Detection method for in-vitro transdermal penetration of ripdicaine emulsifiable paste

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting in-vitro transdermal penetration of a riptacaine cream by using an animal in-vitro skin instead of an artificial membrane through a diffusion cell method. The method comprises the following steps: based on an open diffusion cell system, putting in-vitro skin between quantitative loops, loading a sample on the quantitative loops by taking riptacaine cream, adding a permeation medium, setting test temperature, medium volume and stirring speed, sampling for a fixed time, and analyzing the permeation sample, the epidermal residual sample and the intradermal retention sample by using a high performance liquid chromatography method, so as to determine the content of riptacaine in the liptacaine cream in the liptacaine cream in the liptacaine cream in the liptacaine cream. A reversed-phase liquid chromatographic column is adopted, the flow velocity, the column temperature, the detection wavelength and the sample injection volume are set, and a mixed phase of a water phase and an organic phase is used as a mobile phase for isocratic operation. The invention discloses a post-treatment process of a riptacaine in-vitro transdermal sample, and the disclosed detection method is high in sensitivity, good in accuracy and strong in distinguishing ability, can be used for in-vitro transdermal evaluation of riptacaine cream, and has positive effect and practical application value for quality control of the riptacaine cream.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting the in vitro skin permeation of leprocaine cream by using a diffusion cell method with animal ex vivo skin replacing an artificial membrane. Background Art

[0002] Lidocaine is a local anesthetic and antiarrhythmic drug. It is a derivative of cocaine, but it does not have the hallucinations and addictive components of cocaine. Lidocaine hydrochloride is a white crystalline powder, easily soluble in water, and has the same toxicity as procaine, but the local anesthetic effect is stronger and longer-lasting, with good surface penetration. It can be injected or used as a surface anesthesia. Lidocaine is a very good local anesthetic. It usually takes effect one to three minutes after application and the effect lasts one to three hours. Its structural formula is as follows:

[0003] Prilocaine is an amide local anesthetic. Its anesthetic intensity and speed are similar to those of lidocaine, but its duration is longer and its vasodilatory effect is weaker. Its toxicity is lower than that of lidocaine. It is clinically used for local anesthesia, especially for patients who cannot take epinephrine. Its structural formula is as follows:

[0004] Lidocaine cream is a topical local anesthetic containing a mixture of lidocaine and prilocaine. Since most nerve endings in the skin are usually located in the subcutaneous tissue, local anesthetics applied to the skin surface must pass through the stratum corneum and reach the skin before they can exert an anesthetic effect. The stratum corneum contains keratinocytes and absorbs water. There are intercellular lipids between keratinocytes and act as a barrier for water-soluble substances. In order for the local anesthetic to pass through the stratum corneum and reach the subcutaneous tissue, a preparation containing a high concentration of a hydrophobic local anesthetic that can pass through the lipid layer and has a high water content is required.

[0005] Due to the complexity of the prilocaine cream process, in vitro transdermal permeation, as a key quality attribute, its research can not only distinguish preparations with different prescription processes, but also compare the quality level of this product with that of the original research, meeting the quality control requirements of this product. At present, the pharmacopoeias of various countries do not include the detection method for in vitro transdermal permeation of prilocaine cream. Through literature research, it is found that there are many studies on the transdermal permeation of lidocaine alone, but few on the in vitro transdermal permeation of compound lidocaine. The main methods are as follows: gas chromatography-mass spectrometry was used to simultaneously determine prilocaine and lidocaine in the transdermal receiving solution, Yang Yingying, etc., "Chinese Chromatographic Impurities", Volume 27, Issue 1, Pages 74-77 (Document 1); J. Klein, et al., Journal of Chromatography B: Biomedical Sciences and Applications Volume 655, Issue 1, 22 April 1994, Pages 83-88 (Document 2). When detecting lidocaine and prilocaine in the receiving solution in Document 1, the receiving solution was extracted twice and analyzed by GC-MS. This process is complex and time-consuming, and there are losses during the extraction process. The extraction recovery rate is between 85.3% and 91.5%. Document 2 discloses a method for detecting the content of lidocaine and prilocaine in plasma. This method detects the content of lidocaine and prilocaine in plasma, and the linear relationship between lidocaine and prilocaine is poor, r < 0.998.

