Analysis method for detecting related substances in lidocaine and prilocaine aerosol by high performance liquid chromatography

Abstract

The invention discloses an analysis method for detecting related substances in lidocaine and prilocaine aerosol by a high performance liquid chromatography, the high performance liquid chromatography is adopted, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises an ammonium phosphate buffer solution and acetonitrile, and the volume ratio of the ammonium phosphate buffer solution to the acetonitrile is (83-87): (17-13); the mobile phase B contains an ammonium phosphate buffer solution and acetonitrile in a volume ratio of (28-32): (72-68); the pH value of the ammonium phosphate buffer solution ranges from 7.0 to 8.0; and a chromatographic column adopts a phenyl column. The invention provides a high performance liquid chromatography for detecting related substances in the lidocaine and prilocaine aerosol, and good quality control of the product can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis methods, and particularly relates to an analysis method for detecting related substances in lidocaine and prilocaine aerosols by a high performance liquid phase method. Background Art

[0002] Lidocaine prilocaine aerosol is a local anesthetic drug, mainly used to treat primary premature ejaculation in adult men, with clear clinical efficacy and easy use. The quality standard of this product is not included in domestic and foreign pharmacopoeias. It is found that lidocaine prilocaine cream is included in the United States Pharmacopoeia. After reproducing the related substance inspection method, the related substance method has problems such as poor separation of lidocaine and adjacent impurity peaks, interference of diluent with impurity detection, and poor durability of chromatographic columns, which cannot be well controlled for the quality of this product.

[0003] Impurities that may arise from lidocaine and prilocaine include:

[0004]

[0005] Among them, lidocaine A and B, and prilocaine B are listed in the British Pharmacopoeia.

[0006] The sources of impurities are:

[0007] Lidocaine impurity A: It is a degradation impurity produced by the hydrolysis of the amide bond of lidocaine. The generation pathway is:

[0008]

[0009] Lidocaine impurity B: It is a degradation impurity produced by oxidation of lidocaine tertiary amine. The generation pathway is:

[0010]

[0011] Prilocaine impurity B: It is a degradation impurity produced by hydrolysis of the amide bond of prilocaine. The generation pathway is:

[0012]

[0013] The formula of lidocaine prilocaine aerosol contains lidocaine, prilocaine, and tetrafluoroethane, and is packaged in a pressure-resistant container with a valve.

[0014] The relevant substances are mainly the starting materials, intermediates, polymers, side reaction products introduced during the production process, and degradation products produced during storage and transportation. In this project, the introduction of moisture or oxygen may cause the active ingredients in lidocaine prilocaine aerosol to degrade, thereby producing the above-mentioned impurities.

[0015] The detection of related substances is an important indicator for controlling drug quality. One of the contents of drug analysis is to detect related impurities sensitively and accurately once they are produced.

[0016] In order to ensure the quality of lidocaine prilocaine aerosol and ensure the safety and effectiveness of the drug, it is urgently necessary to provide a method for detecting related substances in lidocaine prilocaine aerosol with high separation, good sensitivity, strong specificity and good separation between impurities. Summary of the invention

[0017] In order to overcome the shortcomings of the prior art, the present invention provides an analytical method for detecting related substances in lidocaine prilocaine aerosol by high performance liquid chromatography, and the technical solution of the present invention is as follows:

[0018] The high performance liquid chromatography method is adopted, and the mobile phase includes mobile phase A and mobile phase B. The mobile phase A contains ammonium phosphate buffer and acetonitrile in a volume ratio of 83-87:17-13; the mobile phase B contains ammonium phosphate buffer and acetonitrile in a volume ratio of 28-32:72-68; the pH value range of the ammonium phosphate buffer is 7.0-8.0; and the chromatographic column adopts a phenyl column.

[0019] Preferably, the ammonium phosphate salt is diammonium hydrogen phosphate or ammonium dihydrogen phosphate, and further, the concentration range of the ammonium phosphate salt is 0.01 to 0.03 mol / L.

