Quality control method of body resistance strengthening and detoxification granules

Abstract

The invention provides a quality control method of body resistance strengthening and detoxification granules, which is characterized by comprising a qualitative method and a quantitative method, the qualitative method is used for controlling product quality by establishing a characteristic spectrum of the body resistance strengthening and detoxification granules; according to the quantitative method, the product quality is controlled by controlling the contents of diester alkaloid, monoester alkaloid and glycyrrhizic acid. According to the method, dry ginger, honey-fried licorice root, honeysuckle, spina gleditsiae, radix fici simplicissimae and pericarpium citri reticulatae in the granules are identified by adopting a thin-layer chromatography method, a specific chromatogram with relatively good separation degree and reproducibility is established by adopting a high-performance liquid chromatography method, and the content of diester-type alkaloids is detected by adopting a high-performance liquid chromatography-mass spectrometry method. The glycyrrhizic acid is quantitatively studied through high performance liquid chromatography, and the monoester alkaloid is quantitatively studied through high performance liquid chromatography-mass spectrometry. According to the quality standard research method for the body resistance strengthening and detoxification granules, the overall quality of the granules can be comprehensively reflected, and then the product quality is controlled and evaluated.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a quality control method for Fuzheng Jiedu granules. Background Art

[0002] The prescription of Fuzheng Jiedu Granules is composed of eight Chinese medicines: Danfu Pian, Ganjiang, Zhigancao, Wuzhimaotao, Gleditsia sinensis, Honeysuckle, Patchouli and Tangerine Peel. It is a Chinese medicine preparation with the main effects of invigorating qi and warming yang, removing dampness and detoxifying. It is an empirical prescription for clinical auxiliary treatment of novel coronavirus infection, especially suitable for critically ill patients with deficiency syndrome. Danfu Pian, Ganjiang and Zhigancao in the prescription are derived from Sini Decoction in Treatise on Febrile Diseases, which has the function of warming and nourishing the lower abdomen; Wuzhimaotao nourishes the middle and replenishes qi, and the four herbs are used together to strengthen the positive energy. Zhigancao and Wuzhimaotao strengthen the spleen and replenish qi, and are used together with Patchouli and Tangerine Peel to aromaticize the evil of dampness and turbidity; Honeysuckle clears heat and detoxifies, and expels evil; Wuzhimaotao and Gleditsia sinensis are used together to increase the effect of clearing epidemic toxins and removing turbidity. The combination of these medicines has the effect of strengthening the body, detoxifying, invigorating the spleen and removing dampness.

[0003] However, there is no research report on the quality research method of Fuzheng Jiedu Granules. Therefore, it is very necessary to establish its quality standard to comprehensively evaluate the quality of this preparation. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a quality control method for Fuzheng Jiedu granules. The quality control method provided by the present invention can comprehensively control the quality of the product, and the method has good stability, precision and repeatability.

[0005] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0006] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0007] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0008] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the following processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0009] The present invention provides a quality control method for Fuzheng Jiedu granules, including qualitative and quantitative methods. The qualitative method is to control the product quality by establishing a characteristic spectrum of Fuzheng Jiedu granules; specifically, the method is established by controlling 3 to 10 characteristic peaks, wherein the characteristic peaks include chlorogenic acid, liquiritin, hesperidin, ammonium glycyrrhizinate and 6-gingerol;

[0010] The quantitative method controls the product quality by controlling the contents of diester type alkaloids, monoester type alkaloids and glycyrrhizic acid.

[0011] The present invention provides a characteristic spectrum of Fuzheng Jiedu granules, characterized in that a method for establishing the characteristic spectrum of Fuzheng Jiedu granules comprises:

[0012] A) extracting the Fuzheng Jiedu granules with a solvent to obtain a test solution;

[0013] B) taking chlorogenic acid, liquiritin, hesperidin, ammonium glycyrrhizinate and 6-gingerol standard products, dissolving them in a solvent to obtain a reference substance solution;

[0014] C) using high performance liquid chromatography to obtain chromatograms of the test solution in step A) and the reference substance in step B) under the same detection conditions, and the specific chromatographic parameters are as follows:

[0015] The chromatographic column was a C18 column, and the detection wavelength was 237 nm; the mobile phase A was acetonitrile, and the mobile phase B was a 0.1% phosphoric acid aqueous solution;

[0016] The elution procedure is:

[0017]

[0018] D) obtaining a characteristic spectrum of Fuzheng Jiedu Granules according to the reference substance concentration, the peak area of ​​the reference substance solution in the chromatogram, and the peak area of ​​the component in Fuzheng Jiedu Granules corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C).

[0019] The quality control method of the Fuzheng Jiedu granules provided by the present invention first extracts the Fuzheng Jiedu granules with a solvent to obtain a test solution.

[0020] The present invention does not specifically limit the Fuzheng Jiedu granules, which may be commercially available or prepared using the method of the present invention.

[0021] In some embodiments, the preparation process of the Fuzheng Jiedu granules is: weigh 2 parts of Aconiti Lateralis Preparata, 3 parts of dried ginger, 4 parts of roasted licorice, 4 parts of Eleutherodactyla quinquefolia, 2 parts of Gleditsia sinensis, 2 parts of honeysuckle, 2 parts of patchouli, and 1 part of tangerine peel according to the weight ratio, add 160 parts of water, extract three times, each time for 1 hour, filter, concentrate, and granulate.

[0022] In some embodiments, the preparation of the Fuzheng Jiedu granule test solution is as follows: take Fuzheng Jiedu granule powder, add 70% ethanol, ultrasonically dissolve, cool, dilute to the scale with 70% ethanol, shake well, centrifuge at high speed, and take the supernatant to obtain the test solution.

[0023] The ultrasonic extraction time of the present invention is 10 to 20 minutes.

[0024] In some embodiments, the mass volume ratio of the Fuzheng Jiedu granules and 70% ethanol is 1:25.

[0025] Take chlorogenic acid, liquiritin, hesperidin, ammonium glycyrrhizinate and 6-gingerol standard products, dissolve them in a solvent to obtain a reference substance solution;

[0026] The concentration of chlorogenic acid in the present invention is 40 μg / mL, the concentration of liquiritin is 40 μg / mL, the concentration of hesperidin is 40 μg / mL, the concentration of ammonium glycyrrhizinate is 40 μg / mL, and the concentration of 6-gingerol is 10 μg / mL. Using high performance liquid chromatography, the chromatograms of the test solution in step A) and the reference substance in step B) are obtained under the same detection conditions. The specific chromatographic parameters are as follows:

[0027] The chromatographic column is a C18 chromatographic column, and the detection wavelength is 237 nm; the mobile phase A is acetonitrile, and the mobile phase B is a 0.1% phosphoric acid aqueous solution; the mobile phase flow rate is 1 ml / min, the chromatographic column temperature is 30° C., and the injection volume is 10 μL;

[0028] The elution procedure is:

[0029]

[0030] According to the reference substance concentration, the peak area of ​​the reference substance solution in the chromatogram and the peak area of ​​the components in the Fuzheng Jiedu Granules corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C), a characteristic spectrum of the Fuzheng Jiedu Granules is obtained.

[0031] The characteristic peaks include chlorogenic acid, liquiritin, hesperidin, ammonium glycyrrhizinate and 6-gingerol.

[0032]

[0033]

[0034] The present invention compares the whole formula of Fuzheng Jiedu Granule, the extracts of Radix Aconiti Lateralis, dried ginger, roasted licorice, Rhizoma Cibotii, Gleditsia sinensis, Flos Lonicerae, Pogostemon cablin, and dried orange peel with the negative samples in sequence, and the chromatogram is shown in FIG. Figure 7-14 The attribution results of the characteristic peaks of the characteristic spectrum are as follows: in the characteristic spectrum, peak 1 is chlorogenic acid from honeysuckle, peaks 2 and 4 are from roasted liquorice, and peak 2 is liquiritin and peak 4 is glycyrrhizic acid; peak 3 is hesperidin from dried tangerine peel, and peak 5 is 6-gingerol from dried ginger; there are no characteristic peaks of Aconiti Lateralis Preparata, Prunus Armeniaca, Gleditsia sinensis, and Pogostemon cablin.

[0035] The quality control method of the present invention also includes any one or more of the following determination methods:

[0036] i) HPLC determination of the limit content of diester-type alkaloids in Fuzheng Jiedu Granules;

[0037] The diester type alkaloids of the present invention are selected from the group consisting of mesaconitine, mesaconitine and aconitine;

[0038] ii) HPLC determination of the content of monoester alkaloids in Fuzheng Jiedu Granules; the monoester alkaloids are selected from benzoyl aconitine, benzoyl mesaconitine and benzoyl mesaconitine;

[0039] iii) HPLC determination of glycyrrhizic acid content in Fuzheng Jiedu granules;

[0040] iv) Identification of dried ginger using thin layer chromatography;

[0041] v) Identification of Licorice using thin layer chromatography;

[0042] vi) Identification of honeysuckle by thin layer chromatography;

[0043] vii) Identification of Gleditsia sinensis thorns by thin layer chromatography;

[0044] viii) using thin layer chromatography to identify the five-fingered peach;

[0045] ix) Identification of dried tangerine peel using thin layer chromatography.

