyridinium chloride lozenges are oral local antibacterial agents that can be used as an adjuvant treatment for oral infectious diseases, such as acute and subacute pharyngitis and gingivitis. They are used to slowly dissolve in the mouth and gradually dissolve in the mouth. They cannot be chewed orally. There are many commercially available gargles, and they are included in the 2020 edition of the Chinese Pharmacopoeia, but there are no related substance inspection items in the inspection items. Considering that lozenges enter the body and have higher safety requirements than gargles, cetylpyridinium chloride lozenges are inspected for related substances.

[0005] The synthetic route of cetylpyridinium chloride is simple. It is only produced by the reaction of pyridine and hexadecane chloride. The degradation impurities are only pyridine and hexadecane chloride. These two substances are checked by gas chromatography-mass spectrometry, and other impurities are checked by high performance liquid chromatography. Cetylpyridinium chloride tablets are large in size and small in size, and the amount of auxiliary materials is large, so the quality of the auxiliary materials is greatly affected. The difficulty in detecting related substances also lies in eliminating the interference of the auxiliary material peak. After research, it was found that 5-hydroxymethylfurfural introduced by sucrose is the largest impurity, and the process impurities 1-chlorotetradecylpyridine and 1-chloroheptadecylpyridine were studied as known impurities. Impurity information is shown in Table 1.

[0006] Table 1 Impurities List of Cetylpyridinium Chloride Tablets

[0007]

[0008] Cetylpyridinium chloride raw materials have been included in the 2020 edition of the Chinese Pharmacopoeia and USP43. There are no detection methods for related substances in cetylpyridinium chloride lozenges in existing pharmacopoeias and literature. In order to ensure the quality and safety of drugs, it is very necessary to establish a detection method for related substances in cetylpyridinium chloride lozenges and control process impurities and degradation impurities. Summary of the invention

[0009] The invention aims to provide a method for detecting related substances of cetylpyridinium chloride lozenges, which realizes effective separation of impurities of cetylpyridinium chloride lozenges, has simple operation, high precision and good durability.

[0010] The present invention is specifically implemented through the following technical solutions:

[0011] A method for detecting related substances in cetylpyridinium chloride lozenges uses a reverse phase chromatographic column with silane bonded silica gel as filler, trifluoroacetic acid aqueous solution as mobile phase A, and acetonitrile solution containing trifluoroacetic acid as mobile phase B for gradient elution.

[0012] Preferably, the volume proportion of trifluoroacetic acid in the mobile phase A is 0.1% to 0.2%.

[0013] Further preferably, the volume proportion of trifluoroacetic acid in the mobile phase A is 0.12% to 0.13%.

[0014] The volume proportion of trifluoroacetic acid in the mobile phase B is 0.05-0.15%.

[0015] Further preferably, the volume proportion of trifluoroacetic acid in the mobile phase B is 0.1%.

[0016] Preferably, a reverse phase chromatography column is used which uses silane bonded silica gel with polar amide groups embedded in long alkyl chains as filler.

[0017] Further preferably, the chromatographic column is Agilent ZORBAX Bonus-RP.

[0018] Preferably, the elution gradient is:

[0019]

[0020] Further preferably, the elution gradient is:

[0021]

[0022] Preferably, the detection wavelength is 258 nm.

[0023] Preferably, the flow rate during gradient elution is 0.9-1.1 mL / min.

[0024] Preferably, the column temperature during gradient elution is 25-35°C.

[0025] Preferably, the injection volume is 20-30 μl.

[0026] The following content further elaborates on the detection method of related substances in cetylpyridinium chloride lozenges.

[0027] A method for detecting related substances in cetylpyridinium chloride lozenges, specifically comprising the following steps:

[0028] Take cetylpyridinium chloride tablets, grind them into powder, accurately weigh an appropriate amount of fine powder, and use diluent (V 流动相A / V 流动相B =60:40) to prepare a sample solution;

[0029] Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine, and 1-chloroheptadecylpyridine reference substances, add diluent to dissolve and dilute to prepare reference substance solutions.

