Detection method and application of related substances in lidocaine hydrochloride preparation

Abstract

The invention relates to the technical field of medicine detection, in particular to a method for detecting related substances in a lidocaine hydrochloride preparation and application. According to the detection method disclosed by the invention, octadecylsilane chemically bonded silica is adopted as a filling agent of a chromatographic column; the mobile phase A is a mixed solution of 0.015 to 0.025 mol / L of monopotassium phosphate solution and acetonitrile; and the mobile phase B is acetonitrile. According to the detection method disclosed by the invention, all related substances can be detected in only 30 minutes, so that the detection speed is remarkably increased. According to the method disclosed by the invention, 11 impurities and added auxiliary materials can be detected, the separation degree between impurity peaks is greater than 1.5 and can be accurately quantified, and a water-soluble high-molecular compound in the preparation has no influence on impurity detection. The method disclosed by the invention is good in specificity, high in reproducibility, high in sensitivity and accuracy, suitable for determining related substances in the lidocaine hydrochloride preparation, and efficient, accurate and reliable in result.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a detection method and application of related substances in a lidocaine hydrochloride preparation. Background Art

[0002] Lidocaine hydrochloride is an amide local anesthetic primarily used for block anesthesia and epidural anesthesia, and is also used to treat arrhythmias. Related substances in lidocaine hydrochloride primarily originate from starting materials, intermediates, side reaction products that may be introduced during the production process, and degradation products during storage. High-performance liquid chromatography is currently used to detect related substances in lidocaine hydrochloride preparations.

[0003] Currently, lidocaine hydrochloride can be prepared in a variety of dosage forms, including injections, gels, oral solutions, aerosols, tablets, ointments, suppositories, patches, plasters, and ophthalmic preparations (eye drops, eye ointments, eye creams, and eye gels). However, depending on the dosage form, various excipients, such as solvents, matrices, and additives, are added. Certain water-soluble polymers, when added as excipients, can remain in the chromatographic column during testing, severely impacting column efficiency.

[0004] Furthermore, existing methods for detecting related substances in lidocaine preparations generally take too long. For example, patent application CN115141115A, "Impurities in lidocaine hydrochloride, preparation method, and detection method thereof," uses gradient elution conditions with an elution time of 65 minutes. Patent application CN111208236A, "A method for determining related substances in lidocaine hydrochloride by high-performance liquid chromatography," detects 11 impurities with an elution time of 50 minutes, and the retention time of the last impurity is 40.312 minutes.

[0005] Therefore, there is an urgent need for a rapid and effective method for detecting related substances in lidocaine hydrochloride preparations. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a method for detecting related substances in lidocaine hydrochloride preparations and its application.

[0007] The present invention provides a method for detecting related substances in a lidocaine hydrochloride preparation. The chromatographic conditions adopted in the detection method include: using octadecylsilane bonded silica gel as a filler in a chromatographic column;

[0008] Mobile phase: Mobile phase A is a mixture of 0.015-0.025 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 6.5-7.5:2.5-3.5; mobile phase B is acetonitrile;

[0009] The elution method adopts gradient elution, and the gradient elution procedure is as follows:

[0010] 0-10 minutes: mobile phase A uses a first concentration, and mobile phase B uses a second concentration; the first concentration is any value from 95% to 100%, the second concentration is any value from 0 to 5%, and the sum of the first and second concentrations is 100%;

[0011] 10-23 minutes: Mobile phase A changes from the first concentration to the third concentration at a constant rate, and mobile phase B changes from the second concentration to the fourth concentration at a constant rate;

[0012] 23-33 minutes: mobile phase A uses the third concentration, and mobile phase B uses the fourth concentration; the third concentration is any value from 55% to 65%, the fourth concentration is any value from 35% to 45%, and the sum of the third concentration and the fourth concentration is 100%.

[0013] Optionally, after the detection is completed, a process of returning the mobile phase to its original equilibrium is also included, preferably from 33.1 minutes to 38 to 42 minutes, with mobile phase A at 95% to 100% and mobile phase B at 0 to 5%.

[0014] The present invention also proposes the application of the above-mentioned detection method in detecting lidocaine hydrochloride preparations, which include liquid preparations and semisolid preparations, and water-soluble polymer compounds are added to the preparations; liquid preparations include oral liquid preparations and injections, and semisolid preparations include gels, gel patches or ointments.

[0015] The technical solution provided by the embodiment of the present invention has the following advantages compared with the existing technology:

[0016] The detection method of the present invention significantly improves the detection speed, and all relevant substances can be detected in just 30 minutes.

[0017] The detection method of the present invention avoids the column efficiency attenuation caused by the water-soluble high molecular compound in the auxiliary materials added to the preparation remaining in the chromatographic column.

[0018] The detection method of the present invention can detect 11 known impurities and 2 unknown impurities related to lidocaine hydrochloride, and the separation between each impurity peak is greater than 1.5, and the correlation coefficient R 2 The average recovery rates of all impurities were between 95% and 105%, indicating that the method of the present invention has good specificity and high reproducibility, is suitable for the determination of related substances in lidocaine hydrochloride preparations, and the results are accurate and reliable.

[0019] The detection method of the present invention has higher sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the HPLC chromatogram of blank solvent;

[0021] Figure 2 is the HPLC chromatogram of the mixed reference solution;

[0022] Figure 3 HPLC chromatogram of the test solution (including all impurities);

[0023] Figures 4 to 7 is the HPLC chromatogram of each positioning solution;

[0024] Figure 8 This is the linearity test result of impurity B;

[0025] Figure 9 is the linear test result of impurity H;

[0026] Figure 10 This is the linearity test result of impurity A;

[0027] Figure 11 This is the result of lidocaine hydrochloride linearity test;

[0028] Figure 12 HPLC chromatogram of the reference solution D1 in Comparative Example 1;

[0029] Figure 13 is the HPLC chromatogram of the test solution in Comparative Example 1;

[0030] Figure 14 is the HPLC chromatogram of the test solution in Comparative Example 2;

[0031] Figure 15 is the HPLC chromatogram of the test solution in Comparative Example 3;

[0032] Figure 16 is the HPLC chromatogram of the test solution in Comparative Example 4;

[0033] Figure 17 is the HPLC chromatogram of the test solution in Comparative Example 5;

[0034] Figure 18 is the HPLC chromatogram of the test solution in Comparative Example 6;

[0035] Figure 19 is the HPLC chromatogram of the test solution in Comparative Example 7;

[0036] Figure 20 is the HPLC chromatogram of the test solution in Comparative Example 8;

[0037] Figure 21 This is the HPLC chromatogram of the test solution in Comparative Example 9. DETAILED DESCRIPTION

[0038] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features therein can be combined with each other.

[0039] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all the embodiments.

