A method for drawing the dissolution curve of perphenazine tablets by high performance liquid chromatography

Abstract

The application discloses a method for drawing the dissolution curve of perphenazine tablets by high performance liquid chromatography, comprising the following steps: adding perphenazine tablets and preheated dissolution medium into a plurality of dissolution cups in sequence under the light-proof environment; using a dissolution instrument to obtain dissolution liquid by sampling the plurality of dissolution cups for multiple times, 5mL of the dissolution liquid is sampled each time and the isothermal dissolution medium is supplemented at the same time; filtering the dissolution liquid, taking the filtered liquid to obtain the test sample solution, and storing the test sample solution for use in the dark; preparing the control sample solution with the perphenazine tablet concentration of 2.012ug / mL and 4.024ug / mL respectively, and storing the control sample solution for use in the dark; using a high performance liquid chromatograph, sucking the test sample solution and the control sample solution into the high performance liquid chromatograph according to preset chromatographic conditions; recording the peak area, and calculating the dissolution amount at each time point according to the formula; taking the dissolution time as the horizontal coordinate and the accumulated dissolution amount at each time as the vertical coordinate, and drawing the dissolution curve.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography. Background Technology

[0002] Perphenazine tablets are a commonly used antipsychotic drug. Their dissolution curve is a key indicator for evaluating the rationality of the drug formulation process and the consistency of quality, directly affecting the drug's in vivo absorption and clinical efficacy. In existing technologies, the detection of the dissolution curve of perphenazine tablets mainly relies on the pharmacopoeia standard using ultraviolet-visible spectrophotometry. Using pH 6.8 phosphate buffer, pH 4.5 acetate buffer, and 0.1 mol / L hydrochloric acid solution as dissolution media, absorbance is measured at a wavelength of 254 nm, and the amount dissolved is calculated. However, this method has significant drawbacks: firstly, insufficient specificity, as excipients and degradation impurities in the dissolution solution can easily cause absorption interference in the ultraviolet region, leading to measurement deviations; secondly, limited quantitative accuracy, with insufficient sensitivity for low-concentration dissolution solutions, failing to accurately reflect the early dissolution characteristics of the drug; and thirdly, weak anti-interference ability, still easily affected by environmental factors even under light-protected operation requirements, and its stability needs improvement. Summary of the Invention

[0003] The technical problem this invention aims to solve is the poor specificity, susceptibility to interference, and insufficient accuracy of existing UV-Vis spectrophotometric methods for detecting the dissolution curve of perphenazine tablets. This invention provides a method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography (HPLC). This method effectively separates perphenazine from excipients and impurities through chromatographic separation, eliminating absorption interference problems in the UV method, resulting in more accurate and reliable measurement results. It features a wide linear range (1-10 μg / mL), a correlation coefficient r≥0.9998, a detection limit as low as 15 ng / mL, and a quantitation limit of 40 ng / mL, enabling precise determination of drug concentration at different dissolution stages. The average recovery rate is 98.0%-102.0%, the relative standard deviation (RSD) is ≤1.5%, and the test solution is stable within 24 hours, meeting the requirements for batch sample testing. The dissolution conditions are consistent with existing pharmacopoeia standards; only the detection method needs to be changed, without adjusting the formulation dissolution test device, facilitating rapid adoption by enterprises and overcoming the shortcomings of existing technologies.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography, comprising the following steps: Under light-protected conditions, the paddle method (method 2 for dissolution and release determination) was used. Perphenazine tablets (2mg and 4mg film-coated tablets) and preheated dissolution medium were added sequentially to multiple dissolution vessels. After placing the dissolution vessels into the dissolution apparatus, the apparatus was started, and multiple samples were taken to obtain the dissolution solution. Each sample was 5mL, and isothermal dissolution medium was added simultaneously. After filtering the above dissolution solution, the filtrate was collected to obtain the test solution, which was stored in the dark for later use. Reference solutions of perphenazine tablets with concentrations of 2.012 μg / mL and 4.024 μg / mL were prepared, respectively. Turn on the high-performance liquid chromatograph (HPLC) according to the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution amount at each time point using the following formula (external standard method): Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference standard × Tablet labeling amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0005] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography, wherein the perphenazine tablets are 2 mg / tablet or 4 mg / tablet.