[0006] In summary, there is an urgent need to develop a detection method for in vitro transdermal permeation of prilocaine cream with simple operation, high precision and good repeatability. Summary of the Invention

[0007] The purpose of the present invention is to solve the problems of poor reproducibility of in vitro transdermal permeation detection and great difficulty in residual detection in the prior art, and provide a detection method for in vitro transdermal permeation of prilocaine cream with strong discrimination ability, high sensitivity, good repeatability and accuracy, which is used for evaluating the in vitro transdermal permeation of prilocaine.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention discloses a detection method for in vitro transdermal permeation of prilocaine cream. According to the embodiments of the present invention, based on an open diffusion cell system, the excised skin is placed between quantitative loops, prilocaine cream is sampled on the quantitative loops, a permeation medium is added, the test temperature, medium volume, and stirring speed are set, samples are taken after a fixed time, and then the permeated samples, epidermal residual samples, and intradermal retention samples are analyzed by high performance liquid chromatography. A reversed-phase chromatographic column is used, and an isocratic elution is performed with a mixed phase of an aqueous phase and an organic phase as the mobile phase. The flow rate, column temperature, detection wavelength, and injection volume are set; wherein the diffusion cell system is a Logan DHC-6TD diffusion cell.

[0009] According to an embodiment of the present invention, the ex vivo skin is selected from mouse skin or pig skin. Since the skin permeability of pigs is more similar to that of human skin, the ex vivo skin is selected from pig skin. Preferably, it is selected from the skin of miniature pigs at 30 days old or 45 days old.

[0010] More preferably, the ex vivo skin is selected from the skin of miniature pigs at 30 days old.

[0011] According to an embodiment of the present invention, the intradermal retention samples used in the present invention must be post-treated. The post-treatment methods are selected from different skin treatment methods and different ultrasonic durations, as shown in the following table. When the skin after in vitro percutaneous absorption test is cleaned and cut into pieces, and then medium and acetonitrile are added, and the ultrasonic duration is selected as 40 min, the intradermal retention samples can be effectively measured to ensure material balance.

[0012] Table 1 Post-treatment methods and results of different intradermal retention samples

[0013] According to an embodiment of the present invention, the medium is selected from normal saline and phosphate buffer solution, preferably phosphate buffer solution.

[0014] Preferably, the pH value of the phosphate buffer solution is selected from 5.0 - 8.0, preferably 7.4.

[0015] According to an embodiment of the present invention, the dosing amount of lidocaine cream in the detection method disclosed in the present invention is 1.5 g / 10 cm 2 ~ 2 g / 10 cm 2 Preferably, it is 1.5 g / 10 cm 2 The area of the diffusion cell is 1.76 cm 2 The corresponding dosing amount of the drug is 0.3 g; the temperature of the diffusion cell is 25 - 37 °C, preferably 32 °C.

[0016] According to an embodiment of the present invention, the analytical method used in the present invention is high performance liquid chromatography. The reverse phase chromatographic column is selected from Phenomenex Titank C18, and its specifications are 150 mm × 4.6 mm, 3 µm.

[0017] According to an embodiment of the present invention, the mobile phase is a mixture of an aqueous phase and an organic phase, selected from phosphate buffer solution - acetonitrile.

[0018] Preferably, the volume ratio of phosphate buffer solution to acetonitrile is selected from 35∶65.

[0019] More preferably, the phosphate buffer solution is selected from sodium dihydrogen phosphate - disodium hydrogen phosphate buffer solution and potassium dihydrogen phosphate solution, and the pH value is adjusted to 7.2 with 5 mol / L sodium hydroxide.

[0020] According to an embodiment of the present invention, in the liquid chromatography method disclosed by the present invention, the column temperature is selected from 30°C, the flow rate is selected from 1.0 ml / min, the wavelength is selected from 232 nm, and the injection volume is selected from 10 μl.