[0040] The embodiment of the present invention provides a method for detecting related substances in a lidocaine hydrochloride preparation. The chromatographic conditions used in the detection method include: using octadecylsilane bonded silica gel as a filler in the chromatographic column;

[0041] Mobile phase: Mobile phase A is a mixture of 0.015-0.025 mol / L potassium dihydrogen phosphate solution and acetonitrile at a volume ratio of 6.5-7.5:2.5-3.5; mobile phase B is acetonitrile; gradient elution is adopted:

[0042] 0-10 minutes: mobile phase A uses a first concentration, and mobile phase B uses a second concentration; the first concentration is any value from 95% to 100%, the second concentration is any value from 0 to 5%, and the sum of the first and second concentrations is 100%;

[0043] 10-23 minutes: Mobile phase A changes from the first concentration to the third concentration at a constant rate, and mobile phase B changes from the second concentration to the fourth concentration at a constant rate;

[0044] 23-33 minutes: mobile phase A uses the third concentration, and mobile phase B uses the fourth concentration; the third concentration is any value from 55% to 65%, the fourth concentration is any value from 35% to 45%, and the sum of the third concentration and the fourth concentration is 100%.

[0045] Further preferably, the first concentration can be 96%, 97%, 98%, 99%, 100%, further preferably any value from 98% to 100%, and the second concentration can be 4%, 3%, 2%, 1%, 0; further preferably any value from 0 to 2%.

[0046] Further preferably, the third concentration may be 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, or 64%; further preferably, it may be any value from 58% to 62%, and the fourth concentration may be 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, or 44%; further preferably, it may be any value from 38% to 42%, and more preferably, the third concentration may be any value from 59% to 61%, and the fourth concentration may be any value from 39% to 41%.

[0047] In the embodiment of the present invention, the main component (lidocaine hydrochloride) peak is controlled to appear within the second stage. The main component (lidocaine hydrochloride) peak is allowed to appear as quickly as possible through gradient change. At the same time, in order to avoid a baseline rise caused by a too rapid gradient change, the end time of the second stage is set to 23 minutes. This takes into account both shortening the detection and analysis time and maintaining a relatively stable baseline, making the detection more efficient and accurate.

[0048] Since lidocaine hydrochloride preparations may contain a small amount of water-soluble high molecular weight compounds, they may adversely affect the column efficiency of high performance liquid chromatography. Existing technologies for improving column efficiency include the following technical means:

[0049] 1. Reduce the flow rate of the mobile phase. The disadvantage is that it will prolong the analysis time.

[0050] 2. Reduce the amount of stationary phase. The disadvantage is that the sample loading amount in the chromatographic column is also reduced, and the detection of trace related substances cannot be ensured.

[0051] 3. Reduce the particle size of the stationary phase. The disadvantage is that if the particle size is too small, the permeability of the chromatographic column will also decrease.

[0052] 4. Choose a low-viscosity mobile phase to facilitate rapid mass transfer. The disadvantage is that it is not conducive to multi-component analysis.

[0053] 5. Appropriately increase the column temperature to reduce the viscosity of the flow. The disadvantage is that the column efficiency and separation degree will also decrease.

[0054] 6. Try to reduce the volume of stagnant mobile phase. The disadvantage is that the flow rate of the mobile phase needs to be increased.

[0055] Due to the large number of related substances in lidocaine hydrochloride preparations, the aforementioned methods for improving column efficiency are insufficient when aiming to increase detection speed. Through innovative research, the inventors discovered that mobile phase A, a mixture of a low-concentration potassium dihydrogen phosphate solution and acetonitrile in a specific ratio, can elute water-soluble polymers, thereby avoiding the column efficiency degradation caused by residual water-soluble polymers and significantly improving column efficiency.

[0056] As an improvement to the embodiment of the present invention, the gradient elution procedure may be:

[0057] 0-10 minutes, mobile phase A is 100%, mobile phase B is 0;

[0058] From 10 to 23 minutes, mobile phase A was changed from 100% to 60% at a constant speed, and mobile phase B was changed from 0% to 40% at a constant speed;

[0059] 23-33 minutes, mobile phase A is 60%, mobile phase B is 40%.

[0060] As an improvement to the embodiment of the present invention, after the detection is completed, a process of returning the mobile phase to its original equilibrium is preferably adopted: from 33.1 minutes to 38-42 minutes, the mobile phase A is 95%-100%, and the mobile phase B is 0-5%. The equilibrium process can further be adopted: from 33.1 minutes to 40 minutes, the mobile phase A is 100% and the mobile phase B is 0.

[0061] As an improvement to the embodiment of the present invention, mobile phase A is a mixed solution of 0.018-0.022 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 6.5-7.5:2.5-3.5.

[0062] As an improvement to the embodiment of the present invention, mobile phase A is a mixed solution of 0.018-0.022 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 6.8-7.2:2.8-3.2.

[0063] As an improvement to the embodiment of the present invention, mobile phase A is a mixed solution of 0.02 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 6.8-7.2:2.8-3.2.

[0064] As a specific implementation of the embodiment of the present invention, mobile phase A is a mixed solution of 0.02 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 7:3.

[0065] The pH value of the potassium dihydrogen phosphate solution in mobile phase A is 7.8 to 8.1, preferably 8.0, and can be adjusted using a 10 mol / L sodium hydroxide solution.

[0066] The technical solution consisting of the mobile phase and gradient elution conditions of the embodiment of the present invention not only improves the detection speed, but also completes the detection of all related substances in only 30 minutes, and has a stable baseline and good separation. The separation between lidocaine hydrochloride, 11 related substances and other added excipients in the preparation is greater than 1.5, ensuring the sensitivity and accuracy of the detection.

[0067] As an improvement of the embodiment of the present invention, the particle size of the chromatographic column is 3-4 μm; the diameter of the chromatographic column is 4-5 mm, the column length is 150 mm, and the pore size is For example, Xtimate C18, 4.6 mm × 150 mm, with a particle size of 3 μm and a pore size of

[0068] As an improvement to the embodiment of the present invention, the detection wavelength of the detection method of the embodiment of the present invention is 225 nm to 235 nm, and can further be 228 nm to 232 nm, for example, 230 nm.

[0069] As an improvement to the embodiment of the present invention, the flow rate of the detection method of the embodiment of the present invention is 0.8-1.2 mL / min, for example, 1.0 mL / min can be used.

[0070] As an improvement to the embodiment of the present invention, the column temperature of the detection method of the embodiment of the present invention is 30-40°C, for example, 35°C can be used.

[0071] As an improvement to the embodiment of the present invention, the injection volume of the detection method of the embodiment of the present invention is 10 to 30 μL, and can further be 15 to 25 μL, for example, 20 μL.

[0072] As a specific implementation of the embodiment of the present invention, the test solution is diluted with mobile phase A to prepare a solution containing 1 to 10 mg of lidocaine hydrochloride per 1 mL.

