Method for determining contents of 12 components in Shangqing tablet by adopting high performance liquid chromatography

Abstract

The invention relates to the technical field of chemical analysis, and discloses a method for determining the content of 12 components in Shangqing tablets by adopting high performance liquid chromatography, an Agilent C18 chromatographic column is adopted, a mobile phase A is methanol, a mobile phase B is a 0.1% phosphoric acid solution, gradient elution is adopted, and the content of 12 components in the Shangqing tablets is determined under the conditions that the column temperature is 30 DEG C, the flow rate is 1.0 mL / min, the detection wavelength is 254 nm and the sample size is 10 L. A high performance liquid chromatography method is established for simultaneously determining the contents of geniposide, 3, 5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol and physcion in the Shangqing tablets, and the method is sensitive, accurate, strong in specificity, good in reproducibility, relatively stable in base line, symmetric in chromatographic peak, high in accuracy and high in accuracy. The separation degree is good, the determination time is greatly shortened, methanol is used as an extraction solvent, ultrasonic extraction is adopted, the method is simple, rapid and effective, impurity peaks are few, and theoretical basis and reference can be provided for quality control of the Shangqing tablets.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and specifically relates to a method for determining the contents of 12 components in Shangqing Tablets by high performance liquid chromatography. Background Art

[0002] Shangqing Tablets are composed of twelve traditional Chinese medicines such as Flos Chrysanthemi, Herba Menthae, Rhizoma Chuanxiong, Radix Angelicae Dahuricae, Herba Schizonepetae, Radix Saposhnikoviae, Radix Platycodonis, Fructus Forsythiae, Fructus Gardeniae, Radix Scutellariae (processed with wine), Cortex Phellodendri (processed with wine), and Radix et Rhizoma Rhei (processed with wine). The efficacy of Shangqing Tablets is to clear heat and disperse wind, and detoxify and relieve constipation. It is mainly used for dizziness and tinnitus caused by wind-heat, red eyes, yellow nasal discharge, sores and ulcers in the mouth and tongue, swollen and painful gums, and constipation. In the prescription of Shangqing Tablets, Flos Chrysanthemi is the monarch drug, and its functions and indications are to disperse wind and clear heat, soothe the liver and improve eyesight, and detoxify and resolve toxins. It is clinically used for wind-heat colds, headache and dizziness, red and swollen eyes, blurred vision, and sores and carbuncles. Radix et Rhizoma Rhei (processed with wine) is good at clearing heat and detoxifying the blood heat in the upper jiao, and is used for red eyes, swollen throat, and swollen and painful gums. The efficacy of Radix Scutellariae (processed with wine) is to clear heat and dry dampness, purge fire and detoxify, stop bleeding, and prevent miscarriage. It is used for damp-warmth, summer dampness, chest distress and nausea, damp-heat stagnation, diarrhea, jaundice, cough due to lung heat, high fever and restlessness, bleeding due to blood heat, sores and carbuncles, and fetal restlessness.

[0003] The implementation standard of Shangqing Tablets is the national drug administration standard WS-10301(ZD-0301)2002-2012Z. However, only the baicalin in Radix Scutellariae is quantitatively investigated in the implementation standard. In the prescription, there is also less research on other active ingredients, mostly pharmacological research in clinical practice. Usually, a single traditional Chinese medicine has multiple components, and compound preparations have even more components, which will have problems of mutual influence and interaction. At the same time, it is very difficult to truly control the product quality of traditional Chinese medicines by quantitative detection of components, and it is impossible to completely prevent phenomena such as adulteration and counterfeiting of traditional Chinese medicines, nor can it objectively reflect and evaluate the consistency of the quality of traditional Chinese medicines. Moreover, the sample pretreatment is cumbersome, the analysis time is long, and the operability is not strong. Therefore, it is particularly important to conduct quantitative analysis on multiple index components in the prescription, which can conduct a more comprehensive quality analysis and evaluation of the prescription.