[0030] Accurately measure the sample solution and inject it into the liquid chromatograph to complete the analysis and detection of related substances in cetylpyridinium chloride tablets;

[0031] in:

[0032] High performance liquid chromatograph: Waters Acquity Arc;

[0033] Chromatographic column: Reversed phase chromatography column (4.6×250mm, 5μm) filled with silane-bonded silica gel with polar amide groups embedded in long alkyl chains;

[0034] Detector: UV detector;

[0035] Detection wavelength: 258nm;

[0036] Column temperature: 25-35°C;

[0037] Flow rate: 0.9~1.1mL / min;

[0038] Mobile phase: Aqueous solution containing 0.1% to 0.2% trifluoroacetic acid is used as mobile phase A, and acetonitrile solution containing 0.05% to 0.15% trifluoroacetic acid is used as mobile phase B;

[0039] The elution gradient is:

[0040]

[0041] In a preferred embodiment, the HPLC detection conditions are as follows:

[0042] High performance liquid chromatograph: Waters Acquity Arc;

[0043] Chromatographic column: Agilent ZORBAX Bonus-RP (4.6×250 mm, 5 μm);

[0044] Detector: UV detector;

[0045] Detection wavelength: 258nm;

[0046] Column temperature: 30°C;

[0047] Flow rate: 1.0 mL / min;

[0048] Mobile phase: Aqueous solution containing 0.12% trifluoroacetic acid was used as mobile phase A, and acetonitrile solution containing 0.10% trifluoroacetic acid was used as mobile phase B;

[0049] The elution gradient is:

[0050]

[0051]

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The invention provides a method for detecting related substances of cetylpyridinium chloride lozenges, which fills the blank of the method for determining related impurities of cetylpyridinium chloride lozenges; the method has strong repeatability, good linear relationship, high precision, stable and reliable results, realizes quality control of cetylpyridinium chloride lozenges, and provides effective guarantee for the quality thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : Embodiment 1 blank solvent HPLC spectrum;

[0055] Figure 2 : Example 1 blank auxiliary material HPLC spectrum;

[0056] Figure 3 : Example 1 System Suitability HPLC Spectrum;

[0057] Figure 4 : HPLC spectrum of the test solution of Example 1;

[0058] Figure 5 : HPLC spectrum of Example 1 reference substance solution;

[0059] Figure 6 : Example 2 System Suitability HPLC Spectrum;

[0060] Figure 7 : Example 3 System Suitability HPLC Spectrum;

[0061] Figure 8 : Example 5 System Suitability HPLC Spectrum;

[0062] Fig. 9 : Comparative Example 1 system suitability HPLC spectrum. DETAILED DESCRIPTION

[0063] The present invention is further illustrated by the following examples. It should be correctly understood that the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Therefore, simple improvements to the present invention based on the method of the present invention all fall within the scope of protection claimed by the present invention.

[0064] Cetylpyridinium chloride lozenges were provided by Shandong Xinshidai Pharmaceutical Co., Ltd.

[0065] Example 1

[0066] (1) Solution preparation

[0067] Diluent: V 流动相A / V 流动相B = 60:40;

[0068] Test solution: Take 20 cefpodoxime proxetil buccal tablets, grind them finely, accurately weigh the fine powder equivalent to 4 mg of cefpodoxime proxetil, add 12 ml of diluent, shake and sonicate for 10 minutes, filter, and take the subsequent filtrate;

[0069] Reference solution: Accurately weigh reference substances of 5 - hydroxymethylfurfural, 1 - chlorotetradecylpyridine, and 1 - chlorooctadecylpyridine, dissolve and dilute with diluent to prepare a mixed solution containing 2 μg of each per 1 ml;

[0070] System suitability solution: Accurately weigh the fine powder of this product equivalent to 4 mg of cefpodoxime proxetil, appropriate amounts of reference substances of 5 - hydroxymethylfurfural, 1 - chlorotetradecylpyridine, and 1 - chlorooctadecylpyridine, place them in the same volumetric flask, add 12 ml of diluent, shake and sonicate for 10 minutes to dissolve and dilute to prepare a solution containing approximately 0.3 mg of cefpodoxime proxetil, approximately 20 μg of 5 - hydroxymethylfurfural, 1 - chlorotetradecylpyridine, and 1 - chlorooctadecylpyridine per 1 ml, filter, and take the subsequent filtrate;

[0071] Blank excipient solution: Take blank excipients (equivalent to the excipient dosage corresponding to 4 mg of cefpodoxime proxetil), add 12 ml of diluent, shake and sonicate for 10 minutes, filter, and take the subsequent filtrate.

