In-vitro transdermal detection method of ripdicaine medicine

Abstract

The invention discloses an in-vitro transdermal detection method of a ripdicaine drug. The in-vitro transdermal detection method of the riptacaine medicine comprises the following steps: a to-be-detected solution comprises a sample collected by transdermal treatment of the riptacaine medicine or the riptacaine medicine; high performance liquid chromatography-mass spectrometry equipment is adopted, and an internal standard method is combined to qualitatively or quantitatively detect lidocaine and prilocaine in a to-be-detected solution. The method has the advantages of short detection time, high detection efficiency, high specific detection property, high accuracy, high precision and high detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis methods, and particularly relates to an in vitro transdermal detection method for prilocaine cream. Background Art

[0002] Prilocaine cream is a commonly used topical anesthetic preparation on the skin surface in clinical practice. Lidocaine and prilocaine in this 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. Prilocaine cream is commonly used in clinical practice for analgesia during intravenous puncture, intravenous cannulation, and various minor surgical operations, especially suitable for pediatric patients.

[0003] Pharmacokinetic studies have shown that when prilocaine cream is applied to intact skin, approximately 5% - 10% of lidocaine and prilocaine are 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] According to the relevant guidelines for reference in in vitro transdermal tests in the "Technical Guidelines for the Research of Topical Chemical Generic Drugs" trial version, the established in vitro transdermal test method needs to be correspondingly verified, mainly for the investigation of the sensitivity and selectivity of the transdermal method. Existing detection methods mainly use high-performance liquid chromatography to detect the collected transdermal samples and analyze the transdermal effect, but the detection time is long, all above 12 minutes, the detection efficiency is low, when dealing with a large number of samples, the detection cycle is long, the detection results between batches are prone to errors, the solvent usage is large, and the detection cost is high.

[0005] Therefore, there is an urgent need in this field to develop a transdermal substance detection method with exclusive detectability for lidocaine and prilocaine, high accuracy, high detection sensitivity and high detection efficiency. Summary of the Invention

[0006] The technical problem to be solved by this application is to overcome the defects in the existing transdermal sample detection methods such as long detection time and low detection efficiency, and provide an in vitro transdermal detection method for prilocaine cream. When the in vitro transdermal detection method adopted in this application is used to detect lidocaine and prilocaine, the detection can be completed within 3.5 minutes. The detection time is at least reduced by 2 / 3 compared with the existing method, significantly increasing the detection efficiency, reducing the solvent consumption, lowering the detection cost, and being applicable to high-throughput detection, meeting the detection requirements of a large number of samples and reducing the detection error between batches. And while ensuring rapid detection, it also has high exclusive detectability, accuracy, precision and detection sensitivity.

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

[0008] The present application provides an in vitro transdermal detection method for prilocaine and lidocaine drugs, which specifically includes the following steps: the test solution includes a sample collected by transdermal treatment of the prilocaine and lidocaine drugs or the prilocaine and lidocaine drugs; a high performance liquid chromatography-mass spectrometry (HPLC-MS) device is used, combined with the internal standard method, to qualitatively or quantitatively detect lidocaine and prilocaine in the test solution.

[0009] In some embodiments, the prilocaine and lidocaine drugs include lidocaine, prilocaine, and excipients.

[0010] Preferably, the mass percentage of lidocaine in the prilocaine and lidocaine drugs is 0.2% to 10%, such as 0.25% or 2.5%.

[0011] Preferably, the mass percentage of prilocaine in the prilocaine and lidocaine drugs is 0.2% to 10%, such as 0.25% or 2.5%.

[0012] Preferably, the excipients include at least one of an emulsifier, a matrix, a pH regulator, and water.

[0013] Among them, the emulsifier is polyoxyethylene hydrogenated castor oil, preferably polyoxyethylene hydrogenated castor oil-54.

[0014] Among them, the matrix is carbomer, preferably carbomer homopolymer type B.

[0015] Among them, the pH regulator is an alkali metal hydroxide, preferably selected from sodium hydroxide and / or potassium hydroxide.

[0016] Among them, the mass percentage of the emulsifier in the prilocaine and lidocaine drugs is 1% to 5%, preferably 1.5% to 2.5%, such as 1.9%.

[0017] Among them, the mass percentage of the matrix in the prilocaine and lidocaine drugs is 0.5% to 5%, preferably 0.5% to 1.5%, such as 1%.

[0018] Among them, the mass percentage of the pH regulator in the prilocaine and lidocaine drugs is 0.2% to 0.8%, preferably 0.4% to 0.6%, such as 0.52%.

[0019] In some embodiments, the test solution further includes a solvent.

[0020] 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.

[0021] Preferably, in the test solution, the volume ratio of the sample collected by transdermal treatment of prilocaine drug to the solvent is 1:100 to 250, such as 1:200.

[0022] In some embodiments, 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.

[0023] In some embodiments, the skin used for the transdermal treatment is animal skin, preferably selected from pig skin, mouse skin or rabbit skin.

[0024] 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.

[0025] In some embodiments, during the transdermal treatment, the transdermal medium is selected from normal saline and / or phosphate buffer solution, preferably an aqueous solution of sodium chloride with a mass percentage of 0.5% to 1.5%, such as an aqueous solution of sodium chloride with a mass percentage of 0.9%.

