HPLC (High Performance Liquid Chromatography) detection method for content of bacteriostatic agent and lidocaine hydrochloride in lidocaine hydrochloride gel

Abstract

The invention relates to the technical related field of pharmaceutical analytical chemistry, and particularly discloses an HPLC (High Performance Liquid Chromatography) detection method for the content of a bacteriostatic agent in lidocaine hydrochloride gel and the content of lidocaine hydrochloride. The detection method comprises the following steps: (1) preparing a reference solution and a test solution; (2) injecting the reference solution and the test solution into a liquid chromatograph, and carrying out separation detection by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler and a mobile phase in an isocratic elution mode; and (3) recording a chromatogram, and calculating the content of the bacteriostatic agent and the content of lidocaine hydrochloride in the test solution by peak area according to an external standard method. The detection method provided by the invention can rapidly and accurately detect the content of the bacteriostatic agent in the lidocaine hydrochloride gel and the content of lidocaine hydrochloride, has the advantages of strong specificity, good separation degree, high accuracy and good reproducibility, and can be used for quality control of the lidocaine hydrochloride gel in actual production.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical analytical chemistry technology, and specifically discloses an HPLC detection method for the content of an antibacterial agent and the content of lidocaine hydrochloride in lidocaine hydrochloride gel. Background Art

[0002] Lidocaine hydrochloride is a common amide local anesthetic that exerts a local anesthetic effect by reversibly blocking sodium channels and blocking the transmission of action potentials on nerve fibers. Due to its low oral bioavailability, common dosage forms in clinical practice are mostly injections, gels, etc. Lidocaine hydrochloride gel (2%) is widely used due to its rapid onset, strong effect, and wide safety range.

[0003] Lidocaine hydrochloride gel (2%) is a sterile gel and is packaged in multiple doses. In order to ensure the quality of the drug after opening and use, antibacterial agents - methylparaben and propylparaben need to be added during the preparation process. In order to effectively control the quality of the drug and ensure the safety of drug use, it is necessary to establish relevant methods for detecting the content of antibacterial agents and lidocaine hydrochloride in lidocaine hydrochloride gel.

[0004] Currently, relevant standards have been used to detect the content of lidocaine hydrochloride, but it is not yet known whether the relevant methods are applicable to the simultaneous detection of the content of antibacterial agents and lidocaine hydrochloride in lidocaine hydrochloride gel. Summary of the invention

[0005] The object of the present invention is to provide a HPLC detection method for the content of antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel.

[0006] The HPLC detection method for the content of antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel provided by the present invention adopts the following technical scheme:

[0007] A HPLC detection method for the content of antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel comprises the following steps:

[0008] (1) Prepare reference solution and test solution;

[0009] (2) Injecting the reference solution and the test solution into a liquid chromatograph, and performing separation and detection using a chromatographic column with octadecylsilane bonded silica gel as a filler and an isocratic elution method of the mobile phase;

[0010] (3) Record the chromatogram and calculate the content of antibacterial agent and lidocaine hydrochloride in the test solution by peak area using the external standard method.

[0011] Furthermore, the octadecylsilane bonded silica gel chromatographic column is C18, specifications are 4.6mm×150mm, 5μm.

[0012] Furthermore, the HPLC detection method is an isocratic elution mode, and the volume ratio of phosphate buffer to acetonitrile in the mobile phase is (55-65:45-35).

[0013] Furthermore, the phosphate buffer is a dipotassium hydrogen phosphate solution with a concentration of 11-12 mmol / L and a pH of 7.8-8.2.

[0014] Furthermore, in the HPLC detection, the column temperature of the chromatographic column is 30-40°C.

[0015] Furthermore, in the HPLC detection, the detection wavelength is 252-256 nm.

[0016] Furthermore, in the HPLC detection, the column flow rate of the chromatographic column is 0.8-1.2 ml / min.

[0017] Furthermore, in the HPLC detection, the injection volume is 18-22 μl.