[0072] (2) Chromatographic conditions:

[0073] Brand and model of the high - performance liquid chromatograph: Waters Acquity Arc;

[0074] Detector: Ultraviolet detector;

[0075] Chromatographic column: Agilent ZORBAX Bonus - RP, 4.6×250 mm, 5 μm;

[0076] Mobile phase A: 0.12% trifluoroacetic acid aqueous solution, Mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0077] Elution gradient:

[0078]

[0079] Flow rate: 1.0 ml / min;

[0080] Detection wavelength: 258 nm;

[0081] Column temperature: 30 °C;

[0082] Injection volume: 30 μl.

[0083] Figure 1 , Figure 2 They are the HPLC spectra of blank solvent and blank excipient, respectively. Neither solvent nor excipient interferes with the detection of related substances; Figure 3 System suitability HPLC spectrum, 3.594min for 5-hydroxymethylfurfural peak, 20.130min for 1-chlorotetradecylpyridine peak, 22.074min for cetylpyridinium chloride peak, 23.129min for 1-chloroheptadecylpyridine peak; Figure 3 It can be seen that cetylpyridinium chloride and each impurity have achieved baseline separation, which meets the requirements of the Chinese Pharmacopoeia; Figure 4 , Figure 5 They are respectively the test solution and the reference solution, which can detect the impurities in the sample and separate them.

[0084] Example 2

[0085] (1) Solution preparation

[0086] Same as Example 1.

[0087] (2) Chromatographic conditions:

[0088] Brand and model of HPLC: Waters Acquity Arc;

[0089] Detector: UV detector;

[0090] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0091] Mobile phase A: 0.13% trifluoroacetic acid aqueous solution, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0092] Elution gradient:

[0093]

[0094] Flow rate: 0.9ml / min;

[0095] Detection wavelength: 258nm;

[0096] Column temperature: 25°C;

[0097] Injection volume: 30 μl.

[0098] Figure 6In the system suitability HPLC spectrum, 3.901min is the 5-hydroxymethylfurfural peak, 20.129min is the 1-chlorotetradecylpyridine peak, 22.044min is the cetylpyridinium chloride peak, and 23.087min is the 1-chloroheptadecylpyridine peak; cetylpyridinium chloride and each impurity are baseline separated, which meets the requirements of the Chinese Pharmacopoeia.

[0099] Example 3

[0100] (1) Solution preparation

[0101] Same as Example 1.

[0102] (2) Chromatographic conditions:

[0103] Brand and model of HPLC: Waters Acquity Arc;

[0104] Detector: UV detector;

[0105] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0106] Mobile phase A: 0.12% trifluoroacetic acid aqueous solution, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0107] Elution gradient:

[0108]

[0109] Flow rate: 1.1 ml / min;

[0110] Detection wavelength: 258nm;

[0111] Column temperature: 35°C;

[0112] Injection volume: 30 μl.

[0113] Figure 7 In the system suitability HPLC spectrum, 3.180min is the 5-hydroxymethylfurfural peak, 19.013min is the 1-chlorotetradecylpyridine peak, 20.890min is the cetylpyridinium chloride peak, and 21.926min is the 1-chloroheptadecylpyridine peak; cetylpyridinium chloride and each impurity are baseline separated, which meets the requirements of the Chinese Pharmacopoeia.

[0114] Example 4

[0115] (1) Solution preparation

[0116] Same as Example 1.

[0117] (2) Chromatographic conditions:

[0118] Brand and model of HPLC: Waters Acquity Arc;

[0119] Detector: UV detector;

[0120] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0121] Mobile phase A: 0.10% trifluoroacetic acid aqueous solution, mobile phase B: 0.12% trifluoroacetic acid acetonitrile solution;

[0122] Elution gradient:

[0123]

[0124] Flow rate: 1.0ml / min;

[0125] Detection wavelength: 258nm;

[0126] Column temperature: 30°C;

[0127] Injection volume: 30 μl.

[0128] Under the above chromatographic conditions, the main peak of cetylpyridinium chloride and the peaks of impurities and excipients were baseline separated, which met the requirements of the Chinese Pharmacopoeia.

[0129] Example 5

[0130] (1) Solution preparation

[0131] Same as Example 1.

