A method for detecting impurities in levocaine cream

Abstract

The present invention belongs to the field of biomedicine technology, relates to the monitoring of drug quality, and specifically to a method for detecting impurities in a leprocaine cream. Leprocaine cream is detected by high performance liquid chromatography; wherein mobile phase A is a mixture of an aqueous solution of dipotassium hydrogen phosphate and acetonitrile, the volume ratio of the aqueous solution of dipotassium hydrogen phosphate to acetonitrile is 60:40-70:30, and the pH of the aqueous solution of dipotassium hydrogen phosphate is 8.5-9.0; mobile phase B is a mixture of phosphate buffer and acetonitrile, the volume ratio of phosphate buffer to acetonitrile is 85:15-95:5; the chromatographic column is XB-C18, 4.6mm×150mm, 5μm. The detection method of the present invention can not only separate and detect the 6 known impurities in leprocaine cream, but also effectively separate the 4 potential degradation impurities therein.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, relates to the monitoring of drug quality, and specifically relates to a method for detecting impurities in leprocaine cream. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Leprocaine cream is a local anesthetic analgesic preparation. According to the inventor's research, 6 impurities are currently known to exist in leprocaine cream. However, further research has found that new impurities will appear in leprocaine cream during long-term storage, and the current detection method is difficult to effectively separate the new impurities. Summary of the invention

[0004] In order to solve the shortcomings of the prior art, the present invention provides a method for detecting impurities in leprocaine cream, which can not only separate and detect the six known impurities in leprocaine cream, but also effectively separate four potential degradation impurities therein.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] On the one hand, a method for detecting impurities in a leprocaine cream is provided, wherein the leprocaine cream is detected by high performance liquid chromatography; wherein mobile phase A is a mixture of an aqueous solution of dipotassium hydrogen phosphate and acetonitrile, the volume ratio of the aqueous solution of dipotassium hydrogen phosphate to acetonitrile is 60:40-70:30, and the pH of the aqueous solution of dipotassium hydrogen phosphate is 8.5-9.0; mobile phase B is a mixture of phosphate buffer and acetonitrile, the volume ratio of phosphate buffer to acetonitrile is 85:15-95:5; the chromatographic column is XB-C18, 4.6 mm×150 mm, 5 μm; the chemical structural formulas of the 10 impurities detected are as follows:

[0007] Impurity 1: (o-Toluidine);

[0008] Impurity 2:

[0009] Impurity 3: (2,6-dimethylaniline);

[0010] Impurity 4:

[0011] Impurity 5:

[0012] Impurity 6:

[0013] Impurity 7:

[0014] Impurity 8:

[0015] Impurity 9:

[0016] Impurity 10:

[0017] Studies have shown that the selection of mobile phase and chromatographic column is a key factor affecting the detection of 10 impurities in leprocaine cream. When the mobile phase and chromatographic column are selected, especially when the pH of the aqueous solution of dipotassium hydrogen phosphate in mobile phase A is 8.5-9.0, lidocaine, prilocaine and 10 impurities in leprocaine cream can be completely separated, thereby achieving effective detection of impurities in leprocaine cream. Among them, impurities 1-6 are 6 impurities known to exist in leprocaine cream, and 7-10 are newly discovered degradation impurities in leprocaine cream according to the present invention.

[0018] Furthermore, the elution procedure is as follows: from 0 to 8 minutes, the volume fraction of mobile phase A is 37%, and the volume fraction of mobile phase B is 63%; from 8 minutes to 38 minutes, the volume fraction of mobile phase A increases from 37% to 100%, and the volume fraction of mobile phase B decreases from 63% to 0; from 38 minutes to 60 minutes, the volume fraction of mobile phase A is 100%; from 60 minutes to 61 minutes, the volume fraction of mobile phase A decreases from 100% to 37%, and the volume fraction of mobile phase B increases from 0 to 63%. Under the premise of the selection of the above mobile phase and chromatographic column, the elution procedure can be used to better separate lidocaine, prilocaine and 10 impurities in the prilocaine cream.

[0019] On the other hand, a method for detecting impurities in the above-mentioned leprocaine cream is used in quality monitoring during the production process of leprocaine cream.

[0020] Since the detection method of the present invention can detect four newly added degradation impurities, in the third aspect, a detection method for impurities in the above-mentioned leprocaine cream is used in monitoring the storage of leprocaine cream.

