Extraction method and detection method of ripdicaine medicine in skin

Abstract

The invention discloses an extraction method and a detection method of a ripdicaine medicine in skin. The extraction method of the ripdicaine medicine in the skin comprises the following steps: performing transdermal treatment on the ripdicaine medicine, removing the residual ripdicaine medicine on the surface of the skin, and performing liquid nitrogen freezing, crushing and ultrasonic extraction on the part of the skin coated with the ripdicaine medicine, thereby obtaining the ripdicaine medicine. The lidocaine and the prilocaine are fully extracted from the skin tissue by adopting a freezing and ultrasonic combined method, so that the accuracy of a detection result is ensured. The invention provides a safe and reliable detection method for detecting drug quality and clinical treatment effect. The invention further provides a rapid detection method, detection can be completed within 3.5 min, the detection efficiency is high, solvent consumption is reduced, the detection cost is reduced, the method can be suitable for high-throughput detection, the detection requirement of a large batch of samples is met, inter-batch detection errors are reduced, and the method further has high exclusive detection performance and is high in accuracy, precision and detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis methods, and particularly relates to a method for extracting and detecting lidocaine and prilocaine drugs in the skin. Background Art

[0002] Lidocaine and prilocaine cream is a commonly used topical anesthetic preparation on the skin surface in clinical practice. Lidocaine and prilocaine in the cream can penetrate the skin to reach the dermis layer, accumulate at the pain receptors and nerve endings in the dermis layer, and stabilize the nerve cell membrane by inhibiting the ion flow required for the generation and transmission of nerve impulses, thereby producing a local anesthetic effect. Lidocaine and prilocaine cream is commonly used in clinical practice for analgesia during intravenous puncture, intravenous intubation, and various minor surgical operations, especially suitable for pediatric patients.

[0003] Pharmacokinetic studies have shown that when lidocaine and prilocaine cream is applied to intact skin, approximately 5% - 10% of lidocaine and prilocaine is absorbed, and the maximum plasma drug concentration (0.1 - 0.2 μg / mL) is below the toxic concentration (5 μg / mL), indicating that this drug can penetrate the skin into the blood circulation to a certain extent.

[0004] In vitro transdermal permeation simulates the transdermal process of drugs under physiological conditions in vitro to reflect the quality of drugs and the effectiveness of clinical treatment. The residual amount of drugs in the skin after transdermal detection is also an important monitoring index. Due to the special structure and composition of skin tissue, there are few reports in the prior art on how to efficiently extract lidocaine and prilocaine drugs from the skin and ensure the accuracy and precision of the detection results.

[0005] Therefore, there is an urgent need in the art to develop an in vitro transdermal test method that can completely extract lidocaine and prilocaine from the skin and ensure high accuracy of the detection results. Summary of the Invention

[0006] This application provides a method for extracting and detecting lidocaine and prilocaine drugs in the skin to solve the problem of the lack of a method for detecting the content of lidocaine and prilocaine in the skin in the prior art. This application uses a method combining freezing and ultrasonic waves to fully extract lidocaine and prilocaine from skin tissue, ensuring the accuracy of the detection results without interference. It provides a safe and reliable extraction method for detecting the quality of drugs and the clinical treatment effect. It also provides a rapid detection method that can complete the detection within 3.5 minutes, with high detection efficiency, reduced solvent consumption, reduced detection costs, and can be applied to high-throughput detection to meet the detection needs of a large number of samples, reduce the detection error between batches, and also has high specificity, accuracy, precision, and detection sensitivity.

[0007] This application solves the above technical problems through the following technical solutions.

[0008] The present application provides a method for extracting prilocaine drug from the skin, which specifically includes the following steps: After the prilocaine drug is subjected to transdermal treatment and the remaining prilocaine drug on the skin surface is removed, the skin at the site where the prilocaine drug is coated is cryopreserved with liquid nitrogen, pulverized, and extracted by ultrasound. That's all.

[0009] In some embodiments, before the cryopreservation, the operation of cutting the skin into pieces may further be included. According to the routine in the art, the operation of cutting the skin into pieces is beneficial to improving the cryopreservation efficiency and shortening the cryopreservation time.

[0010] In some embodiments, the time of cryopreservation with liquid nitrogen is 1 hour or more, preferably 1 - 3 hours.

[0011] In some embodiments, the extraction solution used for ultrasonic extraction is an alcohol solvent, preferably methanol and / or ethanol.

[0012] In some embodiments, during the ultrasonic extraction, the volume of the extraction solution used per unit area of the skin is 8 - 20 mL / cm 2 , for example, 14 mL / cm 2 .

[0013] In some embodiments, the time of ultrasonic extraction is 3 - 20 minutes, preferably 5 - 15 minutes, for example, 10 minutes.

[0014] In some embodiments, the temperature of ultrasonic extraction is room temperature.

[0015] In some embodiments, after the operation of ultrasonic extraction, the operation of centrifugation may further be included.

[0016] Preferably, the rotation speed of centrifugation is 6000 - 10000 rpm, for example, 8000 rpm.

[0017] Preferably, the time of centrifugation is 3 - 10 minutes, for example, 5 minutes.

[0018] Preferably, the temperature of centrifugation is 3 - 10 °C, for example, 5 °C.

[0019] In some embodiments, the prilocaine drug includes lidocaine, prilocaine, and excipients.

[0020] Preferably, the mass percentage of lidocaine in the prilocaine drug is 0.2% - 10%, for example, 0.25% or 2.5%.

[0021] Preferably, the mass percentage of prilocaine in the prilocaine drug is 0.2% - 10%, for example, 0.25% or 2.5%.

[0022] Preferably, the adjuvant includes at least one of an emulsifier, a matrix, a pH regulator, and water.

[0023] Among them, the emulsifier is polyoxyethylene hydrogenated castor oil, preferably polyoxyethylene hydrogenated castor oil-54.

[0024] Among them, the matrix is carbomer, preferably carbomer homopolymer type B.

[0025] Among them, the pH regulator is an alkali metal hydroxide, preferably selected from sodium hydroxide and / or potassium hydroxide.

[0026] Among them, the mass percentage of the emulsifier in the prilocaine drug is 1% to 5%, preferably 1.5% to 2.5%, such as 1.9%.

[0027] Among them, the mass percentage of the matrix in the prilocaine drug is 0.5% to 5%, preferably 0.5% to 1.5%, such as 1%.

[0028] Among them, the mass percentage of the pH regulator in the prilocaine drug is 0.2% to 0.8%, preferably 0.4% to 0.6%, such as 0.52%.

[0029] In some embodiments, the skin used for the transdermal treatment is animal skin, preferably selected from pig skin, mouse skin, or rabbit skin.

[0030] In some embodiments, the device used for the transdermal treatment is selected from Franz diffusion cells. Preferably, the diameter of the Franz diffusion cell is 1 to 3 cm, such as 1.5 cm.

[0031] In some embodiments, during the transdermal treatment, the transdermal medium is selected from normal saline and / or phosphate buffer solution, preferably an aqueous sodium chloride solution with a mass percentage of 0.5% to 1.5%, such as an aqueous sodium chloride solution with a mass percentage of 0.9%.