[0046] The present invention finds corresponding characteristic spots from a series of thin layer chromatographic identifications of medicinal materials and preparations, which provides a basis for tracing the characteristic components in the preparations back to the medicinal materials, and confirms a certain correlation between the medicinal materials and the preparations through content determination.

[0047] The method for determining the contents of the main components diester alkaloids, monoester alkaloids and glycyrrhizic acid established by the invention has good precision, stability and repeatability.

[0048] The thin layer identification method of six medicinal materials (dried ginger, liquorice, honeysuckle, Gleditsia sinensis and Prunus mume) established by the present invention has good durability.

[0049] The present invention can ensure the safety, effectiveness and stability of clinical medication, and also provides a detailed and reliable research basis for subsequent preparation process research, quality standard establishment and stability investigation of Fuzheng Jiedu granule preparations.

[0050] On the one hand, the present invention uses HPLC to determine the limit content of diester-type alkaloids in Fuzheng Jiedu Granules, which is specifically:

[0051] a) extracting the Fuzheng Jiedu granules with a solvent to obtain a test solution; the solvent is a 0.1% formic acid methanol solution.

[0052] The present invention preferably takes Fuzheng Jiedu granules, adds 0.1% formic acid methanol, ultrasonically treats for 20 to 30 minutes, cools, adds 0.1% formic acid methanol to dilute to the scale, shakes well, and passes through a membrane to obtain the Fuzheng Jiedu granules. The ratio of the mass g of the Fuzheng Jiedu granules to the volume mL of the 0.1% formic acid methanol solution is 0.2:5.

[0053] b) using high performance liquid chromatography-mass spectrometry to conduct qualitative and quantitative analysis of the components of the test liquid;

[0054] The specific chromatographic parameters are as follows:

[0055] The chromatographic column was a C18 chromatographic column, the mobile phase A was 0.1% formic acid methanol solution, and the mobile phase B was 0.1% formic acid aqueous solution;

[0056] Gradient elution:

[0057]

[0058] The mobile phase flow rate was 0.2 ml / min; the column temperature was 30 °C, and the injection volume was 1 to 10 μL;

[0059] Mass spectrometry conditions: electrospray ionization positive ion mode; multiple reaction (MRM) detection mode.

[0060] In some embodiments, the parameters of each compound are set as shown in the following table.

[0061]

[0062] The limit requirement in the preparation of the present invention is that the total amount of diester type alkaloids contained in each gram of the product, calculated as neoaconitine, mesaconitine and aconitine, shall not exceed 5.67 μg.

[0063] The standard curves are as follows: neoaconitine Y=421310X-10409.65145, r=0.999, hypoaconitine Y=4971130X-57824.1456, r=0.999, aconitine Y=50980490X+8200.96973, r=0.999, and neoaconitine is linear in the range of 0.00265-2.61525 μg / ml, hypoaconitine is linear in the range of 0.00248-2.47500 μg / ml, and aconitine is linear in the range of 0.00262-2.62350 μg / ml.

[0064] In some embodiments, the content of monoester alkaloids in Fuzheng Jiedu granules determined by HPLC of the present invention is specifically:

[0065] a) extracting the Fuzheng Jiedu granules with a solvent to obtain a test solution; the solvent is a 0.1% formic acid methanol solution;

[0066] The present invention preferably takes Fuzheng Jiedu granules, adds 0.1% formic acid methanol, ultrasonically treats for 20 to 30 minutes, cools, adds 0.1% formic acid methanol to dilute to the scale, shakes well, and passes through a membrane to obtain the Fuzheng Jiedu granules. The ratio of the mass g of the Fuzheng Jiedu granules to the volume mL of the 0.1% formic acid methanol solution is 0.2:5.

[0067] b) using high performance liquid chromatography-mass spectrometry to conduct qualitative and quantitative analysis of the components of the test liquid;

[0068] The specific chromatographic parameters are as follows:

[0069] The chromatographic column was a C18 chromatographic column, the mobile phase A was 0.1% formic acid methanol solution, and the mobile phase B was 0.1% formic acid aqueous solution;

[0070] Gradient elution:

[0071]

[0072] The mobile phase flow rate was 0.2 ml / min; the column temperature was 30 °C, and the injection volume was 1 μL;

[0073] Mass spectrometry conditions: electrospray ionization positive ion mode; multiple reaction (MRM) detection mode.

[0074] In some embodiments, the parameters of each compound are set as shown in the following table.

[0075]

[0076] The standard curves were as follows: benzoylmeaconitine Y=133.3577X+1065.9108, r=0.999, benzoylmeaconitine Y=162.2150X+1470.7474, r=0.999, benzoylhyaconitine Y=142.7351X+1280.5289, r=0.999, and the linearity of benzoylmeaconitine was good in the range of 15.94-1020.00 ng / ml, benzoylmeaconitine was in the range of 15.62-1000.00 ng / ml, and benzoylhyaconitine was in the range of 17.50-1120.00 ng / ml.

[0077] The content range requirement in the preparation is that the total amount of monoester alkaloids, calculated as benzoylacone, benzoylhyaconitine and benzoylmeaconitine, per gram of the product should be 0.017 mg to 0.039 mg.

[0078] The content of glycyrrhizic acid in Fuzheng Jiedu granules determined by HPLC of the present invention is specifically:

[0079] a) extracting the Fuzheng Jiedu granules with 70% ethanol to obtain a test solution; the extraction is preferably ultrasonic extraction; and the ultrasonic extraction time is preferably 20 to 30 minutes;

[0080] The inventors found that among anhydrous ethanol, 95% ethanol, 70% ethanol, 50% ethanol and water as extractants, 70% ethanol as solvent ultrasonic extraction of glycyrrhizic acid content is the highest and the extraction is more complete, so 70% ethanol is selected as the extraction solvent.

[0081] b) using high performance liquid chromatography-mass spectrometry to conduct qualitative and quantitative analysis of the components of the test liquid;

[0082] The specific chromatographic parameters are as follows:

[0083] The chromatographic column is a C18 column, the detection wavelength is 251nm; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution; the mobile phase flow rate is 1.0ml / min; the chromatographic column temperature is 30℃, the injection volume is 10μL; gradient elution:

[0084]

[0085] With glycyrrhizic acid concentration-peak area as coordinate axis, a standard curve was drawn: Y=8434.7X+1367.9, r=0.9999. Cinnamic acid had good linearity in the range of 5.432-678.981 μg / ml.

[0086] The content range requirement in the preparation is that each gram of licorice, calculated as glycyrrhizic acid, should be 4.0mg to 7.5mg.

[0087] In some embodiments, the identification of dried ginger by thin layer chromatography is specifically as follows:

[0088] a) extracting the Fuzheng Jiedu granules, the negative test sample of dried ginger and the control medicinal material of dried ginger with ethyl acetate to obtain a test sample solution, a negative test sample of dried ginger and a control medicinal material solution of dried ginger, respectively;

[0089] Dissolve the 6-gingerol reference substance in ethyl acetate to obtain a 6-gingerol reference substance solution;

[0090] b) performing thin layer chromatography on the test solution, the negative test sample of dried ginger, the control medicinal material solution of dried ginger, and the reference solution of 6-gingerol, wherein the thin layer plate is a silica gel G thin layer plate; the developing solvent is petroleum ether-chloroform-ethyl acetate, and the volume ratio of the petroleum ether-chloroform-ethyl acetate is 2:1:1;

[0091] c) Inspection: If the test sample, the dry ginger control medicinal material, and 6-gingerol have fluorescent spots of the same color at the same Rf value, and the dry ginger negative test sample has no spots, then it is dry ginger;

[0092] The inspection is specifically as follows: spraying with 2% vanillin sulfuric acid solution, heating at 105° C. until the spots are clearly colored; the spotting volume is 6 to 10 μL.

[0093] The present invention uses thin layer chromatography to identify dried ginger. The Fuzheng Jiedu granules, the dried ginger negative test sample and the dried ginger control medicinal material are extracted with ethyl acetate to obtain a test sample solution, a dried ginger negative test sample and a dried ginger control medicinal material solution respectively;

[0094] The above extraction is specifically as follows: take the Fuzheng Jiedu granules to be tested, add water to dissolve them, shake and extract with ethyl acetate, separate the ethyl acetate layer, evaporate to dryness, add 1 ml of ethyl acetate to dissolve the residue, and use it as the Fuzheng Jiedu granules test sample;

[0095] Among them: the mass volume ratio of Fuzheng Jiedu Granules, water and ethyl acetate is 3g:20mL:20mL.

