Method for detecting composition of medicinal-grade sitaammonium chloride alkyl group and application of medicinal-grade sitaammonium chloride alkyl group

Abstract

The invention provides a method for detecting the composition proportion of sitamido chloride alkyl, which is carried out by adopting high performance liquid chromatography, a strong acid type cation exchange chromatographic column is adopted as a chromatographic column, and isocratic elution is carried out by using a mixed solution of a mobile phase A and a mobile phase B according to the volume ratio of 45: 55; wherein the mobile phase A is a mixed solution of 8-45mM of sodium perchlorate and 10mM of potassium hexafluorophosphate (the pH value is adjusted to 3.0-4.0); and the mobile phase B is a mixed solution of acetonitrile and isopropanol in a volume ratio of (60-80): (20-40). The method provided by the invention can be used for distinguishing and detecting three alkyl impurities including the dodecyl dimethyl benzyl ammonium chloride, the tetradecyl dimethyl benzyl ammonium chloride and the octadecyl dimethyl benzyl ammonium chloride, and the detection standard of related substances in the sitaammonium chloride pharmaceutic adjuvant is improved. The detection method disclosed by the invention is high in separation degree of each component, strong in specificity, high in sensitivity and good in repeatability and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the alkyl composition ratio in pharmaceutical-grade cetalkonium chloride and its application. Background Art

[0002] Cetalkonium chloride, with the chemical name of hexadecyl dimethyl benzyl ammonium chloride, English name Cetalkonium chloride, and CAS number 122-18-9. Cetalkonium chloride is a quaternary ammonium salt compound and can be used as an antibacterial agent, fungicide, and disinfectant. In addition, based on its special cationic properties, it can also be used as a pharmaceutical excipient in ophthalmic emulsions. For example, in the commercially available 0.1% cyclosporine eye drops (Ⅲ) (Verkazia), cetalkonium chloride is used as a cationic quaternary ammonium salt surfactant in the prescription, which is beneficial to prolong the residence time of the drug in the eye, increase the ocular tissue distribution of the drug, and enable the active ingredient cyclosporine to have better bioavailability and long-acting properties in the tear film.

[0003] According to the preparation process route of cetalkonium chloride, impurities with different alkyl chain lengths may exist. Benzalkonium chloride is a mixture of dimethyl benzyl alkyl ammonium chlorides, mainly dodecyl dimethyl benzyl ammonium chloride (C12), tetradecyl dimethyl benzyl ammonium chloride (C14), and hexadecyl dimethyl benzyl ammonium chloride (C16, cetalkonium chloride). According to the regulations of the United States Pharmacopeia (USP32), the alkyl composition of benzalkonium chloride should contain no less than 40% of C12, no less than 20% of C14, and the total amount of the two should be no less than 70%. Benzalkonium chloride is often used as a bactericide or preservative in cosmetics, disinfectants, and pharmaceuticals. Some studies have shown that benzalkonium chloride has strong irritation to both the skin and eyes, and excessive use can cause allergic conjunctivitis, visual impairment, contact dermatitis, etc.; while cetalkonium chloride has relatively less irritation and shows different cationic potencies compared to C12 or C14. If the alkyl chain ratio in the product is unreasonable, it may increase the irritation of the product and reduce its safety. Therefore, it is necessary to detect the alkyl composition. The situation of cetalkonium chloride and its alkyl composition substances is shown in the following table:

[0004]

[0005]

[0006] The applicant has consulted a large number of documents and the inclusion of various countries' pharmacopoeias, and has found that there is currently no high-performance liquid chromatography analysis method for the alkyl composition in the pharmaceutical excipients of cetaxel. As a homologue of benzalkonium chloride, it is known that the existing analysis methods are mainly reversed-phase chromatography and ion exchange chromatography with reference to the liquid chromatography analysis method for the alkyl composition of benzalkonium chloride. Among them, reversed-phase chromatography, although it can enhance the retention of each component by adding an ion pair reagent, still has the disadvantages of poor specificity, difficulty in separating each component, low detection sensitivity, and serious tailing of high-concentration samples.

[0007] Regarding the ion exchange chromatography detection method, the document "Yang Yanwei et al., Simultaneous determination of four quaternary ammonium salts in disinfectants by high performance liquid chromatography, Chinese Journal of Health Inspection, July 2006, Vol. 16, No. 7" discloses the use of a C8 chromatographic column, an acetonitrile / water / acetic acid mixture, and a mixed system of ion pair reagent dodecyltrimethylammonium bromide as the mobile phase to establish a high performance liquid chromatography method for the simultaneous determination of four quaternary ammonium salts in disinfectants, namely dodecyldimethylbenzylammonium chloride, miramistine, tetradecyldimethylbenzylammonium chloride and hexadecyldimethylbenzylammonium chloride. The document "Ding Hong et al., Determination of the content of preservatives lauralkonium chloride, benzethonium chloride and cethamidium chloride in cosmetics by HPLC, Chinese Journal of Pharmaceutical Affairs, 2012, 26 (8) 830-834" discloses the use of a cyano chromatographic column, a methanol / ammonium acetate buffer solution as the mobile phase to search for preservatives lauralkonium chloride, benzethonium chloride and cethamidium chloride in cosmetics. In addition, the European Pharmacopoeia EP 10.2 records the analysis method for the alkyl composition of benzalkonium chloride, using a cyano column as the chromatographic column and a mixture of acetonitrile: 0.1 mol / L sodium acetate buffer (adjusted to pH 5.0 with glacial acetic acid) = 45:55 (V / V) as the mobile phase; the United States Pharmacopoeia USP42 and the "Draft of the Publicity of Benzalkonium Chloride National Pharmaceutical Excipients" record the analysis method for the alkyl composition of benzalkonium chloride, and the chromatographic conditions use a cyano column as the chromatographic column and acetonitrile: 0.1 mol / L sodium acetate buffer (adjusted to pH 5.0 with glacial acetic acid) = 9:11 (V / V) as the mobile phase.