[0032] In some embodiments, during the transdermal treatment, the temperature of the transdermal medium is 30 to 40 °C, preferably 30 to 35 °C, such as 32 °C.

[0033] In some embodiments, during the transdermal treatment, the stirring speed of the transdermal medium is 500 to 700 rpm, such as 600 rpm.

[0034] In some embodiments, during the transdermal treatment, the mass-volume ratio of the prilocaine drug to the transdermal medium is 0.01 to 0.05 g / mL, such as 0.0225 g / mL.

[0035] In some embodiments, during the transdermal treatment, the transdermal medium may further include a degassing operation before use.

[0036] The present application also provides a method for detecting prilocaine in the skin. The detection method includes the following steps: the test solution includes the extract obtained by the extraction method described above; a high performance liquid chromatography-mass spectrometry (HPLC-MS) device is used, in combination with the internal standard method, to qualitatively or quantitatively detect lidocaine and prilocaine in the test solution.

[0037] In some embodiments, the test solution further includes a solvent.

[0038] Preferably, in the test solution, the solvent is selected from alcohols or aqueous solutions of alcohols, more preferably an aqueous solution of alcohol with a volume percentage of 40% to 60%, for example, an aqueous solution of alcohol with a volume percentage of 50%. More preferably, the alcohol is a volatile alcohol solvent, and even more preferably methanol and / or ethanol.

[0039] Preferably, in the test solution, the volume ratio of the extract to the solvent is 1:100 to 250, for example 1:200.

[0040] In some embodiments, the concentrations of lidocaine and prilocaine in the extract are each independently 0.2 to 50 μg / mL, more preferably 0.6 to 37.5 μg / mL.

[0041] In some embodiments of the internal standard method, the lidocaine internal standard is a deuterated compound of lidocaine, and the prilocaine internal standard is a deuterated compound of prilocaine; preferably, the lidocaine internal standard is lidocaine-d6; preferably, the prilocaine internal standard is prilocaine-d7; preferably, the concentrations of the lidocaine internal standard and the prilocaine internal standard are each independently 4 to 20 ng / mL, preferably 5 to 10 ng / mL.

[0042] In the high performance liquid chromatography of some embodiments, the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, for example, ACE Excel 2C18-AR 2.1*50mm.

[0043] Preferably, the column length of the octadecylsilane-bonded silica gel chromatographic column is 40 to 150 mm, for example 50 mm.

[0044] Preferably, the inner diameter of the octadecylsilane-bonded silica gel chromatographic column is 1.8 to 4.6 mm, for example 2.1 mm.

[0045] In the high performance liquid chromatography of some embodiments, the mobile phase includes mobile phase A and mobile phase B; mobile phase A is an aqueous solution of a volatile acid, and the mass percentage of the volatile acid in the aqueous solution of the volatile acid is 0.05% to 0.2%; mobile phase B is a volatile alcohol solvent.

[0046] Preferably, the aqueous solution of the volatile acid is selected from aqueous formic acid solution and / or aqueous acetic acid solution.

[0047] Preferably, the mass percentage of the volatile acid in the aqueous solution of the volatile acid is 0.08% to 0.12%, for example, 0.1%.

[0048] Preferably, the volatile alcohol solvent is methanol.

[0049] In the high performance liquid chromatography of some embodiments, the flow rate of the mobile phase is 0.15 to 0.5 mL / min, preferably 0.3 mL / min.

[0050] In the high performance liquid chromatography of some embodiments, the elution method is gradient elution.

[0051] When the gradient elution is adopted, from 0 to 1.1 min, the volume percentage of the mobile phase A is 85% to 95%, and the volume percentage of the mobile phase B is 5% to 15%; from 1.2 to 2.4 min, the volume percentage of the mobile phase A is 8% to 20%, and the volume percentage of the mobile phase B is 80% to 92%; from 2.5 to 3.5 min, the volume percentage of the mobile phase A is 85% to 95%, and the volume percentage of the mobile phase B is 5% to 15%.

[0052] In the high performance liquid chromatography of some embodiments, the temperature of the injector is 4 to 15 °C, for example, 5 °C.

[0053] In the high performance liquid chromatography of some embodiments, the injection volume of the test solution is 0.5 to 5 μL, for example, 2 μL.

[0054] In the high performance liquid chromatography of some embodiments, the column temperature is 30 to 50 °C, for example, 40 °C.

[0055] In the mass spectrometry of some embodiments, the ion mode is positive ion mode.

[0056] In the mass spectrometry of some embodiments, the source temperature is 400 to 600 °C, for example, 500 °C.

[0057] In the mass spectrometry of some embodiments, the scan mode is multiple reaction monitoring mode.

[0058] In the mass spectrometry of some embodiments, the ion source is an electrospray ion source.

[0059] In the mass spectrometry of some embodiments, the drying gas is nitrogen.

[0060] In the mass spectrometry of some embodiments, the curtain gas pressure is 10 to 40 psi, for example, 30 psi.

[0061] In the mass spectrometry of some embodiments, the collision gas pressure is 5 to 15 psi, for example, 8 psi.

[0062] In the mass spectrometry of some embodiments, the voltage applied in the electrospray ionization source is 3500 - 6000 V, preferably 4500 - 6000 V, such as 5500 V.

[0063] In the mass spectrometry of some embodiments, the declustering voltage is 40 - 70 V, such as 50 - 60 V.

[0064] In the mass spectrometry of some embodiments, the collision energy of lidocaine is 50 - 70 eV, preferably 55 - 65 eV, such as 60 eV.

[0065] In the mass spectrometry of some embodiments, the collision energy of the lidocaine internal standard is 16 - 36 eV, preferably 20 - 30 eV, such as 26 eV.

[0066] In the mass spectrometry of some embodiments, the collision energy of prilocaine is 36 - 56 eV, preferably 40 - 50 eV, such as 46 eV.

[0067] In the mass spectrometry of some embodiments, the collision energy of the prilocaine internal standard is 10 - 30 eV, preferably 15 - 25 eV, such as 20 eV.

[0068] In the mass spectrometry of some embodiments, the entrance voltage is 8 - 15 V, such as 10 V.

[0069] In the mass spectrometry of some embodiments, the collision cell exit voltage is 10 - 30 V, preferably 15 - 20 V, such as 16 V or 18 V.

[0070] In the mass spectrometry of some embodiments, the ion pair of lidocaine is 235.2 / 86.2.

[0071] In the mass spectrometry of some embodiments, the ion pair of prilocaine is 221.2 / 86.1.

[0072] In the mass spectrometry of some embodiments, the lidocaine internal standard is lidocaine - d6, and the ion pair of lidocaine - d6 is 241.3 / 86.1.

[0073] In the mass spectrometry of some embodiments, the prilocaine internal standard is prilocaine - d7, and the ion pair of prilocaine - d7 is 228.3 / 86.1.

[0074] Based on common knowledge, the above - mentioned preferred conditions can be combined arbitrarily to obtain the preferred examples of the present application.