[0026] 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.

[0027] In some embodiments, during the transdermal treatment, the stirring speed of the transdermal medium is 500 to 700 rpm, such as 600 rpm.

[0028] 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.

[0029] In some embodiments, during the transdermal treatment, the transdermal medium may further include a degassing operation before use.

[0030] In some embodiments, the samples collected after the transdermal treatment of the prilocaine drug include a receiving solution, an extraction solution, and a collection solution. Among them, the receiving solution is the transdermal medium in which the drug is dissolved in the receiving pool after the transdermal treatment. The extraction solution is the liquid containing the prilocaine drug extracted from the skin after the transdermal treatment. The collection solution is the liquid containing the prilocaine drug remaining on the skin surface collected after the transdermal treatment.

[0031] Preferably, the concentrations of lidocaine and prilocaine in the receiving solution are each independently 0.05 - 50 μg / mL, more preferably 0.15 - 37.5 μg / mL.

[0032] Preferably, the concentrations of lidocaine and prilocaine in the extraction solution are each independently 0.2 - 50 μg / mL, more preferably 0.6 - 37.5 μg / mL.

[0033] Preferably, the concentrations of lidocaine and prilocaine in the collection solution are each independently 2 - 40 μg / mL, more preferably 5 - 30 μg / mL.

[0034] Preferably, the receiving solution may further include a centrifugation operation before use. More preferably, the rotation speed of the centrifugation is 6000 - 10000 rpm, such as 8000 rpm. More preferably, the time of the centrifugation is 3 - 10 min, such as 5 min. More preferably, the temperature of the centrifugation is 3 - 10 °C, such as 5 °C.

[0035] In a preferred embodiment, the method for obtaining the collection solution includes the following steps: after the transdermal treatment, collect the prilocaine drug remaining on the skin surface, treat the skin surface with a washing solution, and mix the washing solution with the prilocaine drug remaining on the collected skin surface.

[0036] Among them, after the mixing operation, it may further include at least one of the operations of ultrasonic treatment, filtration, and centrifugation. Preferably, the time of the ultrasonic treatment is 5 - 20 min, such as 10 min. Preferably, the filter used for filtration is a nylon needle filter. Preferably, the mesh number of the filter screen used for filtration is 0.3 - 1 μm, such as 0.45 μm or 0.8 μm. Preferably, the rotation speed of the centrifugation is 6000 - 10000 rpm, such as 8000 rpm. Preferably, the time of the centrifugation is 3 - 10 min, such as 5 min. Preferably, the temperature of the centrifugation is 3 - 10 °C, such as 5 °C.

[0037] Among them, the washing solution is selected from alcohols or aqueous solutions of alcohols. Preferably, it is an aqueous solution of alcohol with a volume percentage of 40% - 60%. For example, it is an aqueous solution of alcohol with a volume percentage of 50%. Preferably, the alcohol is a volatile alcohol solvent, more preferably methanol and / or ethanol.

[0038] In a preferred embodiment, the method for obtaining the extract comprises the following steps: After removing the remaining prilocaine drug on the skin surface, freeze, crush, and extract lidocaine and prilocaine from the skin at the site where the prilocaine drug is coated.

[0039] Among them, before freezing, the operation of cutting the skin into pieces may further be included. According to the routine in the art, this operation of cutting into pieces is beneficial to improving the freezing efficiency and shortening the freezing time.

[0040] Among them, the freezing method is liquid nitrogen freezing.

[0041] Among them, the freezing time is 1 h or more, preferably 1 - 3 h.

[0042] Among them, the extraction method is ultrasonic extraction.

[0043] Among them, the extraction liquid used for extraction is an alcohol solvent, preferably methanol and / or ethanol.

[0044] Among them, during extraction, the volume of the extraction liquid used per unit area of the skin is 8 - 20 mL / cm 2 , for example, 14 mL / cm 2 .

[0045] Among them, the extraction time is 3 - 20 min, preferably 5 - 15 min, for example, 10 min.

[0046] Among them, the extraction temperature is room temperature.

[0047] Among them, after the extraction operation, the operation of centrifugation may further be included. Preferably, the rotation speed of the centrifugation is 6000 - 10000 rpm, for example, 8000 rpm. Preferably, the centrifugation time is 3 - 10 min, for example, 5 min. Preferably, the centrifugation temperature is 3 - 10 °C, for example, 5 °C.

[0048] In the high performance liquid chromatography of some embodiments, the chromatographic column is an octadecylsilane chemically bonded silica gel chromatographic column, for example, ACE Excel 2C18 - AR 2.1*50 mm.

[0049] Preferably, the column length of the octadecylsilane chemically bonded silica gel chromatographic column is 40 - 150 mm, for example, 50 mm.

[0050] Preferably, the inner diameter of the octadecylsilane chemically bonded silica gel chromatographic column is 1.8 - 4.6 mm, for example, 2.1 mm.

[0051] In the high performance liquid chromatography of some embodiments, the mobile phase comprises mobile phase A and mobile phase B; the 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%; the mobile phase B is a volatile alcohol solvent.

[0052] Preferably, the aqueous solution of the volatile acid is selected from an aqueous solution of formic acid and / or an aqueous solution of acetic acid.

[0053] Preferably, the mass percentage of the volatile acid in the aqueous solution of the volatile acid is 0.08% to 0.12%, such as 0.1%.