[0020] Preferably, the mobile phase adopts gradient elution, and the gradient elution program is:

[0021] Within 0 to 10 minutes, the volume percentage of mobile phase A remains unchanged at 100%, and the volume percentage of mobile phase B remains unchanged at 0%; within 10 to 46 minutes, the volume percentage of mobile phase A linearly changes from 100% to 70%, and the volume percentage of mobile phase B linearly changes from 0% to 30%; within 46 to 50 minutes, the volume percentage of mobile phase A remains unchanged at 70%, and the volume percentage of mobile phase B remains unchanged at 30%; within 50 to 51 minutes, the volume percentage of mobile phase A linearly changes from 70% to 100%, and the volume percentage of mobile phase B linearly changes from 30% to 0%; within 51 to 60 minutes, the volume percentage of mobile phase A remains unchanged at 100%, and the volume percentage of mobile phase B remains unchanged at 0%.

[0022] Preferably, the chromatographic column is Waters XBridge Phenyl, 4.6 mm×150 mm, 3.5 μm or a chromatographic column with equivalent performance.

[0023] Preferably, the detection wavelength is 232nm±2nm.

[0024] Preferably, the column temperature is 36-40°C.

[0025] Preferably, the flow rate of the mobile phase is 1.0 to 1.4 ml / min.

[0026] Furthermore, the analytical method also includes sample preparation:

[0027] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing 0.3 mg of lidocaine and 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution.

[0028] Control solution: Accurately measure 1 ml of the test solution, place it in a 200 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0029] Furthermore, the diluent is mobile phase A.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides a high performance liquid phase method for detecting related substances in lidocaine prilocaine aerosol, which can achieve good quality control of the product.

[0032] 2. The present invention uses one chromatographic condition to simultaneously detect related substances of lidocaine and prilocaine, the method is simple and the detection cost is low.

[0033] 3. The mobile phase of the analytical method provided by the present invention adopts ammonium phosphate, and the chromatographic column adopts a phenyl column. The chromatographic conditions are highly specific and sensitive for the detection of lidocaine and prilocaine-related substances. The main component peak and the impurity peaks are well separated, and the impurities can be effectively detected.

[0034] 4. The method of the present invention is highly durable, and fine-tuning of the chromatographic conditions does not affect the effective detection of known impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] Figure 1 This is the solution chromatogram of the system suitability in Example 1.

[0037] Figure 2 This is the chromatogram of the test sample for related substance detection in Batch 1 in Example 1.

[0038] Figure 3 This is the chromatogram of the test sample for related substance detection in Batch 2 in Example 1.

[0039] Figure 4The chromatograms are of the test solution and the positioning solution in Comparative Example 1, wherein from bottom to top are lidocaine impurity A solution, prilocaine impurity B solution, diluent, and test solution.

[0040] Figure 5 This is the chromatogram of the test in Comparative Example 2 (1) in which the salt in the mobile phase is disodium hydrogen phosphate.

[0041] Figure 6 This is the chromatogram of the test in Comparative Example 2 (2) where the salt in the mobile phase is dipotassium hydrogen phosphate.

[0042] Figure 7 This is the chromatogram of the test in Comparative Example 2 (3) where the salt in the mobile phase is diammonium hydrogen phosphate.

[0043] Figure 8 This is the chromatogram of the (1) pH 6.5 test in Comparative Example 3.

[0044] Fig. 9 This is the chromatogram of the (2) pH 9.0 test in Comparative Example 3.

[0045] Fig.10 This is the chromatogram of the Pheromon C18 column test in (1) in Comparative Example 4.

[0046] Fig.11 This is the chromatogram of the (2) phenylhexyl column test in Comparative Example 4.

[0047] Fig.12 This is the chromatogram of the (3) C8 column test in Comparative Example 4.

[0048] Fig.13 This is the chromatogram of the experiment of (1) gradient program 1 in Comparative Example 5.

[0049] Fig.14 This is the chromatogram of the experiment of (2) gradient program 2 in Comparative Example 5.

[0050] Fig.15 This is the chromatogram of the methodological verification (1) specificity test of Example 2. DETAILED DESCRIPTION

[0051] 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.