[0073] As a specific implementation of the present invention, the reference solution was diluted with mobile phase A to prepare a mixed solution containing approximately 0.25 μg of impurity A, 0.15 μg of impurity H, 5 μg of impurity B, and 5 μg of lidocaine hydrochloride per 1 mL.

[0074] As a specific implementation of an embodiment of the present invention, a method for detecting related substances in a lidocaine hydrochloride preparation comprises the following steps:

[0075] S1. Preparation of reference solution: Accurately weigh impurity A, impurity H, impurity B, and lidocaine hydrochloride reference substance, dissolve them in mobile phase A, and dilute to make a mixed solution containing approximately 0.25 μg of impurity A, 0.15 μg of impurity H, 5 μg of impurity B, and 5 μg of lidocaine hydrochloride per 1 mL to obtain the reference solution.

[0076] S2. Preparation of test solution: Take lidocaine hydrochloride preparation sample and dissolve it in mobile phase A to prepare a solution containing 5 mg of lidocaine hydrochloride per 1 mL to obtain the test solution.

[0077] S3. Use high performance liquid chromatography to detect the test solution and reference solution, and calculate the content of related substances in lidocaine hydrochloride.

[0078] The calculation formula is as follows:

[0079] For impurity A, impurity H, and impurity B:

[0080]

[0081] A 杂 : The peak area of the impurity in the spectrum obtained from the test solution;

[0082] A 对 : The peak area of the impurity in the spectrum obtained from the reference solution;

[0083] C 供 : concentration of test solution, μg / mL;

[0084] C 对 : Concentration of each impurity in the reference solution, μg / mL.

[0085] For unknown impurities:

[0086]

[0087] A 杂 : The peak area of the impurity in the spectrum obtained from the test solution;

[0088] A 对 : The peak area of lidocaine hydrochloride in the spectrum obtained from the reference solution;

[0089] C 供 : concentration of test solution, μg / mL;

[0090] C 对 : Concentration of lidocaine hydrochloride in the reference solution, μg / mL.

[0091] In the step of preparing the test solution, if the viscosity of the prepared test solution is high, a filtration step can be added before sampling. For example, the test solution can be filtered through a 0.45 μm polytetrafluoroethylene (PTFE) filter membrane without discarding the filtrate, thereby filtering out some water-soluble polymer compounds in the test solution. By examining the impurity content of the samples before and after filtration, it was found that the filter membrane had no adsorption effect on various impurities.

[0092] The embodiments of the present invention also relate to the application of the above detection method in detecting lidocaine hydrochloride preparations. The lidocaine hydrochloride preparations can be liquid preparations or semisolid preparations.

[0093] Specifically, lidocaine hydrochloride liquid preparations include oral liquid preparations, external liquid preparations and injections. Lidocaine hydrochloride oral liquid preparations refer to liquid preparations for oral administration composed of lidocaine hydrochloride dispersed in a liquid dispersion medium, such as low molecular weight solutions, polymer solutions or syrups. Lidocaine hydrochloride external liquid preparations refer to liquid preparations for external use composed of lidocaine hydrochloride dispersed in a liquid dispersion medium, and include: skin lotions, skin liniments, ear washes, nasal drops, gargles, tooth drops, enemas, vaginal douches and urethral douches, etc., according to the administration site. The hydrophilic polymer solution of the lidocaine hydrochloride liquid preparation can improve the physical properties of the preparation, enhance its stability and use effect.

[0094] Semisolid preparations of lidocaine hydrochloride specifically refer to uniform semisolid preparations formed by dispersing lidocaine hydrochloride in an aqueous gel matrix, including gels, gel patches, or ointments using an aqueous gel matrix. The gel (jelly / gel) of the embodiments of the present invention refers to a thick liquid or semisolid preparation in the form of a solution, suspension, or emulsion of lidocaine hydrochloride and a gel-forming excipient. Gels are generally used on the skin and body cavities (such as the nasal cavity, vagina, and rectum). Gel ointments, also known as gel patches, are patches made by mixing lidocaine hydrochloride with a hydrophilic matrix and applying it to a backing material. The gel patch consists of a backing layer, a paste layer, and an anti-sticking layer that needs to be removed before use. The gel patch can be used on intact skin surfaces or on skin surfaces with diseases. The ointment of the embodiments of the present invention refers to a semisolid topical preparation with a certain consistency made by uniformly mixing lidocaine hydrochloride with an aqueous gel matrix. Aqueous gel matrices are composed of water-soluble polymers, and can be made from natural, semi-synthetic, and synthetic polymers. Commonly used aqueous gel matrices include alginate, gelatin, pectin, cellulose derivatives, starch and its derivatives, povidone, polyvinyl alcohol and its derivatives, and polyacrylic acids such as carbomer and polyacrylic acid. Cellulose derivatives include methylcellulose and its derivatives, ethylcellulose and its derivatives, and hydroxypropylcellulose and its derivatives.

[0095] The molecular weight of the water-soluble polymer compounds added to lidocaine hydrochloride preparations is typically very large. If not thoroughly eluted, the life of the chromatographic column will be drastically reduced. When the concentration of the water-soluble polymer compounds in the lidocaine hydrochloride preparation is high, a pretreatment method can be used to precipitate the water-soluble polymer compounds, filter them, and then perform detection. However, this pretreatment step increases the workload and affects the recovery rate. Furthermore, when the concentration of the water-soluble polymer compounds in the lidocaine hydrochloride preparation is low, it is difficult to completely precipitate different types of water-soluble polymer compounds using a single method, and this may also affect the accuracy of the detection of related substances in the lidocaine hydrochloride preparation. The detection method of the embodiment of the present invention, when applied to the detection of lidocaine preparations, adopts the technical concept of eluting the water-soluble polymer compounds. This not only greatly simplifies the operating steps, but also has a wider application range and ensures the detection effect of related substances. It can detect 11 known impurities and 2 unknown impurities, and has good separation from other additives. The detection method of the present invention avoids the column efficiency degradation caused by the addition of water-soluble polymers to lidocaine hydrochloride preparations, thereby improving the tolerance of the chromatographic column and reducing detection costs. Furthermore, methodological validation studies have demonstrated that the detection method of the present invention exhibits high accuracy, strong specificity, good reproducibility, stability, and durability.

[0096] Example 1

[0097] This example provides a method for detecting related substances in a lidocaine hydrochloride preparation:

[0098] 1. Preparation of reference solution: Accurately weigh impurity A, impurity H, impurity B, and lidocaine hydrochloride reference substance, dissolve them in mobile phase A, and quantitatively dilute to make a mixed solution containing approximately 0.25 μg of impurity A, 0.15 μg of impurity H, 5 μg of impurity B, and 5 μg of lidocaine hydrochloride per 1 mL.

[0099] 2. Preparation of test solution: Dilute the lidocaine hydrochloride preparation to be tested with mobile phase A to a solution containing approximately 5 mg of lidocaine hydrochloride per 1 mL to obtain the test solution.

