Method for determining methanesulfonate in transdermal patch by derivatization HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) method

Abstract

The invention provides a method for determining methanesulfonate in a transdermal patch by a derivatization HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) method and application, and particularly relates to the technical field of analysis and detection. The method for determining the methanesulfonate in the transdermal patch by the derivatization HPLC-UV method comprises the following steps: a, preparing a test solution: stripping a protective layer of the transdermal patch, adding N, N-dimethylacetamide, sealing and heating, vortex by using a vortex instrument to dissolve a pressure-sensitive adhesive in the transdermal patch and disperse cross-linked povidone, and then carrying out first solid-liquid separation to obtain the test solution; mixing the first supernate with a derivatization reagent, and heating for derivatization; finally, adding acetonitrile to precipitate pressure-sensitive adhesive macromolecules, carrying out second solid-liquid separation, and taking second supernate as a test solution; b, preparing a methanesulfonate standard solution and carrying out derivatization treatment to obtain a reference solution; and c, analyzing the content of methanesulfonate in the test sample solution by using HPLC-UV according to an external standard method.

Description

[0001] This application claims the priority of a Chinese patent application with the application number 202410394455X, titled "Method for Determining Mesylate in Transdermal Patches by Derivatization HPLC-UV Method", filed with the Chinese Patent Office on April 2, 2024, the entire content of which is incorporated herein by reference.

[0002] Technical Field

[0003] The present invention relates to the field of analytical detection technology, and particularly to a method and application for determining mesylate in transdermal patches by derivatization HPLC-UV method. Background Art

[0004] Impurities generated during drug production, preparation, and storage are a major factor in evaluating drug safety. Among them, genotoxic impurities play an important role in drug safety assessment due to their carcinogenic and teratogenic properties. Mesylate is a commonly selected salt form when drug molecules form salts, and lower alcohols (such as methanol, ethanol, isopropanol, etc.) are often used as solvents in the production of active pharmaceutical ingredients, and there is a possibility of generating mesylates. Mesylate genotoxic impurities are strictly regulated by regulatory authorities in various countries due to their genotoxicity. The anti-HIV drug nelfinavir was withdrawn from the market due to excessive mesylates. Therefore, it is necessary to control the quality of mesylate genotoxic impurities during drug research and production.

[0005] The limit for mesylate genotoxic impurities is specified as

[0006] 1.5 μg / day according to the toxicological concern threshold. For the analysis of such trace-level impurities, there are problems with sensitivity. At the same time, the ultraviolet response of lower alcohol sulfonates (such as MMS, EMS, IMS, etc.) themselves is very weak, which brings great difficulties to drug research and analysis.

[0007] Currently, the main detection methods include liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. High-performance liquid chromatography is limited by the sensitivity of the detector and only has a good response to sulfonates with high ultraviolet absorption (such as sulfonates with benzene rings). For lower alcohol sulfonates without ultraviolet response such as MMS, EMS, and IMS, derivatized GC-MS or direct LC-MS / MS methods are mostly used for detection.

[0008] ​Existing methods such as GC-MS and LC-MS / MS have high requirements for instruments and need to be equipped with expensive mass spectrometry instruments. Without a mass spectrometry instrument, testing cannot be carried out. Transdermal patches contain a large amount of components such as polymers and surfactants, with a complex matrix that is difficult to remove. When using mass spectrometry analysis, it will seriously contaminate the mass spectrometry instrument, resulting in poor repeatability or damage to the mass spectrometer. The derivatization method can meet the detection requirements with an ordinary ultraviolet detector, which can meet the detection needs of most laboratories and reduce the detection cost at the same time.

[0009] Existing derivatization systems are all based on the acetonitrile-sodium hydroxide aqueous solution system for the derivatization reaction of mesylate in a single component, and a pH meter is required to accurately adjust the pH; inorganic bases such as sodium hydroxide and sodium bicarbonate have poor solubility in organic solvents, so they are not applicable to some substances that can only be dissolved in the organic system (such as hydrophobic polymers, pressure-sensitive adhesives, etc.). The boiling point of acetonitrile is relatively low, so there are safety hazards when performing derivatization experiments above 80 °C, and the volatilization of the solvent will also cause errors in the detection results; in the prior art, derivatization experiments require multiple preparations of different reagents and sequential addition, which increases additional uncertainties and the risk of operation errors during the experiment. There are also problems with the specificity of the derivatization product method for complex mixtures.

[0010] The detection methods in the prior art are all based on the detection of single-component bulk drugs or reagents, and no relevant reports on the derivatization experiment detection of sulfonates in transdermal patches with a complex matrix have been found.

[0011] In view of this, the present invention is specifically proposed. Summary of the Invention

[0012] One of the purposes of the present invention is to provide a method for determining mesylate in transdermal patches by derivatization HPLC-UV method to solve at least one of the above technical problems.

[0013] Another purpose of the present invention is to provide an application of a method for determining mesylate in transdermal patches by derivatization HPLC-UV method.

[0014] In order to achieve the above purposes of the present invention, the following technical solutions are specifically adopted:

[0015] The first aspect of the present invention provides a method for determining mesylate in transdermal patches by derivatization HPLC-UV method, comprising the following steps:

[0016] a. Preparation of the test solution: After peeling off the protective layer of the transdermal patch, cover the matrix layer with quartz sand, cut it into pieces, add N,N-dimethylacetamide, seal and heat. Use a vortex mixer to vortex to dissolve the pressure-sensitive adhesive in the transdermal patch and disperse the cross-linked polyvinylpyrrolidone. Then perform the first solid-liquid separation. Take the first supernatant and mix it with the derivatization reagent, and heat for derivatization. Finally, add acetonitrile to precipitate the pressure-sensitive adhesive polymer, perform the second solid-liquid separation, and take the second supernatant as the test solution.

[0017] Among them, the derivatization reagent is made of sodium dibenzyldithiocarbamate, an organic base, and N,N-dimethylacetamide.

[0018] b. Prepare a standard solution of methyl sulfonate and perform derivatization treatment to obtain a reference solution.

[0019] c. Analyze the content of methyl sulfonate in the test sample solution by HPLC-UV according to the external standard method.

[0020] The detection conditions for high performance liquid chromatography are as follows:

[0021] Chromatographic column: Octadecylsilane-bonded silica gel is used as the filler.

[0022] Mobile phase A: 0.1% aqueous acetic acid solution; Mobile phase B: 0.1% acetic acid acetonitrile solution.

[0023] Detection wavelength of the ultraviolet detector: 254 - 284 nm.

[0024] Furthermore, the methyl sulfonate includes at least one of methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate, and n-butyl methanesulfonate.

[0025] Preferably, in the derivatization reagent, the mass concentration of the organic base is 10 - 200 mg / mL, preferably 100 mg / mL.

[0026] Preferably, in the derivatization reagent, the mass concentration of sodium dibenzyldithiocarbamate is 10 - 40 mg / mL, preferably 20 mg / mL.

[0027] Preferably, the organic base includes at least one of imidazole, 2,6-dimethylpyridine, pyridine, piperidine, N-methylmorpholine, and N-methylimidazole.

[0028] Preferably, the temperature for heating derivatization is 60 - 110 °C, and the time is 1 - 5 h.

[0029] Furthermore, the temperature for sealed heating is 80 - 120 °C, and the time is 1 - 5 min.

[0030] Preferably, the first solid-liquid separation and the second solid-liquid separation are each independently centrifugation, filtration or sedimentation.