[0075] The reagents and raw materials used in the present application are all commercially available.

[0076] The positive and progressive effects of this application are as follows: The method combining freezing and ultrasound is adopted in this application to fully extract lidocaine and prilocaine from skin tissues, ensuring the accuracy of detection results without interference. It provides a safe and reliable extraction method for detecting drug quality and clinical treatment effects. It also provides a rapid detection method, which can complete the detection within 3.5 minutes, with high detection efficiency, reduced solvent consumption, lower detection costs, and can be applied to high-throughput detection, meeting the detection requirements of a large number of samples, reducing the detection error between batches, and also having high exclusive detectability, accuracy, precision, and detection sensitivity. Description of the Drawings

[0077] Figure 1 It is a comparison chart of the curves of the average cumulative penetration amount of lidocaine in three groups of samples T1, T2, and R over time in Effect Example 8;

[0078] Figure 2 It is a comparison chart of the curves of the average cumulative penetration amount of prilocaine in three groups of samples T1, T2, and R over time in Effect Example 8;

[0079] Figure 3 It is a comparison chart of the curves of the penetration rate per unit area of lidocaine over time after the treatment of three groups of samples T1, T2, and R in Effect Example 8;

[0080] Figure 4 It is a comparison chart of the curves of the penetration rate per unit area of prilocaine over time after the treatment of three groups of samples T1, T2, and R in Effect Example 8;

[0081] Figure 5 It is a comparison chart of the retention amount of lidocaine in pig skin per unit mass after the treatment of three groups of samples T1, T2, and R in Effect Example 8;

[0082] Figure 6 It is a comparison chart of the retention amount of prilocaine in pig skin per unit mass after the treatment of three groups of samples T1, T2, and R in Effect Example 8. Detailed Embodiments

[0083] The present application will be further described below by way of examples, but the present application is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0084] In the following examples and comparative examples, methanol and formic acid are HPLC grade, and water is ultrapure water.

[0085] Equipment information used:

[0086] (1)918-12 Dry Heating Automatic Transdermal System (Logan Company, USA) Configuration: DHC-6TD Dry Heating Transdermal Diffusion Instrument, DHC-PL Automatic Lifting Device, DSC-800 Automatic Controller, SCR-DL Sampler, DHSC-1512 Diffusion Cell and STB-0 Magnetic Stirrer Bar.

[0087] (2) Shimadzu LC-30AD combined with SCIEX API 4000.

[0088] (3) JX-FSTPRP-11 Multi-Sample Cryogenic Grinder (Shanghai Jingxin Technology Co., Ltd.).

[0089] Example 1

[0090] 1. Skin Treatment

[0091] The pig skin of Bama mini-pigs (one-month-old) was used for the transdermal experiment. It was stored frozen in a -20°C refrigerator for later use, ensuring it was used within 2 months. Before use, the whole piece of Bama mini-pig skin was placed in physiological saline and thawed at room temperature. Then, it was cut successively from the back line of the pig skin downwards towards the abdomen, and the skin was cut into circular pieces with a diameter of about 3 cm and placed in physiological saline for standby. Note that adjacent skin was evenly divided into each group of preparations to ensure the consistency of the skin among the preparations.

[0092] 2. Transdermal Experiment Conditions

[0093] After the treated skin was placed on a clean absorbent paper to dry the surface moisture, the stratum corneum side was facing up, and the dosing ring was placed in the center of the skin and fixed. The fixed skin and dosing ring were placed on a balance to weigh, and the weight before drug application was printed. A certain amount of the cream to be tested was extruded at the center of the skin, and the cream was gently spread evenly with a degreased cotton swab wrapped with plastic wrap, then placed on the balance to weigh, and the weight after drug application was printed. The difference between the two weighings was the amount of drug applied (the amount of drug applied was about 0.27 g). Three groups were set up in parallel for each cream to be tested.

[0094] After drug application, the drug-containing skin was installed on the transdermal device, with the drug-containing layer facing up towards the supply pool and the non-drug-containing layer facing down towards the receiving pool. The receiving pool was a Franz diffusion cell with a volume of 12 mL and a diameter of 1.5 cm. The receiving pool was filled with degassed transdermal medium, and the transdermal medium was a 0.9% (mass percentage) sodium chloride aqueous solution (physiological saline). The temperature of the transdermal medium was 32°C, and the electromagnetic stirrer was turned on for continuous stirring at a speed of 600 rpm.

[0095] 3. Sample Collection

[0096] 3.1 Collection of the Receiving Solution

[0097] During the transdermal experiment, all the receiving solutions in the receiving pool were taken out at 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, and 6 h respectively, and an equal amount of transdermal medium at 32 °C was supplemented. The collected receiving solutions were stored at 5 °C for subsequent processing and determination, and the experiment was terminated at 6 h.

[0098] 3.2 Collection of the collected solution

[0099] After the transdermal experiment was completed, the remaining cream on the skin surface was observed. The skin surface was gently scraped with a small spoon, and the remaining cream on the skin surface was scraped as clean as possible into a beaker. Then, 1 mL of a 50% methanol aqueous solution by volume was pipetted into the supply pool with a 1 mL pipette gun, and the skin surface was gently scraped with a small spoon to dissolve the remaining cream on the skin surface in the 50% methanol aqueous solution as much as possible. The washing solution was transferred to the above beaker, and the above operation was repeated 4 times. The skin was removed, and the skin was rinsed with a small amount of 4 mL of 50% methanol aqueous solution multiple times. The rinsing solution was placed in the above beaker. The solution in the beaker was transferred to a 50 mL volumetric flask. The supply pool was placed in the above beaker, and the beaker was washed with a small amount of 50% methanol aqueous solution multiple times and the washing solution was collected into the above volumetric flask. Then, it was made up to the mark with 50% methanol aqueous solution by volume, sonicated for 10 min, and filtered through a 0.8 μm nylon syringe filter to obtain the collected solution.

[0100] 3.3 Collection of the extract

[0101] After the operation of "3.2 Collection of the collected solution", the residual solvent on the skin surface was blotted dry with filter paper. The skin around the outside that did not directly contact the cream was cut off with scissors, and only the skin with an area equivalent to the orifice of the pool (i.e., 1.77 cm 2 ) was retained. The skin was cut into pieces with scissors and put into a cryogenic grinder. The cryogenic grinding cup was placed in liquid nitrogen and frozen for 1 h. After taking it out, it was ground into powder. The powder was mixed with 25 mL of methanol, sonicated for 5 min, shaken well, and sonicated for another 5 min to obtain the extract.

[0102] 4. Preparation of the test solution (sample pretreatment)

[0103] 4.1 Preparation of the test solution from the receiving solution

[0104] Take 10 μL of the receiving solution collected in 3.1 above into a 1.5 mL EP tube, add 1000 μL of a 50% methanol aqueous solution by volume, and vortex to mix evenly. Centrifuge at 8000 rpm for 5 min at 5 °C. Take 200 μL of the centrifuged sample into a 96-well plate, add 200 μL of the internal standard solution, and mix well to obtain the test solution of the receiving solution. Among them, the concentration of lidocaine-d6 in the internal standard solution is 10 ng / mL, the concentration of prilocaine-d7 is 10 ng / mL, and the solvent is a 50% methanol aqueous solution by volume.

