Method for determining contents of multiple index components in Tongfeng pills

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining the content of multiple components in Gout Pill, which simultaneously determines the content of effective components in Gout Pill, such as aesculin, syringin, corilagene, tannic acid and chebulagic acid, by using high performance liquid chromatography, and determines the content of total alkaloids in Gout Pill by using ultraviolet spectrophotometry. The application can effectively shorten the analysis time of main effective components in Gout Pill, has a good linear range, high accuracy, precision and repeatability, and is stable and feasible, and can be applied to the content determination and quality standard of Gout Pill.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine testing technology, specifically relating to a method for determining the content of multiple components in gout pills. Background Technology

[0002] The prescription for Gout Pills originates from the Tibetan medical classic "Secret Treasure Source". The prescription consists of nine medicinal materials: hot-processed gypsum, chebula (without kernel), Zha Xun Gao (a type of herbal paste), senna seeds, kuanjin teng (a type of vine), qinpi (a type of bark), alkali flower (processed), Tibetan madder root, and licorice root. It has the effects of dispelling wind and relieving numbness, reducing inflammation and relieving pain, relaxing muscles and tendons, and drying yellow fluid. In Tibetan medicine, it is used to treat symptoms such as redness, swelling, heat, pain, and limited mobility of the joints in the limbs caused by "Chi Nai" (gout).

[0003] The current quality standards for gout pills lack quantitative detection items for the active ingredients, resulting in a low level of quality control. This invention establishes a method for determining the content of the active ingredients corilagin, fraxin, syringin, ellagic acid, and chebulic acid in gout pills using HPLC, and uses ultraviolet spectrophotometry to determine the content of total alkaloids in gout pills, providing a method and basis for improving the quality standards of Tibetan medicine gout pills. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the content of multiple components in gout pills.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: The gout pills described in this invention are a pharmaceutical preparation composed of gypsum (heat-processed), Terminalia chebula (without kernel), Zha Xun Gao (a type of herbal extract), Asarum sieboldii, Tinospora sinensis, Fraxinus rhynchophylla, processed Alpinia oxyphylla, Rubia cordifolia, and Glycyrrhiza uralensis in the prescribed proportions.

[0006] The method for determining the content of components in the gout pills described in this invention employs high-performance liquid chromatography (HPLC) to simultaneously determine the content of the active ingredients fraxin A, syringin, corilagin, ellagic acid, and chebulic acid in the gout pills. In the gout pill prescription, *Tetrapanax papyriferus* has the effects of promoting blood circulation and removing blood stasis, relaxing muscles and tendons, dispelling wind and relieving pain, regulating imbalances, clearing heat and moistening the lungs, and drying yellow fluid. It is the principal ingredient in the prescription. Modern research shows that phenylpropanoid components such as syringin and syringin in *Tetrapanax papyriferus* are quality markers, possessing hypoglycemic, lipid-lowering, anti-inflammatory, analgesic, and immune-enhancing effects. *Fraxinus rhynchophylla* has the effects of clearing heat and drying dampness, astringing and stopping dysentery, stopping leukorrhea, improving eyesight, and drying yellow fluid. It is the assistant ingredient in the prescription. Modern research shows that fraxin A, fraxin B, and fraxinosin in *Fraxinus rhynchophylla*... Coumarin-like components possess anti-inflammatory, antioxidant, and analgesic properties, with fraxin being the most abundant. Terminalia chebula, known as the "King of Tibetan Medicine," has effects such as clearing heat and detoxifying, astringing and nourishing blood, stopping diarrhea, harmonizing the three factors (body, stomach, and intestines), and harmonizing other medicines. It is one of the most frequently used herbs in Tibetan medicine. Modern research shows that Terminalia chebula contains polyphenols and tannins such as chebulic acid, gallic acid, chebulic biphenyl acid, ellagic acid, and corilagin, which have multiple biological functions including antioxidant, anti-inflammatory, immunomodulatory, and uric acid-lowering effects. Therefore, corilagin, fraxin, syringin, ellagic acid, and chebulic acid were selected as the effective ingredients in the gout pill.

[0007] The high-performance liquid chromatography method described in this invention uses a C10 column. 18 The liquid chromatography column (250 mm × 4.6 mm, 5 μm) is a reversed-phase column with a carbooctadecylsilane chemically bonded silica stationary phase. The silica particles have a diameter of 5 μm, the column length is 250 mm, and the inner diameter of the column is 4.6 mm.