[0008] In the chromatographic conditions of the prior art, methods such as using a C8 chromatographic column, a cyano column, or a reversed-phase ion-pair reagent have, to a certain extent, solved the problem of the retention of cationic compounds. However, the problem of tailing of chromatographic peaks still exists and has not been effectively solved, resulting in poor separation between the main peak and impurity peaks and inaccurate content detection. When the applicant transferred the liquid chromatography detection method for benzalkonium chloride homologues in the prior art to the detection and analysis of cetylpyridinium chloride, it was found that it was not applicable to the separation and detection of alkyl groups in cetylpyridinium chloride. It is speculated that there is a relationship between benzalkonium chloride and the content distribution of different alkyl chain lengths in the pharmaceutical excipients of cetylpyridinium chloride. Cetylpyridinium chloride is a cationic quaternary ammonium salt compound, and the structures of its alkyl impurities are very similar. The main difference lies in the change of the alkyl chain length in the range of C12 - C18. Therefore, there is a certain difficulty in accurately detecting other alkyl impurities in cetylpyridinium chloride by HPLC.

[0009] In summary, the HPLC detection method for the alkyl composition in cetylpyridinium chloride in the prior art has not met the detection requirements, and it is necessary to develop this method to provide a certain basis for the quality of the product, thereby ensuring the safety and effectiveness of the drug, which is of great significance for realizing the controllability of drug quality. Summary of the Invention

[0010] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a detection method for the alkyl composition ratio of cetylpyridinium chloride. Specifically, the present invention provides a method for distinguishing and detecting cetylpyridinium chloride and three impurities with different alkyl lengths (C12, C14, and C18) in cetylpyridinium chloride, which improves the detection standard for related substances in the pharmaceutical excipients of cetylpyridinium chloride. The detection method of the present invention has a large resolution for each component, strong specificity, high sensitivity, good repeatability and accuracy, and can effectively evaluate the alkyl composition in cetylpyridinium chloride.

[0011] The above object of the present invention is achieved through the following technical solutions:

[0012] A detection method for the alkyl composition ratio of cetylpyridinium chloride, the method comprising the following steps:

[0013] (1) Preparation of the sample solution;

[0014] (2) Detecting the sample solution by high performance liquid chromatography;

[0015] Wherein the chromatographic conditions of the high performance liquid chromatography are:

[0016] Chromatographic column: A strong acid cation exchange bonded silica gel is used as the filler;

[0017] Mobile phase: An isocratic elution with a mixed solution of mobile phase A and mobile phase B in a volume ratio of 45:55;

[0018] Mobile phase A is a mixed solution of 8 - 45 mM sodium perchlorate and 10 mM potassium hexafluorophosphate (adjusted to pH 3.0 - 4.0);

[0019] Mobile phase B: A mixed solution of acetonitrile - isopropanol with a volume ratio of 60 - 80:20 - 40

[0020] Flow rate: 0.2 - 1.0 ml / min

[0021] Injection volume: 8 - 25 μl

[0022] Detection wavelength: 205 - 220 nm

[0023] Column temperature: 25 - 45 °C.

[0024] In the detection method provided by the present invention, the chromatographic column, mobile phase A, and the mobile phase system of mobile phase B adopted in the chromatographic conditions are the key factors affecting the detection effect of this application.

[0025] In the detection method provided by the present invention, the selection of the chromatographic column packing material, manufacturer, model, and specification has a great influence on the separation and detection effect of alkyl impurities. After screening a variety of chromatographic columns, it is found that only when a chromatographic column filled with sulfonic acid group cation exchange bonded silica gel as the packing material is selected, the retention can be enhanced, the resolution can be improved, and good symmetry of chromatographic peaks and high column efficiency can be ensured. Further, for the present invention, the corresponding chromatographic column can be a chromatographic column of the Macherey - Nagel, Phenomenex, Hypersil BioBasic SCX, or Capcell Pak SCX series, with better separation and detection effects. Further considering factors such as chromatographic column performance and separation effect comprehensively, the most preferred chromatographic column and specification is Capcell Pak SCX UG80(S5), 4.6 mm * 250 mm, 5 μm, with the best separation and detection effects for alkyl impurities.