[0100] 3. Use high performance liquid chromatography to test the test solution and reference solution, and calculate the content of related substances in lidocaine hydrochloride. The chromatographic conditions are as follows:

[0101] The chromatographic column was filled with octadecylsilane bonded silica gel (Xtimate C18, 4.6 × 150 mm, 3 μm);

[0102] Mobile phase A: a mixture of 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution) and acetonitrile in a volume ratio of 70:30;

[0103] Mobile phase B: acetonitrile.

[0104] The gradient elution program is shown in Table 1:

[0105] Table 1

[0106]

[0107] Detection wavelength: 230nm;

[0108] Column temperature: 35°C;

[0109] Flow rate: 1.0 mL / min;

[0110] Injection volume: 20 μL.

[0111] The calculation formula is as follows:

[0112] For impurity A, impurity H, and impurity B:

[0113]

[0114] A 杂 : The peak area of the impurity in the spectrum obtained from the test solution;

[0115] A 对 : The peak area of the impurity in the spectrum obtained from the reference solution;

[0116] C 供 : concentration of test solution, μg / mL;

[0117] C 对 : Concentration of each impurity in the reference solution, μg / mL.

[0118] For unknown impurities:

[0119]

[0120] A 杂 : The peak area of the impurity in the spectrum obtained from the test solution;

[0121] A 对 : The peak area of lidocaine hydrochloride in the spectrum obtained from the reference solution;

[0122] C 供 : concentration of test solution, μg / mL;

[0123] C 对 : Concentration of lidocaine hydrochloride in the reference solution, μg / mL.

[0124] Example 2

[0125] This example is used to verify the system applicability of the detection method of the embodiment of the present invention:

[0126] 1. Solution preparation

[0127] Blank solvent (same as mobile phase A): 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30).

[0128] Test sample stock solution: Accurately weigh 2 g of lidocaine hydrochloride reference substance, 1 mg of PEG-400, 1 mg of hydroxypropyl methylcellulose, 1 mg of sodium alginate, 0.1 mg of methylparaben, and 0.1 mg of ethylparaben, dissolve them in purified water and quantitatively dilute to 100 mL.

[0129] Impurity A stock solution: accurately weigh 12.5 mg of impurity A reference substance, dissolve it in blank solvent and quantitatively dilute it to 100 mL; Impurity H stock solution: accurately weigh 7.5 mg of impurity H reference substance, dissolve it in blank solvent and quantitatively dilute it to 100 mL; Impurity B stock solution: accurately weigh 10 mg of impurity B reference substance, dissolve it in blank solvent and quantitatively dilute it to 20 mL; Lidocaine hydrochloride stock solution: accurately weigh 10 mg of lidocaine hydrochloride reference substance, dissolve it in blank solvent and quantitatively dilute it to 20 mL; Impurity C stock solution: accurately weigh 2 mg of impurity C reference substance, dissolve it in blank solvent and quantitatively dilute it to 10 mL; Impurity D stock solution: accurately weigh 2 mg of impurity D reference substance, dissolve it in blank solvent and quantitatively dilute it Dilute to 10mL; Impurity E stock solution: accurately weigh 2 mg of impurity E reference substance, dissolve it in blank solvent and quantitatively dilute to 10mL; Impurity F stock solution: accurately weigh 2 mg of impurity F reference substance, dissolve it in blank solvent and quantitatively dilute to 10mL; Impurity G stock solution: accurately weigh 2 mg of impurity G reference substance, dissolve it in blank solvent and quantitatively dilute to 10mL; Impurity I stock solution: accurately weigh 2 mg of impurity I reference substance, dissolve it in blank solvent and quantitatively dilute to 10mL; Impurity J stock solution: accurately weigh 2 mg of impurity J reference substance, dissolve it in blank solvent and quantitatively dilute to 10mL; Impurity K stock solution: accurately weigh 2 mg of impurity K reference substance, dissolve it in blank solvent and quantitatively dilute to 10mL.

[0130] Reference substance stock solution: Accurately measure 1 mL of impurity A stock solution, 1 mL of impurity H stock solution, 5 mL of impurity B stock solution, and 5 mL of lidocaine hydrochloride stock solution, and quantitatively dilute to 100 mL with blank solvent.

[0131] Reference substance solution: Accurately measure 4 mL of reference substance stock solution and quantitatively dilute it to 20 mL with blank solvent.

[0132] Mixed reference solution: Accurately measure 4 mL of reference stock solution, 500 μL each of impurity C to G stock solutions, and 500 μL each of impurity I to K stock solutions, and quantitatively dilute to 20 mL with blank solvent.

[0133] Test solution (including all impurities): Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, add 500 μL each of the impurity C to G stock solutions, add 500 μL each of the impurity I to K stock solutions, and quantitatively dilute to 20 mL with blank solvent.

[0134] Positioning solution: Impurity A positioning solution: accurately measure 2 mL of impurity A stock solution and dilute it to 10 mL with blank solvent; Impurity B positioning solution: accurately measure 400 μL of impurity B stock solution and dilute it to 10 mL with blank solvent; Impurity H positioning solution: accurately measure 2 mL of impurity H stock solution and dilute it to 10 mL with blank solvent; Lidocaine hydrochloride positioning solution: accurately measure 400 μL of lidocaine hydrochloride stock solution and dilute it to 10 mL with blank solvent; Impurity C positioning solution: accurately measure 100 μL of impurity C stock solution and dilute it to 1 mL with blank solvent; Impurity D positioning solution: accurately measure 100 μL of impurity D stock solution and dilute it to 1 mL with blank solvent Impurity E localization solution: accurately measure 100 μL of impurity E stock solution and quantitatively dilute it to 1 mL with blank solvent; Impurity F localization solution: accurately measure 100 μL of impurity F stock solution and quantitatively dilute it to 1 mL with blank solvent; Impurity G localization solution: accurately measure 100 μL of impurity G stock solution and quantitatively dilute it to 1 mL with blank solvent; Impurity I localization solution: accurately measure 100 μL of impurity I stock solution and quantitatively dilute it to 1 mL with blank solvent; Impurity J localization solution: accurately measure 100 μL of impurity J stock solution and quantitatively dilute it to 1 mL with blank solvent; Impurity K localization solution: accurately measure 100 μL of impurity K stock solution and quantitatively dilute it to 1 mL with blank solvent.

[0135] 2. Detection

[0136] Accurately measure 20 μL of blank solvent, reference solution, test solution (including all impurities), and positioning solution, respectively, and inject them into the high performance liquid chromatograph. Detect according to the method of Example 1 and record the chromatogram as shown in the figure below. Figures 1 to 7 shown. Figure 1 is the HPLC chromatogram of blank solvent, Figure 2 is the HPLC chromatogram of the mixed reference solution. Figure 3 is the HPLC chromatogram of the test solution (containing all impurities), Figures 4 to 7 1 and 2 are HPLC chromatograms of each positioning solution.

