Method for determining contents of multi-index components in gout pills

Abstract

The invention 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 pills, which adopts high performance liquid chromatography to simultaneously determine the content of effective components such as aesculin, syringin, corilagin, ellagic acid and chebulilic acid in the gout pills, and adopts ultraviolet spectrophotometry to determine the content of total alkaloids in the gout pills. The method can effectively shorten the analysis time of main effective components in the gout pills, has a good linear range, is high in accuracy, precision and repeatability, is stable and feasible, and can be applied to content determination and quality standard of the gout pills.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining the contents of multiple components in Tongfeng Pills. Background Art

[0002] The prescription of Tongfeng Pills originated from the classic ancient Tibetan medicine book "Secret Treasure Source". Its prescription consists of 9 medicinal materials, namely, Gypsum Fibrosum (processed with heat), Fructus Chebulae (kernel removed), Zhaxun Ointment, Thlaspi arvense L., Tinospora sinensis (Lour.) Merr., Cortex Fraxini, Alkaline Flower (processed), Rubia cordifolia L., and Glycyrrhiza uralensis Fisch., and has the effects of dispelling wind and relieving arthralgia, reducing inflammation and relieving pain, dredging tendons and collaterals, and drying up dampness. In Tibetan medicine clinical practice, it is used for the symptoms such as redness, swelling, heat, pain, and limited movement of the limbs and joints caused by "Chinei" (gout) disease.

[0003] In the current quality standard of Tongfeng Pills, there is no quantitative detection item for the active ingredients, and the quality control level is relatively low. The present invention uses the HPLC method to establish a method for determining the contents of the active ingredients corilagin, aesculin, syringin, ellagic acid, and chebulinic acid in Tongfeng Pills, and uses the ultraviolet spectrophotometry method to determine the content of total alkaloids in Tongfeng Pills, providing a method and basis for improving the quality standard of Tibetan medicine Tongfeng Pills. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for determining the contents of multiple components in Tongfeng Pills.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows: The Tongfeng Pills described in the present invention are pharmaceutical preparations composed of Gypsum Fibrosum (processed with heat), Fructus Chebulae (kernel removed), Zhaxun Ointment, Thlaspi arvense L., Tinospora sinensis (Lour.) Merr., Cortex Fraxini, Alkaline Flower (processed), Rubia cordifolia L., and Glycyrrhiza uralensis Fisch. according to the prescription ratio.

[0006] The method for determining the content of components in the gout pill of the present invention uses high performance liquid chromatography (HPLC) to simultaneously determine the contents of effective components aesculin, syringin, corilagin, ellagic acid, and chebulinic acid in the gout pill. In the prescription of the gout pill, Tinospora sinensis has the effects of promoting blood circulation to remove blood stasis, relaxing tendons and activating collaterals, dispelling wind and relieving pain, regulating and harmonizing disorders, clearing heat and moistening the lungs, and drying up dampness. It is the monarch drug in the formula. Modern research shows that phenylpropanoid components such as syringoside and syringin in Tinospora sinensis are one of the quality markers, and have the effects of reducing blood sugar and lipid, anti-inflammatory and analgesic, and enhancing immunity; Cortex Fraxini in the formula has the effects of clearing heat and drying dampness, astringing diarrhea, arresting vaginal discharge, improving eyesight, and drying up dampness. It is the ministerial drug in the formula. Modern research shows that coumarin components such as aesculin, aesculetin, and fraxin in Cortex Fraxini have the functions of anti-inflammatory, antioxidant, and analgesic, and among them, the content of aesculin is the highest; Terminalia chebula in the formula is also known as the "king of Tibetan medicine" and has the effects of clearing heat and detoxifying, astringing and nourishing blood, astringing the intestines and stopping diarrhea, regulating the three causes, and harmonizing various drugs. It is the most frequently used medicine in Tibetan medicine. Modern research shows that polyphenol and tannin components such as chebulinic acid, gallic acid, chebulignic acid, ellagic acid, and corilagin contained in Terminalia chebula have multiple biological functions such as antioxidant, anti-inflammatory, immune regulation, and uric acid reduction. Therefore, corilagin, aesculin, syringin, ellagic acid, and chebulinic acid are selected as the effective components of the gout pill.