[0031] Further, the elution conditions are as follows:

[0032] 0 min: mobile phase A is 20% - 55%, mobile phase B is 45% - 80%;

[0033] 0 - 12 min: mobile phase A decreases from 20% - 55% in a gradient to 5%, mobile phase B increases from 45% - 80% in a gradient to 95%;

[0034] 12 - 17 min: mobile phase A remains at 5%, mobile phase B remains at 95%;

[0035] 17 - 17.1 min: mobile phase A increases from 5% in a gradient to 20% - 55%, mobile phase B decreases from 95% in a gradient to 45% - 80%;

[0036] 17.1 - 23 min: mobile phase A remains at 20% - 55%, mobile phase B remains at 45% - 80%.

[0037] Further, the elution conditions are as follows:

[0038] 0 min: mobile phase A is 45%, mobile phase B is 55%;

[0039] 0 - 12 min: mobile phase A decreases from 45% in a gradient to 5%, mobile phase B increases from 55% in a gradient to 95%;

[0040] 12 - 17 min: mobile phase A remains at 5%, mobile phase B remains at 95%;

[0041] 17 - 17.1 min: mobile phase A increases from 5% in a gradient to 45%, mobile phase B decreases from 95% in a gradient to 55%;

[0042] 17.1 - 23 min: mobile phase A remains at 45%, mobile phase B remains at 55%.

[0043] Further, the detection wavelength of the ultraviolet detector is 280 - 284 nm.

[0044] Further, in step b, the detection conditions of the high performance liquid chromatography further include:

[0045] Flow rate: 0.5 - 0.7 mL / min;

[0046] Column temperature: 28 - 32 °C;

[0047] Injection volume: 5 - 50 μL.

[0048] Further, in step b, the detection conditions of high performance liquid chromatography also include:

[0049] Flow rate: 0.6 mL / min;

[0050] Column temperature: 30 °C;

[0051] Sample injection volume: 10 μL.

[0052] Further, a method for determining mesylate in a transdermal patch by derivatization HPLC-UV method includes the following steps:

[0053] a. Preparation of test solution: After peeling off the protective layer of the transdermal patch, cover the matrix layer with quartz sand and cut it into pieces. Add N,N-dimethylacetamide. The volume ratio of N,N-dimethylacetamide to the area of the transdermal patch is 1 mL:8 - 12 cm 2 , seal and heat at 80 - 120 °C for 1 - 5 min, use a vortex mixer to vortex at 2000 - 4000 rpm for 10 - 30 s to disperse the pressure-sensitive adhesive and cross-linked polyvinylpyrrolidone in the transdermal patch into N,N-dimethylacetamide. Then perform the first solid-liquid separation, take the first supernatant and mix it with the derivatization reagent, and heat and derivatize at 80 - 120 °C for 1 - 3 h; finally, add acetonitrile to precipitate the pressure-sensitive adhesive polymer, perform the second solid-liquid separation, and take the second supernatant as the test solution;

[0054] Among them, the derivatization reagent is made of sodium dibenzyldithiocarbamate, organic base and N,N-dimethylacetamide;

[0055] Preparation of reference solution: Dissolve the mesylate standard product with N,N-dimethylacetamide to prepare a solution with a concentration of 0.005 mg / mL, filter, mix it with the derivatization reagent, and heat and derivatize at 80 - 120 °C for 1 - 3 h to prepare the reference solution for use;

[0056] b. Provide the mesylate reference solution and the test solution;

[0057] c. Analyze the content of mesylate in the test sample solution by HPLC-UV according to the external standard method;

[0058] The detection conditions of high performance liquid chromatography are:

[0059] Chromatographic column: Packed with octadecylsilane-bonded silica gel;

[0060] Mobile phase A: 0.1% aqueous acetic acid solution; Mobile phase B: 0.1% acetic acid acetonitrile solution;

[0061] Detection wavelength of ultraviolet detector: 254 - 284 nm;

[0062] Flow rate: 0.5 - 0.7 mL / min;

[0063] Column temperature: 28 - 32 °C;

[0064] Sample injection volume: 5 - 50 μL;

[0065] The elution conditions are as follows:

[0066] 0 min: Mobile phase A is 20% - 55%, and mobile phase B is 45% - 80%;

[0067] 0 - 12 min: Mobile phase A gradually decreases from 20% - 55% to 5%, and mobile phase B gradually increases from 45% - 80% to 95%;

[0068] 12 - 17 min: Mobile phase A remains at 5%, and mobile phase B remains at 95%;

[0069] 17 - 17.1 min: Mobile phase A gradually increases from 5% to 20% - 55%, and mobile phase B gradually decreases from 95% to 45% - 80%;

[0070] 17.1 - 23 min: Mobile phase A remains at 20% - 55%, and mobile phase B remains at 45% - 80%;

[0071] d. According to the external standard method, calculate the content of mesylate in the test sample solution.

[0072] The second aspect of the present invention provides the application of the method for determining mesylate in transdermal patches by the derivatization HPLC - UV method in the monitoring during the production process of transdermal patches and the quality inspection of products.

[0073] Compared with the prior art, the present invention has at least the following beneficial effects:

[0074] The method for determining mesylate in transdermal patches by derivatization HPLC-UV provided by the present invention uses sodium dibenzyldithiocarbamate, an organic base, and N,N-dimethylacetamide to prepare a derivatization reagent, avoiding the use of a pH regulator to precisely adjust the pH, eliminating the need for a pH meter, and only requiring subsequent adjustment of the added volume of the derivatization reagent to meet the detection requirements of different samples; at the same time, it also avoids the introduction of an aqueous phase and can completely dissolve the transdermal patch matrix that is insoluble in water and acetonitrile in the new derivatization reagent, smoothly realizing the nucleophilic substitution reaction in a homogeneous system; this system raises the reaction temperature from 80 °C to 100 °C to increase the reaction rate of large steric hindrance sulfonates (such as isopropyl mesylate), and 100 °C can be achieved by a boiling water bath method to reduce the difference between the use of oven equipment and temperature; a pretreatment method has been developed, using a large proportion of acetonitrile added to precipitate a large amount of polymer substances such as pressure-sensitive adhesives in the original system, thereby avoiding the entry of polymer pressure-sensitive adhesives into the chromatographic system, which may damage the chromatographic system, affect the stability of the system and the lifespan of the chromatographic column, saving the analysis cost, and verifying the analysis method for the residue of sulfonates in transdermal patches, with all data meeting the registration requirements.