[0006] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography, wherein 900 ml of the dissolution medium preheated to 37.0 ± 0.5 °C is added to each dissolution vessel; The melting device rotates at 50 revolutions per minute; Samples were taken at 5, 10, 15, 20, 30, 45, 60 and 90 minutes, with 5 mL of sample taken each time and 5.5 mL of isothermal dissolution medium added simultaneously. The leaching solution was filtered using a 0.22μm aqueous filter membrane. After discarding 4ml, the filtrate was collected to remove particulate impurities. At the same time, by discarding part of the leaching solution and storing it in the dark, the adsorption and degradation of the sample filter membrane were reduced, thus ensuring the stability of the test results.

[0007] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography (HPLC) wherein the dissolution medium is a phosphate buffer solution with pH 6.8, an acetate buffer solution with pH 4.5, or a 0.1 mol / L hydrochloric acid solution, ensuring the compatibility of the detection method with existing standards, and specifying the sampling time for different media to ensure the specificity of the detection.

[0008] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography includes the following method for preparing the reference solution: Accurately weigh 10.06 mg of perphenazine reference standard and place it in a 50 mL volumetric flask. Add the mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL. Accurately transfer 1 mL of the reference stock solution into a 50 mL volumetric flask, add dissolution medium to dilute to the mark, and shake well to obtain a reference solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification). Accurately transfer 1 mL of the reference stock solution into a 100 mL volumetric flask, add dissolution medium to dilute to the mark, and shake well to obtain a reference solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification). The purity of the perphenazine reference standard is ≥99.5%.

[0009] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography (HPLC) includes the following chromatographic conditions: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm), which exhibits good retention characteristics for basic drugs; a mobile phase of methanol-30 mmol / L ammonium acetate solution at a volume ratio of 80:20, which effectively improves the peak shape of perphenazine and avoids tailing; a flow rate of 1.0 mL / min; a column temperature of 35 °C; a detection wavelength of 254 nm; and injection volumes of 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensure baseline separation of the perphenazine peak from interfering peaks and improve detection specificity. The high-performance liquid chromatograph is equipped with an ultraviolet detector. The reagents used are as follows: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0010] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography involves accurately pipetting 20 μL (applicable to 4 mg specification) and 50 μL (applicable to 2 mg specification) of the test solution and the reference solution, respectively, and injecting them into the high performance liquid chromatograph to record the peak areas.

[0011] The method described above for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography further includes calculating a linear regression equation to obtain a linear relationship: Weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; Take the reference stock solution and dilute it with a dissolution medium to prepare a series of reference solutions with concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL; Turn on the high-performance liquid chromatograph (HPLC) according to the preset chromatographic conditions, pipette a series of reference solutions, inject them into the HPLC, and record the peak areas. Calculate the dissolution amount at each time point using the following formula: Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference sample × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference sample × Tablet labeled amount) × 100%. Perform linear regression with concentration (x) as the x-axis and peak area (y) as the y-axis.

[0012] The above-mentioned method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography, wherein the dissolution medium is a phosphate buffer solution with pH 6.8, an acetate buffer solution with pH 4.5, or a 0.1 mol / L hydrochloric acid solution; The chromatographic conditions were as follows: a C18 column, a mobile phase of methanol-30 mmol / L ammonium acetate solution (volume ratio 80:20), a flow rate of 1.0 mL / min, a column temperature of 35 °C, and a detection wavelength of 254 nm.

[0013] This technical solution replaces the traditional UV-Vis spectrophotometry with high-performance liquid chromatography (HPLC), which solves the problems of poor specificity and susceptibility to impurities in the traditional UV method. It exhibits good linearity (r≥0.9998), a recovery rate of 98.0%-102.0%, and RSD≤1.5%, significantly improving detection accuracy and stability. It is suitable for the accurate and stable determination of the dissolution curve of perphenazine tablets, providing reliable technical support for drug quality control.