[0132] (2) Chromatographic conditions:

[0133] Brand and model of HPLC: Waters Acquity Arc;

[0134] Detector: UV detector;

[0135] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0136] Mobile phase A: 0.12% trifluoroacetic acid aqueous solution, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0137] Elution gradient:

[0138]

[0139]

[0140] Flow rate: 1.0ml / min;

[0141] Detection wavelength: 258nm;

[0142] Column temperature: 30°C;

[0143] Injection volume: 20 μl.

[0144] Under the above chromatographic conditions, the impurity peak of 5-hydroxymethylfurfural at about 3.0 min was poorly separated from the adjacent peaks, the peak of cetylpyridinium chloride was at 20.128 min, the peak of 1-chloroheptadecanylpyridine was at 21.643 min, and 1-chlorotetradecylpyridine did not appear, which did not meet the requirements of the Chinese Pharmacopoeia.

[0145] Example 6

[0146] (1) Solution preparation

[0147] Same as Example 1.

[0148] (2) Chromatographic conditions:

[0149] Brand and model of HPLC: Waters Acquity Arc;

[0150] Detector: UV detector;

[0151] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0152] Mobile phase A: 0.12% trifluoroacetic acid aqueous solution, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0153] Elution gradient:

[0154]

[0155] Flow rate: 1.0ml / min;

[0156] Detection wavelength: 258nm;

[0157] Column temperature: 30°C;

[0158] Injection volume: 20 μl.

[0159] Under the above chromatographic conditions, the separation degree between the impurity peak and the adjacent peak at 3.3 min is 1.2, and the separation effect is poor, which does not meet the requirements of the Chinese Pharmacopoeia.

[0160] Example 7

[0161] (1) Solution preparation

[0162] Same as Example 1.

[0163] (2) Chromatographic conditions:

[0164] Brand and model of HPLC: Waters Acquity Arc;

[0165] Detector: UV detector;

[0166] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0167] Mobile phase A: 0.12% trifluoroacetic acid solution, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0168] Elution gradient:

[0169]

[0170] Flow rate: 1.0ml / min;

[0171] Detection wavelength: 258nm;

[0172] Column temperature: 30°C;

[0173] Injection volume: 20 μl.

[0174] Under the above chromatographic conditions, the separation degree between the main peak of cetylpyridinium chloride and the excipient peak was 1.3, and the separation effect was poor, which did not meet the requirements of the Chinese Pharmacopoeia.

[0175] Example 8

[0176] (1) Solution preparation

[0177] Same as Example 1.

[0178] (2) Chromatographic conditions:

[0179] Brand and model of HPLC: Waters Acquity Arc;

[0180] Detector: UV detector;

[0181] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0182] Mobile phase A: 0.12% trifluoroacetic acid solution, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0183] Elution gradient:

[0184]

[0185] Flow rate: 1.0ml / min;

[0186] Detection wavelength: 258nm;

[0187] Column temperature: 30°C;

[0188] Injection volume: 20 μl;

[0189] The separation degree between the degradation impurity peak and the adjacent peak at 3.6min is 2.6; the retention time of the main peak is 24.841min, and the peak emerges later.

[0190] Example 9

[0191] (1) Solution preparation

[0192] Same as Example 1.

[0193] (2) Chromatographic conditions:

[0194] Brand and model of HPLC: Waters Acquity Arc;

[0195] Detector: UV detector;

[0196] Column: Agilent ZORBAX Bonus-RP, 4.6×250 mm, 5 μm;

[0197] Mobile phase A: 0.06% trifluoroacetic acid solution, mobile phase B: 0.04% trifluoroacetic acid acetonitrile solution;

[0198] Elution gradient:

[0199]

[0200] Flow rate: 1.0ml / min;

[0201] Detection wavelength: 258nm;

[0202] Column temperature: 30°C;

[0203] Injection volume: 30 μl.

[0204] Under the above chromatographic conditions, the separation between the main peak of cetylpyridinium chloride and the adjacent impurity peaks was poor, and baseline separation was not achieved, which did not meet the requirements.

[0205] Comparative Example 1

[0206] (1) Solution preparation

[0207] Same as Example 1.