[0008] The high-performance liquid chromatography method described in this invention uses an ultraviolet detector. Ultraviolet full-wavelength scanning of the reference solutions of fraxin, syringin, corilagin, ellagic acid, and chebulic acid showed that each index component had high absorption at 220~300 nm, and each component had a high absorption peak area at 270 nm. Taking all factors into consideration, the detection wavelength of 270 nm was selected.

[0009] In the chromatographic conditions described in this invention, the mobile phase is acetonitrile-0.1% phosphoric acid solution, wherein the 0.1% phosphoric acid solution is an aqueous solution of phosphoric acid with a volume fraction of 0.1%. Under these mobile phase conditions, the theoretical plate numbers of the chromatographic peaks of fraxin, syringin, corilagin, ellagic acid, and chebulic acid are all greater than 3000, the tailing factors are all between 0.95 and 1.05, and the resolution is all greater than 1.5, which meets the requirements for high performance liquid chromatography (HPLC) content determination.

[0010] The gradient elution program for chromatographic conditions described in this invention includes the following steps: a) from 0 to 30 min, the acetonitrile ratio is 10% and the 0.1% phosphoric acid aqueous solution ratio is 90%; b) from 30 to 31 min, the acetonitrile ratio is 10 to 15% and the 0.1% phosphoric acid aqueous solution ratio is 90 to 85%; c) from 31 to 50 min, the acetonitrile ratio is 15 to 27% and the 0.1% phosphoric acid aqueous solution ratio is 85 to 73%. Steps a) to b) are for the separation of fraxin and syringin; steps b) to c) are for the separation of corilagin, chebulic acid and ellagic acid.

[0011] The method for preparing the test solution according to the present invention is as follows: take gout pills, grind them into fine powder, add the fine powder of gout pills and methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) at a weight-volume ratio of 1:10 into an extraction container, use an ultrasonic device with a power of 250 W and a frequency of 50 kHz to ultrasonically treat for 60 minutes, and filter with a 0.45 μm microporous membrane to obtain the test solution.

[0012] The methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) in the test sample solution preparation method of the present invention is the extraction solvent, which is a solution prepared by mixing anhydrous methanol and a 0.1% phosphoric acid aqueous solution in a volume ratio of 7:3. Compared with water and methanol-0.1% phosphoric acid aqueous solutions of different methanol concentrations as extraction solvents: When using water as the extraction solvent, the extraction yields of the active ingredients fraxin, syringin, and ellagic acid in the test sample solution were less than 50%; when using methanol-0.1% phosphoric acid aqueous solutions with a methanol volume fraction of 10–70% as the extraction solvent, the contents of the active ingredients fraxin, syringin, and ellagic acid in the test sample solution increased significantly, while the contents of corilagin and chebulic acid did not change significantly; when using methanol-0.1% phosphoric acid aqueous solutions with a methanol volume fraction of 70–80% as the extraction solvent, the contents of fraxin, syringin, ellagic acid, corilagin, and chebulic acid in the test sample solution did not change significantly; when using methanol-0.1% phosphoric acid aqueous solutions with a methanol concentration exceeding 80% as the extraction solvent, the content of chebulic acid in the test sample solution decreased significantly. Therefore, a methanol-0.1% phosphoric acid aqueous solution with a methanol volume fraction of 70% (7:3, v:v) was selected as the extraction solvent.

[0013] In the test solution preparation method of the present invention, the weight-volume ratio of the fine powder of gout pills to methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) is 1:10. If the weight-volume ratio is greater than 1:10, the resolution of the chromatographic peak of chebulic acid in the test solution is less than 1.0; if the weight-volume ratio is less than 1:10, the resolution of the chromatographic peak of syringin in the test solution is less than 1.0. Therefore, the final material-liquid ratio is confirmed to be 1:10.

[0014] The extraction method for preparing the test solution according to the present invention involves ultrasonic extraction using an ultrasonic device with a power of 250 W and a frequency of 50 kHz. A methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) is used as the extraction solvent. No significant difference was observed in the chromatographic peak areas of the active components in the test solution prepared by ultrasonic and reflux extraction methods; therefore, the simpler ultrasonic extraction method was chosen.