[0021] The beneficial effects of the present invention are:

[0022] The invention studies the impurity spectrum of leprocaine cream and establishes a detection method for impurities in leprocaine cream. The detection method can not only separate and detect six known impurities in leprocaine cream, but also effectively separate four potential degradation impurities therein, thereby providing an efficient and feasible method for the research and development, production and stability study of leprocaine cream. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 is a high performance liquid chromatogram of the test solution of Example 1 of the present invention;

[0025] Figure 2 is a high performance liquid chromatogram of the mixed solution of Example 1 of the present invention;

[0026] Figure 3 This is a trend diagram of impurities changing due to changes in the pH value of the mobile phase in Example 2 of the present invention;

[0027] Figure 4 This is a high performance liquid chromatogram of a mixed solution with a mobile phase pH of 9.0 in Example 2 of the present invention;

[0028] Figure 5 This is a high performance liquid chromatogram of a mixed solution with a mobile phase pH of 8.5 in Example 2 of the present invention;

[0029] Figure 6 This is a high performance liquid chromatogram of a mixed solution with a mobile phase pH of 7.2 in Example 2 of the present invention. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0032] In view of the fact that it is difficult to effectively separate new impurities in leprocaine cream using existing detection methods, the present invention proposes a method for detecting impurities in leprocaine cream.

[0033] A typical embodiment of the present invention provides a method for detecting impurities in a leprocaine cream, wherein the leprocaine cream is detected by high performance liquid chromatography; wherein the mobile phase A is a mixture of an aqueous solution of dipotassium hydrogen phosphate and acetonitrile, the volume ratio of the aqueous solution of dipotassium hydrogen phosphate to acetonitrile is 60:40-70:30, and the pH of the aqueous solution of dipotassium hydrogen phosphate is 8.5-9.0; the mobile phase B is a mixture of a phosphate buffer and acetonitrile, the volume ratio of the phosphate buffer to acetonitrile is 85:15-95:5; the chromatographic column is XB-C18, 4.6 mm×150 mm, 5 μm; the chemical structural formulas of the 10 impurities detected are as follows:

[0034] Impurity 1:

[0035] Impurity 2:

[0036] Impurity 3:

[0037] Impurity 4:

[0038] Impurity 5:

[0039] Impurity 6:

[0040] Impurity 7:

[0041] Impurity 8:

[0042] Impurity 9:

[0043] Impurity 10:

[0044] In some embodiments, the elution procedure is: from 0 to 8 min, the volume fraction of mobile phase A is 37%, and the volume fraction of mobile phase B is 63%; from 8 min to 38 min, the volume fraction of mobile phase A increases from 37% to 100%, and the volume fraction of mobile phase B decreases from 63% to 0; from 38 min to 60 min, the volume fraction of mobile phase A is 100%; from 60 min to 61 min, the volume fraction of mobile phase A decreases from 100% to 37%, and the volume fraction of mobile phase B increases from 0 to 63%.

[0045] In some embodiments, the test solution is obtained after pre-treatment of the levocaine cream, and the test solution is detected by high performance liquid chromatography.

[0046] Specifically, sodium hydroxide solution is used to disperse the leprocaine cream, and then hydrochloric acid is added in an amount equal to that of the sodium hydroxide solution, and then the mobile phase A is added to make up the volume. More specifically, 4 to 6 mL of 4 to 6 mol / L sodium hydroxide solution is used to disperse 0.7 to 0.9 g of the leprocaine cream.

[0047] In some embodiments, the volume ratio of the aqueous solution of dipotassium hydrogen phosphate to acetonitrile is 60:40 to 65:35.

[0048] In some embodiments, during HPLC detection, the flow rate is 1.0-2.0 mL / min.

[0049] In some embodiments, the detection wavelength of HPLC is 230-235 nm.

[0050] In some embodiments, during HPLC detection, the column temperature is 39-41°C.

[0051] In some embodiments, during high performance liquid chromatography detection, the injection volume is 50 to 200 μL.

[0052] Another embodiment of the present invention provides an application of the above-mentioned method for detecting impurities in leprocaine cream in quality monitoring during the production process of leprocaine cream.