[0052] Example 1, an analytical method for detecting related substances in lidocaine and prilocaine aerosol by high performance liquid chromatography

[0053] Waters HPLC

[0054] Chromatographic conditions

[0055] Column: Waters XBridge Phenyl, 4.6 mm × 150 mm, 3.5 μm

[0056] Mobile phase A: 0.02 mol / L diammonium hydrogen phosphate solution (pH 7.5)-acetonitrile (85:15)

[0057] Mobile phase B: 0.02 mol / L diammonium hydrogen phosphate solution (pH 7.5)-acetonitrile (30:70)

[0058] Wavelength: 232nm

[0059] Column temperature: 40°C

[0060] Injection volume: 50 μl

[0061] Flow rate: 1.2ml / min

[0062] Diluent: Mobile phase A

[0063] Gradient elution program:

[0064] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 46 70 30 50 70 30 51 100 0 60 100 0

[0065] Sensitivity solution: Take an appropriate amount of prilocaine reference substance and prepare a solution containing 0.1 μg of prilocaine per 1 ml.

[0066] System suitability solution: Take appropriate amounts of lidocaine impurity A reference substance, lidocaine impurity B reference substance, prilocaine impurity B reference substance, lidocaine, and prilocaine to prepare solutions containing approximately 1.5 μg of lidocaine impurity A, approximately 1.5 μg of lidocaine impurity B, approximately 0.5 μg of prilocaine impurity B, approximately 0.3 mg of lidocaine, and approximately 0.1 mg of prilocaine per 1 ml, respectively.

[0067] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine and 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution.

[0068] Control solution: Accurately measure 1 ml of the test solution, place it in a 200 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0069] Accurately measure 50 μl of the diluent and the above solution, inject them into the liquid chromatograph, and record the chromatogram.

[0070] Calculation formula:

[0071]

[0072] Total impurities (%) Impurities (%)

[0073] Where:

[0074] A i : Impurity peak area in the test solution;

[0075] A r利 : lidocaine peak area in control solution;

[0076] A r丙 : prilocaine peak area in control solution;

[0077] f: impurity correction factor.

[0078] The correction factors for each impurity are as follows:

[0079] Impurities Correction Factor Lidocaine Impurity A 0.29 Lidocaine Impurity B 1.0 Prilocaine Impurity B 0.39

[0080] System suitability solution chromatogram Figure 1 shown.

[0081] The test results of the two batches of samples are shown in the following table. Figure 2 and Figure 3 shown.

[0082] name Batch 1 Batch 2 Lidocaine Impurity A 0.005％ 0.01％ Lidocaine Impurity B 0.01％ 0.01％ Prilocaine Impurity B 0.03％ 0.04％ Other largest single 0.14％ 0.15％ Total Miscellaneous 0.51％ 0.67％

[0083] It can be seen that when the present method is used to detect lidocaine prilocaine aerosol, each impurity is well separated and the impurities present in the sample can be sensitively detected.

[0084] Comparative Example 1,

[0085] Refer to the method for related substances of prilocaine and lidocaine cream in the United States Pharmacopoeia for the detection of lidocaine and prilocaine aerosol. The process is as follows:

[0086] Agilent HPLC

[0087] Chromatographic conditions

[0088] Column: Inertsil ODS-3 4.6×100mm, 3μm

[0089] Mobile phase A: Weigh 2.73 g potassium dihydrogen phosphate and dissolve it in 630 ml water, adjust the pH to 7.20 with 5 mol / L sodium hydroxide, and dilute to 1000 ml with acetonitrile.

[0090] Mobile phase B: Weigh 2.73 g potassium dihydrogen phosphate and dissolve it in 900 ml water, adjust the pH to 7.20 with 5 mol / L sodium hydroxide, and dilute to 1000 ml with acetonitrile.

[0091] Wavelength: 232nm; Flow rate: 1.5ml / min; Column temperature: 40℃; Injection volume: 50μl

[0092] Gradient elution program

[0093] Time (min) Mobile phase A (%) Mobile phase B (%) 0 67 33 11 67 33 22 100 0 32 100 0 33 67 33 40 67 33

[0094] Diluent: Mobile phase A

[0095] The preparation method of impurity localization solution is as follows:

[0096] Lidocaine Impurity A Solution: Take an appropriate amount of Lidocaine Impurity A reference substance and use a diluent to prepare a solution containing approximately 1.5 μg of Lidocaine Impurity A per 1 ml.