[0007] The chromatographic column used in the high performance liquid chromatography method of the present invention is: C 18 18 reversed-phase liquid chromatography column (250 mm × 4.6 mm, 5 μm). This chromatographic column is a reversed-phase chromatographic column with a carbon octadecylsilane chemical bond-bonded silica gel stationary phase. Its silica gel particle size is 5 μm, column length is 250 mm, and column inner diameter is 4.6 mm.

[0008] The detector used in the high performance liquid chromatography method of the present invention is an ultraviolet detector. The ultraviolet full-wavelength scanning of the reference substance solutions of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid shows that each target component has a relatively high absorption at 220 - 300 nm, and each component has a relatively high absorption peak area at a wavelength of 270 nm. Considering comprehensively, the detection wavelength of 270 nm is selected.

[0009] In the chromatographic conditions of the present invention, the mobile phase is acetonitrile - 0.1% phosphoric acid water. Here, 0.1% phosphoric acid water is an aqueous phosphoric acid solution with a phosphoric acid volume fraction of 0.1%. Under this mobile phase condition, the theoretical plate numbers of the chromatographic peaks of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid are all greater than 3000, the tailing factors are all within 0.95 - 1.05, and the resolution is all greater than 1.5, meeting the requirements of high performance liquid content determination.

[0010] The gradient elution program of the chromatographic conditions described in the present invention includes the following steps: a) From 0 to 30 minutes, the proportion of acetonitrile is 10%, and the proportion of 0.1% phosphoric acid aqueous solution is 90%; b) From 30 to 31 minutes, the proportion of acetonitrile is 10 - 15%, and the proportion of 0.1% phosphoric acid aqueous solution is 90 - 85%; c) From 31 to 50 minutes, the proportion of acetonitrile is 15 - 27%, and the proportion of 0.1% phosphoric acid aqueous solution is 85 - 73%. Among them, the step from a) to b) is to achieve the separation of aesculin and syringin; the step from b) to c) is to separate corilagin, chebulinic acid and ellagic acid.

[0011] The preparation method of the test solution described in 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) to the extraction container according to the weight - volume ratio of 1:10, use an ultrasonic device with a power of 250 W and a frequency of 50 kHz, perform ultrasonic treatment for 60 minutes, and filter with a 0.45 - μm microporous filter membrane to obtain the test solution.

[0012] The methanol - 0.1% phosphoric acid aqueous solution (7:3, v:v) in the preparation method of the test solution described in the present invention is the extraction solvent, which is a solution prepared by mixing anhydrous methanol and 0.1% phosphoric acid aqueous solution according to the volume ratio of 7:3. Compared with water and methanol - 0.1% phosphoric acid aqueous solutions with different methanol concentrations as extraction solvents: When water is used as the extraction solvent, the extraction yields of the active ingredients aesculin, syringin, and ellagic acid in the test solution are less than 50%; When methanol - 0.1% phosphoric acid aqueous solutions with a methanol volume fraction of 10 - 70% are used as extraction solvents, the contents of the active ingredients aesculin, syringin, and ellagic acid in the test solution increase significantly, and the changes of corilagin and chebulinic acid are not significant; When methanol - 0.1% phosphoric acid aqueous solutions with a methanol volume of 70 - 80% are used as extraction solvents, the changes in the contents of aesculin, syringin, ellagic acid, corilagin, and chebulinic acid in the test solution are not significant; When methanol - 0.1% phosphoric acid aqueous solutions with a methanol concentration exceeding 80% are used as extraction solvents, the content of chebulinic acid in the test solution decreases significantly. Therefore, methanol - 0.1% phosphoric acid aqueous solution (7:3, v:v) with a methanol volume fraction of 70% is selected as the extraction solvent.