[0021] The beneficial effects of the present invention are as follows: (1) The present invention discloses a post-treatment process for in vitro transdermal samples of prilocaine and lidocaine, which can effectively detect the surface residue and intradermal retention amount of the samples; (2) The present invention discloses a detection method for in vitro transdermal of prilocaine and lidocaine cream. The disclosed method has high sensitivity, good accuracy and strong discrimination ability, can be used for the evaluation of in vitro transdermal of prilocaine and lidocaine cream, and has positive effects and practical application value for the quality control of prilocaine and lidocaine cream. Description of the Drawings

[0022] Figure 1 The figure shows the change of the permeation rate per unit area of lidocaine in the preparation investigated in Example 2 over time; Figure 2 The figure shows the change of the permeation rate per unit area of prilocaine in the preparation investigated in Example 2 over time; Figure 3 The figure shows the high performance liquid chromatography (HPLC) chromatogram of the in vitro transdermal blank solution in Example 4; Figure 4 The figure shows the high performance liquid chromatography (HPLC) chromatogram of the in vitro transdermal blank matrix in Example 4; Figure 5 The figure shows the high performance liquid chromatography (HPLC) chromatogram of the in vitro transdermal blank medium in Example 4; Figure 6 The figure shows the high performance liquid chromatography (HPLC) chromatogram of the in vitro transdermal reference solution in Example 4; Figure 7 The figure shows the high performance liquid chromatography (HPLC) chromatogram of the in vitro transdermal drug-containing matrix solution in Example 4. Detailed Embodiments

[0023] The present invention provides a detection method for in vitro transdermal of prilocaine and lidocaine cream, which comprises the following steps: (1) Skin quality control and skin integrity test Before the experiment starts, the thickness of the porcine skin needs to be measured with a vernier caliper, and the thickness of the porcine skin should be 1.0 mm ± 0.2 mm. The skin integrity test is carried out by the method of transepidermal water loss. The transepidermal water loss (TEWL) value should not exceed 20 g / m 2 / h; (2) Transdermal experiment conditions In vitro percutaneous penetration test was carried out using a modified Franz diffusion cell. The pre-saturated excised skin was fixed between the donor cell and the receptor cell, with the stratum corneum of the skin facing the donor cell. Approximately 0.3 g of the cream was weighed and applied. Using approximately 12 ml of the release medium as the receptor solution, the diffusion cell was placed under a constant temperature condition of 32 °C ± 0.5 °C, and an electromagnetic stirrer was started at a rotation speed of 600 r / min. At 1, 2, 3, 4, 5, 6, 8, 12, and 20 h respectively, 10 ml of the receptor solution was taken and immediately replenished with an equal volume and temperature of the receptor solution. The receptor solution was filtered through a 0.45 μm PP microporous membrane, and the subsequent filtrate was taken for HPLC determination; The release medium was PBS buffer solution with pH 7.4; The excised skin selected was the skin of 30-day-old miniature pigs; The chromatographic column was Phenomenex Titank C18, 150 mm × 4.6 mm, 3 μm; The mobile phase was phosphate buffer solution (pH 7.2) - acetonitrile (35∶65); The detection wavelength was 232 nm; The flow rate was 1.0 ml per minute; Column temperature: 30 °C; Injection volume: 10 μl; After the in vitro percutaneous penetration test was terminated, the residual ointment on the surface of the pig skin was immediately scraped off, placed in a 50 ml volumetric flask, 2.5 ml of 5 mol / L sodium hydroxide solution was added to disperse the cream evenly, 2.5 ml of 5 mol / L hydrochloric acid solution was added, and it was dissolved and diluted to the scale with water - acetonitrile (80∶20), filtered (it is recommended to use a 0.45 μm filter head made of PP material), and the subsequent filtrate was taken for HPLC determination to obtain the surface residue amount; After the in vitro percutaneous penetration test was terminated, the residual ointment on the surface of the pig skin was immediately scraped off, the pig skin was rinsed with normal saline, the moisture on the skin was blotted dry with filter paper, the excess skin was cut off, and the skin with an area of about 1.76 cm 2 in contact with the paste was cut into pieces, transferred to a 10 ml centrifuge tube, 0.9 ml of the receptor solution was added, 8.1 ml of acetonitrile was added, ultrasonic extraction was carried out for 40 min, centrifuged at a rotation speed of 4000 r / min for 15 min, the supernatant was decanted, filtered through a 0.45 μm PP membrane, 1 ml of the filtrate was transferred to a 10 ml volumetric flask, and diluted to the scale with water - acetonitrile (80∶20) to obtain the retention rate sample, and HPLC determination was carried out to obtain the intradermal retention amount.