[0075] The application of the method for determining mesylate in transdermal patches by derivatization HPLC-UV provided by the present invention provides a better analysis method for process control monitoring and product quality detection in the production of transdermal patches, shortening the analysis time, simultaneously determining five mesylate compounds, and improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0077] Figure 1 It is a corresponding diagram of the mass spectrometry extracted ion current and the peak in the liquid phase ultraviolet detector;

[0078] Figure 2 It is an overlay diagram of representative spectra of five sulfonate derivatives in the method;

[0079] Figure 3 It is the linear spectrum obtained in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0080] The following will describe the embodiments of the present invention in detail in combination with the implementation manners and examples. However, those skilled in the art will understand that the following implementation manners and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0081] The first aspect of the present invention provides a method for determining mesylate in a transdermal patch by derivatization HPLC-UV, comprising the following steps:

[0082] a. Prepare a test solution: After peeling off the protective layer of the transdermal patch, cover the matrix layer with quartz sand, cut it into pieces, add N,N-dimethylacetamide, seal and heat, use a vortex mixer to vortex to disperse the pressure-sensitive adhesive and polyvinylpyrrolidone in the transdermal patch into N,N-dimethylacetamide, then perform the first solid-liquid separation to remove insoluble cross-linked polyvinylpyrrolidone and quartz sand, take the first supernatant and mix it with a derivatization reagent, and heat for derivatization; finally, add acetonitrile to precipitate the pressure-sensitive adhesive polymer, perform the second solid-liquid separation, and take the second supernatant as the test solution;

[0083] Wherein, the derivatization reagent is made of sodium dibenzyldithiocarbamate, an organic base and N,N-dimethylacetamide;

[0084] b. Prepare a mesylate standard solution, mix it with a derivatization reagent, and heat for derivatization; finally, add acetonitrile and mix well to obtain a reference solution;

[0085] c. Analyze the content of mesylate in the test sample solution by HPLC-UV according to the external standard method;

[0086] The detection conditions for high performance liquid chromatography are as follows:

[0087] Chromatographic column: Packed with octadecylsilane-bonded silica gel;

[0088] Mobile phase A: 0.1% aqueous acetic acid solution; Mobile phase B: 0.1% acetic acid acetonitrile solution;

[0089] Detection wavelength of the ultraviolet detector: 254 - 284 nm.

[0090] The method for determining mesylate in transdermal patches by derivatization HPLC-UV provided by the present invention uses sodium dibenzyldithiocarbamate, an organic base, and N,N-dimethylacetamide to prepare a derivatization reagent, avoiding the use of sodium hydroxide solution to adjust the pH. Subsequently, the detection requirements of different samples can be achieved only by changing the added volume of the derivatization reagent. At the same time, the introduction of the aqueous phase is avoided, and the transdermal patch matrix that is insoluble in water and acetonitrile can be completely dissolved in the new derivatization reagent, and the nucleophilic substitution reaction is successfully achieved under a homogeneous system. Moreover, a pretreatment method is developed. A large proportion of acetonitrile is added to precipitate a large amount of polymer substances such as pressure-sensitive adhesives in the original system, thereby avoiding the damage to the chromatographic system caused by the entry of the polymer pressure-sensitive adhesive into the chromatographic system, affecting the stability of the system and the life of the chromatographic column, saving the analysis cost, and verifying the analysis method for the sulfonate residue in the transdermal patch. All data meet the registration requirements.

[0091] In the process of preparing the test solution, the present invention optimizes the pretreatment method of the transdermal patch, which can remove the polymer matrix to the greatest extent without affecting the recovery rate, reduce interference and improve the life of the chromatographic system.

[0092] The organic base and N,N-dimethylacetamide in the derivatization reagent, as a high-boiling organic base and a high-boiling solvent respectively, enable the derivatization reagent to derivatize safely at a temperature above 100 °C without exceeding the solvent boiling point of the derivatization system, greatly saving the derivatization time of mesylate and improving the detection efficiency. At the same time, the organic base and N,N-dimethylacetamide simplify the complex work such as adjusting the pH in the derivatization conditions. The organic base itself is a near-neutral weak basic compound, so only the added amount of the derivatization reagent needs to be adjusted to reach the final reaction recovery rate, reducing the error introduced in the experiment.

[0093] The present invention uses sodium dibenzyldithiocarbamate as a derivatization reagent. Through the SN2 nucleophilic substitution reaction, using the mesylate group as a good leaving group, and using the strong ultraviolet absorption and strong nucleophilic property of dithiocarbamate, the nucleophilic substitution reaction of mesylate is carried out under alkaline conditions to convert the mesylate with weak ultraviolet absorption into a derivative with strong ultraviolet absorption, so as to detect the residue of mesylate on the ultraviolet detector.

[0094] During the R & D process, the inventor found that if sodium diethyldithiocarbamate is used as the derivatization reagent, since sodium diethyldithiocarbamate has two fewer benzyl groups than sodium dibenzyldithiocarbamate, its hydrophobic interaction is less than that of sodium dibenzyldithiocarbamate, resulting in a shorter retention time and being more susceptible to interference. At the same time, due to its small steric hindrance and high reaction activity, the reaction rate with isopropyl methanesulfonate is higher, but it is also more susceptible to interference. Therefore, more costs are required for subsequent optimization of the chromatographic method. Sodium diethyldithiocarbamate system deteriorates after being placed at room temperature for 3 hours, while the present invention improves the instability of the derivatization reagent in the acetonitrile system. After preparation, it can be stored under refrigeration conditions for more than six months and maintain its original derivatization activity, meeting the storage requirements of subsequent commercial derivatization reagents.

[0095] In addition, the inventor separately used TPO (sodium benzenethiolate), PEX (potassium ethyl xanthate), PAX (potassium n - amyl xanthate), and MBS (sodium 2 - mercaptobenzothiazole) to replace sodium dibenzyldithiocarbamate as the derivatization reagent. During the test, it was found that TPO produced more by - products during the derivatization reaction, making method optimization difficult; PEX and PAX are thermally unstable and decompose upon heating, while isopropyl methanesulfonate requires heating to increase the conversion rate, resulting in insufficient conversion rate of isopropyl methanesulfonate when using PEX; the purity of MBS itself is too low, resulting in the inability to eliminate interference.

[0096] Furthermore, the methanesulfonate ester includes at least one of methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate, and n - butyl methanesulfonate.

[0097] Preferably, in the derivatization reagent, the mass concentration of the organic base is 10 - 200 mg / mL, preferably 100 mg / mL.

[0098] When the mass concentration of the organic base is lower than 10 mg / mL, there are more side reactions and the buffering concentration of the system is insufficient; when the mass concentration of the organic base is higher than 200 mg / mL, the yield of the method derivatization does not increase, but the usage cost of the organic base is increased. At 100 mg / mL, the derivatization reaction has a better reaction rate and fewer by - products.

[0099] Typical but non - limiting, the mass concentration of the organic base can be, for example, 10 mg / mL, 90 mg / mL, 100 mg / mL, 140 mg / mL, 180 mg / mL, or 200 mg / mL, or any value within the range of 10 - 200 mg / mL.

[0100] Preferably, the organic base includes at least one of imidazole, 2,6 - dimethylpyridine, pyridine, piperidine, N - methylmorpholine, and N - methylimidazole.

[0101] Preferably, the temperature for heat-induced derivatization is 60 - 110 °C, and the time is 1 - 5 h.

[0102] If the temperature and time for heat-induced derivatization are not within this range, it will affect the accuracy of subsequent HPLC-UV and prolong the testing time.

[0103] Typically but not restrictively, the temperature for heat-induced derivatization can be, for example, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C or 110 °C, or any value within the range of 60 - 110 °C; the time can be, for example, 1 h, 2 h, 3 h, 4 h or 5 h, or any value within the range of 1 - 5 h.

[0104] The derivatization reaction is shown in Reaction Formula I as follows:

[0105] Reaction Formula I

[0106]

[0107] Among them, R is methyl, ethyl, propyl, isopropyl or butyl.

[0108] Preferably, the temperature for sealed heating is 80 - 120 °C, and the time is 1 - 5 min.

[0109] Preferably, the rotation speed of vortex is 2000 - 4000 rpm, and the time is 10 - 30 s.

[0110] Preferably, the first solid-liquid separation and the second solid-liquid separation are each independently centrifugation, filtration or sedimentation.