[0014] According to the above technical solution, a method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography has the following beneficial effects: Significantly improved specificity: Chromatographic separation effectively separates perphenazine from excipients and impurities, eliminating absorption interference issues in the UV method, resulting in more accurate and reliable test results; Higher detection accuracy: Wide linear range (1-10 μg / mL), correlation coefficient r≥0.9998, detection limit as low as 15 ng / mL, and quantitation limit of 40 ng / mL, enabling precise determination of drug concentration at different dissolution stages; Good stability and reproducibility: Average recovery rate of 98.0%-102.0%, relative standard deviation (RSD) ≤1.5%, and the test solution is stable within 24 hours, meeting the needs of batch sample testing; Strong operational compatibility: Dissolution conditions are consistent with existing pharmacopoeia standards, requiring only a change in the detection method without adjusting the formulation dissolution test device, facilitating rapid promotion and application by enterprises. Attached Figure Description

[0015] Figure 1This is a flowchart of the technical solution; Figure 2 The linear regression equation graph is obtained from a series of control solutions diluted with phosphate buffer at pH 6.8 in the linear relationship experiment. Figure 3 The linear regression equation graph is obtained from a series of control solutions diluted with acetate buffer at pH 4.5 in the linear relationship experiment. Figure 4 The graph shows the linear regression equations obtained from a series of reference solutions diluted with 0.1 mol / L hydrochloric acid solution in the linear relationship experiment. Figure 5 The figure shows a comparison of the dissolution curves of perphenazine film-coated tablets determined by this method and the UV method in pH 6.8 phosphate buffer. Figure 6 This is a comparison chart of the cumulative dissolution amounts of the sample and the original reference, measured by high performance liquid chromatography (HPLC) in pH 6.8 phosphate buffer.

[0016] Figure 7 This is a comparison chart showing the cumulative dissolution of samples with different specifications in pH 6.8 phosphate buffer, as determined by high performance liquid chromatography. Detailed Implementation

[0017] In order to make the technical means, inventive features, objectives and effects of the invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific illustrations. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0018] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings of this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0020] Furthermore, the terms such as "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity of description and are not intended to limit the scope of the invention. Any changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0021] This invention provides a method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography (HPLC). The aim is to achieve effective separation of perphenazine from excipients and impurities through chromatographic separation, eliminating the absorption interference problem of the ultraviolet (UV) method, and resulting in more accurate and reliable measurement results. HPLC has advantages such as high separation efficiency, strong specificity, and high sensitivity. Applying HPLC to the detection of the dissolution curve of perphenazine tablets can overcome the technical shortcomings of existing UV methods, improving the accuracy and reliability of drug quality control.

[0022] like Figure 1 As shown, Example 1: Preparation of the test solution: Under light-protected conditions, add 6 tablets of 2mg / tablet perphenazine, preheated to 37.0±0.5℃, and 900ml of pH 6.8 phosphate buffer to multiple dissolution vessels sequentially. Place the dissolution vessels in the dissolution apparatus and start the apparatus at 50 rpm. Take samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes, taking 5mL of sample each time and simultaneously adding 5.5mL of isothermal pH 6.8 phosphate buffer to obtain the dissolution solution. Filter the dissolution solution using a 0.22μm aqueous filter membrane to remove particulate impurities. Discard 4ml and collect the filtrate to obtain the test solution, which should be stored in the dark for later use. Preparation of reference solutions: Accurately weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; accurately transfer 1 mL of the reference standard stock solution into a 50 mL volumetric flask, add pH 6.8 phosphate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification); accurately transfer 1 mL of the reference standard stock solution into a 100 mL volumetric flask, add pH 6.8 phosphate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification); the purity of perphenazine reference standard is ≥99.5%.