[0105] 4.2. Preparation of the sample solution to be measured from the collected liquid

[0106] Vortex and mix well the collected liquid obtained in 3.2 above, and centrifuge at 5 °C and 8000 rpm for 5 min. Take 10 μL of the centrifuged sample and transfer it into a 1.5 mL EP tube, then add 1000 μL of a methanol aqueous solution with a volume percentage of 50%, and vortex and mix well. Take 200 μL of the centrifuged sample and transfer it into a 96-well plate, then add 200 μL of the internal standard solution and mix well to obtain the sample solution to be measured from the collected liquid. Among them, the concentration of lidocaine-d6 in the internal standard solution is 10 ng / mL, the concentration of prilocaine-d7 is 10 ng / mL, and the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0107] 4.3. Preparation of the sample solution to be measured from the extract

[0108] Vortex and mix well the extract obtained in 3.3 above, and centrifuge at 5 °C and 8000 rpm for 5 min. Take 10 μL of the centrifuged sample and transfer it into a 1.5 mL EP tube, then add 1000 μL of a methanol aqueous solution with a volume percentage of 50%, and vortex and mix well. Then add 200 μL of the internal standard solution and mix well to obtain the sample solution to be measured from the extract. Among them, the concentration of lidocaine-d6 in the internal standard solution is 10 ng / mL, the concentration of prilocaine-d7 is 10 ng / mL, and the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0109] 5. High performance liquid chromatography - mass spectrometry (HPLC - MS) detection

[0110] Using a high performance liquid chromatography - mass spectrometry (HPLC - MS) instrument and combining with the internal standard method, detect the concentrations of lidocaine and prilocaine in the sample solutions to be measured from the receiving liquid, the collected liquid and the extract prepared in 4.1 - 4.3 above. Lidocaine-d6 is the internal standard for the analyte lidocaine, and prilocaine-d7 is the internal standard for the analyte prilocaine.

[0111] 5.1. HPLC conditions

[0112] Injector temperature: 5 °C;

[0113] Chromatographic column: ACE Excel 2C18-AR 2.1*50 mm;

[0114] Column temperature: 40 °C;

[0115] Mobile phase flow rate: 0.3 mL / min;

[0116] Injection volume: 2 μL;

[0117] Mobile phase A: Formic acid aqueous solution with a volume percentage of 0.1%; Mobile phase B: Methanol;

[0118] The gradient elution conditions are shown in Table 1 below:

[0119] Table 1

[0120]

[0121] The retention times of lidocaine, prilocaine, lidocaine-d6, and prilocaine-d7 are 2.0 min.

[0122] 5.2, Mass Spectrometry Conditions

[0123] Ion source: ESI+;

[0124] Source temperature: 500 °C;

[0125] Ionization mode: positive ion;

[0126] Monitoring mode: MRM;

[0127] Curtain gas pressure: 30 psi;

[0128] Collision gas pressure: 8 psi;

[0129] Ion spray voltage: 5500 V;

[0130] Gas1 pressure: 45 psi;

[0131] Gas2 pressure: 45 psi;

[0132] Lidocaine: Ion pair is 235.2 / 86.2, dwell time is 200 ms, declustering voltage is 60 V, collision energy is 60 eV, entrance voltage is 10 V, and collision cell exit voltage is 16 V;

[0133] Lidocaine-d6: Ion pair is 241.3 / 86.1, dwell time is 200 ms, declustering voltage is 60 V, collision energy is 26 eV, entrance voltage is 10 V, and collision cell exit voltage is 16 V;

[0134] Prilocaine: Ion pair is 221.2 / 86.1, dwell time is 200 ms, declustering voltage is 50 V, collision energy is 46 eV, entrance voltage is 10 V, and collision cell exit voltage is 18 V;

[0135] Prilocaine-d7: Ion pair is 228.3 / 86.1, dwell time is 200 ms, declustering voltage is 50 V, collision energy is 20 eV, entrance voltage is 10 V, and collision cell exit voltage is 16 V.

[0136] 6. Data Processing and Statistics

[0137] 6.1, Cumulative Permeation Amount of the Receiver Solution

[0138] The cumulative permeation amount Q per unit area at time t is calculated according to the following formula:

[0139] Q = (Cn·V + ∑Ci·Vi) / S

[0140] Q is the cumulative permeation amount per unit area at time t (ng·cm -2 ), V is the volume of the solution in the receiving cell, Vi is the volume of the sample taken, Cn is the measured concentration of the nth sample, Ci is the measured concentration of the (n - 1)th sample, and S is the effective permeation area.

[0141] 6.2. Permeation rate

[0142] Using the cumulative permeation amount per unit area (Q) as the ordinate and time (t) as the abscissa, draw the percutaneous permeation curves of each preparation respectively. Perform linear regression on the Q~t data, and the slope of the obtained straight line is the permeation rate J (μg·cm -2 ·h -1 ).

[0143] 6.3. Skin retention amount

[0144] The retention amount A of the drug in the skin is calculated according to the following formula:

[0145] A = V'·Ct / M

[0146] A is the content of the drug in the skin per unit mass (μg·g -1 ), Ct is the concentration measured after treating the skin sample at time t (i.e., the test solution prepared from the extract), V' is the volume of dilution after skin extraction, and M is the skin mass.

[0147] System suitability of the detection method in Effect Example 1

[0148] System suitability acceptance criteria: The coefficient of variation of the retention time of the analyte obtained from 3 repeated analyses ≤ 10.00%; the coefficient of variation of the internal standard retention time ≤ 10.00%; the coefficient of variation of the ratio of the analyte chromatographic peak area to the internal standard chromatographic peak area should ≤ 20.00%.

[0149] Lidocaine stock solution: Weigh 47.37 mg of lidocaine reference substance precisely and place it in a brown glass bottle. After calibration, it is 47.23 mg. Add 2361.5 μL of methanol to dissolve and shake well to prepare a lidocaine stock solution containing 20.00 mg / mL of lidocaine, and store it at -20°C.

[0150] Prilocaine stock solution: Weigh 41.54 mg of prilocaine reference substance precisely and place it in a brown glass bottle. After calibration, it is 39.71 mg. Add 1985.5 μL of methanol to dissolve and shake well to prepare a prilocaine stock solution containing 20.00 mg / mL of prilocaine, and store it at -20°C.

[0151] Blank matrix: The blank matrix (including blank receiving solution, blank collecting solution, and blank extraction solution) was prepared by the method of steps 1 to 3 in Example 1 above. Specifically, the customs declaration in step 1 was replaced with blank cream.

[0152] Lidocaine dilution 1 / prilocaine dilution 1 (receiving solution): Dilute the lidocaine stock solution / prilocaine stock solution with 50% methanol-water to prepare a working solution with a final concentration of lidocaine / prilocaine of 1 μg / mL; take 10 μL of the working solution and mix it with 190 μL of the blank receiving solution to obtain lidocaine dilution 1 / prilocaine dilution 1 with a concentration of 0.05 μg / mL.