[0026] In the detection method provided by the present invention, the selection and dosage of sodium perchlorate and potassium hexafluorophosphate in mobile phase A are one of the keys. Although inorganic salts are often used as buffers in the prior art to improve the peak shape of compounds, such as acetates, phosphates, citrates, and perchlorates, etc., technicians have tried to use a single inorganic salt as a buffer, including sodium acetate, ammonium acetate, sodium perchlorate, potassium citrate, dipotassium hydrogen phosphate, etc., and found that the effect of improving the peak shape of cetylpyridinium chloride and its alkyl impurities is not good and fails to meet the detection requirements. Technicians occasionally found that when using a mixed system of sodium perchlorate and potassium hexafluorophosphate as mobile phase A, not only can the peak shape of basic substances be improved, making the peak shape sharper and more symmetrical, but also the accuracy and sensitivity of analysis are improved. In this application, potassium hexafluorophosphate and sodium perchlorate jointly provide the mobile phase buffer system. It is speculated that the addition of sodium perchlorate plays a certain ion-pairing role, so the retention behavior of ions can be improved, and the components can be better separated and detected on the chromatographic column. Further, technicians surprisingly found that when the concentration of potassium hexafluorophosphate is 10 mM and remains unchanged, the concentration of sodium perchlorate has an obvious effect on the separation effect of each component. The lower the concentration, the better the separation effect. Specifically, in this application, the mobile phase A is a mixed solution of 8-45 mM sodium perchlorate and 10 mM potassium hexafluorophosphate; preferably, the mobile phase A is a mixed solution of 10-30 mM sodium perchlorate and 10 mM potassium hexafluorophosphate; more preferably, the mobile phase A is a mixed solution of 10-20 mM sodium perchlorate and 10 mM potassium hexafluorophosphate; specifically, it can be a mixed solution of 10 mM sodium perchlorate and 10 mM potassium hexafluorophosphate.

[0027] Regarding the pH value of mobile phase A, when detecting samples, when the type of mobile phase remains unchanged, the pH value has an obvious effect on the tailing factor of each component. Too high or too low pH value will cause the tailing factor to increase, and the mobile phase with a high pH value is likely to damage the analytical column and shorten the service life of the analytical column, thereby affecting the detection effect. The inventor comprehensively concluded that when the pH value of mobile phase A is 3.0-4.0, specifically 3.6, it is beneficial to achieve the best separation of impurities in each test solution for detection. The pH value of the mobile phase can be adjusted with common organic acids or inorganic acids in the art, such as acetic acid, phosphoric acid, sulfuric acid, or hydrochloric acid, etc., preferably phosphoric acid, and the concentration of the phosphoric acid can be 10%-15%.

[0028] In the detection method provided by the present invention, mobile phase B adopts a mixed solution of acetonitrile and isopropanol with a volume ratio of 60-80:20-40, which can increase the resolution and effectively separate cetylpyridinium chloride from other alkyl impurities. Through preliminary research, technicians found that the resolution between cetylpyridinium chloride and each alkyl impurity decreases with the increase of the acetonitrile ratio, and the retention time of each component peak decreases; when using a single isopropanol, no component peak can be detected within 20 minutes. Technicians creatively selected a mixed solution of acetonitrile and isopropanol, which can achieve a resolution greater than 1.5 between the peaks of cetylpyridinium chloride, alkyl impurities and solvent peaks, with appropriate retention times for each component peak, and all components can be detected within about 30 minutes. Specifically, in this application, mobile phase B preferably adopts a mixed solution of acetonitrile and isopropanol with a volume ratio of 65-75:25-35; more preferably, when the mixed solution of acetonitrile and isopropanol with a volume ratio of 70:30 is used as mobile phase B, the resolution between cetylpyridinium chloride and each alkyl impurity reaches the maximum, and the retrieval effect is better.

[0029] In the detection method provided by the present invention, the column temperature is controlled at 25-45°C. When the temperature is lower than 25°C, the separation efficiency is relatively low; when the temperature is too high, it is not conducive to the service life of the chromatographic column. Considering comprehensively, the separation effect is better when the column temperature is 30-40°C, and specifically it can be 35°C.

[0030] In the detection method provided by the present invention, the flow rate of the mobile phase is preferably controlled at 0.2-1.0 ml / min. Specifically, the control of the flow rate of the mobile phase should take into account both the detection efficiency and the resolution. If the flow rate of the mobile phase is too high, it will accelerate the elution speed, shorten the peak emergence time, which is not conducive to achieving the separation effect and will damage the chromatographic column. If the flow rate is too low, it will reduce the elution speed, widen the peak widths of each component while delaying the peak emergence time, which is also not conducive to achieving the separation effect and will correspondingly reduce the detection efficiency. The preferred flow rate is 0.3-0.8 ml / min; the more preferred flow rate is 0.4-0.6 ml / min, and the detection effect is the best.

[0031] In the detection method provided by the present invention, the injection volume needs to be controlled at 8-25 μL, and at this time, there is a linear relationship between the concentration and the peak area; preferably, the injection volume needs to be controlled at 10-20 μL, and the sample amount per unit detection can maximize the effect of mobile elution, which is conducive to the separation of each component peak and the detection result is the most accurate.

[0032] In the detection method provided by the present invention, considering the maximum absorption of cetylpyridinium chloride and each alkyl at different wavelengths, the sensitivity requirements of the analysis method and the applicability of the instrument, the wavelength in this application is preferably 208-215 nm, and specifically preferably 210 nm as the detection wavelength.

[0033] In the detection method provided by the present invention, it is preferably to use the above-mentioned elution mobile phase, combined with specific chromatographic column and flow rate and other separation conditions, which belong to the overall technical solution and cooperate with each other to achieve better separation of the components and impurities in the chromatogram; at the same time, by using the method of the present invention, the results are accurate, reliable, and have good stability and reproducibility.