[0096] Take the negative prescription of dried ginger and process it in the same way to obtain the negative test sample of dried ginger;

[0097] Take another dried ginger control medicinal material, add ethyl acetate, ultrasonically treat for 10 minutes, filter, and take the filtrate as the dried ginger control medicinal material solution;

[0098] Among them, the mass volume ratio of the dried ginger reference medicinal material and ethyl acetate is 1g:20mL.

[0099] Take 6-gingerol reference substance and add ethyl acetate to make a solution containing 0.5 mg per 1 ml as the reference substance solution;

[0100] According to the thin layer chromatography test, the developing solvent is 2:1:1 petroleum ether-chloroform-ethyl acetate at 60-90°C, and 6μl of the above test sample solution, 10μl of the reference medicinal material solution, and 6μl of the reference substance solution are taken, spotted on the silica gel G plate, developed, taken out, dried, sprayed with 2% vanillin sulfuric acid solution, and heated at 105°C until the spots are clearly colored; if the test sample, the dry ginger reference medicinal material and the 6-gingerol reference substance have spots of the same color at the same Rf value, and the dry ginger negative test sample has no spots, the quality is qualified.

[0101] According to the present invention, the identification of liquorice by thin layer chromatography is specifically as follows:

[0102] a) After dissolving Fuzheng Jiedu Granules, licorice negative test sample and licorice control medicinal material in water, adjusting the pH value to 11-12 with 5% sodium hydroxide, filtering, adding acid to adjust the pH value to 4-5, adding ethyl acetate for extraction, evaporating to dryness and then extracting with methanol to obtain a test sample solution, a licorice negative test sample and a licorice control medicinal material solution, respectively;

[0103] In some embodiments, the mass volume ratio of Fuzheng Jiedu granules, water, ethyl acetate and methanol is: 2.5g:20mL:20mL:5mL.

[0104] Dissolve the liquiritin reference substance in methanol to obtain a liquiritin reference substance solution.

[0105] In some embodiments, a liquiritin reference substance was taken and methanol was added to prepare a solution containing 1 mg per 1 ml, which was used as the reference substance solution.

[0106] The preparation method of the licorice control medicinal material solution of the present invention is preferably as follows: taking a licorice control medicinal material, adding water, heating and refluxing for 1 hour, filtering, adjusting the pH value of the filtrate to 11-12 with a 5% sodium hydroxide solution, filtering, adding concentrated hydrochloric acid to the filtrate to adjust the pH value to 4-5, shaking and extracting with ethyl acetate twice, combining the ethyl acetate solution, evaporating to dryness, and adding methanol to the residue to dissolve it as the licorice control medicinal material solution.

[0107] b) performing thin layer chromatography on the test solution, the licorice negative test sample, the liquiritin reference substance and the licorice reference medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is ethyl acetate-formic acid-glacial acetic acid-water, and the volume ratio of ethyl acetate-formic acid-glacial acetic acid-water is 12:1:1:2;

[0108] c) Inspection: If the test sample, licorice control medicinal material, and liquiritin have spots of the same color at the same Rf value, and the licorice negative test sample has no spots, then it is licorice;

[0109] The inspection is performed by spraying with 10% sulfuric acid ethanol solution and heating at 105° C. until the spots are clearly colored; the spotting volume is 2 μL.

[0110] According to the present invention, the identification of honeysuckle by thin layer chromatography is specifically as follows:

[0111] a) Extract the Fuzheng Jiedu Granules and the honeysuckle negative test sample with methanol, filter, dissolve in water, extract with ether, discard the ether layer, extract with saturated n-butanol, evaporate the n-butanol layer, and dissolve in methanol to obtain a test sample solution and a honeysuckle negative test sample solution, respectively;

[0112] The extraction of the present invention is preferably ultrasonic extraction, and the time of the ultrasonic extraction is preferably 10 to 20 minutes;

[0113] In some embodiments, the mass volume ratio of Fuzheng Jiedu granules, methanol, water, ether, and n-butanol is 0.85g: 10mL, 10mL, 20mL, and 10mL.

[0114] The honeysuckle control medicinal material is extracted with methanol to obtain a honeysuckle control medicinal material solution;

[0115] In some embodiments, the mass volume ratio of honeysuckle control medicinal material and methanol is 0.5h:8mL; the time of the ultrasonic extraction is preferably 10min.

[0116] Dissolve the chlorogenic acid reference substance in methanol to obtain a chlorogenic acid reference substance solution;

[0117] In some embodiments, the concentration of the chlorogenic acid reference solution is 1 mg / mL.

[0118] b) performing thin layer chromatography on the test solution, the honeysuckle negative test product, the honeysuckle control medicinal material solution and the chlorogenic acid reference solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is butyl acetate-formic acid-water, and the volume ratio of butyl acetate-formic acid-water is 14:5:5;

[0119] c) Inspection: If the test sample, the honeysuckle control medicinal material, and chlorogenic acid have spots of the same color at the same Rf value, and the honeysuckle negative test sample has no spots, then it is honeysuckle;

[0120] The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 1 μL.

[0121] According to the present invention, the identification of Gleditsia sinensis by thin layer chromatography specifically comprises:

[0122] a) Extract the Fuzheng Jiedu Granules and the Gleditsia sinensis negative test sample with methanol, filter, dissolve in water, and then extract with ether, discard the ether layer and extract with saturated n-butanol, evaporate the n-butanol layer and dissolve in methanol to obtain a test sample solution and a Gleditsia sinensis negative test sample solution, respectively;

[0123] In some embodiments, the mass volume ratio of Fuzheng Jiedu granules, methanol, water, ether and n-butanol is: 3.4 g: 10 mL: 10 mL: 20 mL: 20 mL.

[0124] The Gleditsia sinensis reference medicinal material was extracted with methanol to obtain a Gleditsia sinensis reference medicinal material solution;

[0125] In some embodiments, the mass volume ratio of the Gleditsia sinensis control medicinal material and methanol is 0.5 g:8 mL. The ultrasonic time is 10 min.

[0126] b) performing thin layer chromatography on the test solution, the negative test sample of Gleditsia sinensis and the control medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is petroleum ether-ethyl acetate-formic acid, and the volume ratio of petroleum ether-ethyl acetate-formic acid is 10:10:0.5;

[0127] c) Inspection: If the test sample and the Gleditsia sinensis control medicinal material have fluorescent spots of the same color at the same Rf value, and the Gleditsia sinensis negative test sample has no spots, then it is Gleditsia sinensis;

[0128] The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 5 μL.

[0129] According to the present invention, the method of identifying the five-fingered peach by thin layer chromatography is as follows:

[0130] a) extracting the Fuzheng Jiedu granules, the negative test sample of Eleutherodendron chinense and the Eleutherodendron chinense control medicinal material solution with ethyl acetate to obtain a test sample solution, a negative test sample solution of Eleutherodendron chinense and a Eleutherodendron chinense control medicinal material solution, respectively;

[0131] In some embodiments,

[0132] Take Fuzheng Jiedu Granules, add ethyl acetate, ultrasonically treat, filter, evaporate the filtrate to dryness at low temperature, add ethyl acetate to dissolve the residue, and use it as the test sample of Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules are treated in the same way.

[0133] In some embodiments, the mass ratio of Fuzheng Jiedu granules to ethyl acetate is 6 g:25 mL; and the ultrasonic treatment time is 20 to 30 min.

[0134] Take the negative prescription of Prunus armeniaca and process it in the same way to obtain the negative test sample of Prunus armeniaca;

[0135] Take another 1g of Prunus armeniaca control medicinal material, and prepare Prunus armeniaca control medicinal material solution in the same way;

[0136] Take the psoralen reference substance and add ethyl acetate to make a solution containing 1 mg per 1 ml as the reference substance solution;

[0137] Extracting psoralen with ethyl acetate to obtain a psoralen control medicinal material solution;

[0138] b) performing thin layer chromatography on the test solution, the negative test sample of Psoralea corylifolia, the psoralen reference solution and the Psoralea corylifolia reference medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is n-hexane-butyl acetate, and the volume ratio of n-hexane-butyl acetate is 4:1;

[0139] c) Inspection: If the test sample, the reference medicinal material of Eleutherodactylus quinquefolius, and psoralen have fluorescent spots of the same color at the same Rf value, and the Eleutherodactylus quinquefolius negative test sample has no spots, then it is Eleutherodactylus quinquefolius;

[0140] The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 1 μL.

[0141] According to the present invention, the identification of dried orange peel by thin layer chromatography is specifically as follows:

[0142] a) extracting the Fuzheng Jiedu granules and the tangerine peel negative test solution with 0.05% sodium hydroxide, adding acid to adjust the pH value to 4-5, adding water to the precipitate and centrifuging it, dissolving the precipitate after centrifugation with methanol, evaporating it to dryness and dissolving it with methanol to obtain a test solution and a tangerine peel negative test solution, respectively;

[0143] In some embodiments, 10 g of Fuzheng Jiedu Granules was added with 25 ml of 0.05% sodium hydroxide solution, ultrasonically treated for 15 minutes, centrifuged for 2 minutes, the supernatant was taken, concentrated hydrochloric acid was added to adjust the pH to 4-5, centrifuged for 5 minutes, the supernatant was discarded, 12 ml of water was added to the precipitate, centrifuged for 2 minutes, the supernatant was discarded, 12 ml of methanol was added to the precipitate, ultrasonically treated for 15 minutes, filtered, the filtrate was evaporated to dryness, and 3 ml of methanol was added to the residue to dissolve it as a test sample of Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules were treated in the same way.