[0153] Lidocaine dilution 2 / prilocaine dilution 2 (collecting solution): Dilute the lidocaine stock solution / prilocaine stock solution with 50% methanol-water to prepare a working solution with a final concentration of lidocaine / prilocaine of 40 μg / mL; take 10 μL of the working solution and mix it with 190 μL of the blank collecting solution to obtain lidocaine dilution 2 / prilocaine dilution 2 with a concentration of 2 μg / mL.

[0154] Lidocaine dilution 3 / prilocaine dilution 3 (extraction solution): Dilute the lidocaine stock solution / prilocaine stock solution with 50% methanol-water to prepare a working solution with a final concentration of lidocaine / prilocaine of 4 μg / mL; take 10 μL of the working solution and mix it with 190 μL of the blank extraction solution to obtain lidocaine dilution 3 / prilocaine dilution 3 with a concentration of 0.2 μg / mL.

[0155] Replace the receiving solution in the method of "4.1 Preparation of the receiving solution for the test solution" in Example 1 with the above-mentioned lidocaine dilution 1 or prilocaine dilution 1, and perform pretreatment to obtain the test solution; similarly, replace the collecting solution in the method of "4.2 Preparation of the collecting solution for the test solution" in Example 1 with the above-mentioned lidocaine dilution 2 or prilocaine dilution 2, and perform pretreatment to obtain the test solution; replace the extraction solution in the method of "4.3 Preparation of the extraction solution for the test solution" in Example 1 with the above-mentioned lidocaine dilution 3 or prilocaine dilution 3 for pretreatment, and perform pretreatment to obtain the test solution.

[0156] Detect the above-mentioned test solution by the method of "5 High-performance liquid chromatography-mass spectrometry detection" in Example 1 above. Each test solution was injected continuously 3 times, and the coefficient of variation of the peak area ratio and retention time of the analyte and internal standard in each test solution was examined. The results are shown in Table 2.

[0157] Table 2

[0158]

[0159]

[0160] Measured results:

[0161] Lidocaine: The coefficient of variation of the retention time of the analyte is ≤ 0.15%, the coefficient of variation of the retention time of the internal standard is ≤ 0.15%, and the coefficient of variation of the area ratio is ≤ 5.25%. Prilocaine: The coefficient of variation of the retention time of the analyte is ≤ 0.10%, the coefficient of variation of the retention time of the internal standard is ≤ 0.15%, and the coefficient of variation of the area ratio is ≤ 3.55%. All meet the above system suitability acceptance criteria, indicating that the test method of this application can ensure high stability and accuracy of the test results.

[0162] Effect Example 2 Standard Curve and Quantitative Range

[0163] The calibration curve samples are freshly prepared every day. Using a method similar to that in Effect Example 1, dilute the lidocaine stock solution or prilocaine stock solution to prepare calibration curve working solutions with different concentrations. Take 10.00 μL of the corresponding calibration curve working solution and add 190.0 μL of blank matrix for dilution and mixing to obtain the calibration curve samples. The concentrations of the calibration curve samples for lidocaine and prilocaine are as follows:

[0164] Receiving solution: 0.05000, 0.1000, 0.2000, 0.5000, 2.000, 10.00, 20.00, 50.00 μg / mL;

[0165] Extract: 0.2000, 0.5000, 2.000, 5.000, 10.00, 20.00, 50.00 μg / mL;

[0166] Collection solution: 2.000, 4.000, 8.000, 16.00, 25.00, 32.00, 40.00 μg / mL.

[0167] The receiving solution standard curve consists of 8 concentration levels, and the extract and collection solution standard curves consist of 7 concentration levels.

[0168] Acceptance criteria: Correlation coefficient R 2 Not less than 0.99 (R 2 ≥ 0.98); The concentration recalculated from the calibration curve samples should be within ± 15.00% of the theoretical concentration, and the lower limit of quantification should be within ± 20.00%; At least 75% of the calibration curve concentration points should be included, at least 6 concentration points should meet the above requirements, and at least one LLOQ (lower limit of quantification) and ULOQ (upper limit of quantification) concentration point should be included.

[0169] The calibration curves of all analytical batches will be calculated uniformly, including the average concentration, standard deviation (SD), coefficient of variation (%CV), and accuracy deviation (%RE).

[0170] The above samples were pretreated by using the method of "Preparation of the test solution" in the above Example 1 to obtain the test solution, and then the test solution was detected by using the method of "High performance liquid chromatography-mass spectrometry detection" in the above Example 1, and the standard curve was drawn.

[0171] The summary of the standard curve parameters corresponding to lidocaine and prilocaine in the receiving solution is shown in Tables 3 and 4 respectively, the summary of the standard curve parameters corresponding to lidocaine and prilocaine in the extraction solution is shown in Tables 5 and 6 respectively, and the summary of the standard curve parameters corresponding to lidocaine and prilocaine in the collection solution is shown in Tables 7 and 8 respectively.

[0172] Table 3

[0173] R for calibration curve fitting 2 Acceptance requirement: R 2 ≥0.98

[0174]

[0175] Concentration unit: μg·mL -1

[0176] Table 4 R for calibration curve fitting Acceptance requirement: R2≥0.98

[0177]

[0178] Concentration unit: μg·mL

[0179] Table 5

[0180] R for calibration curve fitting 2 Acceptance requirement: R 2 ≥0.98

[0181]

[0182] Concentration unit: μg·mL -1

[0183] Table 6

[0184] R for calibration curve fitting 2 Acceptance requirement: R 2 ≥0.98

[0185]

[0186] Concentration unit: μg·mL -1

[0187] Table 7

[0188] R for calibration curve fitting 2 Acceptance requirement: R 2 ≥0.98

[0189]

[0190] Concentration unit: μg·mL -1

[0191] Table 8

[0192] R value of calibration curve fitting 2 Acceptance requirement: R 2 ≥0.98

[0193]

[0194] Concentration unit: μg·mL -1

[0195] According to the above results, the R values of all standard curves 2 are all greater than 0.99, meeting the requirements. The standard curve samples were back-calculated. The results showed that the accuracy deviation between the back-calculated concentration and the theoretical concentration of lidocaine and prilocaine samples in the standard curves of the receiving solution, extraction solution, and collection solution was between -2.42% and 2.28%, and all data met the standard within ±15.00%. The accuracy deviation between the back-calculated concentration of the lower limit of quantification and the theoretical concentration was between -1.3% and 0.52%, and all data met the standard within ±20.00%. It can be seen that the linear relationship of the standard curve is good, ensuring high accuracy of the results analyzed using this standard curve. It can be seen that the linear relationship of the standard curve is good, ensuring high accuracy of the results analyzed using this standard curve.