[0004] The present invention uses high performance liquid chromatography to simultaneously analyze 12 index components including geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion in Fructus Gardeniae, Flos Chrysanthemi, Cortex Phellodendri (processed with wine), Radix Scutellariae (processed with wine), and Radix et Rhizoma Rhei (processed with wine), so as to further improve the quality standard control of this prescription. Summary of the Invention

[0005] (I) Technical Problems to be Solved To solve the above technical problems, the present invention provides a method for determining the contents of 12 components in Shangqing Tablets by high performance liquid chromatography, and quantitatively determines 12 chemical components in Shangqing Tablets by ultra high performance liquid chromatography. This method is simple, reliable, fast, has good repeatability, excellent precision, and relatively accurate results, and can provide a theoretical basis and reference for the quality control of Shangqing Tablets.

[0006] (2) Technical solution To achieve the above object, the present invention discloses a method for determining the contents of 12 components in Shangqing Tablets by high performance liquid chromatography, including the following steps: (1) Preparation of the test solution: Take Shangqing Tablets, remove the coating, crush and grind them finely, and treat them with an organic solvent to obtain the test solution; (2) Preparation of the mixed reference solution: Weigh Gardenoside reference substance, 3,5-O-dicaffeoylquinic acid reference substance, Berberine hydrochloride reference substance, Baicalin reference substance, Wogonoside reference substance, Baicalein reference substance, Wogonin reference substance, Aloe-emodin reference substance, Rhein reference substance, Emodin reference substance, Chrysophanol reference substance, Physcion reference substance into a volumetric flask, add an organic solvent, and perform ultrasonic treatment to obtain the mixed reference solution; (3) Preparation of the negative control solution: According to the method of step (1), treat the negative control substances without Flos Chrysanthemi, Fructus Gardeniae, Scutellaria baicalensis Georgi stir-fried with wine, Phellodendron amurense Rupr. stir-fried with wine, Rheum palmatum L. stir-fried with wine respectively to obtain the negative control solutions lacking Flos Chrysanthemi, Fructus Gardeniae, Scutellaria baicalensis Georgi stir-fried with wine, Phellodendron amurense Rupr. stir-fried with wine, Rheum palmatum L. stir-fried with wine; (4) Detection: Precisionly pipette 10 μL of the mixed reference solution, the test solution and the negative control solution respectively, inject them into the high performance liquid chromatography for analysis, record the chromatogram and analyze it; The high performance liquid chromatography conditions are as follows: Chromatographic column: Agilent C18 chromatographic column, with a specification of 4.6 mm×250 mm, 5 μm; Mobile phase: Mobile phase A is methanol, and mobile phase B is 0.1% phosphoric acid solution. Gradient elution is adopted, and the proportions of the mobile phases are all volume percentages. Among them, from 0 to 50 min, 40%-90% mobile phase A and 60%-10% mobile phase B; from 50 to 55 min, 90%-40% mobile phase A and 10%-60% mobile phase B; from 55 to 60 min, 40% mobile phase A and 60% mobile phase B; the injection is delayed by 2 min for each injection; Column temperature: 30 °C; Flow rate: 1.0 mL / min; Detection wavelength: 254 nm; Injection volume: 10 μL.

[0007] Preferably, the specific preparation process of the test sample solution in step (1) includes taking 20 supernatant tablets, removing the coating, crushing and grinding them finely, taking 1.0 g, weighing accurately, placing it in a stoppered conical flask, adding 50 mL of an organic solvent, where the organic solvent is methanol, weighing, performing ultrasonic treatment for 45 min, with the power of the ultrasonic treatment being 500 W and the frequency of the ultrasonic treatment being 40 kHz, cooling, making up the reduced weight with methanol, immediately shaking well and filtering, and taking the subsequent filtrate to obtain the test sample solution.