[0054] Preferably, the volatile alcohol solvent is methanol.

[0055] 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.

[0056] In the high performance liquid chromatography of some embodiments, the elution method is gradient elution.

[0057] 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%.

[0058] In the high performance liquid chromatography of some embodiments, the temperature of the injector is 4 to 15 °C, such as 5 °C.

[0059] In the high performance liquid chromatography of some embodiments, the injection volume of the test solution is 0.5 to 5 μL, such as 2 μL.

[0060] In the high performance liquid chromatography of some embodiments, the column temperature is 30 to 50 °C, such as 40 °C.

[0061] In the mass spectrometry of some embodiments, the ion mode is positive ion mode.

[0062] In the mass spectrometry of some embodiments, the source temperature is 400 to 600 °C, such as 500 °C.

[0063] In the mass spectrometry of some embodiments, the scanning mode is multiple reaction monitoring mode.

[0064] In the mass spectrometry of some embodiments, the ion source is an electrospray ionization source.

[0065] In the mass spectrometry of some embodiments, the drying gas is nitrogen.

[0066] In the mass spectrometry of some embodiments, the curtain gas pressure is 10 to 40 psi, such as 30 psi.

[0067] In the mass spectrometry of some embodiments, the collision gas pressure is 5 to 15 psi, such as 8 psi.

[0068] In the mass spectrometry of some embodiments, the voltage applied in the electrospray ionization source is 3500 to 6000 V, preferably 4500 to 6000 V, such as 5500 V.

[0069] In the mass spectrometry of some embodiments, the declustering voltage is 40 to 70 V, such as 50 to 60 V.

[0070] In the mass spectrometry of some embodiments, the collision energy of lidocaine is 50 to 70 eV, preferably 55 to 65 eV, such as 60 eV.

[0071] In the mass spectrometry of some embodiments, the collision energy of the lidocaine internal standard is 16 to 36 eV, preferably 20 to 30 eV, such as 26 eV.

[0072] In the mass spectrometry of some embodiments, the collision energy of prilocaine is 36 to 56 eV, preferably 40 to 50 eV, such as 46 eV.

[0073] In the mass spectrometry of some embodiments, the collision energy of the prilocaine internal standard is 10 to 30 eV, preferably 15 to 25 eV, such as 20 eV.

[0074] In the mass spectrometry of some embodiments, the entrance voltage is 8 to 15 V, such as 10 V.

[0075] In the mass spectrometry of some embodiments, the collision cell exit voltage is 10 to 30 V, preferably 15 to 20 V, such as 16 V or 18 V.

[0076] In the mass spectrometry of some embodiments, the ion pair of lidocaine is 235.2 / 86.2.

[0077] In the mass spectrometry of some embodiments, the ion pair of prilocaine is 221.2 / 86.1.

[0078] 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;

[0079] 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.

[0080] Based on common knowledge, the above preferred conditions can be combined arbitrarily to obtain the preferred examples of the present application.

[0081] The reagents and raw materials used in this application are all commercially available.

[0082] The positive and progressive effects of this application are as follows: When the in vitro transdermal detection method provided by this application is used to detect lidocaine and prilocaine, the detection can be completed within 3.5 minutes. The detection time is at least reduced by 2 / 3 compared with the existing method, significantly increasing the detection efficiency, reducing the solvent consumption, lowering the detection cost, and being applicable to high-throughput detection, meeting the detection requirements of a large number of samples and reducing the detection error between batches. Moreover, while ensuring rapid detection, it also has high specific detectability, accuracy, precision, and detection sensitivity. Description of the Drawings

[0083] Figure 1 It is a comparison graph of the curves of the average cumulative penetration amount of lidocaine in the three groups of samples T1, T2, and R over time in Effect Example 8;

[0084] Figure 2 It is a comparison graph of the curves of the average cumulative penetration amount of prilocaine in the three groups of samples T1, T2, and R over time in Effect Example 8;

[0085] Figure 3 It is a comparison graph of the curves of the penetration rate per unit area of lidocaine over time after the three groups of samples T1, T2, and R are processed in Effect Example 8;

[0086] Figure 4 It is a comparison graph of the curves of the penetration rate per unit area of prilocaine over time after the three groups of samples T1, T2, and R are processed in Effect Example 8;

[0087] Figure 5 It is a comparison graph of the retention amount of lidocaine in the pig skin per unit mass after the three groups of samples T1, T2, and R are processed in Effect Example 8;

[0088] Figure 6 It is a comparison graph of the retention amount of prilocaine in the pig skin per unit mass after the three groups of samples T1, T2, and R are processed in Effect Example 8. Detailed Embodiments

[0089] 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 noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0090] In the following examples and comparative examples, methanol and formic acid are of HPLC grade, and water is ultrapure water.

[0091] Equipment information used:

[0092] (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 Stirring Bar.

[0093] (2) Shimadzu LC-30AD combined with SCIEX API 4000.

[0094] (3) JX-FSTPRP-11 Multi-Sample Cryogenic Grinder (Shanghai Jingxin Technology Co., Ltd.).

[0095] Example 1

[0096] 1. Skin Treatment

[0097] Porcine skin from 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 round 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.