[0013] In the preparation method of the test solution described in 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. When the weight - volume ratio is greater than 1:10, the resolution of the chromatographic peak of chebulinic acid in the test solution is less than 1.0; when 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] In the method for preparing the test solution according to the present invention, the extraction method is ultrasonic extraction using an ultrasonic device with a power of 250 W and a frequency of 50 kHz. Methanol - 0.1% phosphoric acid aqueous solution (7:3, v:v) is used as the extraction solvent. There is no significant difference in the chromatographic peak areas of each active ingredient in the test solutions prepared by ultrasonic extraction and reflux extraction. Therefore, the ultrasonic extraction method, which is more convenient to handle, is selected.

[0015] In the method for preparing the test solution according to the present invention, the ultrasonic extraction time is 30 - 120 minutes, preferably 60 minutes. When the ultrasonic time is 5 - 60 minutes, the chromatographic peak areas of each active ingredient in the test solution change significantly; when the ultrasonic time is 60 - 120 minutes, the chromatographic peak areas of the active ingredients aesculin, syringin, ellagic acid, corilagin, and chebulinic acid in the test solution do not change significantly; when the ultrasonic time exceeds 120 minutes, the chromatographic peak area of chebulinic acid in the test solution decreases significantly. Therefore, the preferred extraction time is 60 minutes.

[0016] In the method for determining the content of multiple active ingredients according to the present invention, the injection volume is 10 - 15 μL, preferably 10 μL. When the injection volume is less than 10 μL, the resolution of the chromatographic peaks of the active ingredients aesculin and syringin in the test solution of Tongfeng Pills is less than 1.0; when the injection volume is 10 - 15 μL, the resolution of the chromatographic peaks of the active ingredients is greater than 1.5; when the injection volume exceeds 15 μL, the resolution of the chromatographic peak of the active ingredient chebulinic acid in the test solution is less than 1.0. Therefore, the injection volume is 10 μL.

[0017] The high - performance liquid chromatography method according to the present invention also includes a method for preparing a reference solution. The method is as follows: Accurately weigh aesculin, syringin, corilagin, and chebulinic acid respectively, dissolve them with methanol - 0.1% phosphoric acid aqueous solution (7:3, v:v) and make up the volume to obtain each reference stock solution. Accurately weigh the reference substance of ellagic acid, dissolve it with methanol and make up the volume, shake well to prepare the reference stock solution of ellagic acid. Accurately pipette appropriate amounts of the reference stock solutions of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid according to the volume ratio (15:0.1:1.3:6:0.5) into the same volumetric flask, dilute and make up the volume with methanol - 0.1% phosphoric acid aqueous solution (7:3, v:v), shake well to prepare a mixed reference solution.

[0018] The high - performance liquid chromatography method according to the present invention also includes a method for preparing a blank solution: The method for the blank solution is: Without adding Tongfeng Pills, and the remaining operations are the same as those for preparing the test solution to obtain the blank solution.

[0019] In the method for determining the content of multiple components in Tongfeng Pills according to the present invention, the ultraviolet spectrophotometry is used to determine the content of the total alkaloids, the main active ingredients in Tongfeng Pills. Palmatine hydrochloride is a typical isoquinoline alkaloid. In the present invention, palmatine hydrochloride is used as a reference to detect the content of the total alkaloids.

[0020] For the ultraviolet spectrophotometry described in the present invention, a full-wavelength scan was performed on the reference solution of palmatine hydrochloride. The results showed that the reference solution had a maximum absorption at 433 nm. Therefore, 433 nm was selected as the detection wavelength.

[0021] For the ultraviolet detection method described in the present invention, the ratio of the reflux extraction solvent was investigated. When the volume ratio of hydrochloric acid to ethanol was 1:20, the highest content of the total alkaloids, which were the target components, was measured in the test solution.