[0008] Preferably, the specific preparation process of the mixed reference substance solution in step (2) includes weighing 5.542 mg of geniposide reference substance, 5.464 mg of 3,5-O-dicaffeoylquinic acid reference substance, 4.152 mg of berberine hydrochloride reference substance, 15.872 mg of baicalin reference substance, 11.171 mg of wogonoside reference substance, 5.261 mg of baicalein reference substance, 5.026 mg of wogonin reference substance, 1.266 mg of aloe-emodin reference substance, 1.094 mg of rhein reference substance, 1.120 mg of emodin reference substance, 4.963 mg of chrysophanol reference substance, and 4.988 mg of physcion reference substance and placing them in a 100 mL volumetric flask, adding an organic solvent, where the organic solvent is methanol, performing ultrasonic treatment, with the power of the ultrasonic treatment being 250 W and the frequency of the ultrasonic treatment being 40 kHz, dissolving and making up to the scale to prepare a mixed reference substance solution. The mixed reference substance solution includes geniposide reference substance solution, 3,5-O-dicaffeoylquinic acid reference substance solution, berberine hydrochloride reference substance solution, baicalin reference substance solution, wogonoside reference substance solution, baicalein reference substance solution, wogonin reference substance solution, aloe-emodin reference substance solution, rhein reference substance solution, emodin reference substance solution, chrysophanol reference substance solution, and physcion reference substance solution. Among them, the mass concentration of the geniposide reference substance solution is 54.090 μg / mL, the mass concentration of the 3,5-O-dicaffeoylquinic acid reference substance solution is 52.400 μg / mL, the mass concentration of the berberine hydrochloride reference substance solution is 37.783 μg / mL, the mass concentration of the baicalin reference substance solution is 154.276 μg / mL, the mass concentration of the wogonoside reference substance solution is 110.034 μg / mL, the mass concentration of the baicalein reference substance solution is 51.505 μg / mL, the mass concentration of the wogonin reference substance solution is 47.345 μg / mL, the mass concentration of the aloe-emodin reference substance solution is 12.344 μg / mL, the mass concentration of the rhein reference substance solution is 10.863 μg / mL, the mass concentration of the emodin reference substance solution is 11.054 μg / mL, the mass concentration of the chrysophanol reference substance solution is 49.332 μg / mL, and the mass concentration of the physcion reference substance solution is 45.391 μg / mL.

[0009] Preferably, the specific preparation process of the negative control solution in step (3) comprises weighing 1.0 g of a negative control substance that does not contain chrysanthemum, gardenia, wine-fried scutellaria, wine-fried phellodendron, and wine-fried rhubarb, accurately weighing, placing in a stoppered conical flask, adding 50 mL of an organic solvent, wherein the organic solvent is methanol, weighing, ultrasonically treating for 45 min, wherein the power of the ultrasonic treatment is 500 W, and the frequency of the ultrasonic treatment is 40 kHz, cooling, supplementing the reduced weight with methanol, immediately shaking and filtering, and taking the filtrate to obtain a negative control solution lacking chrysanthemum, gardenia, wine-fried scutellaria, wine-fried phellodendron, and wine-fried rhubarb.

[0010] 3. Beneficial technical effects (1) In the present invention, Shangqing Tablets were taken as the research object, and the active ingredients of its components, namely, chrysanthemum, gardenia, wine-fried rhubarb, wine-fried scutellaria, and wine-fried phellodendron, were selected as the research targets. The contents of 12 active ingredients were simultaneously determined by high performance liquid chromatography with a diode array detector. This method is simple, reliable, and rapid, with good repeatability and precision. The results are relatively accurate, and can provide a theoretical basis and reference for the quality control of Shangqing Tablets.

[0011] (2) The present invention selects the mobile phase. Since there are many components, a suitable gradient elution is selected. Methanol and 0.1% phosphoric acid solution are selected as the mobile phase, which can effectively perform elution separation, the baseline is relatively stable, the chromatographic peaks are symmetrical, the separation is good, and the measurement time is greatly shortened. Methanol is used as the extraction solvent, and ultrasonic extraction is simple, fast and effective, with few impurity peaks. When the detection wavelength is 254nm, the absorbance of the twelve targets to be measured is good, and the separation also meets the requirements.