[0098] 2. Transdermal Experiment Conditions

[0099] After the treated skin was placed on a clean absorbent paper to blot the surface moisture, the stratum corneum side was facing up, and a dosing ring was placed in the center of the skin to fix it. 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 test cream was extruded onto the center of the skin, and a degreased cotton swab wrapped with plastic wrap was used to gently spread the cream evenly. Then, it was 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 test cream.

[0100] 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, which was a 0.9% (mass percentage) sodium chloride aqueous solution (physiological saline). The temperature of the transdermal medium was 32°C, and an electromagnetic stirrer was turned on for continuous stirring at a speed of 600 rpm.

[0101] 3. Sample Collection

[0102] 3.1 Collection of Receiver Fluid

[0103] 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 replenished. The collected receiving solutions were stored at 5 °C for subsequent treatment and determination, and the experiment was terminated at 6 h.

[0104] 3.2 Collection of the collected solution

[0105] 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 the 50% methanol aqueous solution by volume several times with a total volume of 4 mL. 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 the 50% methanol aqueous solution by volume several times and the washing solution was collected into the above volumetric flask. Then, it was made up to the mark with the 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.

[0106] 3.3 Collection of the extract

[0107] 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 periphery 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 placed in 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.

[0108] 4. Preparation of the test solution (sample pretreatment)

[0109] 4.1 Preparation of the test solution from the receiving solution

[0110] 10 μL of the receiving solution collected in 3.1 above was taken into a 1.5 mL EP tube, and 1000 μL of a 50% methanol aqueous solution by volume was added, and vortexed thoroughly. Centrifuged at 8000 rpm at 5 °C for 5 min. 200 μL of the centrifuged sample was taken into a 96-well plate, and 200 μL of the internal standard solution was added and mixed well to obtain the test solution from the receiving solution. Among them, the concentration of lidocaine-d6 in the internal standard solution was 10 ng / mL, the concentration of prilocaine-d7 was 10 ng / mL, and the solvent was a 50% methanol aqueous solution by volume.

[0111] 4.2 Preparation of the liquid to be measured in the collected liquid

[0112] Vortex and mix the collected liquid collected in 3.2 above, and centrifuge at 5 °C and 8000 rpm for 5 min. Take 10 μL of the centrifuged sample into a 1.5 mL EP tube, add 1000 μL of a 50% (v / v) methanol aqueous solution, and vortex and mix. 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 liquid to be measured in 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 50% (v / v) methanol aqueous solution.

[0113] 4.3 Preparation of the liquid to be measured in the extract

[0114] Vortex and mix the extract collected in 3.3 above, and centrifuge at 5 °C and 8000 rpm for 5 min. Take 10 μL of the centrifuged sample into a 1.5 mL EP tube, add 1000 μL of a 50% (v / v) methanol aqueous solution, and vortex and mix. Add 200 μL of the internal standard solution, and mix well to obtain the liquid to be measured in 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 50% (v / v) methanol aqueous solution.

[0115] 5. High performance liquid chromatography-mass spectrometry (HPLC-MS) detection

[0116] Use an HPLC-MS instrument and the internal standard method to detect the concentrations of lidocaine and prilocaine in the receiving liquid to be measured, the collected liquid to be measured, and the extract to be measured 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.

[0117] 5.1 HPLC conditions

[0118] Injector temperature: 5 °C;

[0119] Chromatographic column: ACE Excel 2C18-AR 2.1*50 mm;

[0120] Column temperature: 40 °C;

[0121] Mobile phase flow rate: 0.3 mL / min;

[0122] Injection volume: 2 μL;

[0123] Mobile phase A: 0.1% (v / v) formic acid aqueous solution; Mobile phase B: methanol;

[0124] The gradient elution conditions are shown in Table 1 below:

[0125] Table 1

[0126]

[0127]

[0128] The retention times of lidocaine, prilocaine, lidocaine-d6 and prilocaine-d7 are 2.0 min.

[0129] 5.2, Mass Spectrometry Conditions

[0130] Ion source: ESI+;

[0131] Source temperature: 500 °C;

[0132] Ionization mode: positive ion;

[0133] Monitoring mode: MRM;

[0134] Curtain gas pressure: 30 psi;

[0135] Collision gas pressure: 8 psi;

[0136] Ion spray voltage: 5500 V;

[0137] Gas1 pressure: 45 psi;

[0138] Gas2 pressure: 45 psi;

[0139] Lidocaine: Ion pair is 235.2 / 86.2, dwell time 200 ms, declustering voltage is 60 V, collision energy is 60 eV, entrance voltage is 10 V, collision cell exit voltage is 16 V;

[0140] Lidocaine-d6: Ion pair is 241.3 / 86.1, dwell time 200 ms, declustering voltage is 60 V, collision energy is 26 eV, entrance voltage is 10 V, collision cell exit voltage is 16 V;

[0141] Prilocaine: Ion pair is 221.2 / 86.1, dwell time 200 ms, declustering voltage is 50 V, collision energy is 46 eV, entrance voltage is 10 V, collision cell exit voltage is 18 V;

[0142] Prilocaine-d7: Ion pair is 228.3 / 86.1, dwell time 200 ms, declustering voltage is 50 V, collision energy is 20 eV, entrance voltage is 10 V, collision cell exit voltage is 16 V.