[0018] Furthermore, the preparation method of the reference solution is: take appropriate amounts of lidocaine hydrochloride, methylparaben and propylparaben reference substances, dissolve them in mobile phase and quantitatively dilute them to prepare reference solutions containing 100 μg of lidocaine hydrochloride, 3.5 μg of methylparaben and 1.5 μg of propylparaben per 1 ml.

[0019] Furthermore, the preparation method of the test solution is: take about 2.5g of lidocaine hydrochloride gel, put it in a 50ml volumetric flask, dissolve it with the mobile phase and dilute it to the scale.

[0020] The present invention uses a high performance liquid chromatography external standard method to simultaneously detect the contents of antibacterial agents methylparaben, propylparaben and lidocaine hydrochloride in lidocaine hydrochloride gel under the same chromatographic conditions. Compared with multiple detections, the detection method of the present invention can synchronously, quickly and accurately detect the contents of the three substances, greatly improving the detection efficiency and saving relevant resources. The detection method of the present invention is verified by systematic methodologies such as specificity, system applicability, solution stability, linearity and range, accuracy, precision, and durability, proving that the method is scientific and accurate, and is suitable for the detection of antibacterial agents and lidocaine hydrochloride contents in the production process of lidocaine hydrochloride gel, achieving effective, rapid and accurate control of product quality and ensuring drug safety. DETAILED DESCRIPTION

[0021] The present invention provides an HPLC detection method for the content of an antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel, comprising the following steps:

[0022] (1) Prepare reference solution and test solution;

[0023] (1-1) Preparation of reference solution: Take appropriate amount of lidocaine hydrochloride, methylparaben and propylparaben reference substances, weigh accurately, add mobile phase to dissolve and quantitatively dilute to make a solution containing 100 μg of lidocaine hydrochloride, 3.5 μg of methylparaben and 1.5 μg of propylparaben per 1 ml.

[0024] (1-2) Preparation of test solution: Take an appropriate amount of lidocaine hydrochloride gel (equivalent to 5 mg of lidocaine hydrochloride), accurately weigh it, place it in a 50 ml volumetric flask, add an appropriate amount of mobile phase to dissolve it, and then dilute it to the scale with mobile phase.

[0025] (2) Injecting the reference solution and the test solution into a liquid chromatograph, and performing separation and detection using a chromatographic column with octadecylsilane bonded silica gel as a filler and an isocratic elution method of the mobile phase; during the separation and detection process, the mobile phase is a potassium hydrogen phosphate solution-acetonitrile (60:40) (55-65:45-35), wherein the concentration of potassium hydrogen phosphate is 11-12 mmol / L, and the pH is 7.8-8.2; the column temperature is 30-40° C.; the detection wavelength is 252-256 nm; the flow rate is 0.8-1.2 ml / min; and the injection volume is 18-22 μl.

[0026] (3) Record the chromatogram and calculate the content of antibacterial agent and lidocaine hydrochloride in the test solution by peak area using the external standard method.

[0027] The materials and equipment used in the present invention are all known products, and the remaining reagents, solvents, etc. can be purchased commercially.

[0028] The present invention will be further described below in conjunction with specific embodiments.

[0029] Embodiment 1:

[0030] (1) Prepare reference solution and test solution;

[0031] (1-1) Preparation of reference solution: Take appropriate amount of lidocaine hydrochloride, methylparaben and propylparaben reference substances, weigh accurately, add mobile phase to dissolve and quantitatively dilute to make a solution containing 100 μg of lidocaine hydrochloride, 3.5 μg of methylparaben and 1.5 μg of propylparaben per 1 ml.

[0032] (1-2) Preparation of test solution: Take an appropriate amount of lidocaine hydrochloride gel (equivalent to 5 mg of lidocaine hydrochloride), accurately weigh it, place it in a 50 ml volumetric flask, add an appropriate amount of mobile phase to dissolve it, and then dilute it to the scale with mobile phase.