[0137] 3. Test results

[0138] The retention times in each chromatogram are shown in Table 2.

[0139] Table 2

[0140]

[0141] exist Figures 1 to 3 In the spectrum shown, the separation between the impurity peaks is shown in Table 3:

[0142] Table 3

[0143]

[0144]

[0145] As shown in Tables 2 and 3, the blank solvent and blank excipient solutions did not interfere with impurity detection, and the separation between each component and adjacent peaks was greater than 1.5. Among them, the separation between impurity A and impurity H in the reference solution was greater than 8, which met the test requirements.

[0146] Example 3

[0147] This example is used to verify the quantitative limit and detection limit of the detection method of the embodiment of the present invention:

[0148] 1. Solution preparation

[0149] The preparation methods of blank solvent, impurity A stock solution, impurity H stock solution, impurity B stock solution, and lidocaine hydrochloride stock solution are the same as those in Example 2.

[0150] Impurity B intermediate solution: Accurately measure 1 mL of Impurity B stock solution and quantitatively dilute it to 100 mL with blank solvent;

[0151] Impurity H intermediate solution: Accurately measure 1 mL of the impurity H stock solution and quantitatively dilute it to 50 mL with blank solvent;

[0152] Impurity A intermediate solution: Accurately measure 1 mL of the impurity A stock solution and quantitatively dilute it to 50 mL with blank solvent;

[0153] Lidocaine hydrochloride intermediate solution: Accurately measure 1 mL of lidocaine hydrochloride stock solution and quantitatively dilute it to 100 mL with blank solvent;

[0154] Mixed intermediate solution: Accurately measure 1.1 mL of impurity B intermediate solution, 7.5 mL of impurity H intermediate solution, 1.4 mL of impurity A intermediate solution, and 2.5 mL of lidocaine hydrochloride intermediate solution, and quantitatively dilute to 50 mL with blank solvent;

[0155] Quantitation limit solution: accurately measure 4 mL of the mixed intermediate solution and quantitatively dilute it to 20 mL with blank solvent;

[0156] Detection limit solution: Accurately measure 3 mL of quantification limit solution and quantitatively dilute it to 10 mL with blank solvent.

[0157] 2. Detection

[0158] Accurately measure 20 μL each of blank solvent, quantitative limit solution, and detection limit solution, inject them into the high performance liquid chromatograph, and record the chromatogram.

[0159] 3. Test results

[0160] The test results of detection limit and quantification limit are shown in Table 4:

[0161] Table 4

[0162] name Detection limit (μg / mL) Limit of quantification (μg / mL) Peak area RSD of 6 consecutive injections Impurity B 0.0065 0.0217 3.24％ Impurity H 0.0136 0.0452 3.84％ Impurity A 0.0049 0.0165 3.98％ Lidocaine hydrochloride 0.0142 0.0472 1.63％

[0163] According to the above test results, the signal-to-noise ratio of the detection limit solution of each component was >3, the signal-to-noise ratio of the quantification limit solution was >10, the quantification limit solution was injected continuously for 6 times, the peak area RSD was <10%, and the sensitivity met the quantitative requirements.

[0164] Example 4

[0165] This example is used to verify the linear relationship of the detection method of the embodiment of the present invention:

[0166] 1. Solution preparation

[0167] The preparation method of blank solvent and reference substance stock solution is the same as that of Example 2. The preparation method of quantitative limit solution is the same as that of Example 3.

[0168] Linear solution-LOQ: The quantification limit solution is used as the linear solution-LOQ.

[0169] Linear solution-1: Accurately measure 2 mL of the reference stock solution and quantitatively dilute it to 20 mL with blank solvent;

[0170] Linear solution-2: Accurately measure 3 mL of the reference stock solution and quantitatively dilute it to 20 mL with blank solvent;

[0171] Linear solution-3: Accurately measure 4 mL of the reference stock solution and quantitatively dilute it to 20 mL with blank solvent;

[0172] Linear solution-4: Accurately measure 6 mL of the reference stock solution and quantitatively dilute it to 20 mL with blank solvent;

[0173] Linear solution-5: Accurately measure 8 mL of the reference stock solution and quantitatively dilute it to 20 mL with blank solvent.

[0174] 2. Detection

[0175] Accurately measure 20 μL of blank solvent and each linear solution, inject them into high performance liquid chromatography, perform detection according to the method of Example 1, and record the chromatogram.

[0176] 3. Test results

[0177] The linear ranges of impurity B, impurity H, impurity A and lidocaine hydrochloride are shown in Table 5, and the linear relationship diagram is shown in Figures 8 to 11 shown.

[0178] Table 5

[0179] name Linear range (μg / mL) Correlation coefficient Impurity B 0.0217～9.8708 R＝0.9999 Impurity H 0.0452～0.3016 R＝0.9977 Impurity A 0.0165～0.5884 R＝0.9999 Lidocaine hydrochloride 0.0472～9.4497 R＝0.9999

[0180] according to Figures 8 to 11 It can be seen that the correlation coefficient r of impurity B in the range of 0.0217-9.8708 μg / mL (equivalent to 0.4%-197.4% of the limit concentration) is 0.9999 (>0.990), the correlation coefficient r of impurity H in the range of 0.0452-0.3016 μg / mL (equivalent to 30.1%-201.1% of the limit concentration) is 0.9977 (>0.990), and the correlation coefficient r of impurity A in the range of 0.0165-0.5884 μg / mL is 0. The correlation coefficient r in the range of impurity B (equivalent to 6.6% to 235.4% of the sample concentration) is 0.9999 (>0.990), and the correlation coefficient r in the range of lidocaine hydrochloride 0.0472 to 9.4497 μg / mL (equivalent to 0.9% to 189.0% of the sample concentration) is 0.9999 (>0.990), both of which meet the test requirements, indicating that impurity B, impurity H, impurity A and lidocaine hydrochloride have a good linear relationship within the above concentration range.

[0181] Example 5

[0182] This example is used to verify the accuracy of the detection method of the embodiment of the present invention:

[0183] 1. Solution preparation

[0184] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution are the same as those in Example 2.

[0185] The preparation method of the mixed intermediate solution is the same as that of Example 3.

[0186] Reference substance solution: Accurately measure 4 mL of reference substance stock solution and quantitatively dilute it to 20 mL with blank solvent.

[0187] LOQ-Accuracy Test Solution: Accurately pipette 5 mL of the test sample stock solution, add 4 mL of the mixed intermediate solution, and quantitatively dilute to 20 mL with blank solvent; prepare three parallel aliquots.

[0188] 50%-accuracy test solution: Accurately pipette 5 mL of the test sample stock solution into a 20 mL volumetric flask, add 2 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent; prepare three replicates.

[0189] 100%-Accuracy Test Solution: Accurately pipette 5 mL of the test sample stock solution into a 20 mL volumetric flask, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent; prepare three replicates.