[0023] Turn on the high-performance liquid chromatograph (HPLC) under the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution amount at each time point using the following formula (external standard method): Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of pH 6.8 phosphate buffer) / (Peak area of ​​reference standard × Tablet label amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0024] The chromatographic conditions were as follows: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm) was used, which exhibits good retention characteristics for basic drugs; the mobile phase was methanol-30 mmol / L ammonium acetate solution, with a volume ratio of 80:20, which effectively improved the peak shape of perphenazine and avoided tailing; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensured baseline separation of the perphenazine peak from interfering peaks and improved detection specificity; the high-performance liquid chromatograph was equipped with an ultraviolet detector, and the following reagents were used: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0025] like Figure 1 As shown, Example 2: Preparation of the test solution: Under light-protected conditions, add 6 tablets of 2mg / tablet perphenazine, preheated to 37.0±0.5℃, and 900ml of pH 4.5 acetate buffer to multiple dissolution vessels sequentially. Place the dissolution vessels in the dissolution apparatus and start the apparatus at 50 rpm. Take samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes, taking 5mL of sample each time and simultaneously adding 5.5mL of isothermal pH 4.5 acetate buffer to obtain the dissolution solution. Filter the dissolution solution using a 0.22μm aqueous filter membrane to remove particulate impurities. Discard 4ml and collect the filtrate to obtain the test solution, which should be stored in the dark for later use. Preparation of reference solutions: Accurately weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; accurately transfer 1 mL of the reference standard stock solution into a 50 mL volumetric flask, add pH 4.5 acetate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification); accurately transfer 1 mL of the reference standard stock solution into a 100 mL volumetric flask, add pH 4.5 acetate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification); the purity of perphenazine reference standard is ≥99.5%.

[0026] Turn on the high-performance liquid chromatograph (HPLC) under the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution rate at each time point using the following formula (external standard method): Dissolution rate (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of pH 4.5 acetate buffer) / (Peak area of ​​reference standard × Tablet labeling amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0027] The chromatographic conditions were as follows: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm) was used, which exhibits good retention characteristics for basic drugs; the mobile phase was methanol-30 mmol / L ammonium acetate solution, with a volume ratio of 80:20, which effectively improved the peak shape of perphenazine and avoided tailing; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensured baseline separation of the perphenazine peak from interfering peaks and improved detection specificity; the high-performance liquid chromatograph was equipped with an ultraviolet detector, and the following reagents were used: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0028] like Figure 1 As shown, Example 3: Preparation of the test solution: Under light-protected conditions, add 6 tablets of 2mg / tablet perphenazine, preheated to 37.0±0.5℃, and 900ml of 0.1mol / L hydrochloric acid solution to multiple dissolution vessels sequentially. Place the dissolution vessels in the dissolution apparatus and start the apparatus at 50 rpm. Take samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes, taking 5mL of sample each time and simultaneously adding 5.5mL of isothermal 0.1mol / L hydrochloric acid solution to obtain the dissolution solution. Filter the dissolution solution using a 0.22μm aqueous filter membrane to remove particulate impurities. Discard 4ml and collect the filtrate to obtain the test solution, which should be stored in the dark for later use. Preparation of reference solutions: Accurately weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference stock solution with a concentration of 201.20 μg / mL; accurately transfer 1 mL of the reference stock solution into a 50 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dilute to the mark, and shake well to obtain a reference solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification); accurately transfer 1 mL of the reference stock solution into a 100 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dilute to the mark, and shake well to obtain a reference solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification); the purity of perphenazine reference standard is ≥99.5%.

[0029] Turn on the high-performance liquid chromatograph (HPLC) under the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution amount at each time point using the following formula (external standard method): Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of 0.1 mol / L hydrochloric acid solution) / (Peak area of ​​reference standard × Tablet label amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0030] The chromatographic conditions were as follows: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm) was used, which exhibits good retention characteristics for basic drugs; the mobile phase was methanol-30 mmol / L ammonium acetate solution, with a volume ratio of 80:20, which effectively improved the peak shape of perphenazine and avoided tailing; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensured baseline separation of the perphenazine peak from interfering peaks and improved detection specificity; the high-performance liquid chromatograph was equipped with an ultraviolet detector, and the following reagents were used: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0031] like Figure 1 As shown, Example 4: Preparation of the test solution: Under light-protected conditions, add 6 tablets of 4mg / tablet perphenazine, preheated to 37.0±0.5℃, and 900ml of pH 6.8 phosphate buffer to multiple dissolution vessels sequentially. Place the dissolution vessels in the dissolution apparatus and start the apparatus at 50 rpm. Take samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes, taking 5mL of sample each time and simultaneously adding 5.5mL of isothermal pH 6.8 phosphate buffer to obtain the dissolution solution. Filter the dissolution solution using a 0.22μm aqueous filter membrane to remove particulate impurities. Discard 4ml and collect the filtrate to obtain the test solution, which should be stored in the dark for later use. Preparation of reference solutions: Accurately weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; accurately transfer 1 mL of the reference standard stock solution into a 50 mL volumetric flask, add pH 6.8 phosphate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification); accurately transfer 1 mL of the reference standard stock solution into a 100 mL volumetric flask, add pH 6.8 phosphate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification); the purity of perphenazine reference standard is ≥99.5%.