[0015] In the preparation method of the test solution described in this invention, the ultrasonic extraction time is 30–120 minutes, preferably 60 minutes. When the ultrasonic time is 5–60 minutes, the peak areas of each active ingredient in the test solution change significantly; when the ultrasonic time is 60–120 minutes, the peak areas of the active ingredients fraxin, syringin, ellagic acid, corilagin, and chebulic acid in the test solution do not change significantly; when the ultrasonic time exceeds 120 minutes, the peak area of ​​chebulic acid in the test solution decreases significantly. Therefore, the preferred extraction time is 60 minutes.

[0016] The injection volume of the multi-active ingredient content determination method described in this invention is 10–15 μL, preferably 10 μL. When the injection volume is less than 10 μL, the chromatographic peak resolution of the active ingredients fraxinus and syringin in the gout pill test solution is less than 1.0; when the injection volume is 10–15 μL, the chromatographic peak resolution of the active ingredients is greater than 1.5; when the injection volume exceeds 15 μL, the chromatographic peak resolution of the active ingredient chebulic acid in the test solution is less than 1.0, therefore, the injection volume is 10 μL.

[0017] The high-performance liquid chromatography method of this invention also includes a method for preparing reference solutions, which is as follows: Fraxinoside, syringin, corilagin, and chebulic acid are accurately weighed, dissolved in methanol-0.1% phosphoric acid aqueous solution (7:3, v:v), and diluted to volume to prepare stock solutions of each reference standard. Ellagic acid reference standard is accurately weighed, dissolved in methanol, diluted to volume, and shaken well to prepare an ellagic acid reference standard stock solution. Appropriate volumes of the fraxinoside, syringin, corilagin, ellagic acid, and chebulic acid reference standard stock solutions are accurately transferred to the same volumetric flask according to a volume ratio (15:0.1:1.3:6:0.5), diluted to volume with methanol-0.1% phosphoric acid aqueous solution (7:3, v:v), and shaken well to prepare a mixed reference solution.

[0018] The high performance liquid chromatography method of the present invention also includes a method for preparing a blank solution: the blank solution is prepared by not adding gout pills, and the remaining operations are the same as the preparation method of the test solution.

[0019] The method for determining the content of multiple components in gout pills described in this invention employs ultraviolet spectrophotometry to determine the content of the total alkaloids, the main active ingredient, in gout pills. Palmatine hydrochloride is a typical isoquinoline alkaloid; this invention uses palmatine hydrochloride as a control to determine the content of total alkaloids.

[0020] The ultraviolet spectrophotometry method described in this invention performs a full-wavelength scan on the reference solution of palmatine hydrochloride. The results show that the reference solution has the maximum absorption at 433 nm, so 433 nm is selected as the detection wavelength.

[0021] The ultraviolet detection method described in this invention investigated the ratio of reflux extraction solvent. When the volume ratio of hydrochloric acid to ethanol was 1:20, the total alkaloid content of the indicator component in the test sample solution was the highest.

[0022] After determining the extraction method, extraction solvent, and solvent ratio, this invention compared the effects of extraction time and solid-liquid ratio on the active ingredients through single-factor experiments. The results showed that after reflux extraction for 30, 60, 90, and 120 min, the total alkaloid content of the indicator component in the test sample gradually increased within the range of 30-60 min, and decreased after 60 min. Therefore, the optimal extraction time was 60 min. Subsequently, different solid-liquid ratios were compared, and the highest total alkaloid content of the indicator component was observed when the solid-liquid ratio was 1:40.

[0023] The beneficial effects of this invention are: (1) This invention establishes a high performance liquid chromatography method for the simultaneous quantitative determination of fraxin, syringin, corilagin, ellagic acid and chebulic acid in gout pills, and an ultraviolet detection method for the quantitative determination of total alkaloids in gout pills. The study was verified by methodology, and the results show that the method is scientific and feasible. (2) Compared with separate analysis, the present invention can effectively reduce the number of sample injections and analysis time of the instrument, with short detection time, accurate detection results, and simple and easy operation; (3) The present invention has a wide linear range, good repeatability, stable and feasible sample processing method, and the obtained test solution has good stability at room temperature. (4) The method of the present invention is stable and accurate, and can be used for the quality evaluation of gout pills, which can provide a basis for improving the quality standards of gout pills. Attached Figure Description