[0034] As a preferred embodiment of the present invention, a method for detecting the alkyl composition ratio of cetylpyridinium chloride includes the following steps:

[0035] (1) Preparation of sample solution;

[0036] (2) Detecting the sample solution by high performance liquid chromatography;

[0037] Among them, the chromatographic conditions of the high performance liquid chromatography are as follows:

[0038] Chromatographic column: A chromatographic column filled with sulfonic acid group cation exchange bonded silica gel

[0039] Mobile phase: Isocratic elution with a mixed solution of mobile phase A and mobile phase B in a volume ratio of 45:55;

[0040] Among them, mobile phase A: A mixed solution of 10 - 20 mM sodium perchlorate and 10 mM potassium hexafluorophosphate (adjusted to a pH value of 3.0 - 4.0 with 10% - 15% phosphoric acid)

[0041] Mobile phase B: A mixed solution of acetonitrile - isopropanol with a volume ratio of 65 - 75:25 - 35

[0042] Flow rate: 0.3 - 0.8 ml / min

[0043] Injection volume: 10 - 20 μl

[0044] Detection wavelength: 208 - 215 nm

[0045] Column temperature: 30 - 40 °C.

[0046] The results of the methodological verification experiment show that the detection method of the present invention for detecting the alkyl composition ratio in cetylpyridinium chloride has strong specificity, good system suitability, stable control solution and spiked test solution, and C18, cetylpyridinium chloride, C14, and C12 have good linear relationships in the concentration ranges of 0.5152 μg / ml - 10.30 μg / ml, 0.4709 μg / ml - 9.418 μg / ml, 0.5096 μg / ml - 10.19 μg / ml, and 0.4953 μg / ml - 9.907 μg / ml respectively, with good repeatability, high precision, high accuracy, and good durability.

[0047] In the method for detecting the alkyl composition ratio in cetylpyridinium chloride by the detection method of the present invention, C18, cetylpyridinium chloride, C14, and C12 elute in sequence. Among them, the relative retention time of C18 is 0.88 ± 0.05, the relative retention time of cetylpyridinium chloride is 1.00, the relative retention time of C14 is 1.14 ± 0.05, and the relative retention time of C12 is 1.31 ± 0.05.

[0048] In the present invention, unless otherwise specified, the term "resolution" refers to the separation of the front and rear compounds. Generally, a resolution of not less than 1.5 is considered to have good separation and the determination method is accurate and available.

[0049] Advantages of the present invention:

[0050] 1) The present invention uses a strongly acidic cation chromatography column. By optimizing the high-performance liquid chromatography conditions, the solvent peak does not interfere with the determination during the detection process, and it can achieve effective separation of cetylpyridinium chloride and its alkyl impurities, and the resolution between each peak is greater than 1.5.

[0051] 2) This method has strong specificity, high accuracy, and is easy to operate, thus providing a technical basis for the quality control of cetylpyridinium chloride as a pharmaceutical excipient, and ensuring the safety of R & D, production, and medication. Description of the Drawings

[0052] Figure 1 HPLC chromatogram of the mixed reference solution with different mobile phase A.

[0053] Figure 2 HPLC chromatogram of the mixed reference solution with different mobile phase B.

[0054] Figure 3 HPLC chromatogram of the sensitivity solution detected by a cyanide chromatography column.

[0055] Figure 4 HPLC chromatogram of the sensitivity solution detected by a sulfonic acid group cation exchange column.

[0056] Figure 5 HPLC chromatogram of the specificity and system suitability spiked test solution.

[0057] Figure 6 HPLC chromatogram of the alkyl composition analysis method of reference benzalkonium chloride in Comparative Example 1.

[0058] Figure 7 HPLC chromatogram of the mixed reference solution with 50 mM sodium acetate as mobile phase A and the blank solvent.

[0059] Figure 8 HPLC chromatogram of the mixed reference solution with 100 mM sodium acetate as mobile phase A and the blank solvent. Detailed implementation mode

[0060] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation modes of the invention are not limited thereto. Unless otherwise specified, the reagents in the following examples are all analytical pure reagents available on the market.

[0061] Unless otherwise specified, the detection method in the present invention is high performance liquid chromatography, and the detector is an ultraviolet detector.

[0062] Unless otherwise specified, the blank solvent used in the chromatographic detection of the present invention is a 50% acetonitrile aqueous solution.

[0063] Unless otherwise specified, for the preparation of sample solutions such as the positioning solution, reference substance solution, mixed reference substance solution, resolution solution, spiked test sample solution, and test sample solution in the present invention, those skilled in the art can prepare them according to experience. Unless otherwise specified, the sample solutions involved in the embodiments of the present invention are prepared as follows.

[0064] Resolution solution: Weigh appropriate amounts of benzalkonium chloride dodecyl (C12), benzalkonium chloride tetradecyl (C14), benzalkonium chloride octadecyl (C18), and cetylpyridinium chloride reference substance accurately, and quantitatively dilute with the blank solvent to prepare a solution containing about 1 mg of cetylpyridinium chloride, 0.005 mg of C12, 0.005 mg of C14, and 0.005 mg of C18 per 1 ml as the resolution solution.

[0065] Mixed reference substance solution: Accurately weigh benzalkonium chloride dodecyl (C12), benzalkonium chloride tetradecyl (C14), cetylpyridinium chloride, and benzalkonium chloride octadecyl (C18), dissolve them by ultrasonic treatment with the blank solvent, and dilute and make up the volume to prepare a mixed reference substance solution with a concentration of 0.5 mg / ml or 1.0 mg / ml.