[0144] Take the tangerine peel negative prescription and process it in the same way to obtain the tangerine peel negative test sample;

[0145] Extract the tangerine peel control medicinal material with methanol to obtain a tangerine peel control medicinal material solution; preferably, the method comprises the following steps: taking 0.3 g of the tangerine peel control medicinal material, adding 10 ml of methanol, ultrasonically treating for 20 minutes, filtering, taking 5 ml of the filtrate, and concentrating the filtrate to 1 ml to obtain the tangerine peel control medicinal material solution;

[0146] Then take the hesperidin reference substance and add methanol to make a saturated solution as the reference substance solution;

[0147] b) performing thin layer chromatography on the test solution, the tangerine peel negative test solution, the hesperidin reference solution and the tangerine peel reference medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is ethyl acetate-methanol-water, and the volume ratio of ethyl acetate-methanol-water is 100:17:13;

[0148] c) Inspection: If the test sample, the tangerine peel control medicinal material, and hesperidin have fluorescent spots of the same color at the same Rf value, and the tangerine peel negative test sample has no spots, then it is tangerine peel;

[0149] The inspection is specifically as follows: spraying with 1% aluminum chloride solution and placing under ultraviolet light for inspection; the wavelength of the ultraviolet light is 365nm; and the sample spotting volume is 4 to 6 μL.

[0150] The present invention provides a characteristic spectrum of the Fuzheng Jiedu granules, which evaluates the types, transfer rules and contents of the internal chemical substances of the Fuzheng Jiedu granules as a whole.

[0151] The present invention finds corresponding characteristic spots from a series of thin layer chromatographic identifications of medicinal materials, decoction pieces and granules, which provides a basis for tracing the characteristic components in the granules back to the medicinal materials. The content determination confirms that there is a certain correlation between the medicinal materials, decoction pieces and granules.

[0152] The method for determining the contents of the two main components, monoester alkaloid and glycyrrhizic acid, established by the invention has good precision, stability and repeatability.

[0153] The present invention can ensure the safety, effectiveness and stability of clinical medication, and also provides a detailed and reliable research basis for the subsequent quality standard establishment and stability investigation of Fuzheng Jiedu Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0154] Figure 1 .TLC chart of dried ginger in Fuzheng Jiedu granules;

[0155] Figure 2 .TLC chart of licorice root in Fuzheng Jiedu granules;

[0156] Figure 3 .TLC chart of honeysuckle in Fuzheng Jiedu granules;

[0157] Figure 4 .TLC chart of Gleditsia sinensis spines in Fuzheng Jiedu granules;

[0158] Figure 5 .TLC chart of Prunus mume in Fuzheng Jiedu Granules;

[0159] Figure 6 .TLC chart of dried orange peel in Fuzheng Jiedu granules;

[0160] Figure 7 .Characteristic spectrum of the extract of Radix Aconiti Lateralis Preparata in Fuzheng Jiedu Granules;

[0161] Figure 8 .Characteristic spectrum of dried ginger extract in Fuzheng Jiedu granules;

[0162] Fig. 9 .Characteristic spectrum of the extract of licorice root in Fuzheng Jiedu granules;

[0163] Fig.10 .Characteristic spectrum of honeysuckle extract in Fuzheng Jiedu granules;

[0164] Fig.11 .Characteristic spectrum of the extract of Gleditsia sinensis thorns in Fuzheng Jiedu Granules;

[0165] Fig.12 .Characteristic spectrum of the extract of Prunus mume in Fuzheng Jiedu Granules;

[0166] Fig.13 .Characteristic spectrum of patchouli extract in Fuzheng Jiedu granules;

[0167] Fig.14 .Characteristic spectrum of tangerine peel extract in Fuzheng Jiedu granules;

[0168] Fig.15 .The common pattern diagram of Fuzheng Jiedu Granules. DETAILED DESCRIPTION

[0169] The present invention provides a quality control method for Fuzheng Jiedu Granules. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0170] In order to further illustrate the present invention, the quality control method of a Fuzheng Jiedu granule provided by the present invention is described in detail below in conjunction with the examples. If no specific conditions are specified in the examples, the method is carried out according to conventional conditions or conditions recommended by the manufacturer. Among them, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased on the market.

[0171] Example 1 The Fuzheng Jiedu granules are obtained by referring to the preparation method of Example 2 in the prior art CN111110824B.

[0172] 2. Study on the quality standard of Fuzheng Jiedu Granules

[0173] 2.1 Qualitative Research

[0174] 2.1.1 Thin layer chromatography studies

[0175] (1) Thin layer identification of dried ginger

[0176] Take 3g of the powder of this product, add 20ml of water to dissolve it, shake and extract with 20ml of ethyl acetate, separate the ethyl acetate layer, evaporate to dryness, add 1ml of ethyl acetate to dissolve the residue, and use it as the test sample of Fuzheng Jiedu Granules. Treat 3 batches of Fuzheng Jiedu Granules test samples in the same way; take the negative prescription of dried ginger, and treat it in the same way to obtain the negative test sample of dried ginger; take 1g of dried ginger reference medicinal material, add 20ml of ethyl acetate, ultrasonically treat for 10 minutes, filter, and take the filtrate as the dried ginger reference medicinal material solution; take 6-gingerol reference substance, add ethyl acetate to make a solution containing 0.5mg per 1ml, as the reference substance solution;

[0177] According to the thin layer chromatography method (General Rules of Part IV 0502), the developing solvent is petroleum ether (60-90°C)-chloroform-ethyl acetate (2:1:1), 6μl of the test solution, 10μl of the reference drug solution, and 6μl of the reference substance solution are taken, spotted on a silica gel G plate, developed, taken out, dried, sprayed with 2% vanillin sulfuric acid solution, and heated at 105°C until the spots are clearly colored;.

[0178] The dried ginger reference material and 6-gingerol reference substance of the three batches of Fuzheng Jiedu Granule test samples had spots of the same color at the same Rf value, while the dried ginger negative test sample had no spots.

[0179] Results Figure 1 (A. 6-gingerol reference substance, B. dried ginger reference medicinal material, C~E. S20200701~3 test samples, F. negative sample lacking dried ginger).

[0180] (2) Thin layer chromatography identification of licorice

[0181] Take 2.5g of the powder of this product, add 20mL of water to dissolve it, adjust the pH value to 11-12 with 5% sodium hydroxide solution, filter, add concentrated hydrochloric acid to the filtrate to adjust the pH value to 4-5, shake and extract with ethyl acetate twice, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, add 5ml of methanol to the residue to dissolve it, and use it as the test sample of Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules test samples are treated in the same way; take the negative prescription of licorice, and treat it in the same way to obtain the negative test sample of licorice; take another 1g of licorice control medicinal material, add 20ml of water, heat and reflux for 1 hour, filter, adjust the pH value of the filtrate to 11-12 with 5% sodium hydroxide solution, filter, add concentrated hydrochloric acid to the filtrate to adjust the pH value to 4-5, shake and extract with ethyl acetate twice, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, add 5ml of methanol to the residue to dissolve it, and use it as the licorice control medicinal material solution; then take the liquiritin reference substance, add methanol to make a solution containing 1mg per 1ml, and use it as the reference substance solution;

[0182] According to the thin layer chromatography method (General Rules of Part IV 0502), the developing solvent is ethyl acetate-formic acid-glacial acetic acid-water (12:1:1:2), 2 μl of each of the above solutions is taken, spotted on a silica gel G plate, developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots are clearly colored.

[0183] The three batches of Fuzheng Jiedu Granule test samples, licorice control medicinal materials and liquiritin control substance had spots of the same color at the same Rf values, while the licorice negative test sample had no spots.

[0184] Results Figure 2 (A. Licorice glycoside reference substance, B. Licorice reference medicinal material, C~E. S20200701~3 test samples, F. Negative sample lacking licorice).

[0185] (3) Thin layer identification of honeysuckle

[0186] Take 0.85g of the powder of this product, add 10ml of methanol, ultrasonically treat twice, each time for 10 minutes, filter, evaporate the filtrate, add 10ml of water to the residue to dissolve it, transfer it to a separatory funnel, extract it twice with ether, 10ml each time, discard the ether layer, and then extract it with 10ml of water-saturated n-butanol, separate the n-butanol layer, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test sample of Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules test samples are treated in the same way; take the honeysuckle negative prescription, treat it in the same way to obtain the honeysuckle negative test sample; take another 0.5g of honeysuckle control medicinal material, add 8ml of methanol, ultrasonically treat it for 10 minutes, filter, and take the filtrate as the honeysuckle control medicinal material solution; then take the chlorogenic acid reference substance, add methanol to make a solution containing 1mg per 1ml as the reference substance solution;

[0187] According to the thin layer chromatography method (General Rules of Part IV 0502), the developing solvent is the upper layer solution of butyl acetate-formic acid-water (14:5:5), aspirate 1 μl of each of the above solutions, spot on the silica gel G plate, develop, take out, dry, and examine under ultraviolet light at 365 nm.