[0190] 150%-Accuracy Test Solution: Accurately pipette 5 mL of the test sample stock solution into a 20 mL volumetric flask, add 8 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent; prepare three replicates.

[0191] Blank excipient stock solution: Accurately weigh 1 mg of PEG-400, 1 mg of hydroxypropyl methylcellulose, 1 mg of sodium alginate, 0.1 mg of methylparaben, and 0.1 mg of ethylparaben, and quantitatively dilute to 100 mL with purified water.

[0192] Blank excipient solution: Accurately transfer 5 mL of blank excipient stock solution and quantitatively dilute to 20 mL with blank solvent; prepare 3 replicates in parallel.

[0193] 2. Detection

[0194] Accurately measure 20 μL each of blank solvent, blank excipient solution, reference solution, and accuracy test solution (12 portions), inject them into a high performance liquid chromatograph, detect according to the method of Example 1, record the chromatogram, and calculate the content of related substances.

[0195] 3. Test results

[0196] 3.1 The results of the accuracy test of impurity B are shown in Table 6:

[0197] Table 6

[0198]

[0199] According to the above test results, the recovery rates at each concentration point were between 90% and 108%, and the RSD was 2.57% (<10%), which met the test requirements and showed that the method had good accuracy.

[0200] 3.2 The results of the impurity H accuracy test are shown in Table 7:

[0201] Table 7

[0202]

[0203]

[0204] According to the above test results, the recovery rates at each concentration point were between 85% and 110%, and the RSD was 2.10% (<10%), which met the test requirements and showed that the method had good accuracy.

[0205] 3.3 The results of the accuracy test of impurity A are shown in Table 8:

[0206] Table 8

[0207]

[0208] According to the above test results, the recovery rates at each concentration point were between 85% and 110%, and the RSD was 2.65% (<10%), which met the test requirements and showed that the method had good accuracy.

[0209] Example 6

[0210] This example is used to verify the stability of the detection method of the embodiment of the present invention.

[0211] 1. Solution preparation

[0212] The preparation methods of blank solvent, test sample stock solution, reference sample stock solution, and reference sample solution are the same as those in Example 2.

[0213] Test solution: Accurately pipette 5 mL of the test sample stock solution and quantitatively dilute it to 20 mL with blank solvent.

[0214] Test solution: Accurately pipette 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent.

[0215] 2. Detection

[0216] Storage conditions: Place the reference solution, test solution, and test solution at room temperature under natural light for 48 hours.

[0217] Sampling time: 0h, 6h, 12h, 16h, 20h, 24h, 35h, 48h.

[0218] Accurately measure 20 μL each of blank solvent, reference solution, test solution, and test solution, and inject them into a high performance liquid chromatograph, perform detection according to the method of Example 1, and record the chromatogram.

[0219] 3. Test results

[0220] 3.1 The results of the stability test of the reference solution are shown in Table 9:

[0221] Table 9

[0222]

[0223]

[0224] According to the above test results, when the reference solution was placed at room temperature for 48 hours and then injected, the RSD of the peak areas of impurity B and lidocaine hydrochloride were no more than 3%, and the RSD of the peak areas of impurity H and impurity A were no more than 4%, which met the test requirements and showed that the reference solution was stable when placed at room temperature for 48 hours and then injected.

[0225] 3.2 The results of the stability test of the test solution are shown in Table 10:

[0226] Table 10

[0227]

[0228] According to the above test results, the test solution was placed at room temperature for 48 hours and sampled. Compared with 0 hours, the content changes at each inspection point were all within the range of ±10% of the 0 hour content, which met the test requirements, indicating that the test solution was stable after being placed at room temperature for 48 hours.

[0229] 3.3 The results of the test solution stability test are shown in Table 11:

[0230] Table 11

[0231] Inspection time Impurity B content (%) Impurity H content (%) Impurity A content (%) Unknown impurity 1 content (%) Unknown impurity 2 content (%) 0h 0.099 0.0030 0.0060 0.0123 0.0084 6h 0.098 0.0027 0.0060 0.0121 0.0084 12h 0.098 0.0027 0.0060 0.0123 0.0088 16h 0.097 0.0027 0.0060 0.0120 0.0086 20h 0.098 0.0029 0.0060 0.0124 0.0086 24h 0.098 0.0030 0.0060 0.0125 0.0085 35h 0.097 0.0029 0.0059 0.0119 0.0086 48h 0.097 0.0028 0.0059 0.0119 0.0086 0h±10％ 0.089％～0.109％ 0.0027％～0.0033％ 0.0054％～0.0066％ 0.0111％～0.0135％ 0.0076％～0.0092％

[0232] According to the above test results, the test solution was placed at room temperature for 48 hours and sampled. Compared with 0 hours, the content changes at each inspection point were all within the range of ±10% of the 0 hour content, which met the test requirements, indicating that the test solution was stable after being placed at room temperature for 48 hours.

[0233] Example 7

[0234] This example is used to verify the applicability of the detection method of this example.

[0235] 1. Solution preparation

[0236] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution are the same as those in Example 2.

[0237] Test solution: Accurately pipette 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent. Prepare mobile phase A according to the composition and pH shown in Table 12.

[0238] 2. Detection

[0239] Accurately measure 20 μL of the test solution and inject it into a high performance liquid chromatograph. Detection was performed using the chromatographic conditions shown in Table 12 (column temperature change ±5°C, flow rate change ±0.2 mL / min, wavelength change ±5 nm, pH change in mobile phase A 7.8-8.1, ratio change in mobile phase A ±0.2, salt concentration change in mobile phase A 0.002 mol / L) (the other conditions were the same as in Example 1), record the chromatogram, and calculate the impurity content.

[0240] 3. Results

[0241] The calculated impurity content is shown in Table 12:

[0242] Table 12

[0243]

[0244] According to the above test results, when the chromatographic conditions are slightly changed, the impurities are separated well and there is no significant difference in the amount of impurities detected, indicating that the chromatographic conditions of the embodiment of the present invention are durable.

[0245] Comparative Example 1

[0246] 1. Chromatographic conditions

[0247] Chromatographic column: Infinity Lab Poroshell 120EC-C18 column (150×4.6 mm, 4 μm);

[0248] Mobile phase: Mobile phase A is 0.035 mol / L potassium dihydrogen phosphate solution; mobile phase B is acetonitrile;

[0249] Elution mode: isocratic elution, mobile phase A: mobile phase B = 70:30;

[0250] Detection wavelength: 230nm;

[0251] Column temperature: 35°C;

[0252] Flow rate: 1.0 mL / min;

[0253] Injection volume: 20 μL.

[0254] 2. Solution preparation

[0255] The preparation methods of the test sample stock solution and the reference sample stock solution are the same as those in Example 2.