[0012] (3)In the present invention, a high performance liquid chromatography (HPLC) method was established for the simultaneous determination of the contents of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion in Shangqing tablets. The linear relationships between the injection volume and the peak area of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion were good in the ranges of 5.409 - 54.090 μg / mL (r = 0.9999), 5.240 - 52.400 μg / mL (r = 0.9997), 3.778 - 37.783 μg / mL (r = 0.9993), 15.428 - 154.276 μg / mL (r = 0.9997), 11.003 - 110.034 μg / mL (r = 0.9997), 5.151 - 51.505 μg / mL (r = 0.9998), 4.734 - 47.345 μg / mL (r = 0.9998), 1.234 - 12.344 μg / mL (r = 0.9997), 1.086 - 10.863 μg / mL (r = 0.9997), 1.105 - 11.054 μg / mL (r = 0.9996), 4.933 - 49.332 μg / mL (r = 0.9998), and 4.539 - 45.391 μg / mL (r = 0.9997), respectively. The average recoveries by addition were 97.54%, 98.76%, 100.53%, 96.67%, 98.12%, 99.63%, 100.32%, 101.44%, 97.25%, 98.63%, 99.51%, and 101.03%, respectively, and the RSD values were 0.63% - 1.42%. This method is sensitive, accurate, highly specific, and has good reproducibility, and can be used as a method for the determination of the contents of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion in Shangqing tablet preparations for the quality control of Shangqing tablets. Description of the Drawings

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0014] Figure 1 is the HPLC chromatogram of the mixed reference solution in the present invention; Figure 2 It is the high performance liquid chromatography (HPLC) chromatogram of the test sample solution in the present invention; Figure 3 It is the high performance liquid chromatography (HPLC) chromatogram of the negative control solution without Flos Chrysanthemi in the present invention; Figure 4 It is the high performance liquid chromatography (HPLC) chromatogram of the negative control solution without Fructus Gardeniae in the present invention; Figure 5 It is the high performance liquid chromatography (HPLC) chromatogram of the negative control solution without Radix Scutellariae Praeparata cum Alumine in the present invention; Figure 6 It is the high performance liquid chromatography (HPLC) chromatogram of the negative control solution without Cortex Phellodendri Chinensis Praeparata cum Alumine in the present invention; Figure 7 It is the high performance liquid chromatography (HPLC) chromatogram of the negative control solution without Radix et Rhizoma Rhei Praeparata cum Alumine in the present invention.

[0015] In the figure: 1, Geniposide; 2, 3,5-O-dicaffeoylquinic acid; 3, Berberine hydrochloride; 4, Baicalin; 5, Wogonoside; 6, Baicalein; 7, Wogonin; 8, Aloe-emodin; 9, Rhein; 10, Emodin; 11, Chrysophanol; 12, Physcion. Detailed implementation manners

[0016] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0017] The relevant instruments used in the present invention are as follows: Waters 2695-2998 type high performance liquid chromatography instrument and chromatographic workstation; XPE165 type electronic analytical balance (Mettler Toledo); AR224CN type electronic analytical balance (Shanghai Ohaus); 700VDE type ultrasonic instrument (Hefei Jinnick Machinery Manufacturing Co., Ltd.); UPR-II-20T type pure water machine (Chengdu Ultra-pure Technology Co., Ltd.); SHZ-DIII type circulating water multi-purpose vacuum pump (Beijing Yongguang Medical Instrument Co., Ltd.).

[0018] The relevant drugs and reagents used in the present invention are as follows: geniposide reference substance (batch number: 110749-201718, purity 97.6%); 3,5-O-dicaffeoylquinic acid reference substance (batch number: 111782-202208, purity 95.9%); berberine reference substance (batch number: 110713-202316, purity 91.0%); baicalin reference substance (batch number: 110715-202223, purity 97.2%); wogonoside reference substance (batch number: 112002-202303, purity 98.5%); baicalein reference substance (batch number: 111595-201808, purity 97.9%); wogonin reference substance (batch number: 111514-201706, purity 94.2%); aloe-emodin reference substance (batch number: 110795-202011, purity 97.5%); rhein reference substance (batch number: 110757-201607, purity 99.3%); emodin reference substance (batch number: 110756-201512, purity 98.7%); chrysophanol reference substance (batch number: 110796-201922, purity 99.4%); physcion reference substance (batch number: 110758-202218, purity 91.0%); the above reference substances were all purchased from the National Institutes for Food and Drug Control; Shangqing Tablets were purchased from Chongqing Huasen Pharmaceutical Co., Ltd., batch numbers: 230106, 230504, 230902, sugar-coated tablets, tablet core weight 0.3 g); methanol was chromatographically pure and purchased from Sinopharm Chemical Reagent Co., Ltd.; water was ultrapure water; the negative control medicinal materials chrysanthemum, mint, chuanxiong, angelica dahurica, schizonepeta, ledebouriella root, platycodon root, forsythia, gardenia, scutellaria baicalensis (stir-fried with wine), phellodendron amurense (stir-fried with wine), rhubarb (stir-fried with wine) required for the high-performance liquid content determination were all the remaining samples from the daily sampling tasks; other reagents were all of analytical purity.