[0143] 6. Data Processing and Statistics

[0144] 6.1 Cumulative Permeation Amount of the Receiver Solution

[0145] The cumulative permeation amount Q per unit area at time t is calculated by the following formula:

[0146] Q = (Cn·V + ∑Ci·Vi) / S

[0147] Q is the cumulative permeation amount per unit area at time t (ng·cm -2 ), V is the volume of the solution in the receiver 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.

[0148] 6.2 Permeation Rate

[0149] Taking the cumulative permeation amount per unit area (Q) as the ordinate and time (t) as the abscissa, the percutaneous permeation curves of each preparation are plotted respectively. The Q~t data are linearly regressed, and the slope of the obtained straight line is the permeation rate J (μg·cm -2 ·h -1 ).

[0150] 6.3 Drug Retention Amount in Skin

[0151] The retention amount A of the drug in the skin is calculated by the following formula:

[0152] A = V'·Ct / M

[0153] A is the content of the drug in the skin per unit mass (μg·g -1 ), Ct is the concentration measured after processing 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.

[0154] System Suitability of the Detection Method in Effect Example 1

[0155] 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 to the internal standard chromatographic peak area should ≤ 20.00%.

[0156] Lidocaine stock solution: Accurately weigh 47.37 mg of lidocaine reference substance 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.

[0157] Prilocaine stock solution: Accurately weigh 41.54 mg of prilocaine reference substance 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.

[0158] Blank matrix: Prepare the blank matrix (including blank receiving solution, blank collecting solution, and blank extraction solution) by the method of steps 1 - 3 in Example 1 above. Specifically, replace the incoming cream in step 1 with blank cream.

[0159] Lidocaine dilution solution 1 / Prilocaine dilution solution 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 a lidocaine dilution solution 1 / prilocaine dilution solution 1 with a concentration of 0.05 μg / mL.

[0160] Lidocaine dilution solution 2 / Prilocaine dilution solution 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 a lidocaine dilution solution 2 / prilocaine dilution solution 2 with a concentration of 2 μg / mL.

[0161] Lidocaine dilution solution 3 / Prilocaine dilution solution 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 a lidocaine dilution solution 3 / prilocaine dilution solution 3 with a concentration of 0.2 μg / mL.

[0162] Replace the receiving solution in the method of "4.1 Preparation of receiving solution test solution" in Example 1 with the above - mentioned lidocaine dilution solution 1 or prilocaine dilution solution 1, and perform pretreatment to obtain the test solution; Similarly, replace the collecting solution in the method of "4.2 Preparation of collecting solution test solution" in Example 1 with the above - mentioned lidocaine dilution solution 2 or prilocaine dilution solution 2, and perform pretreatment to obtain the test solution; Replace the extraction solution in the method of "4.3 Preparation of extraction solution test solution" in Example 1 with the above - mentioned lidocaine dilution solution 3 or prilocaine dilution solution 3 for pretreatment, and perform pretreatment to obtain the test solution.

[0163] Detect the above - mentioned test solutions by the method of "5 High - performance liquid chromatography - mass spectrometry detection" in Example 1 above. Inject each test solution continuously for 3 times, and examine the coefficient of variation of the peak area ratio and retention time of the analyte and internal standard in each test solution. The results are shown in Table 2.

[0164] Table 2

[0165]

[0166]

[0167] Measured results:

[0168] Lidocaine: The coefficient of variation of the retention time of the analyte ≤ 0.15%, the coefficient of variation of the retention time of the internal standard ≤ 0.15%, and the coefficient of variation of the area ratio ≤ 5.25%. Prilocaine: The coefficient of variation of the retention time of the analyte ≤ 0.10%, the coefficient of variation of the retention time of the internal standard ≤ 0.15%, and the coefficient of variation of the area ratio ≤ 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.

[0169] Effect Example 2 Standard Curve and Quantitative Range

[0170] The standard 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 standard curve working solutions with different concentrations. Respectively take 10.00 μL of the corresponding standard curve working solution, add 190.0 μL of blank matrix for dilution, and mix to obtain the standard curve samples. The concentrations of the standard curve samples of lidocaine and prilocaine are respectively:

[0171] Receiving solution: 0.05000, 0.1000, 0.2000, 0.5000, 2.000, 10.00, 20.00, 50.00 μg / mL;

[0172] Extract: 0.2000, 0.5000, 2.000, 5.000, 10.00, 20.00, 50.00 μg / mL;

[0173] Collection solution: 2.000, 4.000, 8.000, 16.00, 25.00, 32.00, 40.00 μg / mL.

[0174] The standard curve of the receiving solution consists of 8 concentration levels, and the standard curves of the extract and the collection solution consist of 7 concentration levels.

[0175] Acceptance criteria: Correlation coefficient R 2 not less than 0.99 (R 2 ≥ 0.98); The concentration recalculated from the standard curve samples should be within ±15.00% of the theoretical concentration, and the lower limit of quantification should be within ±20.00%; It should include at least 75% of the standard curve concentration points, at least 6 concentration points meet the above requirements, and at least include one LLOQ (lower limit of quantification) and ULOQ (upper limit of quantification) concentration point.

[0176] The calibration curves of all analysis batches will be calculated uniformly, including the average concentration, standard deviation (SD), coefficient of variation (%CV), and accuracy deviation (%RE).