[0256] Reference substance stock solution D1: Accurately measure 1 mL of impurity A stock solution, 1 mL of impurity H stock solution, and 5 mL of lidocaine hydrochloride stock solution, and quantitatively dilute to 100 mL with blank solvent.

[0257] Blank solvent D1: 0.035 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30);

[0258] Reference solution D1: Accurately measure 4 mL of reference stock solution D1 and quantitatively dilute it to 20 mL with blank solvent D1;

[0259] Test solution: Accurately measure 5 mL of the test sample stock solution, add 14 mL of the reference sample stock solution D, and quantitatively dilute to 20 mL with blank solvent D1.

[0260] 3. Detection

[0261] Accurately measure 20 μL of reference solution D1 and test solution, inject them into high performance liquid chromatography for detection, and record the chromatogram.

[0262] 4. Results

[0263] The HPLC chromatogram of the reference solution D1 is as follows: Figure 12 The HPLC chromatogram of the test solution is shown in Figure 13 According to the test results, the elution time is long under this method, sodium carboxymethyl cellulose is difficult to elute, and the column efficiency drops sharply.

[0264] Comparative Example 2

[0265] 1. Chromatographic conditions

[0266] Chromatographic column: ChromCore C18 (150 × 4.6 mm, 3 μm);

[0267] Mobile phase: Mobile phase A is 0.035 mol / L potassium dihydrogen phosphate solution; mobile phase B is acetonitrile;

[0268] Elution mode: isocratic elution, mobile phase A: mobile phase B = 65:35;

[0269] Run time: 40 minutes;

[0270] Detection wavelength: 230nm;

[0271] Column temperature: 35°C;

[0272] Flow rate: 1.0 mL / min;

[0273] Injection volume: 20 μL.

[0274] 2. Solution preparation

[0275] The preparation methods of the test sample stock solution and the reference sample stock solution are the same as those in Example 2.

[0276] Blank solvent D1: 0.035 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30).

[0277] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent D1.

[0278] 3. Detection

[0279] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0280] 4. Results

[0281] The HPLC chromatogram of the test solution is as follows: Figure 14 According to the test results, the separation degree of impurities A and H from the auxiliary materials is good under this method, but the peak shape of the main peak is poor. During the repeated test, the column efficiency dropped sharply.

[0282] Comparative Example 3

[0283] 1. Chromatographic conditions

[0284] Column: Xtimate C18, 4.6 × 150 mm, 3 μm;

[0285] Mobile phase: Mobile phase A is 0.01 mol / L potassium dihydrogen phosphate solution; mobile phase B is acetonitrile;

[0286] Elution mode: isocratic elution, mobile phase A: mobile phase B = 55:45;

[0287] Running time: 50 minutes;

[0288] Detection wavelength: 230nm;

[0289] Column temperature: 35°C;

[0290] Flow rate: 1.0 mL / min;

[0291] Injection volume: 20 μL.

[0292] 2. Solution preparation

[0293] The preparation methods of the test sample stock solution and the reference sample stock solution are the same as those in Example 2.

[0294] Blank solvent D2: 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (50:50).

[0295] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent D2.

[0296] 3. Detection

[0297] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0298] 4. Results

[0299] The HPLC chromatogram of the test solution is as follows: Figure 15 According to the test results, impurity C overlaps with methylparaben under this method and is not suitable for the detection of related substances.

[0300] Comparative Example 4

[0301] 1. Chromatographic conditions

[0302] Column: Xtimate C18, 4.6 × 150 mm, 3 μm;

[0303] Mobile phase: Mobile phase A is 0.02 mol / L potassium dihydrogen phosphate solution; mobile phase B is acetonitrile;

[0304] Elution mode: isocratic elution, mobile phase A: mobile phase B = 7:3;

[0305] Running time: 50 minutes;

[0306] Detection wavelength: 230nm;

[0307] Column temperature: 35°C;

[0308] Flow rate: 1.0 mL / min;

[0309] Injection volume: 20 μL.

[0310] 2. Solution preparation

[0311] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution are the same as those in Example 2.

[0312] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent.

[0313] 3. Detection

[0314] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0315] 4. Results

[0316] The HPLC chromatogram of the test solution is as follows: Figure 16According to the test results, the main peak retention time under this method is 45 minutes, and the overall running time is 130 minutes, which is too long.

[0317] Comparative Example 5

[0318] 1. Chromatographic conditions

[0319] Column: Xtimate C18, 4.6 × 150 mm, 3 μm;

[0320] Mobile phase: Mobile phase A is 0.035 mol / L potassium dihydrogen phosphate solution; mobile phase B is acetonitrile;

[0321] The gradient elution program is shown in Table 13:

[0322] Table 13

[0323] Time (min) A(％) B(％) 0 60 40 10 60 40 20 30 70 25 30 70 25.1 60 40 30 60 40

[0324] Running time: 30 minutes;

[0325] Detection wavelength: 230nm;

[0326] Column temperature: 35°C;

[0327] Flow rate: 1.0 mL / min;

[0328] Injection volume: 20 μL.

[0329] 2. Solution preparation

[0330] The preparation methods of the test sample stock solution and the reference sample stock solution are the same as those in Example 2.

[0331] Blank solvent D1: 0.035 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30).

[0332] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent D1.

[0333] 3. Testing:

[0334] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0335] 4. Results:

[0336] The HPLC chromatogram of the test solution is as follows: Figure 17 According to the test results, all impurities were baseline separated, and the excipients did not interfere with the peaks of known impurities and lidocaine hydrochloride, but the column efficiency was significantly attenuated.

[0337] Comparative Example 6

[0338] 1. Chromatographic conditions

[0339] Column: Xtimate C18, 4.6 × 150 mm, 3 μm;

[0340] Mobile phase: Mobile phase A is 0.02 mol / L potassium dihydrogen phosphate solution; mobile phase B is acetonitrile;

[0341] The gradient elution program is shown in Table 14:

[0342] Table 14

[0343] Time (min) A(％) B(％) 0 70 30 10 70 30 20 30 70 25 30 70 25.1 70 30 30 70 30

[0344] Running time: 30 minutes;

[0345] Detection wavelength: 230nm;

[0346] Column temperature: 35°C;

[0347] Flow rate: 1.0 mL / min;

[0348] Injection volume: 20 μL.

[0349] 2. Solution preparation

[0350] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution were the same as those in Example 2.

[0351] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent.

[0352] 3. Detection

[0353] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0354] 4. Results

[0355] The HPLC chromatogram of the test solution is as follows: Figure 18 According to the test results, all known impurities were baseline separated, but there was interference at the retention time of lidocaine hydrochloride.

[0356] Comparative Example 7

[0357] 1. Chromatographic conditions

[0358] Column: Xtimate C18, 4.6 × 150 mm, 3 μm;

[0359] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30);

[0360] Mobile phase B: acetonitrile;

[0361] The gradient elution program is shown in Table 15:

[0362] Table 15

[0363] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 20 60 40 30 60 40 30.1 100 0 40 100 0

[0364] Detection wavelength: 230nm;

[0365] Column temperature: 35°C;

[0366] Flow rate: 1.0 mL / min;

[0367] Injection volume: 20 μL.