[0066] Reference substance solution (5 μg / ml): Weigh an appropriate amount of cetylpyridinium chloride reference substance accurately, and quantitatively dilute with the blank solvent to prepare a solution containing about 5 μg of cetylpyridinium chloride per 1 ml as the reference substance solution.

[0067] Test sample solution (1.0 mg / ml): Take an appropriate amount of this product, quantitatively dilute with the blank solvent to prepare a solution containing about 1 mg of cetylpyridinium chloride per 1 ml as the test sample solution.

[0068] Spiked test sample solution: Take about 25 mg of the test sample, weigh it accurately, place it in a 25 ml volumetric flask, add an appropriate amount of the blank solvent to dissolve it, then accurately measure 5 ml of the impurity mixed reference substance stock solution (25 μg / ml) and place it in the same volumetric flask as above, dilute to the mark with the blank solvent, and shake well to obtain it.

[0069] Investigation on the Concentration of Sodium Perchlorate in Mobile Phase A of Example 1

[0070] The specific experimental methods and steps are as follows:

[0071] (1) Prepare a mixed reference solution (0.5 mg / ml) as the sample solution;

[0072] (2) Analyze the sample solution by high performance liquid chromatography;

[0073] (3) Organize the analysis results;

[0074] Among them, the analysis conditions of the high performance liquid chromatography are as follows:

[0075] Chromatographic column: Sulfonic acid group cation exchange bonded silica gel as the filler (Capcell Pak SCX UG80(S5), 4.6 mm * 250 mm, 5 μm)

[0076] Mobile phase: The mobile phase A and mobile phase B are isocratically eluted with a mixed solution in a volume ratio of 45:55;

[0077] Among them, mobile phase A: A mixed solution of sodium perchlorate with different concentrations and 10 mM potassium hexafluorophosphate;

[0078] Mobile phase B: A mixed solution of acetonitrile - isopropanol with a volume ratio of 70:30;

[0079] Detection wavelength: 210 nm;

[0080] Column temperature: 25 °C;

[0081] Flow rate: 0.5 ml / min;

[0082] Injection volume: 20 μl.

[0083] Table 1 Influence of Different Mobile Phase A on Resolution

[0084] Number Mobile phase A Resolution between cetyltrimethylammonium chloride and C18 Sample 1 20 mM sodium perchlorate + 10 mM potassium hexafluorophosphate 2.356 Sample 2 30 mM sodium perchlorate + 10 mM potassium hexafluorophosphate 1.901 Sample 3 50 mM sodium perchlorate + 10 mM potassium hexafluorophosphate 1.314 Sample 4 50 mM sodium perchlorate 1.183

[0085] Under this chromatographic condition, C18, cetyltrimethylammonium chloride, C14 and C12 elute in turn. The HPLC chromatograms of the mixed reference solution samples 1 - 4 with different mobile phase A are as Figure 1 shown. Combining the data in the above table, it can be found that in mobile phase A, whether potassium hexafluorophosphate is added and the concentration of sodium perchlorate have obvious effects on the separation effect between cetyltrimethylammonium chloride and C18. In sample 4, without adding potassium hexafluorophosphate and only adding 50 mM sodium perchlorate, the resolution between the main component of cetyltrimethylammonium chloride and the previous impurity C18 alkyl group is 1.183, which is lower than 1.5 and cannot achieve effective separation.

[0086] When the concentration of potassium hexafluorophosphate remains unchanged at 10 mM, as the concentration of sodium perchlorate decreases, the resolution between cetyltrimethylammonium chloride and alkyl impurities increases. When the concentration of sodium perchlorate is 20 mM to 30 mM, the resolution between the main component of cetyltrimethylammonium chloride and the previous impurity C18 alkyl is greater than 1.5, enabling effective separation.

[0087] Investigation of the proportion of acetonitrile in mobile phase B in Example 2

[0088] The specific experimental methods and steps are as follows:

[0089] (1) Prepare a mixed reference solution (0.5 mg / ml) as the sample solution;

[0090] (2) Analyze the sample solution by high performance liquid chromatography;

[0091] (3) Organize the analysis results;

[0092] The analysis conditions of the high performance liquid chromatography are as follows:

[0093] Chromatographic column: Sulfonic acid group cation exchange bonded silica gel as the packing material (Capcell Pak SCX UG80 (S5), 4.6 mm * 250 mm, 5 μm);

[0094] Mobile phase: The mobile phase A and mobile phase B are isocratically eluted with a mixed solution in a volume ratio of 45:55;

[0095] Among them, mobile phase A: A mixed solution of 50 mM sodium perchlorate and 10 mM potassium hexafluorophosphate;

[0096] Mobile phase B: A mixed solution of acetonitrile - isopropanol with different volume ratios;

[0097] Detection wavelength: 210 nm;

[0098] Column temperature: 25 °C;

[0099] Flow rate: 0.5 ml / min;

[0100] Injection volume: 20 μl.