[0177] The above samples were pretreated by the method of "4 Preparation of the test solution" in Example 1 above to obtain the test solution, and then the test solution was detected by the method of "5 High performance liquid chromatography - mass spectrometry detection method" in Example 1 above, and the standard curve was drawn.

[0178] The summary of the standard curve parameters for lidocaine and prilocaine corresponding to the receiving solution is shown in Tables 3 and 4 respectively, the summary of the standard curve parameters for lidocaine and prilocaine corresponding to the extract is shown in Tables 5 and 6 respectively, and the summary of the standard curve parameters for lidocaine and prilocaine corresponding to the collection solution is shown in Tables 7 and 8 respectively.

[0179] Table 3

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

[0181]

[0182] Concentration unit: μg·mL -1

[0183] Table 4

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

[0185]

[0186] Concentration unit: μg·mL -1

[0187] Table 5

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

[0189]

[0190] Concentration unit: μg·mL -1

[0191] Table 6

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

[0193]

[0194] Concentration unit: μg·mL -1

[0195] Table 7

[0196] R value of calibration curve fitting 2 Acceptance requirement: R 2 ≥0.98

[0197]

[0198] Concentration unit: μg·mL- 1

[0199] R value of calibration curve fitting in Table 8 2 Acceptance requirement: R 2 ≥0.98

[0200]

[0201] Concentration unit: μg·mL -1

[0202] 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, and 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.

[0203] Precision / Accuracy within / between batches in Effect Example 3

[0204] 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. The quality control concentrations of lidocaine and prilocaine were respectively:

[0205] Receiving solution: 0.05000 (LLOQ QC), 0.1500 (LQC), 1.500 (MQC), 37.50 μg / mL (HQC); DQC: 200.0 μg / mL;

[0206] Extract: 0.2000 (LLOQQC), 0.6000 (LQC), 4.000 (MQC), 37.50 μg / mL-l (HQC); DQC: 200.0 μg / mL;

[0207] Collection solution: 2.000 (LLOQQC), 5.000 (LQC), 15.00 (MQC), 30.00 μg / mL (HQC).

[0208] The precision / accuracy verification was passed through 3 verification batches and completed in at least two days. The three batches of samples included analytical QC samples from four concentrations of LLOQQC, LQC, MQC, and HQC. Six parallel samples were analyzed for each concentration. The above samples were pretreated by the method of "Preparation of 4 test solutions" in Example 1 above to obtain test solutions, and then the above test solutions were detected by the method of "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.

[0209] Table 9

[0210]

[0211]

[0212] 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 ≤15.00% (LLOQ should ≤20.00%).

[0213] 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.

[0214] According to the above results, all results meet the above standards. It can be seen that the detection method of the present application has ideal precision and accuracy.

[0215] Effect Example 4 Residual Analysis

[0216] A total of three batches of samples were made, and the blank matrix after the two highest calibration curve points in each analytical batch was used for evaluation.

[0217] Acceptance criteria: The average peak area in the residual sample does not exceed 20.00% of the average peak area at the lower limit of quantitation (LLOQ), and does not exceed 5.00% of the average internal standard peak area within the LLOQ.

[0218] The results showed that the residual percentages of lidocaine and prilocaine were 0% and ≤0.11% respectively, and the residual percentages of the corresponding internal standards were both 0%, meeting the above criteria.

[0219] Screening of the receiving solution in Effect Example 5

[0220] Principle for screening the types of receiving solutions: Related to the drug solubility and the stability of the drug in solution, meeting the requirements of the sink condition test (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 examined according to the receiving solution concentration not exceeding 10% - 30% of the maximum solubility of the analyte.

[0221] 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, and the percentage of the permeation amount at each time point to the saturated solubility was calculated to evaluate whether the receiving solution met the sink condition. The results are shown in Table 10 below. Among them, the saturated solubility of lidocaine was 2.806 ± 0.106 mg / mL; the saturated solubility of prilocaine was 4.781 ± 0.069 mg / mL.

[0222] Table 10

[0223]

[0224] 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 method.

[0225] Effect Example 6 Investigation on the adsorption of the filter

[0226] The cream collecting solution was determined to be 50% aqueous methanol solution. Therefore, a solution containing 2.000 μg / mL of lidocaine and prilocaine each was prepared with 50% aqueous methanol 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). The filtrate was collected once every 1 mL pushed and injected into a 1.5 mL EP tube. The peak area ratio was used as the investigation index. The results are shown in Table 11.

[0227] Table 11

[0228]

[0229] It can be seen from the change results of the peak area ratio of the 1st - 4th mL continuous filtrate that the nylon syringe filter (0.8μm) has no adsorption on lidocaine and prilocaine solutions prepared with 50% methanol aqueous solution.

[0230] Taking all into consideration, the nylon syringe filter has no effect on lidocaine and prilocaine solutions.

[0231] Feasibility verification of the extraction method of drugs in skin in Effect Example 7

[0232] 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).

[0233] After 24 h, the skins were taken out, the solutions on the skins were 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 multiple times, sonicated for 10 min, and the extract and the remaining solution were collected. The method of "4.3 Preparation of the test solution of the extract" in Example 1 above was used to prepare the test solution, and the method of "5 High - performance liquid chromatography - mass spectrometry" in Example 1 was used to detect the contents of lidocaine and prilocaine in the test solution, and the recovery rate was calculated. The results are shown in Table 12.