[0033] (2) Accurately measure 20 μl of each of the blank solution, reference solution and test solution, and inject them into a liquid chromatograph for separation and detection. During the separation and detection process, the chromatographic column is: C18, specification is 4.6mm×150mm, 5μm; mobile phase is 10mmol / L potassium dihydrogen phosphate solution (pH 8.0)-acetonitrile (60:40); column temperature is 35℃; detection wavelength is 254nm; flow rate is 1.0ml / min; injection volume is 20μl.

[0034] The preparation method of the dipotassium hydrogen phosphate solution is as follows: 1.74 g of dipotassium hydrogen phosphate is weighed, dissolved in water and diluted to 1000 ml, and sodium hydroxide solution is added to adjust the pH value to 8.0.

[0035] (3) Record the chromatogram and calculate the content of the antibacterial agent and lidocaine hydrochloride in the test solution by the external standard method using the following formula based on the peak area:

[0036]

[0037] Where:

[0038] A 对 : Peak area of ​​reference solution; m 对 : The weight of the reference substance, mg;

[0039] A 供 : Peak area of ​​test solution; m 供 : The weight of the test sample, mg;

[0040] V 对 : dilution multiple of reference solution; V 供 : dilution multiple of the test solution;

[0041] P: Lidocaine hydrochloride reference substance content; Label amount: 2%

[0042]

[0043] Where:

[0044] A 对 : Peak area of ​​reference solution; m 对 : The weight of the reference substance, mg;

[0045] A 供 : Peak area of ​​test solution; m 供 : The weight of the test sample, mg;

[0046] V 对 : dilution multiple of reference solution; V 供 : dilution multiple of the test solution;

[0047] P: content of antibacterial reference substance;

[0048] (4) Test results: The blank solution, reference solution and test solution were tested using the above method. The test results are as follows:

[0049] Table 1 HPLC test results

[0050]

[0051]

[0052] According to the test results in Table 1, the content detection method provided by the present invention can accurately detect the content of the antibacterial agent and lidocaine hydrochloride in the lidocaine hydrochloride gel, and the blank solution does not interfere with the detection. The peak separations of methylparaben, propylparaben and lidocaine hydrochloride in the antibacterial agent are all greater than 1.5, and the method has strong specificity.

[0053] Exclusive

[0054] (1) Prepare reference solution and test solution;

[0055] (2) Accurately measure 20 μl of each of the above solutions, inject them into a liquid chromatograph, and record the chromatogram.

[0056] (3) Test results are shown in Table 1.

[0057] According to the test results in Table 1, the content detection method provided by the present invention can accurately detect the content of the antibacterial agent and lidocaine hydrochloride in the lidocaine hydrochloride gel, and the blank solution does not interfere with the detection. The peak separations of methylparaben, propylparaben and lidocaine hydrochloride in the antibacterial agent are all greater than 1.5, and the method has strong specificity.

[0058] System suitability

[0059] (1) Accurately measure 20 μl of the reference solution, inject it into a liquid chromatograph, perform detection according to the detection method provided in Example 1, and record the chromatogram.

[0060] (2) Test results are shown in Table 2.

[0061] Table 2 System suitability test results

[0062]

[0063] According to the test results in Table 2, the reference solution was injected 5 times repeatedly, and the peak retention time RSDs of methylparaben, propylparaben and lidocaine hydrochloride were all within 1.0%, and the peak area RSDs were all within 2.0%, which met the requirements, indicating that the content detection method provided by the present invention has good system applicability.

[0064] Solution stability

[0065] (1) At different time points, accurately measure 20 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0066] (2) Test results are shown in Table 3.

[0067] Table 3. Results of stability test of reference solution

[0068]

[0069]

[0070] Table 4 Solution stability test results of the test solution

[0071]

[0072] According to the test results in Tables 3 and 4, after the reference solution was placed at room temperature for 48 hours, the RD of each component at each time point was less than 2% compared with the corresponding peak area at 0 hour; after the test solution was placed at room temperature for 48 hours, the RD of each component at each time point was less than 2% compared with the corresponding peak area at 0 hour. Therefore, the reference solution and the test solution were stable within 48 hours at room temperature.