[0053] A third embodiment of the present invention provides an application of the above-mentioned method for detecting impurities in the leprocaine cream in monitoring the storage of the leprocaine cream.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0055] Example 1

[0056] Chromatographic column: Luna (4.6mm×100mm, 3μm)

[0057] Mobile phase A: Dissolve 2.73 g of potassium dihydrogen phosphate in 630 mL of water, adjust the pH to 7.20 ± 0.02 with 5 mol / L sodium hydroxide solution, and make up to 1000 mL with acetonitrile.

[0058] Mobile phase B: Dissolve 2.73 g potassium dihydrogen phosphate in 900 mL water, adjust the pH to 7.20 ± 0.02 with 5 mol / L sodium hydroxide solution, and make up to 1000 mL with acetonitrile.

[0059] Flow rate: 1.5mL / min

[0060] Wavelength: 232nm

[0061] Column temperature: 40°C

[0062] Injection volume: 50 μL

[0063]

[0064] Test solution: Take 0.8 g of the product, accurately weigh it, put it in a 100 mL volumetric flask, add 5 mL of 5 mol / L sodium hydroxide solution, disperse the cream, add 5 mL of 5 mol / L hydrochloric acid solution, dilute to the scale with mobile phase A, shake well, and take the filtrate.

[0065] Mixed solution: a mixed solution containing 0.01 mg / mL each of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9 and impurity 10 and 0.2 mg / mL each of lidocaine and prilocaine.

[0066] The chromatogram obtained by the detection in this embodiment is as follows Figures 1-2 As shown, in the test solution and the mixed solution prepared from the reference preparation, the circles indicate that the lidocaine peak contains impurity 8.

[0067] Embodiment 2:

[0068] Chromatographic column: XB-C18 (4.6mm×150mm, 5μm)

[0069] Mobile phase A: Take 6.85 g of potassium dihydrogen phosphate, add 1000 mL of water to dissolve it, and adjust the pH value to 6.5, 7.0, 7.2, 7.5, 8.0, 8.5, 9.0 respectively with phosphoric acid)-acetonitrile (63:37).

[0070] Mobile phase B: phosphate buffer-acetonitrile (90:10).

[0071] Flow rate: 1.5mL / min

[0072] Wavelength: 232nm

[0073] Column temperature: 40°C

[0074] Injection volume: 100 μL

[0075]

[0076] Take the mixed solution, inject it into the liquid chromatograph, and record the chromatogram.

[0077] Method study chromatograms such as Figures 3 to 6As shown, taking the mobile phase pH 8.5 as an example, the order of peaks is impurity 1, impurity 2, impurity 3, impurity 5, prilocaine, impurity 6, impurity 7, impurity 8, lidocaine, impurity 9, impurity 10, and impurity 4.

[0078] The retention time of the chromatographic peaks of impurity 1, impurity 2, impurity 3, impurity 5, impurity 7, impurity 8, impurity 9, and impurity 10 does not change with the change of the pH of the mobile phase; the chromatographic peaks of lidocaine, prilocaine, impurity 4, and impurity 6 move forward with the decrease of pH value.

[0079] When the mobile phase pH is 9.0, the chromatographic data are as follows:

[0080] Element Retention time Peak area Peak area% high Separation Tailing Factor Theoretical plate number Impurity 1 6.046 235860 0.477 16234 -- 1.119 4166 Impurity 2 8.665 62014 0.125 3162 5.909 1.049 4589 Impurity 3 11.012 199825 0.404 10326 4.597 1.018 7483 Impurity 5 19.898 57641 0.117 2522 15.755 0.9 16540 Prilocaine 20.859 32765827 66.25 1318570 1.495 1.257 15690 Impurity 6 23.349 186201 0.376 6921 3.635 1.058 17602 Impurity 7 28.868 94713 0.192 3526 7.874 0.974 27360 Impurity 8 33.117 16939 0.034 605 6.048 0.942 35126 Lidocaine 35.369 15503369 31.347 502424 2.919 1.475 28626 Impurity 9 47.547 196673 0.398 5134 13.196 0.982 35487 Impurity 10 50.079 17325 0.035 449 2.469 0.879 37009 Impurity 4 54.252 121485 0.246 2368 3.483 0.997 25666

[0081] When the mobile phase pH is 8.5, the chromatographic data are as follows:

[0082]

[0083]

[0084] When the mobile phase pH is 8.5-9.0, the separation between the main component and each impurity is good (not less than 1.5). At pH 8.0, prilocaine interferes with the determination of impurity 5, at pH 7.5, lidocaine interferes with the determination of impurity 8, at pH 7.0, lidocaine interferes with the determination of impurity 7, and at pH 6.5, lidocaine interferes with the determination of impurity 5. At pH 7.2, the main component does not interfere with the determination of each impurity, but impurity 9 and impurity 4 are not baseline separated.