[0111] Furthermore, the elution conditions are as follows:

[0112] 0 min: Mobile phase A is 20% - 55%, and mobile phase B is 45% - 80%;

[0113] 0 - 12 min: Mobile phase A gradually decreases from 20% - 55% to 5%, and mobile phase B gradually increases from 45% - 80% to 95%;

[0114] 12 - 17 min: Mobile phase A remains at 5%, and mobile phase B remains at 95%;

[0115] 17 - 17.1 min: Mobile phase A gradually increases from 5% to 20% - 55%, and mobile phase B gradually decreases from 95% to 45% - 80%;

[0116] 17.1 - 23 min: Mobile phase A remains at 20% - 55%, and mobile phase B remains at 45% - 80%.

[0117] Furthermore, the elution conditions are as follows:

[0118] At 0 min: Mobile phase A is 45%, and mobile phase B is 55%.

[0119] From 0 to 12 min: Mobile phase A decreases from 45% to 5% in a gradient manner, and mobile phase B increases from 55% to 95% in a gradient manner.

[0120] From 12 to 17 min: Mobile phase A remains at 5%, and mobile phase B remains at 95%.

[0121] From 17 to 17.1 min: Mobile phase A increases from 5% to 45% in a gradient manner, and mobile phase B decreases from 95% to 55% in a gradient manner.

[0122] From 17.1 to 23 min: Mobile phase A remains at 45%, and mobile phase B remains at 55%.

[0123] Furthermore, the detection wavelength of the ultraviolet detector is 280 - 284 nm, more preferably 282 nm.

[0124] Furthermore, in step c, the detection conditions of the high - performance liquid chromatography further include:

[0125] Flow rate: 0.5 - 0.7 mL / min;

[0126] Column temperature: 28 - 32 °C;

[0127] Sample injection volume: 5 - 50 μL.

[0128] The sample injection volume has been investigated. This parameter can improve the final detection limit and ensure the accuracy of the test results within the range of 5 - 50 μL.

[0129] The needle - washing solution can be a mixed solution of acetonitrile and water, and the volume ratio to water is 1:1.

[0130] Furthermore, in step c, the detection conditions of the high - performance liquid chromatography further include:

[0131] Flow rate: 0.6 mL / min;

[0132] Column temperature: 30 °C;

[0133] Sample injection volume: 10 μL.

[0134] The method for determining mesylate in transdermal patches by the derivatization HPLC - UV method includes the following steps:

[0135] a. Prepare the test solution: After peeling off the protective layer of the transdermal patch, cover the matrix layer with quartz sand, cut it into pieces, add N,N - dimethylacetamide, and the volume ratio of N,N - dimethylacetamide to the area of the transdermal patch is 1 mL:8 - 12 cm 2, seal and heat at 80 - 120 °C for 1 - 5 min, use a vortex mixer to disperse the pressure-sensitive adhesive in the transdermal patch into N,N-dimethylacetamide, then perform the first solid-liquid separation to remove the insoluble cross-linked polyvinylpyrrolidone and quartz sand. Take the first supernatant and mix it with the derivatization reagent, and heat for derivatization at 80 - 120 °C for 1 - 3 h; finally, add acetonitrile to precipitate the pressure-sensitive adhesive polymer, perform the second solid-liquid separation, and take the second supernatant as the test sample solution;

[0136] Among them, the derivatization reagent is made of sodium dibenzyldithiocarbamate, an organic base, and N,N-dimethylacetamide;

[0137] Prepare the reference solution: Dissolve the mesylate standard with N,N-dimethylacetamide to make a solution with a concentration of 0.005 mg / mL. Take this solution and mix it with the derivatization reagent, and heat for derivatization; finally, add acetonitrile and mix well for use as the reference solution;

[0138] b. Provide the mesylate reference solution and the test sample solution;

[0139] c. Analyze the mesylate content in the test sample solution by external standard method using HPLC-UV;

[0140] The detection conditions for high-performance liquid chromatography are as follows:

[0141] Chromatographic column: Filled with octadecylsilane-bonded silica gel;

[0142] Mobile phase A: 0.1% aqueous acetic acid solution; Mobile phase B: 0.1% acetic acid acetonitrile solution;

[0143] Detection wavelength of the ultraviolet detector: 280 - 284 nm;

[0144] Flow rate: 0.5 - 0.7 mL / min;

[0145] Column temperature: 28 - 32 °C;

[0146] Injection volume: 5 - 50 μL;

[0147] The elution conditions are as follows:

[0148] 0 min: Mobile phase A is 20% - 55%, and mobile phase B is 45% - 80%;

[0149] 0 - 12 min: Mobile phase A gradually decreases from 20% - 55% to 5%, and mobile phase B gradually increases from 45% - 80% to 95%;

[0150] 12 - 17 min: Mobile phase A remains at 5%, and mobile phase B remains at 95%;

[0151] 17 - 17.1 min: Mobile phase A is gradiently increased from 5% to 20% - 55%, and mobile phase B is gradiently decreased from 95% to 45% - 80%;

[0152] 17.1 - 23 min: Mobile phase A is maintained at 20% - 55%, and mobile phase B is maintained at 45% - 80%;

[0153] d. Calculate the content of mesylate in the test sample solution by the external standard method.

[0154] The second aspect of the present invention provides the application of the method for determining mesylate in transdermal patches by the derivatization HPLC - UV method in the in - process control monitoring and product quality inspection during the production process of transdermal patches.

[0155] The application of the method for determining mesylate in transdermal patches by the derivatization HPLC - UV method provided by the present invention provides a better analysis method for in - process control monitoring and product quality inspection during the production process of transdermal patches, shortens the analysis time, simultaneously determines five mesylate compounds, and improves the detection efficiency.

[0156] The present invention will be further illustrated by specific examples and comparative examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form. For the raw materials used in the examples and comparative examples of the present invention, those without specific conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0157] Example 1

[0158] 1. Development of the mesylate derivatization analysis method

[0159] 1.1 Instruments:

[0160] Agilent 1260 liquid chromatograph (including on - line vacuum degasser, quaternary gradient pump, auto - sampler, column oven, VWD detector and CDS2 chromatographic workstation);

[0161] Sciex X500R with Waters H - Class premier LC - QTOF liquid chromatography tandem high - resolution mass spectrometer (including on - line vacuum degasser, high - pressure binary gradient pump, auto - sampler, column oven, electrospray (ESI) interface and SciexOS 3.0 workstation);

[0162] METTLER TOLEDO XPR205DU analytical balance (Mettler, Sweden);

[0163] Binder FD260 forced - air drying oven (Binder, Germany);

[0164] Xiangyi H1750 High-Speed Centrifuge (Hunan Xiangyi Instrument Development Co., Ltd.);

[0165] Titan VM-T2 Vortex Mixer (Titan Technology Co., Ltd., China);

[0166] millipore Direct16 Ultra-Pure Water Instrument (Sigma-Aldrich (Shanghai) Trading Co., Ltd.).