[0019] Example 1: Preparation of a mixed reference solution: Weigh 5.542 mg of geniposide reference substance, 5.464 mg of 3,5-O-dicaffeoylquinic acid reference substance, 4.152 mg of berberine hydrochloride reference substance, 15.872 mg of baicalin reference substance, 11.171 mg of wogonoside reference substance, 5.261 mg of baicalein reference substance, 5.026 mg of wogonin reference substance, 1.266 mg of aloe-emodin reference substance, 1.094 mg of rhein reference substance, 1.120 mg of emodin reference substance, 4.963 mg of chrysophanol reference substance, and 4.988 mg of physcion reference substance and place them in a 100 mL volumetric flask. Add methanol and perform ultrasonic treatment. The power of the ultrasonic treatment is 250 W, and the frequency is 40 kHz. After dissolution, make up the volume to the mark to obtain a mixed reference solution. The mixed reference solution includes geniposide reference solution, 3,5-O-dicaffeoylquinic acid reference solution, berberine hydrochloride reference solution, baicalin reference solution, wogonoside reference solution, baicalein reference solution, wogonin reference solution, aloe-emodin reference solution, rhein reference solution, emodin reference solution, chrysophanol reference solution, and physcion reference solution. The mass concentration of the geniposide reference solution is 54.090 μg / mL, the mass concentration of the 3,5-O-dicaffeoylquinic acid reference solution is 52.400 μg / mL, the mass concentration of the berberine hydrochloride reference solution is 37.783 μg / mL, the mass concentration of the baicalin reference solution is 154.276 μg / mL, the mass concentration of the wogonoside reference solution is 110.034 μg / mL, the mass concentration of the baicalein reference solution is 51.505 μg / mL, the mass concentration of the wogonin reference solution is 47.345 μg / mL, the mass concentration of the aloe-emodin reference solution is 12.344 μg / mL, the mass concentration of the rhein reference solution is 10.863 μg / mL, the mass concentration of the emodin reference solution is 11.054 μg / mL, the mass concentration of the chrysophanol reference solution is 49.332 μg / mL, and the mass concentration of the physcion reference solution is 45.391 μg / mL.

[0020] The chromatographic conditions include: chromatographic column: Agilent C18 chromatographic column, with a specification of 4.6 mm × 250 mm, 5 μm; Mobile phase: Mobile phase A is methanol, and mobile phase B is 0.1% phosphoric acid solution. Gradient elution is adopted, and the proportions of the mobile phases are all in volume percentages. Among them, from 0 to 50 min, 40% - 90% mobile phase A and 60% - 10% mobile phase B; from 50 to 55 min, 90% - 40% mobile phase A and 10% - 60% mobile phase B; from 55 to 60 min, 40% mobile phase A and 60% mobile phase B; the injection is delayed by 2 min for each injection; column temperature: 30 °C; flow rate: 1.0 mL / min; detection wavelength: 254 nm; injection volume: 10 μL.

[0021] Example 2: Preparation of the test solution: Take 20 supernatant tablets with the batch number 230106, remove the coating, crush and grind them finely. Take 1.0 g, accurately weigh it, place it in a stoppered conical flask, add 50 mL of methanol, weigh it, perform ultrasonic treatment for 45 min. The power of the ultrasonic treatment is 500 W, and the frequency of the ultrasonic treatment is 40 kHz. Let it cool, make up the reduced weight with methanol, shake it immediately and filter it. Take the continuous filtrate to obtain the test solution.

[0022] The chromatographic conditions are the same as those in Example 1.