[0024] The present invention will be described below in conjunction with specific examples, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1 The present invention measures the saturation solubility of three receiving media with an ionic strength of 0.05 and pH values of 5.0, 6.0, and 7.0 at 32°C, and the measurement results all meet the sink conditions. The above three media are respectively used for in vitro release experiments. The experimental results show that when the ionic strength of the release medium is 0.05, the release rate (slope) is low, and the cumulative release amount at 8 hours is only about 50%, which does not meet the experimental requirements. The ionic strength of the medium is increased to 1.5, and referring to the simulated body fluid environment in the in vitro transdermal method, the pH of the medium is set to 7.4. The cumulative release amounts of prilocaine and lidocaine at 8 hours are both about 90%. Therefore, the receiving medium is confirmed to be a phosphate buffer solution with an ionic strength of 1.5 and a pH of 7.4. The screening results are shown in Tables 2 to 3.

[0026] Table 2 Comparison of saturation solubility of media with different pH values

[0027] Table 3 Comparison of release rate and cumulative release amount of media with different ionic strengths

[0028] Example 2 Transdermal experiments were carried out on the skin of miniature pigs of different months of age using the developed preparation, and the epidermal residue, intradermal retention, and permeated sample amount were measured respectively. The results are shown in Tables 4 to 7.

[0029] Table 4 In vitro transdermal results of different miniature pig skins - lidocaine

[0030] Table 5 In vitro transdermal results of different miniature pig skins - prilocaine

[0031] Table 6 Drug permeation amount per unit area at different time points of different miniature pig skins - lidocaine, unit (mg / cm 2 )

[0032] Table 7 Drug permeation amount per unit area at different time points of different miniature pig skins - prilocaine, unit (mg / cm 2 )

[0033] Conclusion: (1) In vitro transdermal experiments were carried out on the skin of two-month-old pigs at the same time, and the results showed that the measured results of various parameters were basically the same. Since the skin of 30-day-old pigs has better skin adhesion and is more convenient for sample loading, the skin of 30-day-old miniature pigs was selected as the experimental skin.

[0034] (2) From Figure 1 , Figure 2It can be seen that the absorption rate tends to be stable after 12 hours. After 24 hours, due to the long skin immersion time, the absorption rate decreases. Therefore, the running time can be shortened to 20 hours, so that the absorption rate tends to be stable for 4 hours without damaging the skin structure. Example 3

[0035] Examine the ratio of the aqueous phase to the organic phase in the mobile phase. The screening results are as follows in the table: Table 8 Screening of Mobile Phase Ratio

[0036] Conclusion: For Method 1, since the two peaks are relatively close and the baseline is uneven. Considering the detection time and the distance between the two chromatographic peaks comprehensively, the ratio of the aqueous phase to the organic phase is selected as 35:65.

[0037] Example 4 Examine the influence of different chromatographic columns on the resolution and peak shape of the two main components when sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is used as mobile phase A. The results are as follows: Table 9 Screening of Chromatographic Columns

[0038] Note: In the above table, "P" represents prilocaine and "L" represents lidocaine.

[0039] When using a 10 cm chromatographic column, the sample elutes too early and the column efficiency is poor. Therefore, a 15 cm chromatographic column is selected. Finally, Phenomenex Titank C18 150 mm × 4.6 mm, 3 μm column with the same column efficiency as ACE excel 3 superC18 4.6*150 mm column is determined. Since the preparation is relatively cumbersome with the mixture of two phosphates, according to the actually measured pH value (7.2) of the buffer salt, the aqueous phase in the mobile phase is adjusted to phosphate buffer solution (take 2.73 g of potassium dihydrogen phosphate, dissolve it in 1000 ml of water, and adjust the pH value to 7.2 with 5 mol / L sodium hydroxide). Example 5

[0040] Prepare sample solutions with different concentrations, inject samples respectively to determine the detection limits and quantification limits of lidocaine and prilocaine. The results are shown in Tables 10 - 11.

[0041] Table 10 Detection Limits and Quantification Limits of Lidocaine and Prilocaine

[0042] Table 11 Results of Repeatability Experiment of Quantification Limit

[0043] The results show that the detection limits and quantification limits of lidocaine and prilocaine are much lower than the concentration of the sample solution, indicating that the liquid chromatography detection method used in the present invention has high detection sensitivity and good repeatability, meeting the detection requirements for in vitro transdermal of lidocaine and prilocaine cream. Compared with the GC-MS analysis method used in Comparative Document 1, the operation is simpler and the equipment cost is lower. Example 6

[0044] In this example, the linear relationship of the above detection method was investigated, and the results are as follows: Under the conditions of this method, a test solution with an appropriate concentration was prepared and diluted with a diluent to form a series of concentrations as the linear solutions of each component. The results show that the linear relationships of lidocaine and prilocaine are good.