[0196] Precision / Accuracy within / between 3 batches of Effect Example

[0197] The quality control samples were freshly prepared every day. 10.00 μL of the corresponding quality control working solution was taken and diluted with 190.0 μL of blank matrix (the preparation method is referred to Effect Example 1), and then mixed to obtain. The quality control concentrations of lidocaine and prilocaine were respectively:

[0198] Receiving solution: 0.05000 (LLOQQC), 0.1500 (LQC), 1.500 (MQC), 37.50 μg / mL (HQC); DQC: 200.0 μg / mL;

[0199] Extraction solution: 0.2000 (LLOQQC), 0.6000 (LQC), 4.000 (MQC), 37.50 μg / mL-l (HQC); DQC: 200.0 μg / mL;

[0200] Collection solution: 2.000 (LLOQQC), 5.000 (LQC), 15.00 (MQC), 30.00 μg / mL (HQC).

[0201] Precision / accuracy verification was completed with 3 verification batches over at least two days. The three batches of samples included analytical QC samples at four concentrations from LLOQ QC, LQC, MQC, and HQC, and 6 parallel samples were analyzed for each concentration. The above samples were pretreated using the method of "Preparation of 4 test solutions" in Example 1 above to obtain test solutions, and then the above test solutions were detected using the "5 High Performance Liquid Chromatography-Mass Spectrometry Detection Method" in Example 1 above. The within-batch precision (%CV), between-batch precision (%CV), and accuracy deviation (%RE) were calculated, and the results are shown in Table 9.

[0202] Table 9

[0203]

[0204]

[0205] Acceptance criteria: For the QC samples at each concentration level within and between batches, at least 2 / 3 of the QC samples and at least 50% of the samples at each concentration level should have a %RE not exceeding ±15.00% (the %RE of the LLOQ QC samples should not exceed ±20.00%). The average within-batch / between-batch %RE of the QC samples at each concentration level should not exceed ±15.00% (LLOQ should not exceed ±20.00%), and the %CV should be ≤15.00% (LLOQ should be ≤20.00%).

[0206] In addition, when analyzing batches other than the analytical accuracy and precision analysis batches, the acceptance criteria are: the %RE of the QC samples should not exceed ±15.00%. In each analytical batch, at least 2 / 3 of the QC samples and at least 50% of the samples at each concentration level need to meet the acceptance criteria.

[0207] Based on the above results, all results meet the above standards. It can be seen that the detection method of this application has ideal precision and accuracy.

[0208] Effect Example 4 Residual Analysis

[0209] A total of three batches of samples were prepared, and the evaluation was carried out using the blank matrix after the two highest calibration curve points in each analytical batch.

[0210] Acceptance criteria: The average peak area in the residual samples does not exceed 20.00% of the average peak area of the lower limit of quantitation, and does not exceed 5.00% of the average internal standard peak in the lower limit of quantitation.

[0211] The results showed that the residual percentages of the analytes lidocaine and prilocaine were 0% and ≤0.11% respectively, and the corresponding residual percentages of the internal standard were both 0%, meeting the above standards.

[0212] Effect Example 5 Screening of Receiving Liquids

[0213] Screening principle for the type of receiving solution: It is related to the solubility of the drug and the stability of the drug in the solution, and meets the requirements of the sink condition (in vivo, after the drug penetrates through the skin, it can be quickly transferred by the microcirculation to form a sink condition). The sink condition is generally investigated according to the receiving solution concentration not exceeding 10%-30% of the maximum solubility of the analyte.

[0214] The transdermal test was carried out using the method of Example 1 above. The concentrations of lidocaine and prilocaine in the receiving solution at each time point were measured. Six groups were tested in parallel. The percentage of the permeation amount at each time point to the saturated solubility was calculated to evaluate whether the receiving solution meets the sink condition. The results are shown in Table 10 below. Among them, the saturated solubility of lidocaine is 2.806±0.106 mg / mL; the saturated solubility of prilocaine is 4.781±0.069 mg / mL.

[0215] Table 10

[0216]

[0217]

[0218] The results of the investigation on the transdermal selectivity sensitivity showed that the maximum concentration of lidocaine in the receiving solution was 9.825 μg / mL, accounting for 0.35% of the saturated solubility of normal saline (calculated as 2.806 mg / mL); the maximum concentration of prilocaine was 12.67 μg / mL, accounting for 0.27% of the saturated solubility of normal saline (calculated as 4.781 mg / mL). The receiving solution concentration that meets the sink condition does not exceed the requirement of 10%-30% of the maximum solubility of the analyte. Therefore, it is feasible to select normal saline as the receiving solution.

[0219] Effect Example 6 was directed to the investigation of filter adsorption

[0220] The cream collecting solution was determined to be 50% methanol aqueous solution. Therefore, a solution containing 2.000 μg / mL of lidocaine and prilocaine each was prepared with 50% methanol aqueous solution to investigate the adsorption of the nylon syringe filter on the solution. 4 mL was aspirated into a syringe and filtered through a nylon syringe filter (0.8 μm). Each time 1 mL was pushed, the filtrate was collected in a 1.5 mL EP tube and then injected. The peak area ratio was used as the investigation index. The results are shown in Table 11.

[0221] Table 11

[0222]

[0223] According to the results of the change in the peak area ratio of the 1st - 4th mL of the subsequent filtrate, it can be seen that the nylon syringe filter (0.8 μm) has no adsorption on the lidocaine and prilocaine solutions prepared with 50% methanol aqueous solution.

[0224] In summary, the nylon needle filter has no effect on lidocaine and prilocaine solutions.

[0225] Feasibility verification of the drug extraction method in skin for Effect Example 7

[0226] Three blank skins with a diameter of 1.5 cm were respectively placed in three 5 mL EP tubes. 2 mL of normal saline solution containing 50 μg / mL of lidocaine and prilocaine each was added to each EP tube, and another control solution was placed at room temperature (without skin).

[0227] After 24 h, the skins were taken out, the solution on the skins was dried with filter paper, the dried skins were cut into pieces, placed in a grinding cup, frozen in liquid nitrogen for 1 h and then ground into powder. The skins were transferred to a 50 mL EP tube with 15 mL of methanol in small amounts for multiple times, sonicated for 10 min, and the extract and the remaining solution were collected. The test solution was prepared by using the method of "4.3 Preparation of the test solution for the extract" in Example 1 above, and the contents of lidocaine and prilocaine in the test solution were detected by using the method of "5 High performance liquid chromatography - mass spectrometry" in Example 1, and the recovery rate was calculated. The results are shown in Table 12.

[0228] Table 12

[0229]

[0230] According to the above results, it can be seen that the extraction method adopted in this application is feasible, has a high recovery rate, and good repeatability.

[0231] Investigation of the selectivity and sensitivity of the in vitro transdermal method for Effect Example 8

[0232] The purpose of this experiment was to compare the differences in the permeation behaviors of lidocaine and prilocaine in the excised skin among the negative control preparation (T1), the test preparation (T2) and the reference preparation (R), and to investigate the selectivity and sensitivity of the transdermal test method.