[0024] Figure 1 The HPLC chromatogram is for the blank solution. Figure 2 This is the HPLC chromatogram of the mixed control solution; Figure 3 The HPLC chromatogram of the test solution is shown below. Figure 4 Linearity graph of fraxin reference standard; Figure 5 The linear relationship graph for syringin reference standard; Figure 6 Linear relationship graph for Corilagin reference standard; Figure 7 The linear relationship graph for ellagic acid reference standard is shown. Figure 8 Linearity graph of chebulic acid reference standard; Figure 9 Linearity graph of palmatine hydrochloride reference standard; Detailed Implementation

[0025] The following experimental examples are used to further illustrate, but are not limited to, the present invention.

[0026] The gout pills used in the following specific embodiments are from Tibet Shenhou Pharmaceutical Co., Ltd., with batch numbers 20241213, 20241215, and 20241217.

[0027] Syringin reference standard (purity 98.54%), fraxin reference standard (purity 99.46%), corilagin reference standard (purity 100.0%), chebulic acid (purity 98.31%), and palmatine hydrochloride (purity 98.56%) were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; ellagic acid reference standard (purity 88.80%) was purchased from the China National Institutes for Food and Drug Control; acetonitrile (chromatographic grade) was purchased from Hubei Futon Biochemical Technology Co., Ltd.; phosphoric acid (analytical grade) and methanol (chromatographic grade) were both purchased from Sinopharm Chemical Reagent Co., Ltd. Example

[0028] 1. The contents of fraxin, syringin, corilagin, ellagic acid, and chebulic acid in gout pills were determined simultaneously by high performance liquid chromatography, including the following steps: Preparation of test solution Take the gout pills, grind them into a fine powder, accurately weigh 2.0 g of the fine powder, place it in a 100 mL volumetric stoppered conical flask, add 20 mL of methanol-0.1% phosphoric acid aqueous solution (7:3, v:v), weigh, sonicate (250 W, 50 kHz) for 60 min, cool, weigh again, make up the reduced weight with methanol-0.1% phosphoric acid aqueous solution (7:3, v:v), shake well, filter, take the filtrate, filter through a 0.45 micrometer microporous membrane to obtain the test solution.

[0029] Preparation of mixed reference solution Accurately weigh 19.26 mg of fraxin reference standard into 20 mL volumetric flasks, add methanol-0.1% phosphoric acid aqueous solution (7:3, v:v), dissolve and dilute to the mark, shake well to obtain a concentration of 0.96 mg·mL⁻¹. -1The reference stock solutions were prepared by accurately weighing 9.75 mg of syringin, 9.25 mg of corilagin, and 10.07 mg of chebulic acid into 10 mL volumetric flasks to obtain mass concentrations of 0.98, 0.93, and 1.01 mg∙mL, respectively. -1 The reference stock solution was prepared by accurately weighing 8.39 mg of ellagic acid reference standard and placing it in a 50 mL volumetric flask. Solvent was added to dissolve and the solution was diluted to volume, then shaken well to obtain a mass concentration of 0.17 mg∙mL⁻¹. -1 The reference stock solution.

[0030] Accurately transfer appropriate amounts of each reference standard stock solution into the same 25 mL volumetric flask, add solvent to make up to volume, and shake well to obtain the mass concentrations of fraxin, syringin, corilagin, ellagic acid, and chebulic acid of 577.80, 3.90, 48.10, 40.27, and 20.14 mg∙mL, respectively. -1 A mixed reference solution.

[0031] (3) Preparation of blank solution Without adding gout pill powder, the blank solution was prepared by the same procedure as in step (1) of the preparation of the test solution.

[0032] (4) Chromatographic conditions The chromatographic column was Ultimate XB-C. 18 (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile-0.1% phosphoric acid solution, with gradient elution (0–30 min, 10% A; 30–31 min, 10%–15% A; 31–50 min, 15%–27% A, injection volume 10 μL, flow rate 1 mL·min). -1 The detection wavelength is 270 nm and the column temperature is 30℃.