[0188] The three batches of Fuzheng Jiedu Granule test samples, honeysuckle control medicinal materials and chlorogenic acid reference substance had spots of the same color at the same Rf values, while the honeysuckle negative test sample had no spots.

[0189] Results Figure 3 (A. Chlorogenic acid reference substance, B. Honeysuckle reference medicinal material, C~E. S20200701~3 test samples, F. Negative sample without honeysuckle).

[0190] (4) Thin layer identification of Gleditsia sinensis thorns

[0191] Take 3.4g of the powder of this product, add 10ml of methanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate, add 10ml of water to the residue to dissolve it, shake and extract with ether twice, 10ml each time, discard the ether layer, and shake and extract with water-saturated n-butanol twice, 10ml each time, combine the n-butanol extracts, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test sample of Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules test samples were treated in the same way; take the negative prescription of Gleditsia sinensis, and treat it in the same way to obtain the negative test sample of Gleditsia sinensis; take another 0.5g of Gleditsia sinensis control medicinal material, add 8ml of methanol, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, and add 1ml of methanol to the residue to dissolve it, and use it as the control medicinal material solution of Gleditsia sinensis.

[0192] According to the thin layer chromatography method (General Rules of Part IV 0502), the developing solvent is petroleum ether (60-90°C)-ethyl acetate-formic acid (10:10:0.5), 1 μl of each of the above solutions is taken, spotted on a silica gel G plate, developed, taken out, dried, sprayed with 10% potassium hydroxide methanol solution, and examined under a UV lamp at 365 nm.

[0193] The three batches of Fuzheng Jiedu Granules test samples and the Gleditsia sinensis control medicinal material had spots of the same color at the same Rf values, while the Gleditsia sinensis negative test sample had no spots.

[0194] Results Figure 4 (A~C. S20200701~3 test samples, D. Gleditsia sinensis control medicinal materials, E. Negative samples lacking Gleditsia sinensis).

[0195] (5) Thin layer identification of five-fingered peach

[0196] Take 6g of the powder of this product, add 25ml of ethyl acetate, ultrasonically treat for 30 minutes, filter, evaporate the filtrate at low temperature, add 0.5ml of ethyl acetate to the residue to dissolve it, and use it as the test sample of Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules test samples are treated in the same way; take the negative prescription of Prunus mume, treat it in the same way to obtain the negative test sample of Prunus mume; take 1g of Prunus mume reference medicinal material, and prepare Prunus mume reference medicinal material solution in the same way; take the psoralen reference substance, add ethyl acetate to prepare a solution containing 1mg per 1ml, as the reference substance solution;

[0197] According to the thin layer chromatography method (General Rules of Part IV 0502), the developing solvent is n-hexane-ethyl acetate (4:1), 5 μl of each of the above solutions is taken, spotted on a silica gel G plate, developed, taken out, dried, and examined under a UV lamp at 365 nm.

[0198] The three batches of Fuzheng Jiedu Granule test samples, Eleutherodactyl quinquefolius control medicinal materials and psoralen control substance had spots of the same color at the same Rf values, while the Eleutherodactyl quinquefolius negative test sample had no spots.

[0199] Results Figure 5(A. Psoralen reference substance, B. Quinquefolius reference medicinal material, C~E. S20200701~3 test samples, F. Quinquefolius negative sample).

[0200] (6) Thin layer identification of tangerine peel

[0201] Take 10g of the powder of this product, add 25ml of 0.05% sodium hydroxide solution, ultrasonically treat for 15 minutes, centrifuge for 2 minutes, take the supernatant, add concentrated hydrochloric acid to adjust the pH to 4-5, centrifuge for 5 minutes, discard the supernatant, add 12ml of water to the precipitate, centrifuge for 2 minutes, discard the supernatant, add 12ml of methanol to the precipitate, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, add 3ml of methanol to dissolve the residue, and use it as a test sample for Fuzheng Jiedu Granules. Three batches of Fuzheng Jiedu Granules test samples are treated in the same way; take the tangerine peel negative prescription and treat it in the same way to obtain the tangerine peel negative test sample; take another tangerine peel reference medicinal material 0.3g, add 10ml of methanol, ultrasonically treat for 20 minutes, filter, take 5ml of filtrate, and concentrate it to 1ml as the tangerine peel reference medicinal material solution; then take the hesperidin reference substance, add methanol to make a saturated solution as the reference substance solution;

[0202] According to the thin layer chromatography method (General Rules of Part IV 0502), the developing solvent is ethyl acetate-methanol-water (100:17:13), 4 μl of the reference solution, 4 μl of the reference medicinal material solution, and 6 μl of the test solution are aspirated and spotted on a silica gel G plate, developed, taken out, dried, sprayed with 1% aluminum chloride solution, and examined under a UV lamp at 365 nm.

[0203] The three batches of Fuzheng Jiedu Granule test samples, dried tangerine peel control medicinal materials and hesperidin control substance had spots of the same color at the same Rf values, while the dried tangerine peel negative test sample had no spots.

[0204] Results Figure 6 (A. Hesperidin reference substance, B. Tangerine peel reference medicinal material, C~E. S20200701~3 test samples, F. Negative sample without tangerine peel).

[0205] (7) Determination of diester alkaloid content

[0206] ①Chromatographic conditions

[0207] Thermo Hypersil GOLD C18 column (100 mm × 2.1 mm, 3 μm), with 0.1% formic acid in methanol as mobile phase A, 0.1% formic acid in water as mobile phase B, gradient elution program: 0-3 min, mobile phase 30% A;

[0208] 3-15min, mobile phase is 30%-80% A;

[0209] 15-18 min, mobile phase is 80%-90% A;

[0210] 18-19 min, mobile phase 90%-30% A;

[0211] The mass spectrometry conditions were electrospray ionization positive ion mode and multiple reaction (MRM) detection mode. The parameters of each compound were set as shown in the table below.

[0212]

[0213] The flow rate was 0.2 mL / min, the column temperature was 30 °C, and the injection volume was 1 to 10 μL;

[0214] ②Specificity inspection

[0215] Weigh the prescription pieces and get the whole prescription test sample according to the test sample treatment; weigh the negative prescription of Radix Aconiti Lateralis Preparata and get the negative test sample of Radix Aconiti Lateralis Preparata in the same way; take 10μL of each of the above test samples and the reference solution of diester alkaloids and analyze them by LC-MS. The results show that the negative of diester alkaloids has no interference.

[0216] ③Linear relationship investigation

[0217] Take the reference extract of aconitum diester type alkaloids, add 0.1% formic acid methanol to dissolve, and obtain solutions with concentrations of 0.1046 mg / ml of neoaconitine, 0.0990 mg / ml of mesaconitine, and 0.1049 mg / ml of aconitine. Use this as the mother solution to prepare solutions with concentrations of 0.00265, 0.01308, 0.06538, 0.65381, and 1. Standard solutions of 30763, 2.61525μg / ml, hypoaconitine 0.00248, 0.01237, 0.06188, 0.618750, 1.23750, 2.47500μg / ml, and aconitine 0.00262, 0.01312, 0.06559, 0.65588, 1.31175, 2.62350μg / ml. 10 μL of the six concentrations of reference solution were taken and injected into the liquid-mass spectrometer, and the standard curves were drawn with the concentration of the component to be measured - the peak area as the coordinate axis: neoaconitine Y = 421310X-10409.65145, r = 0.999, hypoaconitine Y = 4971130X-57824.1456, r = 0.999, aconitine Y = 50980490X+8200.96973, r = 0.999, neoaconitine was in the range of 0.00265-2.61525 μg / ml, hypoaconitine was in the range of 0.00248-2.47500 μg / ml, and aconitine was in the range of 0.00262-2.62350 μg / ml with good linearity.

[0218] ④Precision experiment

[0219] 10 μL of aconitum diester-type alkaloid reference solution with a concentration of 4.405 μg / ml was taken and determined according to the established mass spectrometry analysis method. The sample was repeated 6 times. The results showed that the instrument had good precision.

[0220] ⑤Repeat experiment

[0221] Fuzheng Jiedu Granules were used to prepare the test sample according to the test sample processing method, and 6 parallel samples were used for liquid chromatography-mass spectrometry analysis. The results showed that the method had good repeatability and diester alkaloids were not detected in Fuzheng Jiedu Granules.

[0222] ⑥Stability test

[0223] A reference solution of aconitum diester-type alkaloids with a concentration of 1.857 μg / ml was taken for liquid chromatography-mass spectrometry analysis at 0, 4, 6, 8, and 12 hours after preparation. The results showed that the sample had good stability within 12 hours.