[0233] The reference preparation (R) was eutectic mixture of local anesthetics cream purchased from Aspen Pharma Trading Limited, containing 20 mg of lidocaine and prilocaine per 1 g. The negative control preparation (T1) was a self - made eutectic mixture of local anesthetics cream, containing 2.5 mg of lidocaine and prilocaine, 19 mg of polyoxyethylene hydrogenated castor oil - 54, 10 mg of carbomer homopolymer type B, 5.2 mg of sodium hydroxide and the balance of water per 1 g. The test preparation (T2) was a self - made eutectic mixture of local anesthetics cream, containing 25 mg of lidocaine and prilocaine, 19 mg of polyoxyethylene hydrogenated castor oil - 54, 10 mg of carbomer homopolymer type B, 5.2 mg of sodium hydroxide and the balance of water per 1 g.

[0234] The negative control preparation, the test preparation, and the reference preparation were alternately administered on a continuous diffusion cell to control the consistency of the test conditions between groups.

[0235] ① Grouping: One batch each of the negative control preparation (T1), the test preparation (T2), and the reference preparation (R), with 6 cups in parallel for each group. Cups numbered 1, 4, 7, 10, 13, and 16 were for the T1 preparation, cups numbered 2, 5, 8, 11, 14, and 17 were for the T2 preparation, and cups numbered 3, 6, 9, 12, 15, and 18 were for the R preparation.

[0236] ② Skin distribution: Adjacent skin was evenly distributed into different preparations in turn to ensure the homogeneity of the skin between groups as much as possible.

[0237] Drug application method: To ensure the consistency of the contact time of the cream with the skin and the sampling interval, a cross - drug - application method was used. After applying 1 cup of the negative control preparation, 1 cup of the test preparation and the reference preparation were then applied in the same drug - application manner.

[0238] Cream content test: Weigh about 0.27 g of the cream into a 10 mL EP tube. Each time, wash the EP tube with 5 mL of 50% methanol - water. The washing solution was placed in a 50 mL volumetric flask. After washing 4 times, it was made up to the mark with a 50% methanol - water solution by volume, sonicated for 5 min, and filtered through a 0.8 μm nylon syringe filter to obtain the sample. Refer to the method in Example 1 above to obtain the test solution and measure the contents of lidocaine and prilocaine in each test solution.

[0239] Results:

[0240] (1) Cumulative permeation amount

[0241] The data of the cumulative permeation amount per unit area of the lidocaine -prilocaine cream negative control preparation (T1), the lidocaine -prilocaine cream test preparation (T2), and the lidocaine -prilocaine cream reference preparation (R) are shown in Table 13, Figure 1 and Figure 2 , and the cumulative permeation amounts of lidocaine in T1, R, and T2 at 6 h were 81.65 ± 34.55, 206.7 ± 77.8, and 221.7 ± 94.9 μg / cm respectively. The cumulative permeation amounts of prilocaine in T1, R, and T2 at 6 h were 84.41 ± 35.67, 255.9 ± 97.2, and 277.5 ± 123.4 μg / cm respectively.

[0242] Table 13

[0243]

[0244] (2) Permeation rate

[0245] Taking the average cumulative penetration amount (Q) per unit area as the ordinate and time (t) as the abscissa, draw the percutaneous penetration curve. Perform linear regression on the Q~t data, and the slope of the obtained straight line is the penetration rate J (μg / cm 2 ), and calculate the lag time Tg (h) by dividing the intercept by the slope. The results are shown in Table 14, Figure 3 and Figure 4 .

[0246] Table 14

[0247]

[0248] Within the specified test time (6 h), the average penetration rate ratios of the negative control preparation T1 to the reference preparation R and the negative control preparation T1 to the test preparation T2 for lidocaine and prilocaine are not within the range of 0.7 to 1.3. The penetration rates of the negative control preparation T1 are lower than those of the reference preparation R and the test preparation T2.

[0249] The penetration rate of each actual segment is calculated using J = Cn * V / S / Δt, where Cn is the measured concentration of the nth sample, V is the volume of the solution in the receiving cell, S is the effective penetration area, and Δt is the sampling time interval between adjacent time points. Taking the penetration rate (J) as the ordinate and time (t) as the abscissa, draw the penetration rate curve. The results are shown in Figure 3 、 Figure 4 .

[0250] (3) Skin retention amount

[0251] The skin retention amount results of T1, R and T2 lidocaine and prilocaine cream are as shown in Table 15 below, Figure 5 and Figure 6 as follows:[[]]

[0252] Table 15

[0253]

[0254]

[0255] It can be seen from the results of the skin retention amount per unit mass that compared with the normal specification preparation (T2) and the reference preparation (R), the retention amount of the low specification preparation (T) is significantly lower.

[0256] In summary, when the dose is reduced, the permeation amount, penetration rate and skin retention amount of lidocaine and prilocaine are all lower. Therefore, it can be shown that this method has selective sensitivity.

[0257] (4) Mass balance

[0258] It can be seen from the detection and calculation results that the recovery rates of lidocaine in each preparation are T1 (102.4±1.7%), T2 (99.1±1.6%), R (98.6±2.5%) respectively, and the recovery rates of prilocaine are T1 (101.8±1.9%), T2 (105.1±2.0%), R (102.5±3.5%) respectively. The material balance of each preparation is basically achieved, indicating that the drugs in the dermis are fully extracted. The extraction method of this application is feasible, and the drug determination results of each part (remaining preparation, receiving solution and dermal retention amount) are accurate and reliable.

[0259] Finally, it should also be noted that in this application, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0260] Although the present application has been disclosed above through the description of specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the appended claims. These modifications, improvements or equivalents should also be considered to be included within the scope claimed by the present application.

Claims

1. A method for extracting leprocaine from skin, characterized in that: The method specifically comprises the following steps: after the leprocaine drug is subjected to transdermal treatment to remove the remaining leprocaine drug on the skin surface, the skin at the site where the leprocaine drug is applied is frozen with liquid nitrogen, crushed, and ultrasonically extracted.

2. The extraction method according to claim 1, characterized in that The extraction method satisfies at least one of the following conditions (1) to (7): (1) The step of cutting the skin into pieces before freezing; (2) The liquid nitrogen freezing time is more than 1 hour, preferably 1 to 3 hours; (3) The extraction liquid used in the ultrasonic extraction is an alcohol solvent, preferably methanol and / or ethanol; (4) During the ultrasonic extraction, the volume of the extract per unit area of ​​skin is 8 to 20 mL / cm 2 ; (5) The ultrasonic extraction time is 3 to 20 minutes, preferably 5 to 15 minutes; (6) The temperature of the ultrasonic extraction is room temperature; (7) After the ultrasonic extraction operation, a centrifugal operation is also included.

3. The extraction method according to claim 1 or 2, characterized in that The lidocaine drug comprises lidocaine, prilocaine and auxiliary materials.