[0023] Example 3: Preparation of the test solution: Take 20 supernatant tablets with the batch number 230504, remove the coating, crush and grind them finely. Take 1.0 g, accurately weigh it, place it in a stoppered conical flask, add 50 mL of methanol, weigh it, perform ultrasonic treatment for 45 min. The power of the ultrasonic treatment is 500 W, and the frequency of the ultrasonic treatment is 40 kHz. Let it cool, make up the reduced weight with methanol, shake it immediately and filter it. Take the continuous filtrate to obtain the test solution.

[0024] The chromatographic conditions are the same as those in Example 1.

[0025] Example 4: Preparation of the test solution: Take 20 supernatant tablets with the batch number 230902, remove the coating, crush and grind them finely. Take 1.0 g, accurately weigh it, place it in a stoppered conical flask, add 50 mL of methanol, weigh it, perform ultrasonic treatment for 45 min. The power of the ultrasonic treatment is 500 W, and the frequency of the ultrasonic treatment is 40 kHz. Let it cool, make up the reduced weight with methanol, shake it immediately and filter it. Take the continuous filtrate to obtain the test solution.

[0026] The chromatographic conditions are the same as those in Example 1.

[0027] Linearity test: Accurately pipette 1.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL of the mixed reference solution into 10-mL volumetric flasks respectively, dilute to the mark with methanol solution, inject and determine according to the chromatographic conditions in Example 1, perform linear regression with the peak area (Y) as the ordinate and the mass concentration (X, μg / mL) as the abscissa, and the results are shown in Table 1: Table 1 Regression equation, linear range, and correlation coefficient Index component Regression equation r Linear range (µg / mL) Geniposide =8756.01+1429.16 0.9999 5.409～54.090 3,5-O-dicaffeoylquinic acid =14951.17–2517.36 0.9997 5.240～52.400 Berberine hydrochloride =34800.85–9369.27 0.9993 3.778～37.783 Baicalin =14040.38–21360.79 0.9997 15.428～154.276 Wogonoside =17989.80–7262.03 0.9997 11.003～110.034 Baicalein =19412.15–3485.06 0.9998 5.151～51.505 Wogonin =26853.96+6276.86 0.9998 4.734～47.345 Aloe-emodin =52658.01+5598.30 0.9997 1.234～12.344 Rhein =41835.16+2906.53 0.9997 1.086～10.863 Emodin =39731.35-2581.87 0.9996 1.105～11.054 Chrysophanol =35866.37+7247.84 0.9998 4.933～49.332 Physcion =17869.08+1509.61 0.9997 4.539～45.391 Precision test: Take the mixed reference solution respectively, inject and determine according to the chromatographic conditions in Example 1, perform 6 consecutive injections, 10 μL each time, and record the peak area of the chromatographic peak. The test results show that the RSDs of the peak areas of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion are 0.32%, 1.13%, 0.26%, 0.30%, 0.37%, 0.30%, 0.34%, 0.33%, 0.44%, 0.24%, 0.32%, and 0.38% (n = 6) respectively, indicating that the precision of the instrument is good.

[0028] Repeatability test: Take 6 portions of the test solution prepared in Example 4 (batch number 230902), inject and determine according to its chromatographic conditions, and calculate the content and RSD of each component. The test results show that the average contents of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion are 2.135, 1.306, 1.164, 8.658, 2.032, 0.485, 0.285, 0.237, 0.101, 0.074, 0.935, and 0.274 mg / g respectively, and the RSDs are 0.74%, 0.81%, 0.92%, 0.55%, 1.01%, 0.65%, 1.12%, 0.76%, 0.81%, 0.53%, 0.59%, and 0.41% (n = 6) respectively, indicating that the repeatability of this method is good.

[0029] Stability test: The test sample solution prepared in Example 4 (batch number 230902) was taken, and at 0, 2, 4, 8, 16, and 24 h after preparation, it was injected for determination according to its chromatographic conditions, and the peak areas were recorded. The test results showed that the RSDs of the peak areas of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion were 0.61%, 0.35%, 0.84%, 0.63%, 0.61%, 0.95%, 0.47%, 0.77%, 0.52%, 0.39%, 0.43%, and 0.75% (n = 6), respectively, indicating that the sample solution had good stability within 24 h.