[0101] Table 2 Influence of different mobile phase B on resolution

[0102]

[0103] Under these chromatographic conditions, C18, cetyltrimethylammonium chloride, C14, and C12 elute in sequence. The HPLC chromatograms of the mixed reference solution samples 5 - 8 with different mobile phase B are as Figure 2As shown. From the data in Table 2 above, it can be seen that pure isopropanol (Sample 5) cannot be used as Mobile Phase B in the present invention; while using pure acetonitrile as Mobile Phase B (Sample 8), the resolution of each component peak is poor and there is interference from the solvent peak, making it difficult to meet the detection requirements. When a mixed solution of acetonitrile and isopropanol is used as Mobile Phase B, the resolution and peak shape of each component peak and other separation effects are improved. Specifically, when the volume ratio of acetonitrile - isopropanol is 70:30 or 60:40, the resolution of each component peak of cetyltrimethylammonium chloride, C14, and C12 is appropriate and the peak shape is better.

[0104] Effect of Mobile Phase pH on Resolution Solution in Example 3

[0105] Based on the investigation results of the change in sodium perchlorate concentration in Example 1, when the concentration of potassium hexafluorophosphate remains unchanged at 10 mM potassium hexafluorophosphate, within the range of 20 mM to 50 mM of sodium perchlorate concentration, the resolution between cetyltrimethylammonium chloride and alkyl impurities increases as the sodium perchlorate concentration decreases. Therefore, when investigating the effect of mobile phase pH, a mixed solution of a lower 10 mM sodium perchlorate concentration and 10 mM potassium hexafluorophosphate is selected as Mobile Phase A. The specific experimental methods and steps are as follows:

[0106] (1) Prepare a resolution solution as the sample solution;

[0107] (2) Analyze the sample solution by high performance liquid chromatography;

[0108] The analysis conditions of the high performance liquid chromatography are as follows:

[0109] Chromatographic column: Sulfonic acid group cation exchange bonded silica gel as the packing material (Capcell Pak SCX UG80 (S5), 4.6 mm * 250 mm, 5 μm)

[0110] Mobile phase: Isocratic elution with a mixed solution of Mobile Phase A and Mobile Phase B in a volume ratio of 45:55;

[0111] Among them, Mobile Phase A: A mixed solution of 10 mM sodium perchlorate and 10 mM potassium hexafluorophosphate; Adjust the pH value to 2.6, 3.6, 4.6, and 5.6 respectively using 10% phosphoric acid;

[0112] Mobile Phase B: A mixed solution of acetonitrile - isopropanol with a volume ratio of 70:30

[0113] Detection wavelength: 210 nm

[0114] Column temperature: 40 °C

[0115] Flow rate: 0.5 ml / min

[0116] Injection volume: 10 μl

[0117] Table 3 Effect of Different Mobile Phase pH on Resolution Solution

[0118]

[0119] Note: The pH of the original solution of 10 mM sodium perchlorate is about 5.6.

[0120] From the data in the above table, it can be seen that under each pH condition, the resolution between each component peak is greater than 1.5, while the tailing factor decreases or increases to varying degrees with the change of pH. Especially for the C18 alkyl component peak, in the range of pH 2.6 - 5.6, the tailing factor changes from the range of 1.2 - 1.6, and is the smallest when the pH value of the mobile phase is 3.6.

[0121] Example 4 Investigation of Detection Sensitivity by Different Methods

[0122] The specific experimental methods and steps are as follows:

[0123] (1) Prepare sensitivity solutions with different concentrations as sample solutions;

[0124] (2) Analyze the sample solutions by high performance liquid chromatography;

[0125] Among them, the analysis conditions of the high performance liquid chromatography are as follows:

[0126] Chromatographic condition A: Refer to the prior art for the analysis method of benzalkonium chloride homologues:

[0127] Chromatographic column: Packed with porous silica microspheres bonded with cyano group (ACE Excel 3CN-ES, 150 mm × 4.6 mm 3μm)

[0128] Mobile phase: Isocratic elution with a mixed solution of mobile phase A and mobile phase B in a volume ratio of 35:65;

[0129] Mobile phase A: 0.2 mol / L sodium hexanesulfonate (containing 1% triethylamine, adjusted to pH 5.0 with 10% phosphoric acid)

[0130] Mobile phase B: Methanol;

[0131] Detection wavelength: 214 nm

[0132] Column temperature: 25°C

[0133] Flow rate: 1.0 ml / min

[0134] Injection volume: 20 μl

[0135] Chromatographic condition B: Technical solution within the scope of the present invention of this application:

[0136] Chromatographic column: Sulfonic acid group cation exchange bonded silica gel as filler (Capcell Pak SCX UG80(S5), 4.6mm * 250mm, 5μm)

[0137] Mobile phase: Mobile phase A and mobile phase B are isocratically eluted with a mixed solution in a volume ratio of 45:55;

[0138] Among them, mobile phase A: A mixed solution of 30 mM sodium perchlorate and 10 mM potassium hexafluorophosphate;

[0139] Mobile phase B: A mixed solution of acetonitrile - isopropanol with a volume ratio of 70:30

[0140] Detection wavelength: 210 nm

[0141] Column temperature: 25 °C

[0142] Flow rate: 0.5 ml / min

[0143] Injection volume: 20 μl

[0144] Table 4 Statistical table of sensitivity data under two chromatographic conditions

[0145]

[0146] It can be seen from the sensitivity data of the two methods in the above table that in chromatographic condition A, a cyano chromatographic column is used. Although the so-called ion pair reagent sodium hexanesulfonate is added, the retention time of the longer-chain alkyl groups can be increased, making the resolution of cetylpyridinium chloride and each alkyl impurity meet the requirements, but the detection sensitivity is relatively low. Specifically, based on the evaluation requirements of detection limit S / N ≥ 3 and quantification limit S / N ≥ 10:

[0147] When the concentration of the test solution is 1 mg / ml, detection and analysis are carried out under chromatographic condition A. C12, C14, cetylpyridinium chloride, and C18 elute in sequence. The detection limit is 0.25%, the quantification limit is 0.75%, and the sensitivity is relatively low. The HPLC chromatogram of the sensitivity solution detected by using a cyano chromatographic column and chromatographic condition A is as Figure 3 shown.