[0097] Prilocaine Impurity B Solution: Take an appropriate amount of Prilocaine Impurity B reference substance and use a diluent to prepare a solution containing approximately 1.5 μg of Prilocaine Impurity B per 1 ml.

[0098] Test solution: Take the contents of this product and use a diluent to prepare a solution containing about 0.3 mg of lidocaine and about 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution.

[0099] Take lidocaine impurity A solution, prilocaine impurity B solution, diluent, and test solution and inject them respectively, and record the chromatograms. Figure 4 As shown, the results are as follows.

[0100] Conclusion: The impurity peak at 2.89min in the test solution overlaps with the diluent peak, affecting the detection of impurities; the impurity peak at 7.06min affects the detection of lidocaine impurity A; the prilocaine peak and the 12.14min impurity peak are poorly separated; after multiple tests, the two main component peaks are severely tailed, the chromatographic column performance is reduced, and the durability is poor. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosols.

[0101] Comparative Example 2, different mobile phase phosphate types

[0102] (1) Disodium hydrogen phosphate

[0103] Waters HPLC

[0104] Chromatographic conditions

[0105] Column: Inertsil ODS-3 4.6×150mm, 3μm

[0106] Mobile phase A: 0.01 mol / L disodium hydrogen phosphate solution (pH 8.0)-acetonitrile (85:15)

[0107] Mobile phase B: 0.01 mol / L disodium hydrogen phosphate solution (pH 8.0)-acetonitrile (30:70)

[0108] Wavelength: 232nm; Column temperature: 40℃; Injection volume: 50μl; Flow rate: 1.0ml / min

[0109] Diluent: Mobile phase A

[0110] Gradient elution program:

[0111] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 50 50 50 51 95 5 56 95 5

[0112] Mixed solution: Take the contents of this product and transfer all of it to a volumetric flask to which appropriate amounts of lidocaine impurity A and prilocaine impurity B have been added using a diluent, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine, approximately 0.1 mg of prilocaine, approximately 1.5 μg of lidocaine impurity A, and approximately 0.5 μg of prilocaine impurity B per 1 ml. Shake well to obtain the solution.

[0113] Take the mixed solution and inject it, and record the chromatogram. Figure 5 As shown, the results are as follows.

[0114] Conclusion: The peaks of prilocaine and lidocaine have serious tailing and are poorly separated from the adjacent impurity peaks, which affects the detection of impurities.

[0115] (2) Potassium hydrogen phosphate

[0116] Agilent HPLC

[0117] Chromatographic conditions

[0118] Column: Inertsil ODS-3 4.6×150mm, 5μm

[0119] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution (pH 8.0)-acetonitrile (63:37)

[0120] Mobile phase B: 0.01 mol / L potassium dihydrogen phosphate solution (pH 8.0)-acetonitrile (90:10)

[0121] Wavelength: 232nm; Column temperature: 40℃; Injection volume: 50μl; Flow rate: 1.5ml / min

[0122] Diluent: Mobile phase A

[0123] Gradient elution program:

[0124] Time (min) Mobile phase A (%) Mobile phase B (%) 0 67 33 11 67 33 22 90 10 32 100 0 33 67 33 40 67 33

[0125] Test solution: Take the contents of this product and use a diluent to prepare a solution containing about 0.3 mg of lidocaine and about 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution.

[0126] Take the test solution and inject it, record the chromatogram, such as Figure 6 As shown, the results are as follows.

[0127] Conclusion: The peak shapes of the two main peaks are poor.