[0030] Spiked recovery test: 0.5 g of the supernatant tablets with batch number 230902 were accurately weighed, a total of 9 portions, divided into 3 groups, placed in conical flasks, and immediately accurately added with 0.8 mL, 1.0 mL, and 1.2 mL of the mixed reference substance solution (containing geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion with mass concentrations of 1.021, 0.642, 0.576, 4.117, 0.984, 0.236, 0.137, 0.109, 0.048, 0.041, 0.445, and 0.133 mg / mL), the test sample solution prepared in Example 4 was added, and it was injected for determination according to the chromatographic conditions in Example 4, and the spiked recoveries of 12 components were calculated. The test results showed that the average recoveries of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion were 97.54%, 98.76%, 100.53%, 96.67%, 98.12%, 99.63%, 100.32%, 101.44%, 97.25%, 98.63%, 99.51%, and 101.03%, respectively, and the RSD values were 0.63%, 0.95%, 1.05%, 0.87%, 1.42%, 0.73%, 0.97%, 0.84%, 0.74%, 0.66%, 0.93%, and 1.02%, respectively.

[0031] Determination of sample content: Three parallel samples of the test sample solutions in Example 2, Example 3, and Example 4 were taken, injected and chromatographically analyzed according to the chromatographic conditions in Example 1, and the contents of geniposide, 3,5-O-dicaffeoylquinic acid, berberine hydrochloride, baicalin, wogonoside, baicalein, wogonin, aloe-emodin, rhein, emodin, chrysophanol, and physcion in the samples were calculated by the external standard method. The test results are shown in Table 2: Table 2 Content determination results (mg / g, n = 3) Component Example 2 Example 3 Example 4 Geniposide 2.512 1.986 2.135 3,5-O-dicaffeoylquinic acid 1.469 1.125 1.306 Berberine hydrochloride 0.954 1.326 1.164 Baicalin 8.792 8.554 8.658 Wogonoside 2.492 2.313 2.032 Baicalein 0.568 0.602 0.485 Wogonin 0.302 0.336 0.285 Aloe-emodin 0.264 0.243 0.237 Rhein 0.082 0.078 0.101 Emodin 0.104 0.095 0.074 Chrysophanol 0.913 1.024 0.935 Physcion 0.318 0.265 0.274 Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the application of the present invention shall still fall within the scope covered by the patent of the present invention.

Claims

1. A method for determining the contents of 12 components in a supernatant tablet by high performance liquid chromatography, characterized in that: The following steps are involved: (1) Preparation of test solution: Take the supernatant tablets, remove the coating, grind them into powder, add organic solvent to obtain the test solution; (2) Preparation of mixed reference solution: Weigh gardenoside reference, 3,5-O-dicaffeoylquinic acid reference, berberine hydrochloride reference, baicalin reference, wogonin reference, baicalein reference, wogonin reference, aloe-emodin reference, rhein reference, rhein reference, chrysophanol reference, and physophanol methyl ether reference into a volumetric flask, add an organic solvent, and perform ultrasonic treatment to obtain a mixed reference solution; (3) Preparation of negative control solution: According to the sample prescription process, the method of step (1) is used to prepare a negative control substance that does not contain chrysanthemum, gardenia, wine-fried scutellaria, wine-fried phellodendron, and wine-fried rhubarb, so as to obtain a negative control solution lacking chrysanthemum, gardenia, wine-fried scutellaria, wine-fried phellodendron, and wine-fried rhubarb; (4) Detection: Accurately pipette 10 μL of each mixed reference solution, test solution, and negative control solution, inject them into high performance liquid chromatography, analyze, record the chromatogram, and perform analysis; The HPLC conditions are: Chromatographic column: Agilent C18 column, size 4.6 mm × 250 mm, 5 μm; Mobile phase: Mobile phase A was methanol, mobile phase B was 0.1% phosphoric acid solution, gradient elution was used, and the mobile phase ratios were all volume percentages, where 0-50 min, 40%-90% mobile phase A, 60%-10% mobile phase B; 50-55 min, 90%-40% mobile phase A, 10%-60% mobile phase B; 55-60 min, 40% mobile phase A, 60% mobile phase B; each injection was delayed for 2 min; Column temperature: 30 °C; Flow rate: 1.0 mL / min; Detection wavelength: 254 nm; Injection volume: 10 µL.