[0167] 1.2 Reagents:

[0168] N,N-Dimethylacetamide (DMAc) ≥ 99.0% (GC) (Adamas);

[0169] Methyl Methanesulfonate (MMS) ≥ 99.0% (Adamas);

[0170] Ethyl Methanesulfonate (EMS) ≥ 99.0% (Adamas);

[0171] Isopropyl Methanesulfonate (IMS) ≥ 99.0% (Adamas);

[0172] Propyl Methanesulfonate (PMS) ≥ 99.0% (Adamas);

[0173] n-Butyl Methanesulfonate (BMS) ≥ 99.0% (Adamas);

[0174] Sodium Dibenzyl Dithiocarbamate (DBC), ≥ 98% (RG) (Adamas);

[0175] Imidazole, ≥ 99% (RG) (Adamas);

[0176] Acetonitrile (ACN), HPLC (Merck);

[0177] Glacial Acetic Acid ≥ 99.8% (GR) (Adamas);

[0178] Quartz Sand, 100 - 200 mesh (Adamas);

[0179] 1.3 Samples to be Measured for Method Development and Analysis:

[0180] Rasagiline Mesylate Transdermal Patch, Batch No.: R00115-6-1-L1-E, self-made by Shiling;

[0181] Rasagiline Mesylate Transdermal Patch, Batch No.: R00116-35-1-L1-E, self-made by Shiling;

[0182] Rasagiline Mesylate Transdermal Patch, batch number: R00116-11-1-L1-E, self-made by Shiling;

[0183] Rasagiline Mesylate Transdermal Patch, batch number: R00115-12-2-L1-E, self-made by Shiling;

[0184] Rasagiline Mesylate Transdermal Patch, batch number: 240201-M-01, self-made by Shiling.

[0185] The prescription composition of the Rasagiline Mesylate Transdermal Patch refers to the prescription composition and manufacturing process of the example in WO2024188365A1. The method verification described in the present invention is based on the prescription in Table 1 below. At the same time, this patent also applies to other examples disclosed in patent WO2024188365A1. Specific data are not detailed here one by one.

[0186] Table 1 Prescription Information of Rasagiline Mesylate Transdermal Patch

[0187] Component Prescription Ratio (%) Rasagiline Mesylate 1.75 DURO-TAK 387-2052 74.15 Crospovidone 17.5 Vitamin E 0.1 Triethyl Citrate 2 Polyglycerol Fatty Acid 3 Levulinic Acid 1.5 Total 100 Solvent (Absolute Ethanol) 33

[0188] 1.4 Solution Preparation:

[0189] 1.4.1 Mobile Phase:

[0190] Mobile Phase A: Measure 2 mL of glacial acetic acid, place it in 2000 mL of water, mix well, and ultrasonically degas for 10 min to obtain.

[0191] Mobile Phase B: Measure 2 mL of glacial acetic acid, place it in 2000 mL of acetonitrile, mix well, and ultrasonically degas for 10 min to obtain.

[0192] 1.4.2 Derivatization Reagent:

[0193] Take 200 mg of DBC reagent and 1000 mg of imidazole, accurately weigh them, place both in the same 10 mL volumetric flask, dissolve and dilute to the scale with DMAc, and vortex mix to obtain the derivatization test solution.

[0194] 1.5 Reference Solution:

[0195] Take 50 mg of methyl mesylate reference substance, 50 mg of ethyl mesylate reference substance, 50 mg of propyl mesylate reference substance, 50 mg of isopropyl mesylate reference substance, and 50 mg of n-butyl mesylate reference substance, place them in the same 25 mL volumetric flask, dilute and make up to the scale with DMAc, and shake well. Accurately measure 1 mL of the above solution, place it in a 20 mL volumetric flask, dilute and make up to the scale with DMAc, and shake well to obtain the reference stock solution.

[0196] Precisely measure 1 mL of the reference stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with DMAc, shake well. Precisely pipette 200 μL of this solution and 150 μL of the derivatization test solution into a 1.5 mL injection vial, vortex at 2500 rpm for 10 s to mix evenly, and heat in an oven at 100 °C for 1 h. After taking it out and cooling, precisely add 1000 μL of acetonitrile, vortex at 2500 rpm for 5 min to obtain the reference solution.

[0197] 1.6 Identification of the derivatization product:

[0198] The derivatization product is identified using ultra-high performance liquid chromatography - quadrupole - time-of-flight high-resolution tandem mass spectrometry.

[0199] Liquid phase parameters:

[0200] Liquid chromatography: waters H-class primer / Agilent1260;

[0201] Mobile phase A: 0.1% aqueous acetic acid solution;

[0202] Mobile phase B: 0.1% acetic acid acetonitrile solution;

[0203] Chromatographic column: Agilent poroshell120-EC-C18; 150×3.0 mm, 2.7 μm;

[0204] Flow rate: 0.6 mL / min;

[0205] Detection wavelength: 280 nm;

[0206] Column temperature: 30 °C;

[0207] Injection temperature is 10 °C;

[0208] Injection volume 5 μL.

[0209] The elution process is as follows:

[0210] 0 - 15 min: Mobile phase A is maintained at 20%, and mobile phase B is maintained at 80%.

[0211] Mass spectrometry parameters:

[0212] High-resolution mass spectrometry: Sciex X500R QTOF;

[0213] Ion source: Electrospray ionization source;

[0214] Curtain gas (psi): 45;

[0215] Ion source gas 1 (psi): 45;

[0216] Ion source gas 2 (psi): 45;

[0217] Ion source temperature (°C): 500;

[0218] Scan type: full scan;

[0219] Ion mode: ESI positive ion scan;

[0220] Electrospray voltage (V): 5500;

[0221] Collision gas: 7.

[0222] Extract the corresponding molecular formula for XIC ion current to confirm that the derivatization products are in sequence: MMS derivative: methyl dibenzylthiocarbamate;

[0223] EMS derivative: ethyl dibenzylthiocarbamate;

[0224] IMS derivative: isopropyl dibenzylthiocarbamate;

[0225] PMS derivative: propyl dibenzylthiocarbamate;

[0226] BMS derivative: n-butyl dibenzylthiocarbamate.

[0227] The high-resolution mass spectrometry identification after the reaction is shown in Table 2 below.

[0228] Table 2 Comparison of the theoretical molecular weights and measured molecular weights of the derivative structures

[0229]

[0230] As can be seen from Table 2, the high-resolution mass spectrometry test error does not exceed 1.7 ppm, and at the same time, the mass spectrometry fragments with the corresponding alkyl groups lost neutrally are contained in the second-level mass spectrometry, confirming that the structure is the expected derivatization product.

[0231] Extract the 240.08 (the parent nucleus with the alkyl-substituted mercapto group removed) fragment ion and each M+H ion to obtain the XIC chromatogram as Figure 1 shown.

[0232] From Figure 1 it can be seen that according to the retention properties of different substituted alkyl groups in the reverse chromatographic column, the mass spectrometry extracted ion current corresponds one by one to the peak retention time and elution order in the liquid phase UV detector.

[0233] Figure 2 It is an overlay of the representative chromatograms of five sulfonate derivatives in the method.

[0234] 1.7 Optimization of derivatization conditions

[0235] 1.7.1. Concentration of organic base:

[0236] Prepare derivatization reagents with different concentrations of imidazole: According to the derivatization reagent preparation method in Section 1.3.2, prepare derivatization reagents with imidazole concentrations of 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, and 400 mg / mL respectively. At the same time, use 1 mol / L sodium hydroxide solution as the control group.

[0237] Prepare the control solution: Take 50 mg of methyl methanesulfonate reference substance, 50 mg of ethyl methanesulfonate reference substance, and 50 mg of propyl methanesulfonate reference substance, place them in the same 25 mL volumetric flask, dilute to the scale with DMAc, and shake well. Accurately measure 1 mL of the above solution, place it in a 20 mL volumetric flask, dilute to the scale with DMAc, and shake well to obtain the reference substance stock solution.