[0128] (3) Diammonium hydrogen phosphate

[0129] Waters HPLC

[0130] Chromatographic conditions

[0131] Column: Inertsil ODS-3 4.6×150mm, 3μm

[0132] Mobile phase A: 0.02 mol / L diammonium hydrogen phosphate solution (pH 8.0)-acetonitrile (85:15)

[0133] Mobile phase B: 0.02 mol / L diammonium hydrogen phosphate solution (pH 8.0)-acetonitrile (30:70)

[0134] Wavelength: 232nm; Column temperature: 40℃; Injection volume: 50μl; Flow rate: 1.0ml / min

[0135] Diluent: Mobile phase A

[0136] Gradient elution program:

[0137] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 50 50 50 51 95 5 56 95 5

[0138] Mixed product solution: Take the contents of this product and transfer all of it to a volumetric flask to which appropriate amounts of lidocaine impurity A and prilocaine impurity B have been added using a diluent, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine, approximately 0.1 mg of prilocaine, approximately 1.5 μg of lidocaine impurity A, and approximately 0.5 μg of prilocaine impurity B per 1 ml. Shake well to obtain the solution.

[0139] Take the test solution and inject it, record the chromatogram, such as Figure 7 As shown, the results are as follows.

[0140] Conclusion: The peak shapes of prilocaine and lidocaine were significantly improved, but the prilocaine peak could not be baseline-separated from its adjacent peaks, and the lidocaine peak was significantly delayed and close to the gradient peak, which posed a risk of unknown impurities not being detected.

[0141] Comparative Example 3, different mobile phase pH

[0142] (1) pH 6.5

[0143] Waters HPLC

[0144] Chromatographic conditions

[0145] Column: Waters XBridge Phenyl, 4.6 mm × 150 mm, 3.5 μm

[0146] Mobile phase A: 0.02 mol / L diammonium hydrogen phosphate solution (pH 6.5)-acetonitrile (85:15)

[0147] Mobile phase B: 0.02 mol / L diammonium hydrogen phosphate solution (pH 6.5)-acetonitrile (30:70)

[0148] Wavelength: 232nm; Column temperature: 40℃; Injection volume: 50μl; Flow rate: 1.2ml / min

[0149] Diluent: Mobile phase A

[0150] Gradient elution program:

[0151] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 46 70 30 50 70 30 51 100 0 60 100 0

[0152] Mixed solution: Take the contents of this product and transfer all of it with a diluent into a volumetric flask to which appropriate amounts of lidocaine impurity A reference substance, lidocaine impurity B reference substance, and prilocaine impurity B reference substance have been added, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine, approximately 0.1 mg of prilocaine, approximately 1.5 μg of lidocaine impurity A, approximately 1.5 μg of lidocaine impurity B, and approximately 0.5 μg of prilocaine impurity B per 1 ml. Shake well to obtain the solution.

[0153] Take the mixed solution and inject it, and record the chromatogram. Figure 8 As shown, the results are as follows.

[0154] Conclusion: The peak separation of lidocaine impurity A and prilocaine is poor, which affects the detection of impurities. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosols.

[0155] (2) pH 9.0

[0156] Waters HPLC

[0157] The buffer pH was adjusted to 9.0, and the other chromatographic conditions were the same as "pH 6.5"

[0158] Take the mixed solution and inject it, and record the chromatogram. Fig. 9 As shown, the results are as follows.

[0159] Conclusion: The lidocaine peak is significantly delayed and closer to the gradient peak, which may affect the detection of unknown impurities.

[0160] Comparative Example 4, different chromatographic columns

[0161] (1) C18 column

[0162] Waters HPLC

[0163] Chromatographic conditions

[0164] Chromatographic column: Phenol C18-PFP, 4.6mm×250mm, 3μm

[0165] Mobile phase A: 0.02 mol / L diammonium hydrogen phosphate solution (pH 7.5)-acetonitrile (85:15)

[0166] Mobile phase B: 0.02 mol / L diammonium hydrogen phosphate solution (pH 7.5)-acetonitrile (30:70)

[0167] Wavelength: 232nm; Column temperature: 40℃; Injection volume: 50μl; Flow rate: 1.2ml / min

[0168] Diluent: Mobile phase A

[0169] Gradient elution program:

[0170] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 50 70 30 60 50 50 65 50 50 66 100 0 75 100 0

[0171] Mixed solution: Take the contents of this product and transfer all of it to a volumetric flask to which appropriate amounts of lidocaine impurity A and prilocaine impurity B have been added using a diluent, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine, approximately 0.1 mg of prilocaine, approximately 1.5 μg of lidocaine impurity A, and approximately 0.5 μg of prilocaine impurity B per 1 ml. Shake well to obtain the solution.