[0208] (2) Chromatographic conditions:

[0209] Brand and model of HPLC: Waters Acquity Arc;

[0210] Detector: UV detector;

[0211] Chromatographic column: Agilent SB-CN, 4.6×250 mm, 5 μm;

[0212] Mobile phase A: methanol, mobile phase B: 0.02 mol / L tetramethylammonium hydroxide solution-0.003 mol / L potassium dihydrogen phosphate solution (10:3, pH adjusted to 3.5 with glacial acetic acid);

[0213] Elution gradient:

[0214]

[0215] Flow rate: 1.0ml / min;

[0216] Detection wavelength: 259nm;

[0217] Column temperature: 30°C;

[0218] Injection volume: 50 μl;

[0219] Fig. 9 The peak of cetylpyridinium chloride is at 13.365min, and the theoretical plate number is 2065 based on the cetylpyridinium chloride peak. Cetylpyridinium chloride and the previous impurities have not achieved baseline separation, which does not meet the requirements.

[0220] Comparative Example 2

[0221] (1) Solution preparation

[0222] Same as Example 1.

[0223] (2) Chromatographic conditions:

[0224] Brand and model of HPLC: Waters Acquity Arc;

[0225] Detector: UV detector;

[0226] Column: Angilent ZORBAX Bonus-RP, 4.6×250mm 5μm;

[0227] Mobile phase: 0.1% trifluoroacetic acid aqueous solution: 0.1% trifluoroacetic acid acetonitrile solution = 62.5:37.5;

[0228] Elution procedure: isocratic elution;

[0229] Detection wavelength: 258nm;

[0230] Flow rate: 1.0 mL / min;

[0231] Column temperature: 40°C;

[0232] Injection volume: 10 μl;

[0233] Under the above chromatographic conditions, the separation degree between the main peak of cetylpyridinium chloride and the adjacent impurity peaks was poor, and baseline separation was not achieved. Other auxiliary materials and impurity peaks could not be effectively separated either, which did not meet the requirements.

[0234] Comparative Example 3

[0235] (1) Solution preparation

[0236] Same as Example 1.

[0237] (2) Chromatographic conditions:

[0238] Brand and model of HPLC: Waters Acquity Arc;

[0239] Detector: UV detector;

[0240] Chromatographic column: L78, 4.6×250mm, 5μm;

[0241] Mobile phase: trifluoroacetic acid-water (1:999): acetonitrile-trifluoroacetic acid (999:1) = 62.5:37.5;

[0242] Flow rate: 0.6ml / min;

[0243] Detection wavelength: 258nm;

[0244] Column temperature: 40°C;

[0245] Injection volume: 2 μl;

[0246] Under the above chromatographic conditions, the auxiliary materials and impurity peaks could not be effectively separated, and baseline separation was not achieved, which did not meet the requirements.

[0247] Linearity and range tests

[0248] Reference substance stock solution: accurately weigh 2.5 mg of each of 5-hydroxymethylfurfural, 1-tetradecylpyridine chloride, 1-heptadecylpyridine chloride, and cetylpyridinium chloride reference substances, place them in 10 ml volumetric flasks, add diluent to dissolve by ultrasonication and dilute to the scale, shake well, and use as reference substance stock solution;

[0249] Linear stock solution: Accurately measure 2 ml of the reference stock solution, place it in a 25 ml volumetric flask, add diluent to dilute to the scale, shake well, and use it as the linear stock solution;

[0250] Linear solution: accurately measure 0.05ml, 0.2ml, 0.5ml, 1.0ml, 1.5ml, and 2ml of the linear stock solution respectively, place in a 10ml volumetric flask, add diluent to dilute to the scale, shake well, and use them as linear 2, 3, 4, 5, 6, and 7 solutions; accurately measure 5ml of linear solution 2, place in a 10ml volumetric flask, add diluent to dilute to the scale, shake well, and use them as linear 1 solution.

[0251] Accurately measure 30 μl of each, inject into the liquid chromatograph, measure according to the chromatographic conditions of Example 1, record the chromatogram, and perform linear regression on the concentration C (μg / ml) with the peak area A. The results are shown in Tables 2 to 5.

[0252] Table 2 1-Chlorotetradecylpyridine linearity and range test results

[0253]

[0254] The intercept of the regression equation of 1-chlorotetradecylpyridine is 0.33% of the 100% concentration response value, indicating that 1-chlorotetradecylpyridine has a good linear relationship in the concentration range of 0.1002 μg / ml (approximately 0.03% of the test solution concentration) to 4.0088 μg / ml (approximately 1.2% of the test solution concentration).