[0238] Precisely transfer 200 μL of the reference substance stock solution and 150 μL of the derivatization test solution into a 1.5 mL injection vial, vortex at 2500 rpm for 10 s to mix evenly, and heat in an 80 °C oven for 1 h. After taking it out and cooling, precisely add 1000 μL of acetonitrile, vortex at 2500 rpm for 5 min, and use it as the test sample for injection analysis according to the following parameters to conduct parallel comparison of different derivatization reagents.

[0239] Liquid phase parameters:

[0240] Liquid chromatography: Agilent 1260;

[0241] Mobile phase A: 0.1% aqueous acetic acid solution;

[0242] Mobile phase B: 0.1% acetic acid acetonitrile solution;

[0243] Chromatographic column: Agilent poroshell120 - EC - C18; 150×3.0 mm, 2.7 μm;

[0244] Flow rate: 0.6 mL / min;

[0245] Detection wavelength: 282 nm;

[0246] Column temperature: 30 °C;

[0247] Injection temperature is 10 °C;

[0248] Injection volume: 10 μL.

[0249] The gradient elution process is as follows:

[0250] 0 min: Mobile phase A is 45%, mobile phase B is 55%;

[0251] 0 - 12 min: Mobile phase A is gradient decreased from 45% to 5%, and mobile phase B is gradient increased from 55% to 95%;

[0252] 12 - 17 min: Mobile phase A is maintained at 5%, and mobile phase B is maintained at 95%;

[0253] 17 - 17.1 min: Mobile phase A is gradient increased from 5% to 45%, and mobile phase B is gradient decreased from 95% to 55%;

[0254] 17.1 - 23 min: Mobile phase A is maintained at 45%, and mobile phase B is maintained at 55%.

[0255] The data obtained are shown in Table 3 below.

[0256] Table 3 Relationship between the composition of the derivatizing reagent and the peak area

[0257]

[0258] Table 3 shows the peak areas obtained from the tests of the peak areas of MMS, EMS, and IMS under different alkali concentrations and types. Imidazole has a better reaction rate compared to sodium hydroxide. Even at a lower imidazole concentration, IMS can obtain a better conversion rate. However, during the experiment, the by - product increases after the imidazole concentration is decreased. Therefore, the imidazole concentration needs to be controlled between 10 mg / mL and 200 mg / mL.

[0259] 1.7.2 Derivatization time

[0260] Test solution:

[0261] Take 5 tablets of rasagiline mesylate patch (small - scale experimental batch). After peeling off the protective layer, cover the matrix layer with quartz sand, cut it into pieces and place it in a 20 mL headspace vial. According to the ratio of adding 1 mL for every 10 cm 2 Precisely add DMAc containing the mesylate reference substance, seal it, heat it in an oven at 100 °C for 3 minutes, and use a vortex mixer at 2500 rpm to vortex for 15 s while it is hot to uniformly disperse the pressure - sensitive adhesive and PVPP into DMAc. After cooling to room temperature, centrifuge at 10000 rpm for 5 min to separate the quartz sand and PVPP, and take the supernatant as the test solution.

[0262] Precisely pipette 200 μL of the test solution (this solution has been pre - added with the standard substance of sulfonate) and 150 μL of the derivatization test solution (imidazole concentration is 100 mg / mL) into a 1.5 mL injection vial, vortex at 2500 rpm for 10 s to mix evenly, and heat it in an oven at 100 °C for 0 h, 0.5 h, 1 h, 2.5 h,

[0263] 3.5 h, 5 h or 6 h. After taking out and cooling, precisely add 1000 μL of acetonitrile, vortex at 2500 rpm for 5 min, centrifuge the supernatant at 10000 rpm for 10 min, continue to take the supernatant for injection test, and the obtained data are shown in Table 4 below.

[0264] Table 4 Changes in Derivatization Time and Peak Area

[0265]

[0266] As can be seen from Table 4, when the reaction conditions are set to derivatize at 100 °C for 1 hour, it can basically reach the plateau. It is preferably 1 - 3 hours.

[0267] 1.7.3 Derivatization Temperature

[0268] With the increase of temperature, the derivatization efficiency will be greatly improved. Considering the repeatability of the experiment, the oven temperatures in different laboratories may vary greatly. In order to reduce the experimental differences, the derivatization temperature is set at 100 °C. Even without an incubator, the derivatization experiment can be carried out through a boiling water bath, saving the instrument cost of the laboratory.

[0269] 1.7.4 Stability of Derivatization Reagent

[0270] Place the derivatization reagent under refrigerated conditions. After three months, test the same sample with the newly prepared derivatization reagent, and the result difference is less than 1%.

[0271] 2. Pre - verification of the Liquid Phase Method for Mesylate

[0272] 2.1 Linear Relationship

[0273] Precisely measure the above - mentioned reference solution, place it in a volumetric flask, make it up to the mark with the mobile phase, shake well, and prepare linear solutions with concentrations of 0.04 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1.0 μg / mL. Derivatize the linear solutions according to the derivatization operation of the reference substance; measure according to the above chromatographic conditions, take the concentration (c) as the abscissa and the peak area (A) as the ordinate, draw a standard curve, and calculate the regression equation. The obtained regression equations are as follows:

[0274] MMS derivative: y = 0.1629x - 0.0697, R 2 = 1.0000;

[0275] EMS derivative: y = 0.1554x - 0.3242, R 2 = 0.9999;

[0276] IMS derivative: y = 0.1284x - 0.3423, R 2 = 0.9999;

[0277] PMS derivative: y = 0.1725x - 0.4051, R 2 = 0.9999;

[0278] BMS derivative: y = 0.1151x - 0.3052, R 2 = 1.0000.

[0279] It can be seen that in the range of 0.04 μg / mL - 1.0 μg / mL of the mesylate concentration, there is good linearity, and the correlation coefficient R 2 ≥ 0.9999.

[0280] 2.2. Lower limit of quantitation test

[0281] Accurately weigh an appropriate amount of the test sample solution, place it in a volumetric flask, add the mesylate reference substance to prepare five mesylate solutions containing 0.04 μg / mL, and then add the derivatization reagent for reaction.

[0282] Dissolve each with DMAc and dilute to the mark, shake well. Prepare 3 portions each, and determine the content of the mesylate according to the liquid chromatography conditions, calculate the recovery rate and RSD, and the obtained data are shown in Table 5 below.

[0283] Table 5 Signal-to-noise ratio

[0284] Sulfonate Type MMS EMS IMS PMS BMS Signal-to-Noise Ratio S / N 30.3 27.2 25.7 23.4 20.8

[0285] It can be seen that when the mesylate concentration is 0.04 μg / mL, the signal-to-noise ratio still exceeds 20, meeting the requirement in the pharmacopoeia that the signal-to-noise ratio is greater than 10.

[0286] 2.3. Recovery test

[0287] Accurately weigh an appropriate amount of the test sample solution, place it in a volumetric flask, add the mesylate reference substance in an amount of 1.0 times (100%) of 5 ppm respectively; add the mesylate reference substance in an amount of 1.0 times (100%) of 25 ppm, and then add the derivatization reagent for reaction.

[0288] Dissolve each with DMAc and dilute to the mark, shake well. Prepare 3 portions each, and determine the content of the mesylate according to the liquid chromatography conditions, calculate the recovery rate and RSD, and the obtained data are shown in Table 6 below.