[0148] When the concentration of the test solution is 1 mg / ml, detection and analysis are carried out under chromatographic condition B (the chromatographic column is a sulfonic acid group cation exchange column). C18, cetylpyridinium chloride, C14, and C12 elute in sequence. The detection limit of each impurity can be 0.01%, and the quantification limit can be 0.05%, which can meet the sensitivity requirements for impurity detection. The HPLC chromatogram of the sensitivity solution detected by using a sulfonic acid group cation exchange column and chromatographic condition B is as Figure 4 shown.

[0149] In summary, it can also be shown that compared with the reversed-phase ion pair liquid phase analysis method using a cyano chromatographic column, the detection sensitivity of the ion chromatographic column is higher.

[0150] Example 5 Specificity and System Suitability

[0151] The specific experimental methods and steps are as follows:

[0152] (1) Prepare the resolution solution, impurity localization solutions, reference solution, test solution, and spiked test solution;

[0153] (2) Analyze the sample solution by high performance liquid chromatography;

[0154] The analysis conditions of the high performance liquid chromatography are as follows:

[0155] Chromatographic column: Sulfonic acid cation exchange bonded silica gel as the filler (Capcell Pak SCX UG80(S5), 4.6mm*250mm, 5μm)

[0156] Mobile phase: Isocratic elution with a mixed solution of mobile phase A and mobile phase B in a volume ratio of 45:55;

[0157] Among them, mobile phase A: A mixed solution of 10 mM sodium perchlorate and 10 mM potassium hexafluorophosphate; Adjust the pH value to 3.6 respectively using 10% phosphoric acid;

[0158] Mobile phase B: A mixed solution of acetonitrile - isopropanol in a volume ratio of 70:30;

[0159] Detection wavelength: 210 nm;

[0160] Column temperature: 35 °C;

[0161] Flow rate: 0.5 ml / min;

[0162] Injection volume: 10 μl.

[0163] Table 5 Methodological Verification of the Alkyl Composition Ratio of Cetalkonium Chloride - Experimental Results of Specificity and System Suitability

[0164]

[0165]

[0166] The results in Table 4 show that when analyzing the alkyl composition of cetalkonium chloride under the chromatographic conditions of the present invention, the blank solvent has no interference at the elution positions of each component peak and does not interfere with the determination of the alkyl composition ratio of this product. In the chromatograms of the resolution solution, test solution, and spiked test solution, the resolution between the main peak and the adjacent alkyl impurity peak is greater than 2, the baseline is stable, and the peak shape is good. It can be seen that the specificity and system suitability of this method are good. Among them, the HPLC chromatogram of the spiked test solution in the specificity and system suitability test is as Figure 5 shown.

[0167] Comparative Example 1

[0168] Detection was carried out with reference to the reverse-phase chromatography analysis method of USP42 or the "Draft for Public Comment on National Pharmaceutical Excipients of Benzalkonium Chloride". The specific experimental methods and steps are as follows:

[0169] (1) Prepare a reference substance solution and a mixed reference substance solution (1.0 mg / ml);

[0170] (2) Analyze the sample solution by high performance liquid chromatography;

[0171] Among them, the analysis conditions of the high performance liquid chromatography are as follows:

[0172] Chromatographic column: fully porous silica microspheres bonded with cyano as the filler (Waters Sphereisorb 10μm CN 4.6mm×250mm, 10μm)

[0173] Mobile phase: 0.1 mol / L sodium acetate solution (adjust the pH value to 5.0 with glacial acetic acid)-acetonitrile (53:47) as the mobile phase;

[0174] Detection wavelength: 254 nm;

[0175] Column temperature: 25 °C;

[0176] Flow rate: 2.0 ml / min;

[0177] Injection volume: 10 μl.

[0178] The HPLC chromatogram of the alkyl composition analysis method of benzalkonium chloride with reference to the prior art is as Figure 6 shown. The results show that under these chromatographic conditions, C12, C14, cetylpyridinium chloride, and C18 elute in sequence, and the resolution of each component is poor (<1.5), and the separation effect is slightly worse than the technical solution of the present invention.

[0179] Comparative Example 2

[0180] Detection was carried out using different inorganic salts as mobile phase A. The specific experimental methods and steps are as follows:

[0181] (1) Prepare a blank solvent and a mixed reference substance solution (1.0 mg / ml) as the sample solution;

[0182] (2) Analyze the sample solution by high performance liquid chromatography;

[0183] Among them, the analysis conditions of the high performance liquid chromatography are as follows:

[0184] Chromatographic column: sulfonic acid group cation exchange bonded silica as the filler (Capcell Pak SCX UG80(S5), 4.6mm*250mm, 5μm);

[0185] Mobile phase: The mobile phase A and the mobile phase B are eluted isocratically with a mixed solution with a volume ratio of 45:55;

[0186] Among them, mobile phase A: 50 mM or 100 mM sodium acetate (adjust the pH value to 5.0 with glacial acetic acid);

[0187] Mobile phase B: acetonitrile;

[0188] Detection wavelength: 210 nm;

[0189] Column temperature: 25 °C;

[0190] Flow rate: 1.0 ml / min;

[0191] Injection volume: 10 μl.