[0045] Table 12 Results of the linear experiment

[0046] Compared with Document 2, the linear correlation coefficient r is higher and the linear relationship is better, indicating that the determination of lidocaine and prilocaine by this method has higher accuracy. Example 7

[0047] (1) Blank solution: acetonitrile - water (20∶80); (2) Blank medium: blank release medium; (3) Blank matrix: The excised skin was fixed between the supply cell and the receptor cell with the stratum corneum of the skin facing the supply cell. Approximately 0.3 g of blank excipients were weighed and applied with medicine, and the effective medicine application area was about 1.76 cm 2 , with approximately 12 ml of phosphate buffer solution (pH 7.4) as the receptor solution. The diffusion cell was placed in a constant temperature condition of 32°C ± 0.5°C, and the electromagnetic stirring was started at a rotation speed of 600 r / min. After the experiment ended, the receptor solution was taken, and 3 portions were prepared in parallel, filtered through a 0.45 μm mixed cellulose filter membrane, and the subsequent filtrate was taken; (4) Reference solution: An appropriate amount of lidocaine and prilocaine reference substances were accurately weighed and diluted with the blank solution to a solution containing about 0.1 mg of lidocaine and prilocaine per 1 ml; (5) Medicated matrix: An appropriate amount of lidocaine and prilocaine reference substances were accurately weighed and diluted with the blank matrix to a solution containing about 0.1 mg of lidocaine and prilocaine per 1 ml.

[0048] (6) Chromatographic conditions Chromatographic column: Phenomenex Titank C18 150 mm × 4.6 mm, 3 μm or a chromatographic column with equivalent performance; Column temperature: 30°C Flow rate: 1.0 ml / min Wavelength: 232 nm Injection volume: 10 μl Running time: 6 min; Mobile phase: Phosphate buffer (take 2.73 g of potassium dihydrogen phosphate, dissolve it in 1000 ml of water, and adjust the pH value to 7.2 with 5 mol / L sodium hydroxide) - acetonitrile (35:65); It can be seen from Figure 3 , Figure 4 , Figure 5 that the blank solution, blank matrix, and blank medium do not interfere with the main peak; Figure 6 The in vitro transdermal reference solution, Figure 7 the in vitro transdermal drug-containing matrix solution have basically the same peak emergence time, and the resolution of the two main peaks is good.

[0049] Example 8 Three IVPT runs were performed on three different days, with 6 VDCs in each group. Eutectic mixture of local anesthetics cream (25 mg / g) was used to characterize the precision and reproducibility of the IVPT method; the results are shown in Tables 13 - 14.

[0050] Table 13 Precision and reproducibility - Results of maximum permeation rate investigation

[0051] Table 14 Precision and reproducibility - Results of cumulative permeation amount investigation

[0052] Conclusion: One IVPT run was performed on each of the three days. During a single run, the maximum permeation rate J of lidocaine max had a maximum RSD (n = 6) of 20.9% < 25%, and the cumulative permeation amount A total had a maximum RSD (n = 6) of 15.1% < 25%; the maximum permeation rate J of prilocaine max had a maximum RSD (n = 6) of 19.4% < 25%, and the cumulative permeation amount A total had a maximum RSD (n = 6) of 14.5% < 25%, indicating good within-day precision of this method.

[0053] During a total of three runs, the maximum permeation rate J of lidocaine max had an RSD (n = 18) of 23.0% < 25%, and the cumulative permeation amount A total had an RSD (n = 18) of 17.5% < 25%; the maximum permeation rate J of prilocaine max had a maximum RSD (n = 18) of 15.5% < 25%, and the cumulative permeation amount A total had a maximum RSD (n = 18) of 17.1% < 25%, indicating good between-day reproducibility of this method. Example 9

[0054] After cutting the excised skin, rinse it with normal saline and dry it. Apply the self-developed preparation, with the application amounts being 50% (150 mg), 100% (300 mg), and 150% (450 mg) respectively. Prepare three replicates for each concentration. Immediately after application, scrape off the residual ointment on the surface of the pigskin, place it in a 50 ml volumetric flask, add 2.5 ml of 5 mol / L sodium hydroxide solution to disperse the cream evenly, add 2.5 ml of 5 mol / L hydrochloric acid solution, dissolve and dilute to the mark with water-acetonitrile (80:20), filter (it is recommended to use a 0.45 μm filter head made of PP material), take the subsequent filtrate for injection, and examine the recovery rate of the extraction method for epidermal residue. The results are shown in Tables 15 - 16. The results indicate that the recovery rate of this method for determining epidermal residue is good.