4. The extraction method according to claim 3, characterized in that The leprocaine drug satisfies at least one of the following conditions (1) to (3): (1) The mass percentage of lidocaine to the leprocaine drug is 0.2% to 10%; (2) the mass percentage of the prilocaine to the leprocaine drug is 0.2% to 10%; (3) The auxiliary materials include at least one of an emulsifier, a matrix, a pH regulator and water; preferably, the emulsifier is polyoxyethylene hydrogenated castor oil, more preferably polyoxyethylene hydrogenated castor oil-54; preferably, the matrix is ​​carbomer, more preferably carbomer homopolymer type B; preferably, the pH regulator is an alkali metal hydroxide, more preferably selected from sodium hydroxide and / or potassium hydroxide; preferably, the mass percentage of the emulsifier to the leprocaine drug is 1% to 5%, more preferably 1.5% to 2.5%; preferably, the mass percentage of the matrix to the leprocaine drug is 0.5% to 5%, more preferably 0.5% to 1.5%; preferably, the mass percentage of the pH regulator to the leprocaine drug is 0.2% to 0.8%, more preferably 0.4% to 0.6%.

5. The extraction method according to any one of claims 1 to 4, characterized in that The extraction method satisfies at least one of the following conditions (1) to (7): (1) The skin used for the transdermal treatment is animal skin, preferably selected from pig skin, mouse skin or rabbit skin; (2) The device used for the transdermal treatment is selected from a Franz diffusion cell; preferably, the diameter of the Franz diffusion cell is 1 to 3 cm; (3) During the transdermal treatment, the transdermal medium is selected from physiological saline and / or phosphate buffer, preferably a sodium chloride aqueous solution with a mass percentage of 0.5% to 1.5%; (4) During the transdermal treatment, the temperature of the transdermal medium is 30-40°C, preferably 30-35°C; (5) During the transdermal treatment, the stirring speed of the transdermal medium is 500 to 700 rpm; (6) During the transdermal treatment, the mass volume ratio of the levocaine drug to the transdermal medium is 0.01 to 0.05 g / mL; (7) During the transdermal treatment, the transdermal medium may be degassed before use.

6. A method for detecting leprocaine in the skin, characterized in that: The detection method comprises the following steps: the test liquid comprises an extract obtained by the extraction method as described in any one of claims 1 to 5; and a high performance liquid chromatography-mass spectrometry device is used in combination with an internal standard method to perform qualitative or quantitative detection of lidocaine and prilocaine in the test liquid.

7. The detection method according to claim 6, characterized in that The detection method satisfies at least one of the following conditions (1) to (3): (1) The test liquid further includes a solvent; preferably, in the test liquid, the solvent is selected from alcohol or an aqueous solution of alcohol, more preferably an aqueous solution of alcohol with a volume percentage of 40% to 60%; more preferably, the alcohol is a volatile alcohol solvent, further more preferably methanol and / or ethanol; preferably, in the test liquid, the volume ratio of the extract to the solvent is 1:100 to 250; (2) The concentrations of lidocaine and prilocaine in the extract are independently 0.2 to 50 μg / mL, preferably 0.6 to 37.5 μg / mL; (3) In the internal standard method, the lidocaine internal standard is a deuterated lidocaine compound, and the prilocaine internal standard is a deuterated prilocaine compound; preferably, the lidocaine internal standard is lidocaine-d6; preferably, the prilocaine internal standard is prilocaine-d7; preferably, the concentrations of the lidocaine internal standard and the prilocaine internal standard are each independently 4 to 20 ng / mL, preferably 5 to 10 ng / mL.

8. The detection method according to claim 6 or 7, characterized in that: The detection method satisfies at least one of the following conditions (1) to (7): (1) In high performance liquid chromatography, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, preferably ACE Excel2C18-AR 2.1*50mm; preferably, the column length of the octadecylsilane bonded silica gel chromatographic column is 40 to 150 mm; preferably, the inner diameter of the octadecylsilane bonded silica gel chromatographic column is 1.8 to 4.6 mm; (2) In the high performance liquid chromatography, the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is an aqueous solution of a volatile acid, the mass percentage of the volatile acid in the aqueous solution of the volatile acid is 0.05% to 0.2%; the mobile phase B is a volatile alcohol solvent; preferably, the aqueous solution of the volatile acid is selected from an aqueous formic acid solution and / or an aqueous acetic acid solution; preferably, the mass percentage of the volatile acid in the aqueous solution of the volatile acid is 0.08% to 0.12%; preferably, the volatile alcohol solvent is methanol; (3) In HPLC, the flow rate of the mobile phase is 0.15 to 0.5 mL / min; (4) In high performance liquid chromatography, the elution method is gradient elution; when the gradient elution is used, the volume percentage of the mobile phase A is 85% to 95% and the volume percentage of the mobile phase B is 5% to 15% in 0 to 1.1 min; 1.2-2.4 min, the volume percentage of the mobile phase A is 8%-20%, and the volume percentage of the mobile phase B is 80%-92%; 2.5-3.5 min, the volume percentage of the mobile phase A is 85%-95%, and the volume percentage of the mobile phase B is 5%-15%; (5) In high performance liquid chromatography, the temperature of the injector is 4 to 15°C; (6) In high performance liquid chromatography, the injection volume of the test solution is 0.5 to 5 μL; (7) In high performance liquid chromatography, the column temperature is 30-50°C.

9. The detection method according to any one of claims 6 to 8, characterized in that: The detection method satisfies at least one of the following conditions (1) to (19): (1) In mass spectrometry, the ion mode is positive ion mode; (2) In mass spectrometry, the source temperature is 400-600°C; (3) In mass spectrometry, the scanning mode is multiple reaction monitoring mode; (4) In mass spectrometry, the ion source is an electrospray ion source; (5) In mass spectrometry, the drying gas is nitrogen; (6) In mass spectrometry, the curtain gas pressure is 10 to 40 psi; (7) In mass spectrometry, the collision gas pressure is 5 to 15 psi; (8) In mass spectrometry, the voltage applied in the electrospray ionization source is 3500 to 6000 V, preferably 4500 to 6000 V; (9) In mass spectrometry, the declustering voltage is 40 to 70 V, preferably 50 to 60 V; (10) In the mass spectrometry, the collision energy of lidocaine is 50 to 70 eV, preferably 55 to 65 eV; (11) In the mass spectrometry, the collision energy of the lidocaine internal standard is 16 to 36 eV, preferably 20 to 30 eV; (12) In the mass spectrum, the collision energy of prilocaine is 36 to 56 eV, preferably 40 to 50 eV; (13) In the mass spectrometry, the collision energy of the prilocaine internal standard is 10 to 30 eV, preferably 15 to 25 eV; (14) In mass spectrometry, the inlet voltage is 8–15 V; (15) In mass spectrometry, the collision cell outlet voltage is 10 to 30 V, preferably 15 to 20 V; (16) In the mass spectrum, the ion pair of lidocaine is 235.2 / 86.2; (17) In the mass spectrum, the ion pair of prilocaine is 221.2 / 86.1; (18) In the mass spectrum, the lidocaine internal standard was lidocaine-d6, and the ion pair of lidocaine-d6 was 241.3 / 86.1; (19) In the mass spectrum, the internal standard of prilocaine was prilocaine-d7, and the ion pair of prilocaine-d7 was 228.3 / 86.1.

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