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

[0023] Advantages of the present invention: (1) The present invention established a method for simultaneously quantitatively determining aesculin, syringin, corilagin, ellagic acid, and chebulinic acid in Tongfeng Pills by high performance liquid chromatography (HPLC), and a method for quantitatively determining the total alkaloids in Tongfeng Pills by ultraviolet detection. After research and methodological verification, the results showed that this method was scientific and feasible; (2) Compared with separate analysis, the present invention can effectively reduce the injection times and analysis time of the instrument, with a short detection time, accurate detection results, and being simple and easy to operate; (3) The present invention has a wide linear range, good repeatability, a stable and feasible sample treatment method, and the obtained test solution has good stability at room temperature; (4) The method of the present invention is stable and accurate, can be used for the quality evaluation of Tongfeng Pills, and can provide a basis for improving the quality standard of Tongfeng Pills. Description of the Drawings

[0024] Figure 1 The HPLC chromatogram of the blank solution; Figure 2 The HPLC chromatogram of the mixed reference solution; Figure 3 The HPLC chromatogram of the test solution; Figure 4 The linear relationship diagram of aesculin reference substance; Figure 5 The linear relationship diagram of syringin reference substance; Figure 6 The linear relationship diagram of corilagin reference substance; Figure 7 It is the linear relationship diagram of ellagic acid reference substance; Figure 8 It is the linear relationship diagram of chebulinic acid reference substance; Figure 9 It is the linear relationship diagram of palmatine hydrochloride reference substance; Specific implementation manners

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

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

[0027] Syringin reference substance (purity 98.54%), aesculin reference substance (purity 99.46%), corilagin reference substance (purity 100.0%), chebulinic acid (purity 98.31%), palmatine hydrochloride (purity 98.56%) were purchased from Chengdu Lemeitian Medical Technology Co., Ltd.; ellagic acid reference substance (purity 88.80%) was purchased from the National Institutes for Food and Drug Control; acetonitrile was chromatographically pure and purchased from Hubei Fudun Biochemical Technology Co., Ltd.; phosphoric acid was of superior grade purity and methanol was chromatographically pure, both purchased from Sinopharm Chemical Reagent Co., Ltd. Examples

[0028] 1. Simultaneously determine the contents of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid in gout pills by high performance liquid chromatography, including the following steps: Preparation of test solution Take gout pills, grind them into 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, ultrasonicate (250 W, 50 kHz) for 60 min, let it 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 subsequent filtrate, and filter it through a 0.45 - micron microporous filter membrane to obtain the test solution.

[0029] Preparation of mixed reference substance solution Accurately weigh 19.26 mg of aesculin reference substance in a 20 mL volumetric flask respectively, add methanol - 0.1 phosphoric acid aqueous solution (7:3, v:v), dissolve and make up the volume to the scale, shake well, and prepare a solution with a mass concentration of 0.96 mg∙mL -1Reference substance stock solutions; Weigh accurately 9.75 mg of syringin, 9.25 mg of corilagin, and 10.07 mg of chebulinic acid into 10 mL volumetric flasks respectively, and prepare reference substance stock solutions with mass concentrations of 0.98, 0.93, and 1.01 mg∙mL -1 respectively; Weigh accurately 8.39 mg of ellagic acid reference substance into a 50 mL volumetric flask, add solvent to dissolve and make up the volume to the mark, shake well, and prepare a reference substance stock solution with a mass concentration of 0.17 mg∙mL -1 of the reference substance stock solution.

[0030] Accurately pipette appropriate amounts of each reference substance stock solution into the same 25 mL volumetric flask, add solvent to make up the volume to the mark, shake well, and prepare a mixed reference substance solution with mass concentrations of 577.80, 3.90, 48.10, 40.27, and 20.14 mg∙mL for aesculin, syringin, corilagin, ellagic acid, and chebulinic acid respectively. -1 of the mixed reference substance solution.

[0031] (3) Preparation of blank solution Without adding gout pill powder, prepare the blank solution according to the preparation method of the test solution in step (1) for the remaining operations.

[0032] (4) Chromatographic conditions The chromatographic column is Ultimate XB-C 18 (250 mm×4.6 mm, 5 μm); The mobile phase is 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), the injection volume is 10 μL, and the flow rate is 1 mL·min -1 , the detection wavelength is 270 nm, and the detection column temperature is 30°C.