[0234] Table 12

[0235]

[0236] It can be known from the above results that the extraction method adopted in this application is feasible, has a high recovery rate, and good repeatability.

[0237] Investigation of the selectivity and sensitivity of the in vitro transdermal method in Effect Example 8

[0238] The purpose of this experiment is 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.

[0239] The reference preparation (R) is prilocaine and lidocaine cream purchased from Aspen Pharma Trading Limited, containing 20 mg of lidocaine and prilocaine per 1 g. The negative control preparation (T1) is self-made prilocaine and lidocaine 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) is self-made prilocaine and lidocaine 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.

[0240] The negative control preparation, the test preparation and the reference preparation were alternately administered on a continuous diffusion cell to control the consistency of test conditions between groups.

[0241] 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, 16 contained the T1 preparation, cups numbered 2, 5, 8, 11, 14, 17 contained the T2 preparation, and cups numbered 3, 6, 9, 12, 15, 18 contained the R preparation.

[0242] ② Skin allocation: Adjacent skin was evenly allocated to different preparations in turn to ensure the uniformity of skin between groups as much as possible.

[0243] Medication application method: To ensure the consistency of the contact time of the cream with the skin and the sampling interval, a cross-medication application method was used. After applying 1 cup of the negative control preparation, 1 cup of the test preparation and the reference preparation were applied in the same medication application method.

[0244] Cream content test: Weigh about 0.27 g of the cream into a 10 mL EP tube, wash the EP tube with 5 mL of 50% methanol water each time, place the washing solution in a 50 mL volumetric flask, after washing 4 times, make up the volume with 50% methanol aqueous solution by volume percentage, ultrasonicate for 5 min, filter with a 0.8 μm nylon needle filter to obtain the sample. Refer to the method in Example 1 above to obtain the test solution and test the contents of lidocaine and prilocaine in each test solution.

[0245] Results:

[0246] (1) Cumulative permeation amount

[0247] The data of the cumulative permeation amount per unit area of the negative control preparation (T1) of prilocaine and lidocaine cream, the test preparation (T2) of prilocaine and lidocaine cream and the reference preparation (R) of prilocaine and lidocaine cream are shown in Table 13, Figure 1 and Figure 2, The cumulative permeation amounts of lidocaine T1, R and T2 at 6 h were: 81.65±34.55, 206.7±77.8, 221.7±94.9 μg / cm respectively. The cumulative permeation amounts of prilocaine T1, R and T2 at 6 h were: 84.41±35.67, 255.9±97.2, 277.5±123.4 μg / cm respectively.

[0248] Table 13

[0249]

[0250] (2) Permeation rate

[0251] Using the average cumulative permeation amount per unit area (Q) as the ordinate and time (t) as the abscissa, the percutaneous permeation curve was plotted. The Q~t data were linearly regressed, and the slope of the straight line obtained was the permeation rate J (μg / cm 2 ), and the lag time Tg (h) was calculated by dividing the intercept by the slope. The results are shown in Table 14, Figure 3 and Figure 4 .

[0252] Table 14

[0253]

[0254] For lidocaine and prilocaine, within the specified test time (6 h), the ratios of the average permeation rates of the negative control preparation T1 to the reference preparation R and the negative control preparation T1 to the test preparation T2 were not within the range of 0.7 to 1.3. The permeation rates of the negative control preparation T1 were lower than those of the reference preparation R and the test preparation T2.

[0255] The permeation rate of each actual segment was calculated using J = Cn*V / S / Δt, where Cn was the measured concentration of the nth sample, V was the volume of the solution in the receiving cell, S was the effective permeation area, and Δt was the sampling time interval between adjacent time points. The permeation rate curve was plotted with the permeation rate (J) as the ordinate and time (t) as the abscissa. The results are shown in Figure 3 、 Figure 4 .

[0256] (3) Skin retention amount

[0257] The skin retention amount results of the lidocaine and prilocaine cream of T1, R and T2 are shown in Table 15 below, Figure 5 and Figure 6 as follows:

[0258] Table 15

[0259]

[0260]

[0261] From the results of the skin retention amount per unit mass, it can be seen 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.

[0262] In summary, when the dose is reduced, the permeation amount, permeation rate and skin retention amount of lidocaine and prilocaine are all low. Therefore, it can be shown that this method has selective sensitivity.

[0263] (4) Material balance

[0264] From the detection and calculation results, it can be known 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 materials of each preparation are basically balanced, indicating that the drugs in the skin are fully extracted. The extraction method of this application is feasible, and the drug determination results of each part (remaining preparation, receiving solution and skin retention amount) are accurate and reliable.

[0265] 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 also includes elements inherent to such process, method, article or device.

[0266] 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 solutions. These modifications, improvements or equivalents should also be considered to be included within the scope claimed by the present application.

Claims

1. An in vitro skin permeation detection method for a levocaine drug, characterized in that: The method comprises the following steps: the test liquid comprises a sample collected by transdermal treatment of a prilocaine drug or a prilocaine drug; and a high performance liquid chromatography-mass spectrometry device is used in combination with an internal standard method to perform qualitative or quantitative detection on lidocaine and prilocaine in the test liquid.