[0255] Table 3 1-Chloroheptadecylpyridine linearity and range test results

[0256]

[0257] The intercept of the regression equation of 1-chloroheptadecylpyridine is 1.19% of the 100% concentration response value, indicating that 1-chloroheptadecylpyridine has a good linear relationship in the concentration range of 0.1000 μg / ml (approximately 0.03% of the test solution concentration) to 3.9989 μg / ml (approximately 1.2% of the test solution concentration).

[0258] Table 4 5-Hydroxymethylfurfural linearity and range test results

[0259]

[0260] The intercept of the regression equation for 5-hydroxymethylfurfural is 0.38% of the 100% concentration response value, indicating that 5-hydroxymethylfurfural has a good linear relationship in the concentration range of 0.0503 μg / ml (approximately 0.015% of the test solution concentration) to 4.0259 μg / ml (approximately 1.2% of the test solution concentration).

[0261] Table 5 Cetylpyridinium chloride linearity and range test results

[0262]

[0263] The intercept of the regression equation of cetylpyridinium chloride is 0.93% of the 100% concentration response value, indicating that cetylpyridinium chloride has a good linear relationship in the concentration range of 0.0973 μg / ml (approximately 0.03% of the test solution concentration) to 3.8934 μg / ml (approximately 1.2% of the test solution concentration).

[0264] Precision test

[0265] Accurately measure 0.5 ml, 1.0 ml and 1.5 ml of the linear stock solution under the linearity and range items, respectively, and place them in 10 ml volumetric flasks, dilute to the scale with diluent, shake well, and use them as low, medium and high concentration test solutions. Prepare three copies for each concentration; perform the measurement according to the chromatographic conditions of Example 1, calculate the RSD value of the peak area, and the results are shown in Table 6.

[0266] Table 6 Results of the precision test of impurities in cetylpyridinium chloride tablets

[0267]

[0268]

[0269] The results showed that this method had good precision.

[0270] Accuracy test

[0271] Reference substance stock solution: accurately weigh about 2.5 mg of each of 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine, and 1-chloroheptadecylpyridine reference substances, respectively, and place them in 10 ml volumetric flasks, add diluent to dissolve by ultrasonication and dilute to the scale, shake well, and use them as 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine, and 1-chloroheptadecylpyridine reference substance stock solutions;

[0272] Mixed impurity stock solution: Accurately measure 2 ml of each impurity reference stock solution, place it in a 25 ml volumetric flask, add diluent to dilute to the scale, shake well, and use it as the mixed impurity stock solution;

[0273] Calibration solution: Take an appropriate amount of this product (equivalent to 2 mg of cetylpyridinium chloride), weigh accurately, place in a 10 ml volumetric flask, add 6 ml of diluent, shake, sonicate for 10 minutes, filter, and use as the calibration solution;

[0274] Reference solution: accurately measure 1 ml of the mixed impurity stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, shake well, and use it as the reference solution;

[0275] Test solution: Take an appropriate amount of this product (equivalent to 2 mg of cetylpyridinium chloride), accurately weigh it, place it in a 10 ml volumetric flask, add 5.7 ml of diluent, accurately add 0.3 ml of mixed impurity stock solution, shake, ultrasonic for 10 minutes, filter, as a low concentration test solution; take an appropriate amount of this product (equivalent to 2 mg of cetylpyridinium chloride), accurately weigh it, place it in a 10 ml volumetric flask, add 5.4 ml of diluent, accurately add 0.6 ml of mixed impurity stock solution, shake, ultrasonic for 10 minutes, filter, as a medium concentration test solution; take an appropriate amount of this product (equivalent to 2 mg of cetylpyridinium chloride), accurately weigh it, place it in a 10 ml volumetric flask, add 5.1 ml of diluent, accurately add 0.9 ml of mixed impurity stock solution, shake, ultrasonic for 10 minutes, filter, as a high concentration test solution; prepare three copies for each concentration.

[0276] Accurately measure 30 μl of the test solution, reference solution, and calibration solution, inject them into the liquid chromatograph, measure according to the chromatographic conditions of Example 1, record the chromatogram, and calculate the recovery rate of each impurity by peak area according to the external standard method (the results are also corrected with the blank). The results are shown in Table 7.

[0277] Table 7 Cetylpyridinium Chloride Tablets Impurity Recovery Test Results

[0278]

[0279] The results showed that this method was used for the determination of 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine and 1-chloroheptadecylpyridine with high accuracy.