[0224] ⑦Accuracy experiment

[0225] Take 6 portions of Fuzheng Jiedu granules with known content, add the same concentration of reference solution to each of them, prepare the test solution according to the test sample treatment method, treat 6 portions in parallel, and perform liquid chromatography-mass spectrometry determination. The results show that this method has a high recovery rate and good accuracy in determining the contents of neoaconitine, mesaconitine and aconitine.

[0226] 2.1.2 Feature map research

[0227] (1) Study on the chromatographic conditions of characteristic spectra

[0228] ① Preparation of test samples

[0229] Take 0.4 g of Fuzheng Jiedu granules powder, place it in a 25 mL volumetric flask, add an appropriate amount of 70% ethanol, ultrasonicate for 20 minutes to dissolve, cool, dilute to the scale with 70% ethanol, shake well, centrifuge at high speed, and take the supernatant.

[0230] ②Optimization of chromatographic conditions

[0231] a. Selection of detection wavelength

[0232] Agela Promasil C 18 The chromatographic column (5 μm, 250×4.6 mm) was used with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. The elution was performed according to the ratio in Table 8. The full wavelength scanning at 20 to 400 nm was performed using a PDA detector. Figure 5At 237nm, the peaks are well separated, the peak shape is good, and the response is high. The chromatographic component information can be obtained as much as possible from the spectrum, and the overall picture of the system composition can be reflected. Therefore, 237nm is selected as the characteristic spectrum measurement wavelength.

[0233] Table 8. Mobile phase elution conditions

[0234]

[0235] d. Investigation of mobile phase ratio

[0236] Detection wavelength: 237nm, acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, eluted according to the proportions in Tables 9-13, and compared the chromatograms, see Figure 8 (AE in the figure correspond to elution conditions 1-5 in turn). The experimental results show that the baseline of the chromatogram obtained under the chromatographic conditions of method 3 is relatively stable, the peak shape and separation of each chromatographic peak are good, and it can fully reflect its main components, so this is used as the gradient elution condition.

[0237] Table 9. Mobile phase elution conditions 1-phenomenex GeminiC 18 110A chromatographic column (5μm, 250×4.6mm); flow rate: 1.0ml / min

[0238]

[0239] Table 10. Mobile phase elution conditions 2-phenomenex Gemini C 18 110A chromatographic column (5μm, 250×4.6mm); flow rate: 1.0ml / min

[0240]

[0241] Table 11. Mobile phase elution conditions 3-Agela Promasil C 18 Chromatographic column (5μm, 250×4.6mm); flow rate: 1.0ml / min

[0242]

[0243]

[0244] Table 12. Mobile phase elution conditions 4-Agela Promasil C 18 Chromatographic column (5μm, 250×4.6mm); flow rate: 0.9ml / min

[0245]

[0246] g. Determination of liquid phase conditions

[0247] Agela Promasil C 18 The chromatographic column was (5 μm, 250×4.6 mm), with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, eluted according to the ratio in Table 8, the detection wavelength was 237 nm, the flow rate was 1.0 mL / min; the column temperature was 30° C.; the injection volume was 10 μL.

[0248] ③ Investigation of characteristic graph methodology

[0249] a. Precision experiment

[0250] Accurately pipette 10 μL of a mixed reference solution containing 40 μg chlorogenic acid, 40 μg liquiritin, 40 μg hesperidin, 40 μg ammonium glycyrrhizinate, and 10 μg 6-gingerol per 1 ml, repeat the injection 6 times, and record the chromatogram. The RSD values ​​of the relative retention time and relative peak area of ​​the characteristic chromatographic peaks are less than 1%, proving that the instrument has good precision.

[0251] b. Repeatability experiment

[0252] According to the determined test sample processing method, 6 samples were processed in parallel, and the sample was injected according to the preferred HPLC analysis method, and the chromatogram was recorded. The RSD values ​​of the relative retention time and relative peak area of ​​the characteristic chromatographic peak were less than 1%, proving that the method had good repeatability.

[0253] c. Stability test

[0254] The characteristic spectrum of Fuzheng Jiedu Granules was prepared according to the sample processing method, and the sample was injected according to the preferred HPLC analysis method, and the chromatograms at 0, 6, 12, 18, 24, and 48 hours were recorded. The RSD values ​​of the relative retention time and relative peak area of ​​the characteristic chromatographic peak were less than 1%, indicating that the sample had good stability within 48 hours.

[0255] ④ Attribution of characteristic peaks in characteristic spectra

[0256] The whole formula of Fuzheng Jiedu Granules, the extracts of Radix Aconiti Lateralis, dried ginger, roasted licorice, Rhizoma Cibotii, Gleditsia sinensis, Flos Lonicerae, Pogostemon cablin, and dried tangerine peel in the prescription were compared with the negative samples in turn. The chromatograms are shown in Table 1. Figure 7-14 The attribution results of the characteristic peaks of the characteristic spectrum are as follows: in the characteristic spectrum, peak 1 is chlorogenic acid from honeysuckle, peaks 2 and 4 are from roasted liquorice, and peak 2 is liquiritin and peak 4 is glycyrrhizic acid; peak 3 is hesperidin from dried tangerine peel, and peak 5 is 6-gingerol from dried ginger; there are no characteristic peaks of Aconiti Lateralis Preparata, Prunus Armeniaca, Gleditsia sinensis, and Pogostemon cablin.

[0257] (2) Establishment of a common feature map

[0258] Three batches of Fuzheng Jiedu granules were prepared according to the test sample processing method, and HPLC chromatograms were analyzed to establish a common characteristic spectrum. The results are shown in Fig.15 .

[0259] 2.2 Quantitative research

[0260] 2.2.1 Quantitative Methodological Study of Index Components

[0261] (2) Determination of the content of monoester alkaloids

[0262] (1) Preparation of test samples

[0263] Take 0.2g of Fuzheng Jiedu Granules, put it in a 10ml volumetric flask, add 5ml of 0.1% formic acid methanol, ultrasonically treat for 30 minutes, cool, add 0.1% formic acid methanol to dilute to the scale, shake well, pass through a membrane, and the test sample is obtained.

[0264] ①Chromatographic conditions

[0265] Thermo Hypersil GOLD C18 column (100 mm × 2.1 mm, 3 μm), injection volume of 1 μL, 0.1% formic acid in methanol as mobile phase A, 0.1% formic acid in water as mobile phase B, gradient elution program: 0-3 min, mobile phase 30% A;

[0266] 3-17 min, mobile phase 30%-60% A;

[0267] 17-23 min, mobile phase 60%-90% A;

[0268] 23-24 min, mobile phase 90%-30% A;

[0269] The flow rate was 0.2 mL / min and the column temperature was 30 °C.

[0270] The mass spectrometry conditions were electrospray ionization positive ion mode and multiple reaction (MRM) detection mode. The parameters of each compound were set as shown in the table below.

[0271]

[0272] ②Specificity inspection

[0273] Weigh the prescription pieces and get the whole prescription test sample according to the test sample treatment; weigh the negative prescription of Radix Aconiti Lateralis Preparata and get the negative test sample of Radix Aconiti Lateralis Preparata in the same way; take 1 μL of each of the above test samples and monoester alkaloid reference solution and analyze by LC-MS. The results show that the monoester alkaloid negative has no interference.

[0274] ③Linear relationship investigation

[0275] Take benzoylneaconitine reference substance, benzoylneaconitine reference substance, and benzoylhylaconitine reference substance, add 0.1% formic acid methanol to dissolve, and obtain solutions with concentrations of 0.102 mg / ml benzoylneaconitine, 0.100 mg / ml benzoylneaconitine, and 0.112 mg / ml benzoylhylaconitine, respectively. Use this as the mother solution, and use 0.1% formic acid methanol to prepare benzoylneaconitine with concentrations of 15.94, 31.88, and 63.7 5, 127.50, 255.00, 510.00, 1020.00 ng / ml, benzoylaconitine 15.62, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00 ng / ml, standard solution of benzoylhyaconitine 17.50, 35.00, 70.00, 140.00, 280.00, 560.00, 1120.00 ng / ml. 1 μL of the reference solution of the above seven concentrations was taken and injected into the liquid-mass spectrometer, and the standard curves were drawn with the concentration of the component to be measured-peak area as the coordinate axis: benzoylmeaconitine Y=133.3577X+1065.9108, r=0.999, benzoylmeaconitine Y=162.2150X+1470.7474, r=0.999, benzoylhyaconitine Y=142.7351X+1280.5289, r=0.999, and the linearity of benzoylmeaconitine was good in the range of 15.94-1020.00 ng / ml, benzoylmeaconitine was in the range of 15.62-1000.00 ng / ml, and benzoylhyaconitine was in the range of 17.50-1120.00 ng / ml.