[0032] Turn on the high-performance liquid chromatograph (HPLC) under the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution amount at each time point using the following formula (external standard method): Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of pH 6.8 phosphate buffer) / (Peak area of ​​reference standard × Tablet label amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0033] The chromatographic conditions were as follows: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm) was used, which exhibits good retention characteristics for basic drugs; the mobile phase was methanol-30 mmol / L ammonium acetate solution, with a volume ratio of 80:20, which effectively improved the peak shape of perphenazine and avoided tailing; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensured baseline separation of the perphenazine peak from interfering peaks and improved detection specificity; the high-performance liquid chromatograph was equipped with an ultraviolet detector, and the following reagents were used: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0034] like Figure 1 As shown, Example 5: Preparation of the test solution: Under light-protected conditions, add 6 tablets of 4mg / tablet perphenazine, preheated to 37.0±0.5℃, and 900ml of pH 4.5 acetate buffer to multiple dissolution vessels sequentially. Place the dissolution vessels in the dissolution apparatus and start the apparatus at 50 rpm. Take samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes, taking 5mL of sample each time and simultaneously adding 5.5mL of isothermal pH 4.5 acetate buffer to obtain the dissolution solution. Filter the dissolution solution using a 0.22μm aqueous filter membrane to remove particulate impurities. Discard 4ml and collect the filtrate to obtain the test solution, which should be stored in the dark for later use. Preparation of reference solutions: Accurately weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; accurately transfer 1 mL of the reference standard stock solution into a 50 mL volumetric flask, add pH 4.5 acetate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification); accurately transfer 1 mL of the reference standard stock solution into a 100 mL volumetric flask, add pH 4.5 acetate buffer to dilute to the mark, and shake well to obtain a reference standard solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification); the purity of perphenazine reference standard is ≥99.5%.

[0035] Turn on the high-performance liquid chromatograph (HPLC) under the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution rate at each time point using the following formula (external standard method): Dissolution rate (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of pH 4.5 acetate buffer) / (Peak area of ​​reference standard × Tablet labeling amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0036] The chromatographic conditions were as follows: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm) was used, which exhibits good retention characteristics for basic drugs; the mobile phase was methanol-30 mmol / L ammonium acetate solution, with a volume ratio of 80:20, which effectively improved the peak shape of perphenazine and avoided tailing; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensured baseline separation of the perphenazine peak from interfering peaks and improved detection specificity; the high-performance liquid chromatograph was equipped with an ultraviolet detector, and the following reagents were used: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0037] like Figure 1 As shown, Example 6: Preparation of the test solution: Under light-protected conditions, add 6 tablets of 4mg / tablet perphenazine, preheated to 37.0±0.5℃, and 900ml of 0.1mol / L hydrochloric acid solution to multiple dissolution vessels sequentially. Place the dissolution vessels in the dissolution apparatus and start the apparatus at 50 rpm. Take samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes, taking 5mL of sample each time and simultaneously adding 5.5mL of isothermal 0.1mol / L hydrochloric acid solution to obtain the dissolution solution. Filter the dissolution solution using a 0.22μm aqueous filter membrane to remove particulate impurities. Discard 4ml and collect the filtrate to obtain the test solution, which should be stored in the dark for later use. Preparation of reference solutions: Accurately weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to obtain a reference stock solution with a concentration of 201.20 μg / mL; accurately transfer 1 mL of the reference stock solution into a 50 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dilute to the mark, and shake well to obtain a reference solution with a concentration of 4.024 μg / mL (applicable to 4 mg specification); accurately transfer 1 mL of the reference stock solution into a 100 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dilute to the mark, and shake well to obtain a reference solution with a concentration of 2.012 μg / mL (applicable to 2 mg specification); the purity of perphenazine reference standard is ≥99.5%.