[0073] Linearity and range

[0074] (1) Prepare linear stock solution: accurately weigh 35 mg of methylparaben reference substance and 15 mg of propylparaben reference substance, place in a 100 ml volumetric flask, add mobile phase to dissolve and dilute to scale, and shake well; accurately weigh 2.5 ml of the above solution, place in a 25 ml volumetric flask, accurately add about 27 mg of lidocaine hydrochloride reference substance, add appropriate amount of mobile phase to dissolve and dilute to scale, and shake well.

[0075] Prepare linear solutions: accurately measure 0.6, 0.8, 1.0, 1.2, and 1.4 ml of the linear stock solution respectively, place them in different 10 ml volumetric flasks, add mobile phase to dilute to the scale, shake well, and then you can get 60%, 80%, 100%, 120%, and 140% linear solutions.

[0076] (2) Accurately measure 20 μl of each of the above linear solutions, inject them into a liquid chromatograph, perform detection according to the detection method provided in Example 1, and record the chromatogram.

[0077] (3) Test results are shown in Table 5.

[0078] Table 5 Linear test results

[0079]

[0080]

[0081]

[0082] According to the test results in Table 5, the linear equation of methylparaben in the concentration range of 2.10μg / ml to 4.89μg / ml is y=102561.1566x-1355.5590, the correlation coefficient r is 1.0000, greater than 0.990, and the Y-axis intercept is within 2% of the 100% response value; the linear equation of propylparaben in the concentration range of 0.93μg / ml to 2.17μg / ml is y=86328.7097x- 929.1000, the correlation coefficient r was 1.0000, which was greater than 0.999, and the Y-axis intercept was within 2% of the 100% response value; in the concentration range of 60.73μg / ml to 141.71μg / ml of lidocaine hydrochloride, the linear equation was y=2402.3312x+3960.4407, the correlation coefficient r was 0.9999, which was greater than 0.990, and the Y-axis intercept was within 2% of the 100% response value; the linear relationships of the methods were all good.

[0083] Accuracy

[0084] (1) Prepare recovery rate solution: accurately weigh 0.098g of blank auxiliary material, place in a 20ml volumetric flask, accurately add 1.4ml, 2.0ml, and 2.6ml of linear stock solution respectively, add mobile phase to dissolve and dilute to scale, shake well, and prepare 70%, 100%, and 130% recovery rate solutions. (Prepare 3 copies of each solution in parallel)

[0085] (2) Accurately measure 20 μl of each recovery solution, inject it into a liquid chromatograph, perform detection according to the detection method provided in Example 1, and record the chromatogram.

[0086] (3) Test results are shown in Table 6.

[0087] Table 6 Accuracy test results

[0088]

[0089]

[0090] According to the test results in Table 6, within the concentration range of 70%-130%, the average recovery rate of methylparaben is 99.7%, and the RSD of 9 recovery rate data is 0.44%; the average recovery rate of propylparaben is 100.0%, and the RSD of 9 recovery rate data is 0.32%; the average recovery rate of lidocaine hydrochloride is 99.3%, and the RSD of 9 recovery rate data is 0.14%. The recovery rates are all between 98-101%, and the RSD is no more than 2.0%. This shows that the detection method has good accuracy.

[0091] Repeatability

[0092] (1) Prepare reference solution and test solution according to Example 1 (prepare 6 portions of each test solution in parallel)

[0093] (2) Accurately measure 20 μl of each of the above solutions, inject them into a liquid chromatograph, perform detection according to the detection method provided in Example 1, and record the chromatogram.

[0094] (3) Test results are shown in Table 7.

[0095] Table 7 Repeatability test results

[0096]

[0097]

[0098] According to the test results in Table 7, the RSDs of methylparaben, propylparaben and lidocaine hydrochloride measured in the six test solutions were 0.13%, 0.70% and 0.25%, respectively, all less than 2.0%, indicating that the detection method has good repeatability.