[0280] Durability test

[0281] Reference substance stock solution: accurately weigh about 2.5 mg of each of 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine, and 1-chloroheptadecylpyridine reference substances, respectively, and place them in 10 ml volumetric flasks, add diluent to dissolve by ultrasonication and dilute to the scale, shake well, and use them as 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine, and 1-chloroheptadecylpyridine reference substance stock solutions;

[0282] Mixed impurity stock solution: Accurately measure 2 ml of each impurity reference stock solution, place it in a 25 ml volumetric flask, add diluent to dilute to the scale, shake well, and use it as the mixed impurity stock solution;

[0283] Reference solution: accurately measure 1 ml of the mixed impurity stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, shake well, and use it as the reference solution;

[0284] Sample solution: Accurately weigh an appropriate amount of this product (equivalent to 2 mg of cetylpyridinium chloride), place it in a 10 ml volumetric flask, add 6 ml of diluent, then add 0.6 ml of mixed impurity stock solution, shake, ultrasonicate for 10 min, filter, and use as the sample solution. Prepare two portions in the same way.

[0285] The flow rate, column temperature, mobile phase ratio and detection wavelength in the chromatographic conditions of Example 1 were changed, the chromatographic column was replaced, and the impurity content was determined according to the law. The results are shown in Table 8.

[0286] Table 8 Cetylpyridinium Chloride Tablets Related Substance Durability Test Results

[0287]

[0288] By fine-tuning the flow rate, column temperature, the proportion of trifluoroacetic acid in mobile phase A, and the detection wavelength, and replacing the chromatographic column, there was no significant difference in the content of impurities detected in the test solution, indicating that this method has good durability.

[0289] Limit of Detection and Limit of Quantitation

[0290] Weigh appropriate amounts of cetylpyridinium chloride, 5-hydroxymethylfurfural, 1-chlorotetradecylpyridine, and 1-chloroheptadecylpyridine reference substances, dissolve and dilute with diluent to prepare reference substance stock solutions. Accurately measure appropriate amounts of reference substance stock solutions, dilute stepwise, and use the concentration when the signal-to-noise ratio S / N≈10 as the quantitative limit concentration, the results are shown in Table 9; use the signal-to-noise ratio S / N≈3 as the detection limit, the results are shown in Table 8. Take the solution with the quantitative limit concentration, measure according to the chromatographic conditions of Example 1, inject 6 times continuously, calculate the RSD of the main peak retention time and peak area, the results are shown in Tables 9 and 10.

[0291] Table 9 Results of the quantitative limit and detection limit test of impurities in cetylpyridinium chloride tablets

[0292]

[0293] Table 10 Results of repeatability test of quantitative limit of related substances in cetylpyridinium chloride tablets

[0294]

[0295]

[0296] The results showed that the repeatability of the quantitative limit concentration of each impurity was good, and both the quantitative limit and the detection limit could meet the detection requirements.

Claims

1. A method for detecting related substances in cetylpyridinium chloride lozenges, It is characterized in that A reverse phase chromatographic column with silane bonded silica as filler was used, with trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile solution containing trifluoroacetic acid as mobile phase B, and gradient elution was performed.

2. The detection method according to claim 1, It is characterized in that The volume proportion of trifluoroacetic acid in the mobile phase A is 0.1% to 0.2%; the volume proportion of trifluoroacetic acid in the mobile phase B is 0.05 to 0.15%.

3. The detection method according to claim 1, It is characterized in that The volume proportion of trifluoroacetic acid in the mobile phase A is 0.12% to 0.13%; the volume proportion of trifluoroacetic acid in the mobile phase B is 0.1%.

4. The detection method according to claim 1, It is characterized in that A reverse phase chromatographic column using silane bonded silica gel with polar amide groups embedded in long alkyl chains as filler is used; preferably, the chromatographic column is Agilent ZORBAX Bonus-RP.

5. The detection method according to claim 1, It is characterized in that The elution gradient is:

6. The detection method according to claim 5, It is characterized in that The elution gradient is:

7. The detection method according to claim 1, It is characterized in that The detection wavelength is 258 nm.

8. The detection method according to claim 1, It is characterized in that The flow rate during gradient elution was 0.9-1.1 mL / min.

9. The detection method according to claim 1, It is characterized in that The column temperature was 25-35°C during gradient elution.

10. The detection method according to claim 1, It is characterized in that The injection volume is 20-30 μl.

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