[0038] Turn on the high-performance liquid chromatograph (HPLC) under the preset chromatographic conditions. Inject the test solution and reference solution into the HPLC and record the peak areas. Calculate the dissolution amount at each time point using the following formula (external standard method): Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of 0.1 mol / L hydrochloric acid solution) / (Peak area of ​​reference standard × Tablet label amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

[0039] The chromatographic conditions were as follows: a C18 column (Welch Ultimate XB-C18, 4.6 × 100 mm, 5 μm) was used, which exhibits good retention characteristics for basic drugs; the mobile phase was methanol-30 mmol / L ammonium acetate solution, with a volume ratio of 80:20, which effectively improved the peak shape of perphenazine and avoided tailing; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 20 μL (2 mg specification) and 50 μL (4 mg specification), which ensured baseline separation of the perphenazine peak from interfering peaks and improved detection specificity; the high-performance liquid chromatograph was equipped with an ultraviolet detector, and the following reagents were used: methanol (chromatographic grade), ammonium acetate (analytical grade), hydrochloric acid (analytical grade), potassium dihydrogen phosphate (analytical grade), sodium hydroxide (analytical grade), sodium acetate trihydrate (analytical grade), glacial acetic acid (analytical grade), and purified water.

[0040] The cumulative dissolution amounts detected by high performance liquid chromatography in Examples 1, 2, and 3 are shown in Table 1. The cumulative dissolution amounts detected by high performance liquid chromatography in Examples 4, 5, and 6 are shown in Table 2.

[0041] Table 1

[0042] Table 2 Linear relationship experiment: Preparation of the reference stock solution: Weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well to obtain a reference stock solution with a concentration of 201.20 μg / mL.

[0043] Take the reference standard stock solution and dilute it with phosphate buffer (pH 6.8) to prepare a series of reference standard solutions with concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL. Turn on the high-performance liquid chromatograph (HPLC) according to the preset chromatographic conditions, and inject the series of reference standard solutions diluted with phosphate buffer (pH 6.8) into the HPLC system, recording the peak areas. Calculate the dissolution rate at each time point using the following formula: Dissolution rate (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference standard × Tablet labeled amount) × 100%. Perform linear regression with concentration (x) as the abscissa and peak area (y) as the ordinate, obtaining the regression equation y = 1.532x - 0.0295, r = 0.9998. Figure 2 As shown, this indicates that perphenazine exhibits good linearity in the range of 0.5–5.0 μg / mL.

[0044] Take the reference standard stock solution and dilute it with acetate buffer at pH 4.5 to prepare a series of reference standard solutions with concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL. Turn on the high-performance liquid chromatograph (HPLC) according to the preset chromatographic conditions, and inject the series of reference standard solutions diluted with acetate buffer at pH 4.5 into the HPLC system, recording the peak areas. Calculate the dissolution rate at each time point using the following formula: Dissolution rate (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference standard × Tablet labeled amount) × 100%. Perform linear regression with concentration (x) as the abscissa and peak area (y) as the ordinate, obtaining the regression equation y = 95.006x + 0.2587, r = 1. Figure 3 As shown, this indicates that perphenazine exhibits good linearity in the range of 0.5–5.0 μg / mL.

[0045] Take the reference standard stock solution and dilute it with 0.1 mol / L hydrochloric acid solution to prepare a series of reference standard solutions with concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL. Turn on the high-performance liquid chromatograph (HPLC) according to the preset chromatographic conditions, and inject the series of reference standard solutions diluted with 0.1 mol / L hydrochloric acid solution into the HPLC instrument, recording the peak area. Calculate the dissolution amount at each time point using the following formula: Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference standard × Tablet labeled amount) × 100%. Perform linear regression with concentration (x) as the abscissa and peak area (y) as the ordinate, obtaining the regression equation y = 1.0087x - 0.0085, r = 0.9998. Figure 4 As shown, this indicates that perphenazine exhibits good linearity in the range of 0.5–5.0 μg / mL.