[0289] Table 6 Accuracy data

[0290] Recovery Rate MMS EMS IMS PMS BMS 5ppm 92.7％ 102.3％ 100.0％ 102.7％ 95.9％ 25ppm 101.4％ 100.3％ 100.5％ 100.7％ 101.1％

[0291] It can be seen from Table 6 that the recovery rate of LOQ is between 92.7% and 102.7%; the STD concentration is between 101.4% and 100.3%. The accuracy is good.

[0292] 2.4, Solution Stability

[0293] The 25 ppm test sample solution for the recovery experiment was stored at room temperature for 72 h, and the content of mesylate was redetermined according to the liquid chromatography conditions. The comparison of the changed data with the peak areas determined in 2.2 is shown in Table 7 below.

[0294] Table 7 Stability Data

[0295] Room Temperature Storage Time MMS EMS IMS PMS BMS Initial Value 100.0％ 100.0％ 100.0％ 100.0％ 100.0％ 12h 100.3％ 99.8％ 99.4％ 100.8％ 100.5％ 24h 100.9％ 100.2％ 100.4％ 99.2％ 101.1％ 72h 101.2％ 101.6％ 101.2％ 101.1％ 99.7％

[0296] It can be seen from Table 7 that the change rate of this solution is less than 2%, and the solution can maintain stability for three days at room temperature.

[0297] 2.5, Determination of Pilot-Scale Samples

[0298] Test sample solution:

[0299] Take the rasagiline mesylate patches from the pilot-scale experiment batches (including rasagiline mesylate transdermal patches, batch number: R00116-35-1-L1-E, self-made by Shiling;

[0300] rasagiline mesylate transdermal patches, batch number: R00116-11-1-L1-E, self-made by Shiling;

[0301] rasagiline mesylate transdermal patches, batch number: R00115-12-2-L1-E, self-made by Shiling;),

[0302] 5 pieces. After peeling off the protective layer, the matrix layer was covered with quartz sand, cut into pieces and placed in a 20 mL headspace vial. According to the ratio of adding 1 mL for every 10 cm, 2 precisely add DMAc containing the mesylate reference substance, seal it, heat it in a 100 °C water bath for 3 minutes, and use a vortex mixer at 2500 rpm to vortex for 15 s while it is hot to evenly disperse the pressure-sensitive adhesive and PVPP into DMAc. After cooling to room temperature, centrifuge at 10000 rpm for 5 min to separate the quartz sand and PVPP, and take the supernatant as the test sample solution.

[0303] Precisely pipette 200 μL of the solution to be derivatized and 150 μL of the derivatization test solution into a 1.5 mL injection vial, vortex at 2500 rpm for 10 s to mix evenly, and heat it in a 100 °C oven for 1 h. After taking it out and cooling, precisely add 1000 μL of acetonitrile, vortex at 2500 rpm for 5 min. At this time, the high-molecular pressure-sensitive adhesive will precipitate. Take the supernatant and centrifuge at 10000 rpm for 10 min, and take the supernatant for injection.

[0304] The derivatized test sample solution was determined according to the above chromatographic conditions. By calculation using the external standard method, all sulfonates were not detected in the pilot-scale samples (less than the quantitative detection limit, less than 0.1 μg / patch).

[0305] Example 2

[0306] The patch used in this example was the mesylate rasagiline transdermal patch at the pilot scale-up batch (batch number: 240201-M-01, 10 cm 2 / patch); Detection was carried out according to the same sample preparation method and detection method as in Example 1, and methodological verification was carried out. The quantitative limit of verification was 0.4 μg / mL (10% of the limit standard), and the limit was 4 μg / mL (100% of the limit standard).

[0307] 2.1 Linear relationship

[0308] Precisely measure the reference solution, place it in a volumetric flask, make it up to the mark with the mobile phase, shake well, and obtain linear solutions with concentrations of 0.21 μg / mL - 10.50 μg / mL respectively. Determine according to the above chromatographic conditions. Each linear solution was determined 5 times, and the average peak area was taken. Using the concentration (c) as the abscissa and the peak area (A) as the ordinate, a standard curve was plotted, and the regression equation and correlation coefficient were calculated. The data obtained are shown in Table 8 below:

[0309] Table 8 Linear results

[0310] Component Name Linear Range Regression Equation Correlation Coefficient MMS Derivative 0.40~10.02μg / mL y=17.237x - 0.5234 r＝0.9999 EMS Derivative 0.40~10.05μg / mL y=14.856x + 0.6704 r＝0.9999 IMS Derivative 0.41~10.10μg / mL y=11.866x + 0.5597 r＝0.9999 PMS Derivative 0.40~10.08μg / mL y=17.237x - 0.5234 r＝0.9999 BMS Derivative 0.40~10.12μg / mL y=11.578x - 0.2305 r＝0.9999

[0311] It can be seen that for all sulfonate derivatives in the concentration range of 0.4 μg / mL to 10 μg / mL, this method has good linearity and correlation coefficient, and at the same time, the intercept meets less than 15% of the 100% level. The linear chromatogram is shown in Figure 3 .

[0312] 2.2 Determination results

[0313] The detection levels of the five sulfonates were all less than the quantitative limit (0.4 μg / patch).

[0314] 2.3 Accuracy and repeatability: Recovery test

[0315] Take 5 spiked patches at the corresponding quantitative limit level, 100% level, and 250% level. After peeling off the protective layer, cover the matrix layer with quartz sand, cut it into small pieces and place it in a 20 mL headspace vial. According to the proportion of adding 1 mL per 10 cm 2 precisely add DMAc, seal it, heat it in a 100 °C water bath for 3 minutes, and use a vortex mixer at 2500 rpm to vortex for 15 s while it is hot to evenly disperse the pressure-sensitive adhesive and PVPP into DMAc. After cooling to room temperature, centrifuge at 10000 rpm for 5 min to separate the quartz sand and PVPP, and take the supernatant as the test solution.

[0316] Precisely pipette 200 μL of the solution to be derivatized and 150 μL of the derivatization test solution, place them in a 1.5 mL injection vial, vortex at 2500 rpm for 10 s to mix evenly, and heat in an oven at 100 °C for 1 h. After taking it out and cooling, precisely add 1000 μL of acetonitrile, vortex at 2500 rpm for 5 min. At this time, the high molecular pressure-sensitive adhesive will precipitate. Centrifuge the supernatant at 10000 rpm for 10 min, and inject the supernatant for analysis.

[0317] Prepare 3 portions each, determine the content of mesylate according to the liquid chromatography conditions, calculate the recovery rate and RSD. The data obtained are shown in Table 9 below. The recovery rates all meet the requirements of 80 - 120%.

[0318] Table 9 Results of accuracy verification

[0319]

[0320] 2.4 Intermediate precision

[0321] Different personnel use different liquid phases and chromatographic columns on different days to test the 100% spiked samples. The RSD% of the six results obtained is 1.17%, and the average value difference is 0.57%.

[0322] 2.5 Solution stability

[0323] Store the 100% spiked samples on a 15 °C sample tray, inject samples at different times, and calculate the change in the content of the samples. The results are shown in Table 10.

[0324] Table 10 Results of solution stability test

[0325] Time MMS EMS IMS PMS BMS 0h 100.0％ 100.0％ 100.0％ 100.0％ 100.0％ 8h 100.2％ 100.1％ 100.2％ 100.2％ 100.6％ 24h 100.5％ 99.4％ 99.9％ 99.5％ 100.0％ 72h 99.4％ 99.4％ 100.3％ 100.2％ 100.3％

[0326] The results show that the change in the measured content of the samples is less than 1%.