[0276] ④Precision experiment

[0277] 1 μL of reference solution of the same concentration was accurately aspirated and measured according to the established mass spectrometry method. The injection was repeated 6 times. The results showed that the instrument had good precision.

[0278] ⑤Repeat experiment

[0279] Fuzheng Jiedu Granules were used to prepare the test sample according to the test sample processing method, and 6 parallel samples were used for liquid chromatography-mass spectrometry analysis. The results showed that the method had good repeatability. The average content of benzoylacone was 18.490 μg / g, the average content of benzoylacone was 3.082 μg / g, and the average content of benzoylacone was 7.056 μg / g.

[0280] ⑥Stability test

[0281] The test sample was prepared according to the test sample processing method. The same test sample solution was placed at room temperature for 0, 4, 8, 12, 16, and 24 hours, respectively, and liquid chromatography-mass spectrometry was performed. The experimental results showed that the sample had good stability within 24 hours.

[0282] ⑦ Sample recovery experiment

[0283] Take 6 portions of Fuzheng Jiedu granules with known content, add a reference substance equivalent to 100% of the content of the component to be tested in the test sample to prepare the test sample, and process 6 portions in parallel for liquid chromatography-mass spectrometry determination. The experimental results show that the method has high recovery rate and good accuracy in determining the contents of benzoylmonaconitine, benzoylmonaconitine and benzoylmonaconitine.

[0284] (3) Determination of glycyrrhizic acid content

[0285] ① Chromatographic conditions: octadecylsilane bonded silica gel was used as the filler, the detection wavelength was 251 nm, the injection volume was 10 μL, gradient elution, acetonitrile A-0.1% phosphoric acid aqueous solution B was used as the mobile phase, elution conditions: 0 min, A:B=30:70; 15 min, A:B=35:65; 40 min, A:B=45:95; 45 min, A:B=95:5; 50 min, A:B=30:70; 60 min, A:B=30:70, flow rate was 1.0 mL / min, column temperature was 30°C;

[0286] ②Specificity inspection

[0287] Weigh the prescription pieces and get the whole prescription test sample according to the test sample treatment; weigh the negative prescription of Radix Glycyrrhizae Radix and get the negative test sample of Radix Glycyrrhizae Radix by the same method; take 10μL of each test sample and glycyrrhizic acid reference solution and analyze by HPLC injection. The results show that glycyrrhizic acid negative has no interference.

[0288] ③Linear relationship investigation

[0289] Take ammonium glycyrrhizinate reference substance, add 70% ethanol to dissolve, obtain the glycyrrhizic acid solution with concentration of 678.981μg / ml, take this as mother liquor, prepare the standard solution with concentration of 5.432, 27.159, 54.318, 67.898, 101.847, 135.796, 678.981μg / mL respectively. Take 10μL of the reference solution of the above seven concentrations, inject into HPLC injection system, draw the standard curve Y=8434.7X+1367.9 with glycyrrhizic acid concentration-peak area as coordinate axis, r=0.9999, cinnamic acid has good linearity in the range of 5.432-678.981μg / ml.

[0290] ④ Investigation of the pretreatment method of the test sample

[0291] a. Investigation of extraction methods

[0292] Take 1 portion of Fuzheng Jiedu Granules, place it in a 25ml volumetric flask, add 70% ethanol to the mark, shake to extract, filter, and you are done.

[0293] Take 1 portion of Fuzheng Jiedu Granules, place it in a 25ml volumetric bottle, add 70% ethanol to the scale, ultrasonically treat for 20 minutes, take it out, cool it, dilute it to the scale with 70% ethanol, filter it, and you'll get it. After passing through the membrane, use HPLC injection analysis to determine the glycyrrhizic acid content. The results are shown in Table 18. The results show that the glycyrrhizic acid content is higher and the extraction is more complete under ultrasonic extraction, so ultrasonic extraction is selected.

[0294] Table 18. Results of investigation on glycyrrhizic acid extraction method (n=2)

[0295]

[0296] b. Selection of extraction solvent

[0297] Take 5 portions of Fuzheng Jiedu Granules, place them in a 25ml volumetric bottle, add anhydrous ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and water to the scale, respectively, ultrasonically treat for 20 minutes, take out, cool, dilute to the scale with the above extraction solvents, filter, and obtain. Inject into HPLC injection system, determine the glycyrrhizic acid content, the results are shown in Table 19. The results show that the glycyrrhizic acid content is the highest when 70% ethanol is used as the solvent for ultrasonic extraction, and the extraction is more complete, so 70% ethanol is selected as the extraction solvent.

[0298] Table 19. Results of investigation on glycyrrhizic acid extraction solvents (n=5)

[0299]

[0300] b. Selection of extraction time

[0301] Take 3 portions of Fuzheng Jiedu Granules, place them in a 25ml volumetric bottle, add 70% ethanol to the mark, and ultrasonically treat for 5, 20, and 40 minutes respectively, take them out, cool them, dilute them to the mark with 70% ethanol, and filter them. Inject them into the HPLC injection system to determine the glycyrrhizic acid content. The results are shown in Table 19. The results show that the glycyrrhizic acid extraction is basically balanced and the extraction is relatively complete after ultrasonic extraction for 20 minutes, so 20 minutes of ultrasonic treatment is selected as the extraction time.

[0302] Table 19. Glycyrrhizic acid extraction time investigation results (n = 3)

[0303]

[0304]

[0305] ⑤ Handling of test samples

[0306] Take 0.4 g of Fuzheng Jiedu granules powder, place it in a 25 mL volumetric flask, add an appropriate amount of 70% ethanol, ultrasonicate for 20 min to dissolve, let cool, dilute to the scale with 70% ethanol, shake well, centrifuge at high speed, take the supernatant to obtain the glycyrrhizic acid test sample.

[0307] ⑥Precision experiment

[0308] Accurately pipette 10 μL of reference solution of the same concentration and determine it according to the established HPLC analysis method. The sample is injected 6 times in succession. The results show that the instrument has good precision.

[0309] ⑦Repeat experiment

[0310] Fuzheng Jiedu Granules were used to prepare the test sample according to the test sample processing method, and 6 replicates were prepared and determined according to the established HPLC analysis method. The results showed that the method had good repeatability and the average glycyrrhizic acid content in Fuzheng Jiedu Granules was 4613.67 μg / g.

[0311] ⑧Stability test

[0312] The test sample was prepared according to the test sample treatment method, and the same test sample solution was placed at room temperature for 0, 4, 8, 12, 16, 24, and 50 hours, and then analyzed by HPLC. The results showed that the sample had good stability within 50 hours.

[0313] ⑨Sample recovery experiment

[0314] Take 9 portions of freeze-dried products with known content, add 50%, 100%, and 150% of the glycyrrhizic acid content of the test sample to the reference sample, respectively, prepare the test sample according to the test sample processing method, process 3 portions in parallel, and perform HPLC analysis. The results show that this method has a high recovery rate and good accuracy in determining the glycyrrhizic acid content.

[0315] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quality control method for Fuzheng Jiedu granules, characterized in that: The method includes qualitative and quantitative methods. The qualitative method is to control the product quality by establishing a characteristic spectrum of Fuzheng Jiedu granules; specifically, the method is established by controlling 3 to 10 characteristic peaks, wherein the characteristic peaks include chlorogenic acid, liquiritin, hesperidin, ammonium glycyrrhizinate and 6-gingerol; The quantitative method controls the product quality by controlling the contents of diester alkaloids, monoester alkaloids and glycyrrhizic acid.

2. The quality control method according to claim 1, characterized in that: The method for establishing the characteristic spectrum of the Fuzheng Jiedu granules comprises: A) extracting the Fuzheng Jiedu granules with a solvent to obtain a test solution; B) taking chlorogenic acid, liquiritin, hesperidin, ammonium glycyrrhizinate and 6-gingerol standard products, dissolving them in a solvent to obtain a reference substance solution; C) using high performance liquid chromatography to obtain chromatograms of the test solution in step A) and the reference substance in step B) under the same detection conditions, and the specific chromatographic parameters are as follows: The chromatographic column was a C18 column, and the detection wavelength was 237 nm; the mobile phase A was acetonitrile, and the mobile phase B was a 0.1% phosphoric acid aqueous solution; The elution procedure is: 0~12min, phase A 8%~18%; 12-20min phase A 18%-22%; 20-33min Phase A 22%-30%; 33-40min Phase A 30%-43%; 40~55min Phase A 43%~53%; 55~65 min Phase A 53%~75%; 65~70 min Phase A 75%~95%; 70~75 min Phase A 95%~8%; 75~90 min Phase A 8%; D) obtaining a characteristic spectrum of Fuzheng Jiedu Granules according to the reference substance concentration, the peak area of ​​the reference substance solution in the chromatogram, and the peak area of ​​the component in Fuzheng Jiedu Granules corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C).