[0172] Take the mixed solution and inject it, and record the chromatogram. Fig.10 As shown, the results are as follows.

[0173] Conclusion: The two main peaks have poor peak shapes and severe tailing. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosols.

[0174] (2) Phenyl hexyl column

[0175] Waters HPLC

[0176] The chromatographic conditions and mixed solution were the same as those in "Comparative Example 4 (1) C18 column", except that the chromatographic column was replaced with "Waters Phenyl-Hexyl, 4.6 mm×150 mm, 2.5 μm".

[0177] Take the test solution and inject it, record the chromatogram, such as Fig.11 As shown, the results are as follows.

[0178] Conclusion: There is an unknown impurity peak before the prilocaine peak, which is wrapped by the prilocaine peak. The baseline fluctuates greatly from 34min to 48min. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosol.

[0179] (3) C8 column

[0180] Agilent HPLC

[0181] The chromatographic conditions and mixed solution were the same as those in "Comparative Example 4 (1) C18 column", except that the chromatographic column was replaced with "Agilent ZORBAXSB-C8, 4.6 mm×150 mm, 5 μm".

[0182] Take the test solution and inject it, record the chromatogram, such as Fig.12 As shown, the results are as follows.

[0183] Conclusion: Impurity A of lidocaine did not elute. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosol.

[0184] Comparative Example 5, different gradient elution procedures

[0185] (1) Gradient program 1

[0186] Waters HPLC

[0187] Chromatographic conditions

[0188] Column: Waters XBridge Phenyl, 4.6 mm × 150 mm, 3.5 μm

[0189] Mobile phase A: 0.02 mol / L diammonium hydrogen phosphate solution (pH 7.5)-acetonitrile (85:15)

[0190] Mobile phase B: 0.02 mol / L diammonium hydrogen phosphate solution (pH 7.5)-acetonitrile (30:70)

[0191] Wavelength: 232nm; Column temperature: 40℃; Injection volume: 50μl; Flow rate: 1.2ml / min

[0192] Diluent: Mobile phase A

[0193] Gradient elution program:

[0194] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 6 100 0 40 65 35 45 65 35 46 100 0 55 100 0

[0195] Mixed solution: Take the contents of this product and transfer all of them to a volumetric flask to which appropriate amounts of lidocaine impurity A, lidocaine impurity B and prilocaine impurity B have been added using a diluent, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine, approximately 0.1 mg of prilocaine, approximately 1.5 μg of lidocaine impurity A, approximately 1.5 μg of lidocaine impurity B and approximately 0.5 μg of prilocaine impurity B per 1 ml. Shake well to obtain the solution.

[0196] Prilocaine solution: Take prilocaine raw material and prepare a solution containing about 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution.

[0197] Take prilocaine solution and mixed solution for injection and record the chromatogram. Fig.13 As shown, the results are as follows.

[0198] Conclusion: The 17.55min impurity in prilocaine affects the detection of lidocaine impurity A. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosol.

[0199] (2) Gradient Program 2

[0200] Waters HPLC

[0201] The chromatographic conditions and mixed solution were the same as those in “Gradient Program 1”, and the gradient program was optimized as follows:

[0202] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 90 0 40 70 30 50 70 30 51 100 0 60 100 0

[0203] Take the mixed solution and inject it, and record the chromatogram. Fig.14 As shown, the results are as follows.

[0204] Conclusion: The impurity peak at 8.42 min affects the detection of prilocaine impurity B, and the lidocaine peak is poorly separated from the impurity peak at 41.15 min. This method is not suitable for the detection of related substances in lidocaine and prilocaine aerosol.

[0205] Example 2, Methodology Verification

[0206] Waters HPLC

[0207] (1) Specificity test

[0208] This method has strong specificity: each impurity is located, and the diluent and blank excipients are completely separated from the two active ingredients and known impurities.

[0209] Blank solution: Accurately measure 2 ml of acetonitrile, place in a 25 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0210] Lidocaine reference solution: Take an appropriate amount of lidocaine reference solution to prepare a solution containing approximately 0.3 mg of lidocaine per 1 ml.