[0327] 2.6 Robustness test

[0328] Slightly change the chromatographic condition parameters, and the method still meets the requirements.

[0329] Table 11 Robustness results

[0330]

[0331]

[0332] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining mesylate in a transdermal patch by derivatization HPLC-UV, characterized in that, It includes the following steps: a. Prepare the test sample solution: After peeling off the protective layer of the transdermal patch, cover the matrix layer with quartz sand, cut it into pieces, add N,N-dimethylacetamide, seal and heat, use a vortex mixer to vortex to dissolve the pressure-sensitive adhesive in the transdermal patch and disperse the cross-linked polyvinylpyrrolidone, then perform the first solid-liquid separation, take the first supernatant, mix it with the derivatization reagent, and heat for derivatization; finally, add acetonitrile to precipitate the pressure-sensitive adhesive polymer, perform the second solid-liquid separation, and take the second supernatant as the test sample solution; Among them, the derivatization reagent is made of sodium dibenzyldithiocarbamate, an organic base, and N,N-dimethylacetamide; b. Prepare the methanesulfonate standard solution and perform derivatization treatment to obtain the reference solution; c. Analyze the methanesulfonate content in the test sample solution by HPLC-UV according to the external standard method; The detection conditions of high performance liquid chromatography are as follows: Chromatographic column: Packed with octadecylsilane-bonded silica gel; Mobile phase A: 0.1% aqueous acetic acid solution; Mobile phase B: 0.1% acetic acid acetonitrile solution; Detection wavelength of the ultraviolet detector: 254 - 284 nm.

2. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV according to claim 1, characterized in that, The methanesulfonate includes at least one of methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, propyl methanesulfonate, and n-butyl methanesulfonate; Preferably, in the derivatization reagent, the mass concentration of the organic base is 10 - 200 mg / mL, preferably 100 mg / mL; Preferably, in the derivatization reagent, the mass concentration of sodium dibenzyldithiocarbamate is 10 - 40 mg / mL, preferably 20 mg / mL; Preferably, the organic base includes at least one of imidazole, 2,6-dimethylpyridine, pyridine, piperidine, N-methylmorpholine, and N-methylimidazole; Preferably, the temperature for heating derivatization is 60 - 110 °C, and the time is 1 - 5 h.

3. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV method according to claim 1, wherein The temperature for sealed heating is 80 - 120 °C, and the time is 1 - 5 min; Preferably, the methods of the first solid-liquid separation and the second solid-liquid separation are each independently centrifugation, filtration, or sedimentation.

4. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV according to claim 1, wherein The elution conditions are as follows: 0 min: Mobile phase A is 20% - 55%, and mobile phase B is 45% - 80%; 0 - 12 min: Mobile phase A gradually decreases from 20% - 55% to 5%, and mobile phase B gradually increases from 45% - 80% to 95%; 12 - 17 min: Mobile phase A remains at 5%, and mobile phase B remains at 95%; 17 - 17.1 min: Mobile phase A gradually increases from 5% to 20% - 55%, and mobile phase B gradually decreases from 95% to 45% - 80%; 17.1 - 23 min: Mobile phase A remains at 20% - 55%, and mobile phase B remains at 45% - 80%.

5. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV according to claim 1, characterized in that, The elution conditions are as follows: 0 min: Mobile phase A is 45%, and mobile phase B is 55%; 0 - 12 min: Mobile phase A gradually decreases from 45% to 5%, and mobile phase B gradually increases from 55% to 95%; 12 - 17 min: Mobile phase A remains at 5%, and mobile phase B remains at 95%; 17 - 17.1 min: Mobile phase A is linearly increased from 5% to 45%, and mobile phase B is linearly decreased from 95% to 55%. 17.1 - 23 min: Mobile phase A is maintained at 45%, and mobile phase B is maintained at 55%.

6. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV according to any one of claims 1 to 5, characterized in that, The detection wavelength of the UV detector is 282 nm.

7. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV according to any one of claims 1 to 5, characterized in that, In step b, the detection conditions of the high - performance liquid chromatography further include: Flow rate: 0.5 - 0.7 mL / min; Column temperature: 28 - 32 °C; Injection volume: 5 - 50 μL.

8. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV according to any one of claims 1 to 5, characterized in that, In step b, the detection conditions of the high - performance liquid chromatography further include: Flow rate: 0.6 mL / min; Column temperature: 30 °C; Injection volume: 10 μL.

9. The method for determining mesylate in a transdermal patch by derivatization HPLC-UV method according to any one of claims 1 to 5, characterized in that, It includes the following steps: a. Preparation of the test solution: After peeling off the protective layer of the transdermal patch, cover the matrix layer with quartz sand and cut it into pieces. Add N,N-dimethylacetamide, and the volume ratio of N,N-dimethylacetamide to the area of the transdermal patch is 1 mL: 8 - 12 cm 2 , seal and heat at 80 - 120 °C for 1 - 5 min, use a vortex mixer to vortex at 2000 - 4000 rpm for 10 - 30 s to disperse the pressure-sensitive adhesive and cross-linked povidone in the transdermal patch into N,N-dimethylacetamide. Then perform the first solid-liquid separation, take the first supernatant and mix it with the derivatization reagent, and heat and derivatize at 80 - 120 °C for 1 - 3 h; finally, add acetonitrile to precipitate the pressure-sensitive adhesive polymer, perform the second solid-liquid separation, and take the second supernatant as the test solution; Among them, the derivatization reagent is made of sodium dibenzyldithiocarbamate, organic base and N,N - dimethylacetamide; Prepare the reference solution: Take the mesylate standard product, dissolve it with N,N - dimethylacetamide to make a solution with a concentration of 0.005 mg / mL, filter it, mix it with the derivatization reagent, and heat it for derivatization at 80 - 120 °C for 1 - 3 h to make the reference solution for standby; b. Provide the mesylate reference solution and the test sample solution; c. Analyze the content of mesylate in the test sample solution by HPLC - UV according to the external standard method; The detection conditions of the high - performance liquid chromatography are: Chromatographic column: Packed with octadecylsilyl - bonded silica gel; Mobile phase A: 0.1% aqueous acetic acid solution; Mobile phase B: 0.1% acetic acid acetonitrile solution; Detection wavelength of the UV detector: 254 - 284 nm; Flow rate: 0.5 - 0.7 mL / min; Column temperature: 28 - 32 °C; Injection volume: 5 - 50 μL; The elution conditions are: 0 min: Mobile phase A is 20% - 55%, and mobile phase B is 45% - 80%; 0 - 12 min: Mobile phase A is linearly decreased from 20% - 55% to 5%, and mobile phase B is linearly increased from 45% - 80% to 95%; 12 - 17 min: Mobile phase A is maintained at 5%, and mobile phase B is maintained at 95%; 17 - 17.1 min: Mobile phase A is linearly increased from 5% to 20% - 55%, and mobile phase B is linearly decreased from 95% to 45% - 80%; 17.1 - 23 min: Mobile phase A is maintained at 20% - 55%, and mobile phase B is maintained at 45% - 80%; d. Calculate the content of mesylate in the test sample solution according to the external standard method.

10. Application of the method for determining mesylate in transdermal patches by the derivatization HPLC - UV method according to any one of claims 1 - 9 in the monitoring during the production process of transdermal patches and the quality inspection of products.

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