[0033] (5) Methodology investigation System suitability test investigation: Take the blank solution, the mixed reference substance solution, and the test solution, inject and measure according to the chromatographic conditions, record the chromatogram, and the results are shown in Figure 1 , Figure 2 , Figure 3 . (The peaks numbered 1, 2, 3, 4, and 5 in the figure are aesculin, syringin, corilagin, ellagic acid, and chebulinic acid respectively), and no interfering peaks appear in the chromatogram obtained from the blank solution at the retention times of corilagin, aesculin, syringin, ellagic acid, and chebulinic acid. The resolution between corilagin, aesculin, syringin, ellagic acid, and chebulinic acid and the adjacent chromatographic peaks in the chromatogram of the test solution is greater than 1.5, the tailing factor is between 0.95 and 1.05, the theoretical plate number is greater than 5000, and it is consistent with the retention times of the components in the reference substance solution. The chromatographic system suitability test meets the requirements for content determination.

[0034] Investigation of linear relationship: Accurately pipette 1, 2, 4, 6, 8, and 10 mL of the mixed reference solution into 5-mL volumetric flasks respectively, add methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) to volume, shake well, and prepare mixed reference solutions with different concentrations. Inject and determine according to the chromatographic conditions. Using the mass concentration of the reference solution as the abscissa ( X ), and the peak area as the ordinate ( Y ), perform linear regression. The results show that aesculin has a good linear relationship with the integral value of the peak area within the range of 57.78 - 577.8 μg / mL, and the equation is Y = 4.0938 X + 4.4408 ( r =0.9998). Syringin has a good linear relationship with the integral value of the peak area within the range of 0.39 - 3.9 μg / mL, and the equation is Y = 23.931 X + 1.0286686 ( r =0.9999). Corilagin has a good linear relationship with the integral value of the peak area within the range of 4.81 - 48.1 μg / mL, and the equation is Y = 19.662 X + 3.7093 ( r =0.9998). Ellagic acid has a good linear relationship with the integral value of the peak area within the range of 4.03 - 40.27 μg / mL, and the equation is Y = 46.227 X -47.549 ( r =0.9996). Chebulinic acid has a good linear relationship with the integral value of the peak area within the range of 2.01 - 20.14 μg / mL, and the equation is Y = 17.823 X – 1.4745 ( r =0.9998). The results are shown in Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 .

[0035] Investigation of the limit of quantitation and limit of detection: Take the mixed reference solution under item (2) and dilute it step by step. Use the mass concentrations at signal-to-noise ratios of 3:1 and 10:1 as the limit of detection and limit of quantitation. The results show that the limits of detection of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid are 0.144, 0.039, 0.012, 0.004, 0.050 μg·mL -1 , and the limits of quantitation are 0.058, 0.003, 0.005, 0.0004, 0.02 μg∙mL -1 .

[0036] Precision test: Take the test solution under item (1), inject samples according to the chromatographic conditions, and continuously measure 6 times. The RSD ( n =6) of the peak areas of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid were 1.70%, 1.10%, 0.85%, 1.28%, and 1.87% respectively, indicating good precision of the instrument.

[0037] Repeatability test: Take the same batch of Tongfeng Pills (batch number 20241213), prepare 6 portions of test solutions in parallel according to the preparation method of the test solution, inject samples according to the chromatographic conditions for determination. The RSD of the peak areas of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid were 0.52%, 1.71%, 0.86%, 1.28%, and 0.71% respectively, indicating good repeatability of this method.

[0038] Stability test: Take the same batch of Tongfeng Pills (batch number 20241213), prepare 1 portion of the test solution, inject samples for analysis at 0, 2, 4, 6, 8, 10, 12, and 24 h respectively. The RSD of the peak areas of aesculin, syringin, corilagin, chebulic acid biphenylate, ellagic acid, and chebulinic acid were 0.89%, 1.50%, 1.30%, 1.93%, and 1.82% respectively, indicating that the test solution is stable within 24 h.