[0046] High Performance Liquid Chromatography (HPLC) Precision Test: For the preparation of the test solution, under light-protected conditions, add perphenazine tablets and preheated dissolution medium sequentially to multiple dissolution vessels. The dissolution medium is either a phosphate buffer solution with pH 6.8, an acetate buffer solution with pH 4.5, or a 0.1 mol / L hydrochloric acid solution. After placing the dissolution vessels into the dissolution apparatus, start the dissolution apparatus and take a 5 mL sample to obtain the dissolution solution. After filtration, take the filtrate to obtain the test solution, and store it in the dark for later use. Using a test solution with added pH 6.8 phosphate buffer, the sample was injected six times consecutively, and the peak area was determined to be RSD = 0.54%, indicating that the high performance liquid chromatograph has good precision. The test solution containing pH 4.5 acetate buffer was injected six times consecutively, and the peak area was measured. The RSD was 0.65%, indicating that the high performance liquid chromatograph has good precision. In a 0.1 mol / L hydrochloric acid solution, the same sample solution was injected six times consecutively, and the peak area was measured. The RSD was 0.29%, indicating that the high performance liquid chromatograph has good precision.

[0047] Recovery rate test: Using the blank excipient non-interference method, different concentrations of perphenazine reference standard were added, dissolved and diluted in different media and brought to the mark. According to this method, the average recovery rate was 98.6% in pH 6.8 phosphate buffer, RSD=1.01% (n=18); the average recovery rate was 99.1% in pH 4.5 acetate buffer, RSD=0.98% (n=18); and the average recovery rate was 99.7% in 0.1 mol / L hydrochloric acid solution, RSD=0.83% (n=18), all of which met the requirements for quantitative analysis.

[0048] Stability test: The test solution was placed in the dark for 0, 4, 6, 12, 18 and 24 hours, and the peak area was measured. RSD=1.09%, indicating that the solution was stable within 24 hours.

[0049] like Figure 5 As can be seen, when comparing the dissolution curves of perphenazine film-coated tablets measured by this method with those measured by UV method in pH 6.8 phosphate buffer, the dissolution curve measured by this method more accurately reflects the drug dissolution characteristics and shows no abnormal fluctuations.

[0050] like Figure 6 As can be seen, the cumulative dissolution amounts of the sample and the original reference were compared using high performance liquid chromatography in pH 6.8 phosphate buffer.

[0051] like Figure 7 As can be seen, the cumulative dissolution amount of samples with different specifications in pH 6.8 phosphate buffer was compared using high performance liquid chromatography.

[0052] In summary, the method for plotting the dissolution curve of perphenazine tablets based on high-performance liquid chromatography (HPLC) of this invention can effectively separate perphenazine from excipients and impurities through chromatographic separation, eliminating the absorption interference problem in the ultraviolet method, and making the measurement results more accurate and reliable. It has a wide linear range (1-10 μg / mL), a correlation coefficient r≥0.9998, a detection limit as low as 15 ng / mL, and a quantitation limit of 40 ng / mL, enabling accurate determination of drug concentration at different dissolution stages. The average recovery rate is 98.0%-102.0%, the relative standard deviation (RSD) is ≤1.5%, and the test solution is stable within 24 hours, meeting the requirements for batch sample testing. The dissolution conditions are consistent with existing pharmacopoeia standards; only the detection method needs to be changed, without adjusting the formulation dissolution test device, facilitating rapid promotion and application by enterprises.

[0053] The specific embodiments of the invention have been described above. It should be understood that the invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood to be implemented in a manner common to the art; those skilled in the art can make various modifications or alterations within the scope of the claims, and make several simple deductions, variations or substitutions, which do not affect the substantive content of the invention.