2. The in vitro skin permeation detection method according to claim 1, characterized in that: The prilocaine drug comprises lidocaine, prilocaine and excipients; Preferably, the mass percentage of lidocaine to the leprocaine drug is 0.2% to 10%; Preferably, the mass percentage of the prilocaine to the leprocaine drug is 0.2% to 10%; Preferably, the excipients include at least one of an emulsifier, a matrix, a pH regulator and water; more preferably, the emulsifier is polyoxyethylene hydrogenated castor oil, and further preferably is polyoxyethylene hydrogenated castor oil-54; more preferably, the matrix is ​​carbomer, and further preferably is carbomer homopolymer type B; more preferably, the pH regulator is an alkali metal hydroxide, and further preferably is selected from sodium hydroxide and / or potassium hydroxide; more preferably, the mass percentage of the emulsifier in the leprocaine drug is 1% to 5%, and further preferably is 1.5% to 2.5%; more preferably, the mass percentage of the matrix in the leprocaine drug is 0.5% to 5%, and further preferably is 0.5% to 1.5%; more preferably, the mass percentage of the pH regulator in the leprocaine drug is 0.2% to 0.8%, and further preferably is 0.4% to 0.6%.

3. The in vitro skin permeation detection method according to claim 1 or 2, characterized in that: The in vitro skin permeation detection method satisfies at least one of the following conditions (1) to (10): (1) The test liquid 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, and further preferably methanol and / or ethanol; preferably, in the test liquid, the volume ratio of the sample collected by transdermal treatment of the leprocaine drug to the solvent is 1:100 to 250; (2) 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 independently 4 to 20 ng / mL, preferably 5 to 10 ng / mL; (3) The skin used for the transdermal treatment is animal skin, preferably selected from pig skin, mouse skin or rabbit skin; (4) 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; (5) 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%; (6) During the transdermal treatment, the temperature of the transdermal medium is 30-40°C, preferably 30-35°C; (7) During the transdermal treatment, the stirring speed of the transdermal medium is 500 to 700 rpm; (8) During the transdermal treatment, the mass volume ratio of the levocaine drug to the transdermal medium is 0.01 to 0.05 g / mL; (9) During the transdermal treatment, the transdermal medium further includes a degassing operation before use; (10) The samples collected by transdermal treatment of the leprocaine drug include receiving solution, extraction solution and collection solution.

4. The in vitro skin permeation detection method according to claim 3, characterized in that: The in vitro skin permeation detection method satisfies at least one of the following conditions (1) to (4): (1) The concentrations of lidocaine and prilocaine in the receiving solution are independently 0.05 to 50 μg / mL, preferably 0.15 to 37.5 μg / mL; (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) The concentrations of lidocaine and prilocaine in the collected solution are independently 2 to 40 μg / mL, preferably 5 to 30 μg / mL; (4) The receiving solution further includes a centrifugation operation before use.

5. The in vitro skin permeation detection method according to claim 3 or 4, characterized in that: The method for obtaining the collected liquid comprises the following steps: after the transdermal treatment, collecting the remaining leprocaine drug on the skin surface, treating the skin surface with a lotion, and mixing the lotion with the collected leprocaine drug remaining on the skin surface.

6. The in vitro skin permeation detection method according to claim 5, characterized in that: The method for obtaining the collected liquid satisfies the following conditions (1) and / or (2): (1) After the mixing operation, at least one of ultrasonication, filtration and centrifugation is further performed; preferably, the ultrasonication time is 5 to 20 minutes; preferably, the filter used for filtration is a nylon needle filter; preferably, the filter mesh number used for filtration is 0.3 to 1 μm; (2) The washing liquid is selected from alcohol or an aqueous solution of alcohol, preferably an aqueous solution of alcohol with a volume percentage of 40% to 60%; preferably, the alcohol is a volatile alcohol, more preferably methanol and / or ethanol.

7. The in vitro skin permeation detection method according to claim 3 or 4, characterized in that: The method for obtaining the extract comprises the following steps: after removing the remaining prilocaine drug on the skin surface, freezing and crushing the skin at the site where the prilocaine drug is applied, and extracting lidocaine and prilocaine.

8. The in vitro skin permeation detection method according to claim 7, characterized in that: The method for obtaining the extract satisfies at least one of the following conditions (1) to (9): (1) The step of cutting the skin into pieces before freezing; (2) The freezing method is liquid nitrogen freezing; (3) The freezing time is more than 1 hour, preferably 1 to 3 hours; (4) The extraction method is ultrasonic extraction; (5) The extraction liquid used in the extraction is an alcohol solvent, preferably methanol and / or ethanol; (6) During extraction, the volume of the extract used per unit area of ​​skin is 8-20 mL / cm 2 ; (7) The extraction time is 3 to 20 minutes, preferably 5 to 15 minutes; (8) The extraction temperature is room temperature; (9) After the extraction operation, a centrifugation operation is also included.

9. The in vitro skin permeation detection method according to any one of claims 1 to 8, characterized in that: The in vitro skin permeation 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, in the mobile phase A, 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 high performance liquid chromatography, the flow rate of the mobile phase is 0.15 to 0.5 mL / min, preferably 0.3 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.

10. The in vitro skin permeation detection method according to any one of claims 1 to 9, characterized in that: The in vitro skin permeation 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.

Images