[0055] Table 15 Results of the determination of the recovery rate of epidermal residue (lidocaine)

[0056] Table 16 Results of the determination of the recovery rate of epidermal residue (prilocaine)

[0057] Example 10 (1) Blank matrix: Cut the pigskin into small pieces, transfer it to a 10 ml centrifuge tube, add 0.9 ml of permeation medium, then add 8.1 ml of acetonitrile, extract ultrasonically for 40 min, centrifuge at 4000 r / min for 15 min, pour out the supernatant, filter through a 0.45 μm PP filter membrane, transfer 1 ml of the filtrate to a 10 ml volumetric flask, and dilute to the mark with water-acetonitrile (80:20). Prepare 3 replicates in parallel.

[0058] (2) Recovery stock solution: Take appropriate amounts of lidocaine and prilocaine reference substances, dissolve and dilute with acetonitrile to prepare a solution containing about 0.375 mg of lidocaine and prilocaine in each 1 ml as stock solution I. Transfer 1 ml of stock solution I to a 10 ml volumetric flask and dilute to the mark with acetonitrile as stock solution II.

[0059] (3) 10% recovery sample: Cut the pigskin into small pieces, transfer it to a 10 ml centrifuge tube, add 0.9 ml of permeation medium, add 1 ml of stock solution II, then add 7.1 ml of acetonitrile, extract ultrasonically for 40 min, centrifuge at 4000 r / min for 15 min, pour out the supernatant, filter through a 0.45 μm PP filter membrane, transfer 1 ml of the filtrate to a 10 ml volumetric flask, and dilute to the mark with water-acetonitrile (80:20). Prepare 3 replicates in parallel.

[0060] (4)100% recovery rate sample: Cut the pigskin into small pieces, transfer it to a 10 ml centrifuge tube, add 0.9 ml of permeation medium, add 1 ml of stock solution Ⅰ, then add 7.1 ml of acetonitrile, ultrasonically extract for 40 min, centrifuge at 4000 r / min for 15 min, decant the supernatant, filter through a 0.45 μm PP filter membrane, transfer 1 ml of the filtrate to a 10 ml volumetric flask, and dilute to the mark with water-acetonitrile (80:20). Prepare 3 parallel samples.

[0061] (5)200% recovery rate sample: Cut the pigskin into small pieces, transfer it to a 10 ml centrifuge tube, add 0.9 ml of permeation medium, add 2 ml of stock solution Ⅰ, then add 6.1 ml of acetonitrile, ultrasonically extract for 40 min, centrifuge at 4000 r / min for 15 min, decant the supernatant, filter through a 0.45 μm PP filter membrane, transfer 1 ml of the filtrate to a 10 ml volumetric flask, and dilute to the mark with water-acetonitrile (80:20). Prepare 3 parallel samples.

[0062] Inject the samples to investigate the recovery rate of the extraction method for the intradermal retention amount. The results are shown in Tables 17 - 18. The results indicate that the recovery rate of this intradermal retention amount determination method is good.

[0063] Table 17 Results of the recovery rate determination for the intradermal retention amount (lidocaine)

[0064] Table 18 Results of the recovery rate determination for the intradermal retention amount (prilocaine)

[0065] Example 11 (1)Blank matrix: Load the blank excipients, prepare three parallel samples, conduct the transdermal experiment according to the transdermal experiment conditions, and take the permeate as the blank matrix after the experiment.

[0066] (2)Recovery rate stock solution: Take appropriate amounts of lidocaine and prilocaine reference substances, dissolve and dilute with a solvent to prepare a solution containing about 1 mg of each of lidocaine and prilocaine per 1 ml as the stock solution.

[0067] Prepare the test solutions for the recovery rate according to the following table: Table 19 Preparation of the test solutions for the recovery rate

[0068] Inject the samples to investigate the recovery rate of the permeation amount. The results are shown in Tables 20 - 21. The results indicate that the recovery rate of the permeation amount under the present invention is good, all around 100%, which is better than the recovery rate in Comparative Document 1.