[0039] Spiked recovery test: Accurately weigh 6 portions of the powder of Tongfeng Pills with known content (batch number 20241213), 1 g for each portion, add each reference substance, prepare 6 portions of test solutions in parallel, and measure and calculate the recovery rates respectively. The average spiked recovery rates of aesculin, syringin, corilagin, ellagic acid, and chebulinic acid were 99.27%, 96.22%, 97.52%, 95.63%, and 93.19% respectively, RSD and were 2.1%, 3.6%, 3.3%, 1.2%, and 2.6% respectively, indicating good recovery rate of this method.

[0040] (6) Determination of the content of active ingredients in Tongfeng Pills Take 3 batches of Tongfeng Pills, prepare test solutions respectively, inject samples according to the chromatographic conditions for determination, and the results are shown in Table 1.

[0041] Table 1 Determination results of the contents of 5 components in Tongfeng Pills (mg∙g -1 )

[0042] (2) The content of palmatine hydrochloride in Tongfeng Pills is determined by ultraviolet detection method, 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 under reflux at 90 °C for 1 h, cool, filter, perform reflux extraction twice, combine the filtrates, evaporate the filtrates to dryness, dissolve with anhydrous ethanol and dilute to 50 mL in a volumetric flask. Take 2 mL and further dilute to 25 mL in a volumetric flask to obtain the solution.

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

[0044] Methodological investigation Investigation of linear relationship: Pipette a series of concentrations of palmatine hydrochloride reference stock solution and dilute to 20 mL in a volumetric flask with anhydrous ethanol, shake well. Use anhydrous ethanol solution as the blank control, measure the absorbance at a wavelength of 433 nm. Plot the standard curve with the concentration (mg·L -1 ) as the abscissa and the absorbance value as the ordinate. The results are shown in Figure 9 . After linear regression analysis, the regression equation is Y =0.0141 X +0.0015, indicating that the sample has a good linear relationship in the concentration range of 12.5 - 62.5 mg·L -1 ( r =0.9999).

[0045] Precision test: Pipette 1 mL of the reference stock solution accurately, add solvent and dilute to 10 mL in a volumetric flask, mix well and measure the absorbance continuously for 6 times. The RSD of the measured absorbance is 0.09%, indicating that the instrument precision is good and the experimental results are reliable.

[0046] Repeatability test: Take the same batch of gout pills (batch number 20241213), prepare 6 portions of test solution in parallel, and the measured absorbance RSD value is 0.14% ( n =6), indicating that the method has good repeatability.

[0047] Stability test: Take the same batch of gout pills (batch number 20241213), prepare 1 portion of test solution, measure its absorbance at 0, 10, 20, 40, 60, 80, 100, 120 min. The RSD of the measured absorbance is 0.66%, indicating that the test solution has good stability.

[0048] Spiked recovery test: Accurately weigh 6 portions of the gout pill sample with known content (batch number 20241213), 0.5 g for each portion, accurately add the reference substance solution of palmatine hydrochloride with known content respectively to prepare the test solution, measure the absorbance value at 433 nm, and calculate the recovery rate and RSD , and the results are shown in Table 2.

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

[0050] Determination of palmatine hydrochloride content in gout pills Take three batches of gout pills, prepare the test solution respectively, measure the absorbance value at 433 nm, and the results are shown in Table 3.

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

[0052] The present invention establishes a method for simultaneously determining the contents of indicant components aesculin, syringin, corilagin, ellagic acid, and chebulinic acid in gout pills by high performance liquid chromatography. After methodological verification, the results show that this method is scientific and feasible. Compared with individual analysis, this method can effectively reduce the injection times and analysis time of the instrument, shorten the experimental time, and reduce the experimental cost. The present invention also establishes a method for determining the total alkaloid content by ultraviolet spectrophotometry. The method established by the present invention can accurately detect the contents of indicant components aesculin, syringin, corilagin, ellagic acid, chebulinic acid, and total alkaloids, and can be used for the quality evaluation of gout pills, providing a reference for the improvement of the quality standard 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, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall 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 Tongfeng Pills, characterized in that: The contents of aesculin, syringin, corilagin, ellagic acid and chebulic acid in Tongfeng Pills were determined simultaneously by HPLC, and the content of total alkaloids in Tongfeng Pills was determined by UV spectrophotometry.