[0085] Example 3: Study on the robustness of the elution gradient with mobile phase pH 8.5

[0086] Chromatographic column: XB-C18 (4.6mm×150mm, 5μm)

[0087] Mobile phase A: Take 6.85 g of potassium dihydrogen phosphate, add 1000 mL of water to dissolve it, and adjust the pH to 8.5 with phosphoric acid)-acetonitrile (63:37).

[0088] Mobile phase B: phosphate buffer-acetonitrile (90:10).

[0089] Flow rate: 1.5mL / min

[0090] Wavelength: 232nm

[0091] Column temperature: 40°C

[0092] Injection volume: 100 μL

[0093] Elution gradient 1:

[0094]

[0095]

[0096] Elution gradient 2:

[0097]

[0098] Elution gradient 3:

[0099]

[0100] Elution gradient 4:

[0101]

[0102] Elution gradient 5:

[0103]

[0104]

[0105] Elution gradient 6:

[0106]

[0107] Take the mixed solution separately, inject it into the liquid chromatograph, and record the chromatogram. The separation data of prilocaine peak and adjacent impurity peak are as follows:

[0108]

[0109] The separation data of lidocaine peak and adjacent impurity peak are as follows:

[0110]

[0111] In summary, by appropriately adjusting the mobile phase elution gradient, 10 known impurities can be separated simultaneously. This method has good durability and has established an efficient and feasible liquid chromatography method for the inspection of related substances in leprocaine cream.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting impurities in levocaine cream, characterized in that: High performance liquid chromatography was used to detect the leprocaine cream; wherein, mobile phase A was a mixture of an aqueous solution of dipotassium hydrogen phosphate and acetonitrile, the volume ratio of the aqueous solution of dipotassium hydrogen phosphate to acetonitrile was 60:40-70:30, and the pH of the aqueous solution of dipotassium hydrogen phosphate was 8.5-9.0; mobile phase B was a mixture of phosphate buffer and acetonitrile, the volume ratio of phosphate buffer to acetonitrile was 85:15-95:5; the chromatographic column was XB-C18, 4.6 mm × 150 mm, 5 μm; the chemical structural formulas of the 10 impurities detected were as follows: Impurity 1: ; Impurity 2: ; Impurity 3: ; Impurity 4: ; Impurity 5: ; Impurity 6: ; Impurity 7: ; Impurity 8: ; Impurity 9: ; Impurity 10: ; The elution program was as follows: from 0 to 8 min, the volume fraction of mobile phase A was 37% and the volume fraction of mobile phase B was 63%; from 8 min to 38 min, the volume fraction of mobile phase A increased from 37% to 100%, and the volume fraction of mobile phase B decreased from 63% to 0; from 38 min to 60 min, the volume fraction of mobile phase A was 100%; from 60 min to 61 min, the volume fraction of mobile phase A decreased from 100% to 37%, and the volume fraction of mobile phase B increased from 0 to 63%; A test solution is obtained after pre-treatment of the levocaine cream, and the test solution is detected by high performance liquid chromatography; The leprocaine cream was dispersed with sodium hydroxide solution, and then hydrochloric acid in an amount equal to that of the sodium hydroxide solution was added, and then mobile phase A was added to make up the volume; Use 4-6 mL of 4-6 mol / L sodium hydroxide solution to disperse 0.7-0.9 g of ripracaine cream.

2. The method for detecting impurities in the levulocaine cream according to claim 1, wherein: The volume ratio of the aqueous solution of dipotassium hydrogen phosphate to acetonitrile is 60:40~65:

35.

3. The method for detecting impurities in the levulocaine cream according to claim 1, wherein: When high performance liquid chromatography was used for detection, the flow rate was 1.0~2.0 mL / min.

4. The method for detecting impurities in the levulocaine cream according to claim 1, wherein: The detection wavelength of HPLC was 230~235 nm.

5. Application of the method for detecting impurities in the leprocaine cream according to any one of claims 1 to 4 in quality monitoring during the production process of the leprocaine cream.

6. Use of the method for detecting impurities in the leprocaine cream according to any one of claims 1 to 4 in monitoring the storage of the leprocaine cream.

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