[0211] Prilocaine reference solution: Take an appropriate amount of prilocaine reference and prepare a solution containing approximately 0.1 mg of prilocaine per 1 ml.

[0212] Lidocaine Impurity A Reference Solution: Take an appropriate amount of Lidocaine Impurity A Reference Solution to prepare a solution containing approximately 1.5 μg of Lidocaine Impurity A per 1 ml.

[0213] Lidocaine impurity B reference solution: The preparation method is the same as that of lidocaine impurity A reference solution.

[0214] Prilocaine impurity B reference solution: Take an appropriate amount of prilocaine impurity B to prepare a solution containing approximately 0.5 μg of prilocaine impurity B per 1 ml.

[0215] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine and 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution.

[0216] Blank preparation solution: Take 1 can of blank preparation and prepare it according to the method for preparing the test solution.

[0217] Mixed solution: Take the contents of this product and transfer all of it with a diluent into a volumetric flask to which appropriate amounts of lidocaine impurity A reference substance, lidocaine impurity B reference substance, and prilocaine impurity B reference substance have been added, and dilute to the scale to prepare a solution containing approximately 0.3 mg of lidocaine, approximately 0.1 mg of prilocaine, approximately 1.5 μg of lidocaine impurity A, approximately 1.5 μg of lidocaine impurity B, and approximately 0.5 μg of prilocaine impurity B per 1 ml. Shake well to obtain the solution.

[0218] Accurately measure 50 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. Fig.15 shown.

[0219] Note: From bottom to top, they are diluent, prilocaine reference solution, lidocaine reference solution, mixed solution

[0220] Conclusion: The diluent, blank solution and blank preparation do not produce peaks at the known impurities and main peak, and do not interfere with the main peak and known impurities; the minimum separation between the known impurities and the main peak and the adjacent peaks is greater than 1.5; the purity of the main peak meets the requirements.

[0221] (2) Sensitivity test

[0222] High sensitivity: the detection limit of each impurity is lower than the reporting limit (0.1%);

[0223] The limit of quantification of lidocaine impurity A is 7.5124 ng (accounting for 0.05% of the test sample concentration);

[0224] The limit of quantification of lidocaine impurity B is 7.1862 ng (accounting for 0.05% of the test sample concentration);

[0225] The limit of quantification of prilocaine impurity B is 2.5078 ng (accounting for 0.05% of the test sample concentration);

[0226] The detection limit of lidocaine impurity A is 2.2537 ng (accounting for 0.02% of the test sample concentration);

[0227] The detection limit of lidocaine impurity B is 2.1559 ng (accounting for 0.01% of the test sample concentration);

[0228] The detection limit of prilocaine impurity B is 0.7523 ng (accounting for 0.02% of the test sample concentration).

[0229] (3) Linear relationship

[0230] The linear relationship is good: each impurity is within the range of quantitation limit ~150%, and the correlation coefficient is greater than 0.99.

[0231] Lidocaine impurity A is in the range of 0.1502 μg / ml to 2.2537 μg / ml (10% to 150%), the linear equation is y=148083.8863x+855.8566, and the linear correlation coefficient r is 0.9999;

[0232] Lidocaine impurity B is in the range of 0.1437 μg / ml to 2.1559 μg / ml (10% to 150%), the linear equation is y=37936.0564x+417.7743, and the linear correlation coefficient r is 0.9999;

[0233] The impurity B of prilocaine is in the range of 0.0522 μg / ml to 0.7837 μg / ml (10% to 150%), the linear equation is y=174766.2778x+396.0921, and the linear correlation coefficient r is 0.9999.

[0234] (4) Accuracy test

[0235] High accuracy: the recovery rate of lidocaine impurity A is between 100.2% and 101.0%, and the RSD value is 0.3%;

[0236] The recovery rate of lidocaine impurity B was between 100.2% and 102.0%, and the RSD value was 0.8%;

[0237] The recovery rate of prilocaine impurity B is between 100.0% and 100.9%, and the RSD value is 0.4%. The recovery rates of all impurities meet the requirements of 90% to 108%.