[0069] Table 20 Results of the recovery rate determination for the permeation amount (lidocaine)

[0070] Table 21 Results of Determination of Flux Recovery Rate (Prilocaine)

[0071] Example 12 Three IVPT runs were carried out. For each of the 18 VDCs in the three IVPT runs, the recovery rate was calculated by dividing the sum of the epidermal residue amount, intradermal retention amount, and cumulative permeation amount by the amount of lidocaine and prilocaine cream applied. The overall recovery rate results are shown in Table 22.

[0072] Table 22 Results of Mass Balance Investigation

[0073] In total for the three runs, the minimum overall recovery rate was 78.0% > 70%, indicating that the overall recovery rate of this method is good. Example 13

[0074] Lidocaine and prilocaine creams with different concentrations were prepared. The in vitro transdermal behaviors of the preparations with specifications of 12.5 mg / g (negative preparation) and 25 mg / g (self-developed preparation with standard specification), and 25 mg / g (self-developed preparation with standard specification) and the reference preparation were compared and evaluated respectively, and parallel experiments were carried out. The in vitro transdermal results of the negative preparation and the self-developed preparation with standard specification, and the self-developed preparation and the reference preparation showed that the relative deviations RD of the average epidermal residue rate, intradermal retention rate, and cumulative permeation rate between the negative preparation and the self-developed preparation with standard specification were all greater than 15.0%, showing obvious differentiation. The relative deviations RD of the average epidermal residue rate, intradermal retention rate, and cumulative permeation rate between the self-developed preparation with standard specification and the reference preparation were all less than 15.0%, indicating that the discrimination ability of this in vitro transdermal method is good.

[0075] Table 23 Results of Discrimination Ability between Negative Preparation and Standard Preparation

[0076] Table 24 Results of Discrimination Ability between Self-developed Preparation and Reference Preparation

Claims

1. A method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream, characterized in that, the method is based on an open diffusion cell system. The excised skin is placed between the quantitative rings. Lidocaine and prilocaine cream is sampled on the quantitative rings, and a permeation medium is added. The test temperature, medium volume, and stirring speed are set. Sampling is carried out after a fixed time. Then, the permeated sample, epidermal residual sample, and intradermal retention sample are analyzed by high performance liquid chromatography. A reversed-phase liquid chromatography column is used, and an isocratic elution is performed with a mobile phase that is a mixture of an aqueous phase and an organic phase. The flow rate, column temperature, detection wavelength, and injection volume are set; wherein the diffusion cell system is a Logan DHC-6TD diffusion cell.

2. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, the excised skin is porcine skin, preferably the skin of a 30-day-old miniature xiang pig.

3. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, the intradermal retention sample needs to be post-treated. The method is to cut the skin in contact with the paste into pieces, add a medium and acetonitrile, extract by ultrasonic treatment for 40 min, centrifuge, take the supernatant, dilute it, and then filter and determine for analysis.

4. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, the medium is selected from normal saline and phosphate buffer solution, preferably phosphate buffer solution.

5. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 4, characterized in that, the pH value of the phosphate buffer solution is selected from 5.0 - 8.0, preferably 7.

4.

6. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, The dosing amount of lidocaine and prilocaine cream is 1.5 g / 10 cm 2 ~ 2 g / 10 cm 2 , preferably 1.5 g / 10 cm 2 , the area of the diffusion cell is 1.76 cm 2 , and the corresponding dosing amount of the drug is 0.3 g; the temperature of the diffusion cell is 25~37 °C, preferably 32 °C.

7. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, the reversed-phase liquid chromatography column is selected from Phenomenex Titank C18, with a specification of 150 mm × 4.6 mm, 3 µm.

8. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, the mixture of the aqueous phase and the organic phase of the mobile phase is selected from phosphate buffer solution - acetonitrile, and the volume ratio of phosphate buffer solution to acetonitrile is selected from 35∶65.

9. The in vitro transdermal penetration detection method according to claim 8, characterized in that, the phosphate buffer solution is selected from potassium dihydrogen phosphate solution and sodium dihydrogen phosphate - disodium hydrogen phosphate buffer solution, and the pH value is adjusted to 7.2 with 5 mol / L sodium hydroxide.

10. The method for detecting the in vitro transdermal penetration of lidocaine and prilocaine cream according to claim 1, characterized in that, the column temperature is selected from 30°C, the flow rate is selected from 1.0 ml / min, the wavelength is selected from 232 nm, and the injection volume is selected from 10 μl.

Images