[0192] The HPLC chromatograms of the mixed reference solution with 50 mM or 100 mM sodium acetate as the mobile phase A and the blank solvent are respectively as Figure 6 and Figure 7 shown. It can be seen from the results that when using the same chromatographic column of the present invention, when only 50 mM sodium acetate or 100 mM sodium acetate is used in the mobile phase A, there is interference from the solvent peak. Thus, it can be known that the selection of the type of inorganic salt in the mobile phase A of the present invention has an impact on the detection and separation effect, and the separation effect of using sodium perchlorate as the inorganic salt in the present invention is better than that of sodium acetate.

[0193] In summary, it can be known that the methods disclosed in the prior art are difficult to be directly applied to the detection of the alkyl composition in cetylpyridinium chloride; only when the chromatographic conditions including the chromatographic column, the mobile phase solvent system, etc. simultaneously fall within the protection scope of the present invention, the high-performance liquid chromatography detection method for the alkyl composition ratio of cetylpyridinium chloride can simultaneously achieve effective separation and detection, and the separation effects including resolution, peak shape, tailing situation, etc. are slightly better than those of the prior art analysis methods. The detection method of the present invention can be used for the monitoring of the production process of cetylpyridinium chloride and the quality control of its preparation products.

[0194] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for detecting the alkyl composition ratio of cetaxel chloride, the method comprising the following steps: (1) Sample solution preparation; (2) using high performance liquid chromatography to detect the sample solution; The chromatographic conditions of the high performance liquid chromatography method include: Chromatographic column: Strong acid cation exchange bonded silica gel is used as filler; Mobile phase: isocratic elution with a mixed solution of mobile phase A and mobile phase B in a volume ratio of 45:55; Wherein mobile phase A is a mixed solution of 8-45 mM sodium perchlorate and 10 mM potassium hexafluorophosphate (pH adjusted to 3.0-4.0); Mobile phase B: a mixed solution of acetonitrile and isopropanol with a volume ratio of 60-80:20-40; Flow rate: 0.2~1.0ml / min; Injection volume: 8-25 μl; Detection wavelength: 205~220nm; Column temperature: 25~45℃.

2. The detection method according to claim 1, characterized in that: The strong acid cation exchange chromatography column is a chromatography column of sulfonic acid cation exchange bonded silica gel filler; preferably a chromatography column of Macherey-Nagel, Phenomenex, Hypersil BioBasic SCX or Capcell Pak SCX series; more preferably, the chromatography column is Capcell Pak SCX UG80 (S5), 4.6mm*250mm, 5μm.

3. The detection method according to claim 1, characterized in that: The mobile phase A is preferably a mixed solution of 10-30 mM sodium perchlorate and 10 mM potassium hexafluorophosphate; more preferably a mixed solution of 10-20 mM sodium perchlorate and 10 mM potassium hexafluorophosphate.

4. The detection method according to claim 1, characterized in that: The mobile phase B is a mixed solution of acetonitrile-isopropanol in a volume ratio of 65-75:25-35; more preferably, the mobile phase B is a mixed solution of acetonitrile-isopropanol in a volume ratio of 70:

30.

5. The detection method according to claim 1, characterized in that: The wavelength is 208-215 nm.

6. The detection method according to claim 10, characterized in that: The sample solution includes a resolution solution and a test solution. The resolution solution is prepared as follows: take appropriate amounts of dodecyldimethylbenzylammonium chloride, tetradecyldimethylbenzylammonium chloride, octadecyldimethylbenzylammonium chloride and cephalosporinium chloride reference substances, accurately weigh them, and quantitatively dilute them with 50% acetonitrile aqueous solution to prepare a solution containing approximately 1 mg of cephalosporin chloride, 0.005 mg of C12, 0.005 mg of C14, and 0.005 mg of C18 per 1 ml, as the resolution solution; the test solution has a concentration of 1.0 mg / ml, and is prepared as follows: take an appropriate amount of the product, quantitatively dilute it with 50% acetonitrile aqueous solution to prepare a solution containing approximately 1 mg of cephalosporin chloride per 1 ml, as the test solution.

7. A method for detecting the alkyl composition ratio of cetaxel, comprising the following steps: (1) Sample solution preparation; (2) using high performance liquid chromatography to detect the sample solution; The chromatographic conditions of the high performance liquid chromatography method include: Chromatographic column: Strong acid cation exchange bonded silica gel is used as filler; Mobile phase: isocratic elution with a mixed solution of mobile phase A and mobile phase B in a volume ratio of 45:55; Wherein mobile phase A is a mixture of 10-20 mM sodium perchlorate and 10 mM potassium hexafluorophosphate (adjusted to pH 3.0-4.0 with 10%-15% phosphoric acid) Mobile phase B: a mixed solution of acetonitrile and isopropanol with a volume ratio of 65-75:25-35 Flow rate: 0.3~0.8ml / min Injection volume: 10~20μl Detection wavelength: 208~215nm Column temperature: 30~40℃.

8. The method for detecting the alkyl composition ratio of cetaxelamide chloride according to any one of claims 1 to 7, and its use in detecting the alkyl composition of cetaxelamide chloride in cetaxelamide chloride bulk drug and / or preparation products.

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