[0238] (5) Precision test

[0239] In the 6 sample solutions, the RSD value of lidocaine impurity A was 1.0%; the RSD value of lidocaine impurity B was 1.0%; and the RSD value of prilocaine impurity B was 0.9%. This method has good repeatability for the inspection of related substances of this product.

[0240] In 12 sample solutions, the RSD value of lidocaine impurity A was 1.5%; the RSD value of lidocaine impurity B was 0.9%; and the RSD value of prilocaine impurity B was 1.2%. This method has good reproducibility for the inspection of related substances of this product.

[0241] In summary, the RSD of the repeatability and reproducibility of each impurity content detection was less than 3%, and the precision was good.

[0242] (6) Durability test

[0243] Strong durability: fine-tuning of chromatographic conditions, column temperature (36°C, 40°C), different flow rates (1.0ml / min, 1.4ml / min), different pH values ​​(7.0, 8.0), different batches of chromatographic columns, different brands of liquid phases (Waters, Agilent), the separation degree can meet the requirements.

[0244]

[0245] Under fine-tuning of the chromatographic conditions, in the sensitivity solution, the minimum signal-to-noise ratio of the prilocaine peak was 58.8, which was greater than 10 and met the requirements; the blank preparation had no peaks at the main peak or known impurities, and the minimum separation between the main peak, known impurities and adjacent peaks was 7.43, which was greater than 1.5; the maximum absolute value of the difference between the known impurity content detected under various conditions and the content under normal conditions was 0.014%, which was less than 0.1%.

[0246] 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. An analytical method for detecting related substances in lidocaine prilocaine aerosol by high performance liquid chromatography, characterized in that: The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A contains ammonium phosphate buffer and acetonitrile in a volume ratio of 83-87:17-13; the mobile phase B contains ammonium phosphate buffer and acetonitrile in a volume ratio of 28-32:72-68; the pH value of the ammonium phosphate buffer ranges from 7.0 to 8.0; and the chromatographic column adopts a phenyl column; Impurities in lidocaine prilocaine aerosol include:

2. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The ammonium phosphate salt is diammonium hydrogen phosphate or ammonium dihydrogen phosphate.

3. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The concentration range of ammonium phosphate is 0.01~0.03mol / L.

4. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The mobile phase used was gradient elution, and the gradient elution program was: Within 0 to 10 minutes, the volume percentage of mobile phase A remains unchanged at 100%, and the volume percentage of mobile phase B remains unchanged at 0%; within 10 to 46 minutes, the volume percentage of mobile phase A linearly changes from 100% to 70%, and the volume percentage of mobile phase B linearly changes from 0% to 30%; within 46 to 50 minutes, the volume percentage of mobile phase A remains unchanged at 70%, and the volume percentage of mobile phase B remains unchanged at 30%; within 50 to 51 minutes, the volume percentage of mobile phase A linearly changes from 70% to 100%, and the volume percentage of mobile phase B linearly changes from 30% to 0%; within 51 to 60 minutes, the volume percentage of mobile phase A remains unchanged at 100%, and the volume percentage of mobile phase B remains unchanged at 0%.

5. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The chromatographic column is Waters XBridge Phenyl, 4.6 mm×150 mm, 3.5 μm.

6. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The detection wavelength is 232nm±2nm.

7. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The column temperature is 36-40°C.

8. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The flow rate of the mobile phase is 1.0~1.4ml / min.

9. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 1, characterized in that: The analytical method also includes sample preparation: Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the mark to prepare a solution containing 0.3 mg of lidocaine and 0.1 mg of prilocaine per 1 ml. Shake well to obtain the solution. Control solution: Accurately measure 1 ml of the test solution, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well; The diluent was mobile phase A.

10. The method for analyzing related substances in lidocaine prilocaine aerosol by high performance liquid chromatography according to claim 9, characterized in that: The calculation formula for each impurity is: Total impurities (%) Impurities (%) Where: Ai: impurity peak area in the test solution; ArLi: lidocaine peak area in the control solution; Ar: prilocaine peak area in the control solution; f: Correction factor for each impurity; Among them, the correction factor for lidocaine impurity A is 0.29, the correction factor for lidocaine impurity B is 1.0, and the correction factor for prilocaine impurity B is 0.39.

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