[0033] (5) Methodological investigation System suitability test: Take blank solution, mixed reference solution, and test solution, inject and determine according to chromatographic conditions, record the chromatograms, and the results are shown in [see table below]. Figure 1 , Figure 2 , Figure 3 (Peaks 1, 2, 3, 4, and 5 in the figure represent fraxin, syringin, corilagin, ellagic acid, and chebulic acid, respectively.) No interfering peaks appeared at the retention times of corilagin, fraxin, syringin, ellagic acid, and chebulic acid in the chromatogram of the blank solution. In the chromatogram of the test solution, the resolution of corilagin, fraxin, syringin, ellagic acid, and chebulic acid from adjacent peaks was greater than 1.5, the tailing factor was between 0.95 and 1.05, the theoretical plate number was greater than 5000, and the retention times were consistent with those of the components in the reference solution. The suitability test of the chromatographic system met the requirements for content determination.

[0034] Linearity assessment: Accurately pipette 1, 2, 4, 6, 8, and 10 mL of the mixed reference solution into 5 mL volumetric flasks, add methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) to dilute to volume, shake well, and prepare mixed reference solutions of different concentrations. Inject and determine according to chromatographic conditions. Plot the mass concentration of the reference solution as the x-axis (…). X The peak area is the vertical axis ( Y Linear regression analysis was performed, and the results showed that fraxin exhibited a good linear relationship with the peak area integral value within the range of 57.78–577.8 μg / mL. The equation was as follows: Y = 4.0938 X + 4.4408 ( r =0.9998), syringin showed a good linear relationship with the peak area integral value in the range of 0.39~3.9 μg / mL, and the equation was: Y = 23.931 X + 1.0286686 ( r =0.9999), Corilagin showed a good linear relationship with the peak area integral value in the range of 4.81~48.1 μg / mL, and the equation was: Y = 19.662 X + 3.7093 ( r =0.9998), ellagic acid showed a good linear relationship with the peak area integral value in the range of 4.03~40.27 μg / mL, and the equation was: Y = 46.227 X -47.549 ( r =0.9996), chebulic acid showed a good linear relationship with the peak area integral value in the range of 2.01~20.14 μg / mL, and the equation was: Y = 17.823 X – 1.4745 ( r =0.9998), the result is as follows Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 .

[0035] Limits of quantitation and limits of detection: The mixed reference solution under item (2) was serially diluted, and the mass concentrations at signal-to-noise ratios of 3:1 and 10:1 were used as the limits of detection and limits of quantitation. The results showed that the limits of detection for fraxin, syringin, corilagin, ellagic acid, and chebulic acid were 0.144, 0.039, 0.012, 0.004, and 0.050 μg·mL, respectively. -1 The limits of quantitation were 0.058, 0.003, 0.005, 0.0004, and 0.02 μg∙mL. -1 .

[0036] Precision test: Take the test solution under (1), inject it according to the chromatographic conditions, and determine it 6 times consecutively. Measure the peak areas of fraxin, syringin, corilagin, ellagic acid, and chebulic acid. RSD ( n =6) were 1.70%, 1.10%, 0.85%, 1.28%, and 1.87% respectively, indicating that the instrument has good precision.

[0037] Repeatability test: Six parallel test solutions were prepared using the same batch of gout pills (batch number 20241213) according to the test solution preparation method. These solutions were then injected and analyzed under chromatographic conditions. The peak areas of fraxin, syringin, corilagin, ellagic acid, and chebulic acid were measured. RSD The values ​​were 0.52%, 1.71%, 0.86%, 1.28%, and 0.71%, respectively, indicating that the method has good repeatability.

[0038] Stability test: A freshly prepared test solution was taken from the same batch of gout pills (batch number 20241213) and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 h. The peak areas of fraxin, syringin, corilagin, chebulic biphenyl acid, ellagic acid, and chebulic acid were measured. RSD The concentrations were 0.89%, 1.50%, 1.30%, 1.93%, and 1.82%, respectively, indicating that the test solution was stable within 24 hours.

[0039] Spiking recovery test: Accurately weigh 1 g of 1 g of powder from six portions of gout pills (batch number 20241213) with known content, add each reference standard, and prepare 6 parallel test solutions. Determine and calculate the recovery rate for each solution. The average recoveries of fraxin, syringin, corilagin, ellagic acid, and chebulic acid were 99.27%, 96.22%, 97.52%, 95.63%, and 93.19%, respectively. RSD The recoveries were 2.1%, 3.6%, 3.3%, 1.2%, and 2.6%, respectively, indicating that the method had a good recovery rate.