2. The method for determining the contents of 12 components in a supernatant tablet by high performance liquid chromatography according to claim 1, characterized in that: The specific preparation process of the test solution in step (1) includes taking 20 supernatant tablets, removing the coating, crushing and grinding, taking 1.0 g, accurately weighing, placing in a stoppered conical flask, adding 50 mL of organic solvent, wherein the organic solvent is methanol, weighing, ultrasonic treatment for 45 min, the power of the ultrasonic treatment is 500 W, the frequency of the ultrasonic treatment is 40 kHz, cooling, supplementing the reduced weight with methanol, immediately shaking and filtering, and taking the filtrate to obtain the test solution.

3. The method for determining the contents of 12 components in a supernatant tablet by high performance liquid chromatography according to claim 1, characterized in that: The specific preparation process of the mixed reference solution in step (2) includes weighing 5.542 mg of gardenia glycoside reference, 5.464 mg of 3,5-O-dicaffeoylquinic acid reference, 4.152 mg of berberine hydrochloride reference, 15.872 mg of baicalin reference, 11.171 mg of wogonin reference, 5.261 mg of baicalein reference, 5.026 mg of wogonin reference, 1.266 mg of aloe-emodin reference, 1.094 mg of rhein reference, 1.120 mg of rhein reference, 4.963 mg of chrysophanol reference, and 4.988 mg of physophanol methyl ether reference into a 100 mL volumetric flask, adding an organic solvent, wherein the organic solvent is methanol, performing ultrasonic treatment, the power of the ultrasonic treatment is 250 W, the frequency of the ultrasonic treatment is 40 kHz, and after dissolution, the volume is adjusted to the scale to prepare a mixed reference solution.

4. The method for determining the contents of 12 components in a supernatant tablet by high performance liquid chromatography according to claim 3, characterized in that: The mixed reference solution comprises a gardenoside reference solution, a 3,5-O-dicaffeoylquinic acid reference solution, a berberine hydrochloride reference solution, a baicalin reference solution, a wogonin reference solution, a baicalein reference solution, a wogonin reference solution, an aloe-emodin reference solution, a rhein reference solution, a rhein reference solution, a chrysophanol reference solution, and a physophanol methyl ether reference solution, wherein the mass concentration of the gardenoside reference solution is 54.090 μg / mL, the mass concentration of the 3,5-O-dicaffeoylquinic acid reference solution is 52.400 μg / mL, the mass concentration of the berberine hydrochloride reference solution is 37.783 μg / mL, the mass concentration of the baicalin reference solution is 154.276 μg / mL, and the mass concentration of the wogonin reference solution is 110.034 μg / mL, the mass concentration of baicalin reference solution was 51.505 μg / mL, the mass concentration of wogonin reference solution was 47.345 μg / mL, the mass concentration of aloe-emodin reference solution was 12.344 μg / mL, the mass concentration of rhein reference solution was 10.863 μg / mL, the mass concentration of rhein reference solution was 11.054 μg / mL, the mass concentration of chrysophanol reference solution was 49.332 μg / mL, and the mass concentration of physophanol methyl ether reference solution was 45.391 μg / mL.

5. The method for determining the contents of 12 components in a supernatant tablet by high performance liquid chromatography according to claim 1, characterized in that: The specific preparation process of the negative control solution in step (3) comprises weighing 1.0 g of a negative control substance which does not contain chrysanthemum, gardenia, wine-fried scutellaria, wine-fried phellodendron, and wine-fried rhubarb, accurately weighing the solution, placing the solution in a stoppered conical flask, adding 50 mL of an organic solvent, wherein the organic solvent is methanol, weighing the solution, subjecting the solution to ultrasonic treatment for 45 min, wherein the power of the ultrasonic treatment is 500 W, and the frequency of the ultrasonic treatment is 40 kHz, cooling the solution, supplementing the reduced weight with methanol, immediately shaking and filtering the solution, and taking the filtrate to obtain a negative control solution which lacks chrysanthemum, gardenia, wine-fried scutellaria, wine-fried phellodendron, and wine-fried rhubarb.

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