2. The method for determining the content of multiple components in Tongfeng Pills according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography are: C 18 Liquid chromatography column; mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution; flow rate is 1 mL / min; detection wavelength is 270 nm; column temperature is 30°C.

3. The content determination method according to claim 2, characterized in that: The gradient elution procedure of the chromatographic conditions comprises 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 proportion of acetonitrile was 27%, and the volume proportion of 0.1% phosphoric acid aqueous solution was 73%.

4. The method for determining the contents of multiple components in Tongfeng Pills according to claim 2, characterized in that: C 18 The liquid chromatography column is a reverse phase chromatography column with carbon octadecylsilane chemically bonded to silica gel stationary phase, the silica gel particle size is 5 μm, the column length is 250 mm, and the column inner diameter is 4.6 mm.

5. The method for determining the contents of multiple components in Tongfeng Pills according to claim 1, characterized in that: The method for preparing the test solution in the high performance liquid chromatography method is as follows: take Tongfeng pills, grind them into fine powder, add the Tongfeng pills fine powder and methanol-0.1% phosphoric acid aqueous solution (7:3, v:v) into an extraction container at a weight volume ratio of 1:10, use an ultrasonic device with a power of 250 W and a frequency of 50 kHz, perform ultrasonic treatment for 60 minutes, and filter with a 0.45 μm microporous filter membrane to obtain the test solution.

6. The method for determining the contents of multiple components in Tongfeng Pills according to claim 1, characterized in that: The method for preparing the reference solution in the high performance liquid chromatography method is as follows: accurately weigh the reference substances of aesculin, syringin, corilagin, and chebulic acid, respectively, dissolve and fix the volume with methanol-0.1 phosphoric acid aqueous solution (7:3, v:v), and prepare the reference substance stock solution; accurately weigh the ellagic acid reference substance, dissolve and fix the volume with methanol, and shake well to prepare the ellagic acid reference substance stock solution. Accurately pipette the reference substance stock solutions of aesculin, syringin, corilagin, ellagic acid, and chebulic acid into the same volumetric flask according to the volume ratio (15:0.1:1.3:6:0.5), add methanol-0.1 phosphoric acid aqueous solution (7:3, v:v) to dilute and fix the volume, and shake well to prepare the mixed reference substance solution.

7. The method for determining the contents of multiple components in Tongfeng Pills according to claim 1, characterized in that: The injection volume in the HPLC method was 10 μL.

8. The method for determining the contents of multiple components in Tongfeng Pills according to claim 1, characterized in that: The ultraviolet spectrophotometry absorption wavelength is 433 nm.

9. The method for determining the contents of multiple components in Tongfeng Pills according to claim 1, characterized in that: The ultraviolet spectrophotometry method also includes: (1) Preparation of reference stock solution: Accurately weigh the reference substance of bamipine hydrochloride, dissolve it in anhydrous ethanol, and shake well to prepare the reference stock solution. (2) Preparation of test solution: Weigh the powder of Tongfeng Pills, add hydrochloric acid-ethanol solution (1:20, v:v) at a weight volume ratio of 1:40, heat and reflux at 90 ℃ for 1 h, cool, filter, reflux and extract twice, combine the filtrate, evaporate the filtrate to dryness, dissolve it in anhydrous ethanol and add it to a constant volume flask, shake well to obtain the test solution.

10. The method for determining the contents of multiple components in Tongfeng Pills according to claim 1, characterized in that: The gout pill is a medicine composition consisting of Radix Codonopsis pilosulae, Zhaxun paste, Terminalia chebula (core removed), Semen Impatiens cyrtonema, Radix sylvestris (heat processed), Cortex qinpi, Flos Alkali (processed), Radix madder and Radix Glycyrrhizae, or a preparation made of the medicine composition and a pharmaceutical carrier.

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