[0099] Intermediate precision

[0100] (1) Prepare the reference solution and the test solution according to the repeatability test (prepare 6 copies of each test solution in parallel)

[0101] (2) Accurately measure 20 μl of each of the above solutions, inject them into a liquid chromatograph, perform detection according to the detection method provided in Example 1, and record the chromatogram.

[0102] (3) Test results are shown in Table 8.

[0103] Table 8 Intermediate precision test results

[0104]

[0105] According to the test results in Table 8, the RD values ​​of the mean contents of methylparaben, propylparaben and lidocaine hydrochloride measured in 12 test solutions were 0%, 0% and 0.26%, respectively, all less than 2.0%, indicating that the intermediate precision of the detection method was good.

[0106] Durability

[0107] Embodiment 2-12:

[0108] Examples 2-12 provide HPLC detection methods for the content of antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel, respectively.

[0109] The difference between Example 2-12 and Example 1 is that the HPLC detection conditions are different, as shown in Table 9 for details.

[0110] (1) Prepare reference solution and test solution according to Example 1

[0111] (2) Accurately measure 20 μl of each of the above solutions, inject them into a liquid chromatograph, and record the chromatogram.

[0112] (3) Test results are shown in Table 9.

[0113] Table 9 Durability test results

[0114]

[0115]

[0116]

[0117]

[0118] According to the test results in Table 9, the contents of antibacterial agent and lidocaine hydrochloride in the test solution obtained under the test conditions of Examples 2-12 are not significantly different from the test results of Example 1, indicating that the method has good durability.

[0119] Based on the test results of the above embodiments and comparative examples, it can be clearly seen that the detection method provided by the present invention is scientific and reasonable, has the advantages of rapid, efficient and accurate detection, and is suitable for simultaneously detecting the content of antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel.

[0120] The above-mentioned embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Any changes or improvements made by technicians in the relevant technical field based on the present invention shall fall within the protection scope of the present invention.

Claims

1. A HPLC detection method for the content of antibacterial agent and lidocaine hydrochloride in lidocaine hydrochloride gel, characterized in that: include: (1) Prepare reference solution and test solution; (2) Injecting the reference solution and the test solution into a liquid chromatograph, and performing separation and detection using a chromatographic column with octadecylsilane bonded silica gel as a filler and an isocratic elution method of the mobile phase; (3) Record the chromatogram and calculate the content of antibacterial agent and lidocaine hydrochloride in the test solution by peak area using the external standard method.

2. The detection method according to claim 1, characterized in that: The octadecylsilane bonded silica gel chromatographic column is C18, specifications are 4.6mm×150mm, 5μm.

3. The detection method according to claim 1, characterized in that: The HPLC detection method is an isocratic elution mode, and the volume ratio of phosphate buffer to acetonitrile in the mobile phase is (55-65:45-35).

4. The detection method according to claim 2, characterized in that: The phosphate buffer is a dipotassium hydrogen phosphate solution with a concentration of 11-12 mmol / L and a pH of 7.8-8.

2.

5. The detection method according to claim 1, characterized in that: In the HPLC detection, the column temperature of the chromatographic column is 30-40°C.

6. The detection method according to claim 1, characterized in that: In the HPLC detection, the detection wavelength is 252-256 nm.

7. The detection method according to claim 1, characterized in that: In the HPLC detection, the column flow rate of the chromatographic column is 0.8-1.2 ml / min.

8. The detection method according to claim 1, characterized in that: In the HPLC detection, the injection volume is 18-22 μl.

9. The detection method according to any one of claims 1 to 8, characterized in that: The preparation method of the reference solution is as follows: appropriate amounts of lidocaine hydrochloride, methylparaben and propylparaben reference substances are taken, dissolved in mobile phase and quantitatively diluted to prepare reference solutions containing 100 μg of lidocaine hydrochloride, 3.5 μg of methylparaben and 1.5 μg of propylparaben per 1 ml.

10. The detection method according to any one of claims 1 to 8, characterized in that: The preparation method of the test solution is as follows: take about 2.5 g of lidocaine hydrochloride gel, put it in a 50 ml volumetric flask, dissolve it with mobile phase and dilute it to the scale.

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