[0040] (6) Determination of the content of effective ingredients in gout pills Three batches of gout pills were taken, and test solutions were prepared for each batch. The solutions were then injected and analyzed according to the chromatographic conditions. The results are shown in Table 1.

[0041] Table 1. Content determination results of five components in Gout Pills (mg∙g) -1 )

[0042] 2. The content of palmatine hydrochloride in gout pills was determined by ultraviolet detection, including the following steps: Preparation of test solution Take 1 g of gout pill powder, add 40 mL of hydrochloric acid-ethanol solution (1:20, v:v), heat at 90 ℃ under reflux for 1 h, cool, filter, reflux extract twice, combine the filtrates, evaporate the filtrate to dryness, dissolve in anhydrous ethanol and make up to 50 mL in a volumetric flask, take 2 mL, and make up to 25 mL in a volumetric flask to obtain the final product.

[0043] Preparation of reference stock solution Accurately weigh 12.5 mg of palmatine hydrochloride reference standard, dilute to 10 mL with anhydrous ethanol, mix thoroughly, and prepare the reference standard stock solution.

[0044] Methodological investigation Linearity investigation: A series of concentrations of palmatine hydrochloride stock solutions were diluted to 20 mL in volumetric flasks with anhydrous ethanol, and thoroughly mixed. Anhydrous ethanol solution was used as a blank control. The absorbance was measured at 433 nm. The results were expressed as concentrations (mg·L⁻¹). -1 A standard curve was plotted with absorbance values ​​on the y-axis and X on the x-axis. The results are shown in [reference needed]. Figure 9 After linear regression analysis, the regression equation is: Y =0.0141 X +0.0015 indicates that the sample concentration is between 12.5 and 62.5 mg·L⁻¹. -1 Good linearity within the concentration range ( r =0.9999).

[0045] Precision test: Accurately transfer 1 mL of the reference stock solution, add solvent to make up to 10 mL in a volumetric flask, mix well, and measure the absorbance 6 times consecutively. Measure the absorbance... RSD The result was 0.09%, indicating that the instrument has good precision and the experimental results are reliable.

[0046] Repeatability test: Six test solutions were prepared in parallel from the same batch of gout pills (batch number 20241213), and the absorbance was measured. RSD The value is 0.14% ( n =6), indicating that the method has good repeatability.

[0047] Stability test: Take one sample solution of the same batch of gout pills (batch number 20241213) and measure its absorbance at 0, 10, 20, 40, 60, 80, 100, and 120 min. RSD The value was 0.66%, indicating that the test solution had good stability.

[0048] Spiking recovery test: Accurately weigh 6 portions (batch number 20241213) of gout pills with known content, 0.5 g per portion, and accurately add 1 portion of palmatine hydrochloride reference solution with known content to prepare test solutions. Measure the absorbance at 433 nm and calculate the recovery rate and... RSD The results are shown in Table 2.

[0049] Table 2 Results of the recovery test of palmatine hydrochloride from gout pills ( n =6)

[0050] Determination of palmatine hydrochloride content in gout pills Three batches of gout pills were taken, and test solutions were prepared for each batch. The absorbance values ​​were measured at 433 nm. The results are shown in Table 3.

[0051] Table 3. Results of palmatine hydrochloride content determination in gout pills

[0052] This invention establishes a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the content of indicator components fraxin, syringin, corilagin, ellagic acid, and chebulic acid in gout pills. Methodological validation results show that this method is scientific and feasible. Compared with individual analyses, this method effectively reduces the number of instrument injections and analysis time, shortens experimental time, and lowers experimental costs. This invention also establishes a method for determining the total alkaloid content using ultraviolet spectrophotometry. The method established in this invention can accurately detect the content of indicator components fraxin, syringin, corilagin, ellagic acid, chebulic acid, and total alkaloids, and can be used for the quality evaluation of gout pills, providing a reference for improving the quality standards of this product.