Claims

1. A method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography, characterized in that, Includes the following steps: In a light-protected environment, add phenytoin tablets and preheated dissolution medium to multiple dissolution cups in sequence; The dissolution solution was obtained by taking multiple samples from multiple dissolution vessels using a dissolution apparatus. Each sample was 5 mL and isothermal dissolution medium was added simultaneously. After filtering the above-mentioned dissolution solution, take the filtrate to obtain the test solution, and store it in the dark for later use. Reference solutions of perphenazine tablets with concentrations of 2.012 μg / mL and 4.024 μg / mL were prepared and stored in the dark for later use. High performance liquid chromatography (HPLC) was used. The test solution and the reference solution were injected separately into the HPLC according to the preset chromatographic conditions. Record the peak area and calculate the dissolution amount at each time point according to the following formula: Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference standard × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference standard × Tablet labeled amount) × 100%; Plot a dissolution curve with dissolution time on the x-axis and cumulative dissolution amount at each time on the y-axis.

2. The method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography according to claim 1, characterized in that, The perphenazine tablets are 2 mg / tablet or 4 mg / tablet.

3. The method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography according to claim 2, characterized in that, Add 900 ml of the dissolution medium, preheated to 37.0 ± 0.5 °C, to each dissolution vessel; The melting device rotates at 50 revolutions per minute; Samples were taken at 5, 10, 15, 20, 30, 45, 60 and 90 minutes, with 5 mL of sample taken each time and 5.5 mL of isothermal dissolution medium added simultaneously. The solution was filtered using a 0.22 μm aqueous filter membrane. After discarding 4 ml, the filtrate was collected.

4. A method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography according to any one of claims 1-3, characterized in that, The dissolution medium is a phosphate buffer solution with pH 6.8, an acetate buffer solution with pH 4.5, or a 0.1 mol / L hydrochloric acid solution.

5. The method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography according to claim 4, characterized in that, The preparation method of the reference solution is as follows: Weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; Transfer 1 mL of the reference stock solution into a 50 mL volumetric flask, add dissolution medium to dilute to the mark, and shake well to obtain a reference solution with a concentration of 4.024 μg / mL; Transfer 1 mL of the reference stock solution into a 100 mL volumetric flask, add dissolution medium to dilute to the mark, and shake well to obtain a reference solution with a concentration of 2.012 μg / mL.

6. The method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography according to claim 5, characterized in that, The chromatographic conditions were as follows: a C18 column, a mobile phase of methanol-30 mmol / L ammonium acetate solution (volume ratio 80:20), a flow rate of 1.0 mL / min, a column temperature of 35 °C, a detection wavelength of 254 nm, and injection volumes of 20 μL and 50 μL.

7. The method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography according to claim 6, characterized in that, Pipette 20 μL and 50 μL of the test solution and reference solution respectively, and inject them into the high performance liquid chromatograph to record the peak areas.

8. The method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography according to claim 7, characterized in that, The test solution and the reference solution must be measured within 24 hours after preparation.

9. A method for plotting the dissolution curve of perphenazine tablets using high performance liquid chromatography according to claim 8, characterized in that, It also includes calculating the linear regression equation to obtain a linear relationship: Weigh 10.06 mg of perphenazine reference standard into a 50 mL volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well to obtain a reference standard stock solution with a concentration of 201.20 μg / mL; Take the reference stock solution and dilute it with a dissolution medium to prepare a series of reference solutions with concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, and 5.0 μg / mL; Turn on the high-performance liquid chromatograph (HPLC) according to the preset chromatographic conditions, pipette a series of reference solutions, inject them into the HPLC, and record the peak areas. Calculate the dissolution amount at each time point using the following formula: Dissolution amount (%) = (Peak area of ​​test sample × Concentration of reference sample × Dilution factor × Volume of dissolution medium) / (Peak area of ​​reference sample × Tablet labeled amount) × 100%. Perform linear regression with concentration (x) as the x-axis and peak area (y) as the y-axis.

10. A method for plotting the dissolution curve of perphenazine tablets using high-performance liquid chromatography according to claim 9, characterized in that, The dissolution medium is a phosphate buffer solution with pH 6.8, an acetate buffer solution with pH 4.5, or a 0.1 mol / L hydrochloric acid solution; The chromatographic conditions were as follows: a C18 column, a mobile phase of methanol-30 mmol / L ammonium acetate solution (volume ratio 80:20), a flow rate of 1.0 mL / min, a column temperature of 35 °C, and a detection wavelength of 254 nm.

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