[0368] 2. Solution preparation

[0369] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution were the same as those in Example 2.

[0370] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent.

[0371] 3. Detection

[0372] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0373] 4. Results

[0374] The HPLC chromatogram of the test solution is as follows: Figure 19 As shown in the test results, the components in the test solution are separated from the adjacent peaks, and the blank solvent and blank excipients do not interfere with the detection of impurities. However, the baseline rises significantly, affecting the accuracy.

[0375] Comparative Example 8

[0376] 1. Chromatographic conditions

[0377] The chromatographic column was filled with octadecylsilane bonded silica gel (Xtimate C18, 4.6 × 150 mm, 3 μm);

[0378] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30);

[0379] Mobile phase B: acetonitrile;

[0380] The gradient elution program is shown in Table 16:

[0381] Table 16

[0382] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 0 10 90 0 23 40 60 33 40 60 33.1 90 0 40 90 0

[0383] Detection wavelength: 230nm;

[0384] Column temperature: 35°C;

[0385] Flow rate: 1.0 mL / min;

[0386] Injection volume: 20 μL.

[0387] 2. Solution preparation

[0388] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution were the same as those in Example 2.

[0389] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent.

[0390] 3. Detection

[0391] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0392] 4. Results

[0393] The HPLC chromatogram of the test solution is as follows: Figure 20 The test results show that impurity C and methylparaben are not separated, and impurity A, propylparaben and impurity E are not separated in the test solution. This method is not suitable for the detection of related substances.

[0394] Comparative Example 9:

[0395] 1. Chromatographic conditions

[0396] The chromatographic column was filled with octadecylsilane bonded silica gel (Xtimate C18, 4.6 × 150 mm, 3 μm);

[0397] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 8.0 with 10 mol / L sodium hydroxide solution)-acetonitrile (70:30);

[0398] Mobile phase B: acetonitrile;

[0399] The gradient elution conditions are shown in Table 17:

[0400] Table 17

[0401] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 60 40 35 60 40 35.1 100 0 42 100 0

[0402] Detection wavelength: 230nm;

[0403] Column temperature: 35°C;

[0404] Flow rate: 1.0 mL / min;

[0405] Injection volume: 20 μL.

[0406] 2. Solution preparation

[0407] The preparation methods of blank solvent, test sample stock solution, and reference sample stock solution were the same as those in Example 2.

[0408] Test solution: Accurately measure 5 mL of the test sample stock solution, add 4 mL of the reference sample stock solution, and quantitatively dilute to 20 mL with blank solvent.

[0409] 3. Detection

[0410] Accurately measure 20 μL of the test solution, inject it into the high performance liquid chromatograph for detection, and record the chromatogram.

[0411] 4. Results

[0412] The HPLC chromatogram of the test solution is as follows: Figure 21 The test results show that the components in the test solution are separated from the adjacent peaks, and the blank solvent and blank excipients do not interfere with the detection of each impurity. However, this method takes a long time to detect.

[0413] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments described herein, but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting related substances in a lidocaine hydrochloride preparation, characterized in that: The chromatographic conditions used in the detection method include: the chromatographic column uses octadecylsilane bonded silica gel as a filler; Mobile phase: Mobile phase A is a mixture of 0.015-0.025 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 6.5-7.5:2.5-3.5; mobile phase B is acetonitrile; The elution method adopts gradient elution, and the gradient elution program is: 0-10 minutes: mobile phase A uses the first concentration, and mobile phase B uses the second concentration; The first concentration is any value between 95% and 100%, the second concentration is any value between 0% and 5%, and the sum of the first concentration and the second concentration is 100%; 10-23 minutes: Mobile phase A changes from the first concentration to the third concentration at a constant rate, and mobile phase B changes from the second concentration to the fourth concentration at a constant rate; 23-33 minutes: mobile phase A uses the third concentration, and mobile phase B uses the fourth concentration; The third concentration is any value between 55% and 65%, the fourth concentration is any value between 35% and 45%, and the sum of the third concentration and the fourth concentration is 100%.

2. The detection method according to claim 1, wherein The gradient elution procedure is: 0-10 minutes, mobile phase A is 100%, mobile phase B is 0; From 10 to 23 minutes, mobile phase A was changed from 100% to 60% at a constant speed, and mobile phase B was changed from 0% to 40% at a constant speed; 23-33 minutes, mobile phase A is 60%, mobile phase B is 40%.

3. The detection method according to claim 1 or 2, characterized in that After the detection is completed, a process is included in which the mobile phase returns to its original equilibrium, preferably from 33.1 minutes to 38-42 minutes, with mobile phase A at 95%-100% and mobile phase B at 0-5%.

4. The detection method according to claim 3, characterized in that After the detection was completed, from 33.1 minutes to 40 minutes, mobile phase A was 100% and mobile phase B was 0.

5. The detection method according to claim 1, wherein Mobile phase A is a mixed solution of 0.018-0.022 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 6.8-7.2:2.8-3.2; Preferably, mobile phase A is a mixed solution of 0.02 mol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 7:

3.

6. The detection method according to claim 1 or 5, characterized in that The pH value of the potassium dihydrogen phosphate solution in mobile phase A is 7.8 to 8.1, preferably 8.0; more preferably, it is adjusted using a 10 mol / L sodium hydroxide solution.

7. The detection method according to claim 1 or 5, characterized in that The test solution and the reference solution were diluted with the mobile phase A; Optionally, the test solution is diluted with the mobile phase A to prepare a solution containing 1 to 10 mg of lidocaine hydrochloride per 1 mL; Optionally, the reference solution is diluted with the mobile phase A to prepare a mixed solution containing approximately 0.25 μg of impurity A, 0.15 μg of impurity H, 5 μg of impurity B, and 5 μg of lidocaine hydrochloride per 1 mL.

8. The detection method according to claim 1, wherein The particle size of the chromatographic column is 3-4 μm; the diameter of the chromatographic column is 4-5 mm, the column length is 150 mm, and the pore size is 9. The detection method according to claim 1, wherein Detection wavelength: 225-235 nm, preferably 230 nm; Flow rate: 0.8-1.2 mL / min, preferably 1.0 mL / min; Column temperature: 30-40°C, preferably 35°C; The injection volume is 10-30 μL, preferably 20 μL.

10. Use of the detection method according to any one of claims 1 to 9 in detecting lidocaine hydrochloride preparations, wherein the lidocaine hydrochloride preparations include liquid preparations and semisolid preparations; the liquid preparations include oral liquid preparations and injections; the semisolid preparations include gels, gel patches or ointments; and the preparations contain a water-soluble polymer compound.

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