[0053] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the content of multiple components in gout pills, characterized in that, The contents of fraxin, syringin, corilagin, ellagic acid, and chebulic acid in gout pills were simultaneously determined by high performance liquid chromatography (HPLC), and the total alkaloid content in gout pills was determined by ultraviolet spectrophotometry (UV spectrophotometry). The chromatographic conditions for the HPLC method were: C 18 Liquid chromatography column; mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution; flow rate: 1 mL / min; detection wavelength: 270 nm; column temperature: 30°C; the gradient elution program under the chromatographic conditions includes the following steps: a) At 0 min, the volume ratio of acetonitrile is 10%, and the volume ratio of 0.1% phosphoric acid aqueous solution is 90%. b) At 31 min, the volume ratio of acetonitrile was 15%, and the volume ratio of 0.1% phosphoric acid aqueous solution was 85%. c) At 50 min, the volume ratio of acetonitrile is 27%, and the volume ratio of 0.1% phosphoric acid aqueous solution is 73%. The C 18 The liquid chromatography column is a reversed-phase column with a carbo-octadecylsilane chemically bonded silica stationary phase. The silica particles have a diameter of 5 μm, the column length is 250 mm, and the inner diameter is 4.6 mm. The preparation method of the test solution in the high-performance liquid chromatography is as follows: take gout pills, grind them into fine powder, add the gout pill powder and methanol-0.1% phosphoric acid aqueous solution at a weight-volume ratio of 1:10 into the extraction container, use an ultrasonic device with a power of 250 W and a frequency of 50 kHz to sonicate for 60 minutes, and filter through a 0.45 μm microporous membrane to obtain the test solution; wherein, the methanol-0.1% phosphoric acid aqueous solution is a mixed solution of methanol and 0.1% phosphoric acid aqueous solution at a volume ratio of 7:

3.

2. The method for determining the content of multiple components in gout pills according to claim 1, characterized in that, The method for preparing the reference solutions in the high-performance liquid chromatography (HPLC) method is as follows: Fraxinoside, syringin, corilagin, and chebulic acid reference standards are accurately weighed, dissolved in a 7:3 mixture of methanol and 0.1% phosphoric acid aqueous solution to obtain stock solutions of each reference standard; ellagic acid reference standard is accurately weighed, dissolved in methanol and diluted to volume, and shaken well to prepare an ellagic acid reference standard stock solution; fraxinoside, syringin, corilagin, ellagic acid, and chebulic acid reference standard stock solutions are accurately transferred to the same volumetric flask at a volume ratio of 15:0.1:1.3:6:0.5, diluted to volume with a 7:3 mixture of methanol and 0.1% phosphoric acid aqueous solution, and shaken well to prepare a mixed reference solution.

3. The method for determining the content of multiple components in gout pills according to claim 1, characterized in that, The injection volume in the high-performance liquid chromatography method is 10 μL.

4. The method for determining the content of multiple components in gout pills according to claim 1, characterized in that, The ultraviolet spectrophotometric absorption wavelength is 433 nm.

5. The method for determining the content of multiple components in gout pills according to claim 1, characterized in that, The ultraviolet spectrophotometry method further includes: (1) Preparation of reference stock solution: Accurately weigh palmatine hydrochloride reference standard, dissolve it in anhydrous ethanol, shake well, and prepare reference stock solution; (2) Preparation of test solution: Weigh the powder of gout pills, add hydrochloric acid-ethanol solution at a weight-volume ratio of 1:40, heat and reflux at 90 ℃ for 1 h, cool, filter, reflux extract twice, combine the filtrates, evaporate the filtrate to dryness, add anhydrous ethanol to dissolve and dilute to volume in a volumetric flask, shake well to obtain the test solution; wherein, the hydrochloric acid-ethanol solution is a mixed solution of hydrochloric acid and ethanol solution at a volume ratio of 1:

20.

6. The method for determining the content of multiple components in gout pills according to claim 1, characterized in that, The gout pill is a pharmaceutical composition consisting of *Vine and Rhizoma Cimicifugae*, *Fructus Terminaliae Terminaliae* (without kernels), *Fructus Achyranthis Bidentatae*, *Gypsum Fibrosum* (heat-processed), *Cortex Fraxini*, *Flos Alpiniae Radix et Rhizoma* (processed), *Radix Rubiae Zangensis*, and *Radix Glycyrrhizae*, or a preparation made from the pharmaceutical composition and a pharmaceutical carrier.

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