3. The method according to claim 2, characterized in that The mobile phase flow rate was 1 ml / min, the column temperature was 30°C, and the injection volume was 10 μL; Step A) the solvent is 70% ethanol, the extraction is ultrasonic extraction, and the ultrasonic extraction time is 10 to 20 minutes; The concentration of chlorogenic acid is 40 μg / mL, the concentration of liquiritin is 40 μg / mL, the concentration of hesperidin is 40 μg / mL, the concentration of ammonium glycyrrhizinate is 40 μg / mL, and the concentration of 6-gingerol is 10 μg / mL.

4. The method according to claim 1, characterized in that The quality control method also includes any one or more of the following determination methods: i) HPLC determination of the limit content of diester alkaloids in Fuzheng Jiedu granules; the diester alkaloids are selected from the group consisting of mesaconitine, mesaconitine and aconitine; ii) HPLC determination of the content of monoester alkaloids in Fuzheng Jiedu Granules; the monoester alkaloids are selected from benzoyl aconitine, benzoyl mesaconitine and benzoyl mesaconitine; iii) HPLC determination of glycyrrhizic acid content in Fuzheng Jiedu granules; iv) Identification of dried ginger using thin layer chromatography; v) Identification of Licorice using thin layer chromatography; vi) Identification of honeysuckle by thin layer chromatography; vii) Identification of Gleditsia sinensis thorns by thin layer chromatography; viii) using thin layer chromatography to identify the five-fingered peach; ix) Identification of dried tangerine peel using thin layer chromatography.

5. The method according to claim 4, characterized in that i) The HPLC determination of the diester alkaloid limit content in the Fuzheng Jiedu Granules is specifically as follows: a) extracting the Fuzheng Jiedu granules with a solvent to obtain a test solution; the solvent is a 0.1% formic acid methanol solution; b) using high performance liquid chromatography-mass spectrometry to conduct qualitative and quantitative analysis of the components of the test liquid; The specific chromatographic parameters are as follows: The chromatographic column was a C18 chromatographic column, the mobile phase A was 0.1% formic acid methanol solution, and the mobile phase B was 0.1% formic acid aqueous solution; Gradient elution: 0-3min, phase A 30%; 3-15min Phase A 30%-80%; 15-18min Phase A 80%-90%; 18~19 min Phase A 90%~30%; The mobile phase flow rate was 0.2 ml / min; the column temperature was 30 °C, and the injection volume was 1 to 10 μL; Mass spectrometry conditions: ionization mode was electrospray ionization positive ion mode; multiple reaction (MRM) detection mode; The content of the diester type alkaloid does not exceed 5.67 μg.

6. The method according to claim 5, characterized in that The specific content of monoester alkaloids in Fuzheng Jiedu Granules determined by HPLC is: a) extracting the Fuzheng Jiedu granules with a solvent to obtain a test solution; the solvent is a 0.1% formic acid methanol solution; b) using high performance liquid chromatography-mass spectrometry to conduct qualitative and quantitative analysis of the components of the test liquid; The specific chromatographic parameters are as follows: The chromatographic column was a C18 chromatographic column, the mobile phase A was: 0.1% formic acid methanol solution, and the mobile phase B was 0.1% formic acid aqueous solution; Gradient elution: 0-3min, phase A 30%; 3-17min Phase A 30%-60%; 17-23min Phase A 60%-90%; 23~24 min Phase A 90%~30%; The mobile phase flow rate was 0.2 ml / min; the column temperature was 30 °C, and the injection volume was 1 μL; Mass spectrometry conditions: ionization mode was electrospray ionization positive ion mode; multiple reaction (MRM) detection mode; The content of the monoester alkaloid ranges from 0.017 mg to 0.039 mg.

7. The method according to claim 4, characterized in that iii) The content of glycyrrhizic acid in Fuzheng Jiedu Granules was determined by HPLC as follows: a) extracting Fuzheng Jiedu granules with 70% ethanol to obtain a test solution; b) using high performance liquid chromatography-mass spectrometry to conduct qualitative and quantitative analysis of the components of the test liquid; The specific chromatographic parameters are as follows: The chromatographic column is a C18 column, the detection wavelength is 251nm; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution; the mobile phase flow rate is 1.0ml / min; the chromatographic column temperature is 30℃, the injection volume is 10μL; gradient elution: 0-15min, phase A 30%-35%; 15-40min Phase A 35%-45%; 40-45min Phase A 45%-95%; 45-50min Phase A 95%-30%; 50-60min Phase A 30%; The glycyrrhizic acid content ranges from 4.0 mg to 7.5 mg.

8. The method according to claim 4, characterized in that The specific steps of iv) using thin layer chromatography to identify dried ginger are: a) extracting the Fuzheng Jiedu granules, the negative test sample of dried ginger and the control medicinal material of dried ginger with ethyl acetate to obtain a test sample solution, a negative test sample of dried ginger and a control medicinal material solution of dried ginger, respectively; Dissolve the 6-gingerol reference substance in ethyl acetate to obtain a 6-gingerol reference substance solution; b) performing thin layer chromatography on the test solution, the negative test sample of dried ginger, the control medicinal material solution of dried ginger, and the reference solution of 6-gingerol, wherein the thin layer plate is a silica gel G thin layer plate; the developing solvent is petroleum ether-chloroform-ethyl acetate, and the volume ratio of the petroleum ether-chloroform-ethyl acetate is 2:1:1; c) Inspection: If the test sample, the dry ginger control medicinal material, and 6-gingerol have fluorescent spots of the same color at the same Rf value, and the dry ginger negative test sample has no spots, then it is dry ginger; The inspection is specifically as follows: spraying with 2% vanillin sulfuric acid solution, heating at 105° C. until the spots are clearly colored; the spotting volume is 6 to 10 μL.

9. The method according to claim 4, characterized in that The method of using thin layer chromatography to identify liquorice is specifically as follows: a) After dissolving Fuzheng Jiedu Granules, licorice negative test sample and licorice control medicinal material in water, adjusting the pH value to 11-12 with 5% sodium hydroxide, filtering, adding acid to adjust the pH value to 4-5, adding ethyl acetate for extraction, evaporating to dryness and then extracting with methanol to obtain a test sample solution, a licorice negative test sample and a licorice control medicinal material solution, respectively; Add methanol to dissolve the liquiritin reference substance to obtain a liquiritin reference substance solution; b) performing thin layer chromatography on the test solution, the licorice negative test sample, the liquiritin reference substance and the licorice reference medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is ethyl acetate-formic acid-glacial acetic acid-water, and the volume ratio of ethyl acetate-formic acid-glacial acetic acid-water is 12:1:1:2; c) Inspection: If the test sample, licorice control medicinal material, and liquiritin have spots of the same color at the same Rf value, and the licorice negative test sample has no spots, then it is licorice; The inspection is performed by spraying with 10% sulfuric acid ethanol solution and heating at 105° C. until the spots are clearly colored; the spotting volume is 2 μL.

10. The method according to claim 4, characterized in that The method of using thin layer chromatography to identify honeysuckle is specifically as follows: a) extracting Fuzheng Jiedu granules and honeysuckle negative test samples with methanol, filtering and then extracting with ether, discarding the ether layer and then extracting with saturated n-butanol, evaporating the n-butanol layer and dissolving it with methanol to obtain a test sample solution and a honeysuckle negative test sample solution, respectively; The honeysuckle control medicinal material is extracted with methanol to obtain a honeysuckle control medicinal material solution; Dissolve the chlorogenic acid reference substance in methanol to obtain a chlorogenic acid reference substance solution; b) performing thin layer chromatography on the test solution, the honeysuckle negative test product, the honeysuckle control medicinal material solution and the chlorogenic acid reference solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is butyl acetate-formic acid-water, and the volume ratio of butyl acetate-formic acid-water is 14:5:5; c) Inspection: If the test sample, the honeysuckle control medicinal material, and chlorogenic acid have spots of the same color at the same Rf value, and the honeysuckle negative test sample has no spots, then it is honeysuckle; The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 1 μL.

11. The method according to claim 4, characterized in that The method of using thin layer chromatography to identify Gleditsia sinensis thorns is specifically as follows: a) Extract the Fuzheng Jiedu Granules and the Gleditsia sinensis negative test sample with methanol, filter, dissolve in water, and then extract with ether, discard the ether layer and extract with saturated n-butanol, evaporate the n-butanol layer and dissolve in methanol to obtain a test sample solution and a Gleditsia sinensis negative test sample solution, respectively; The Gleditsia sinensis reference medicinal material was extracted with methanol to obtain a Gleditsia sinensis reference medicinal material solution; b) performing thin layer chromatography on the test solution, the negative test sample of Gleditsia sinensis and the control medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is petroleum ether-ethyl acetate-formic acid, and the volume ratio of petroleum ether-ethyl acetate-formic acid is 10:10:0.5; c) Inspection: If the test sample and the Gleditsia sinensis control medicinal material have fluorescent spots of the same color at the same Rf value, and the Gleditsia sinensis negative test sample has no spots, then it is Gleditsia sinensis; The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 5 μL.

12. The method according to claim 4, characterized in that The method of using thin layer chromatography to identify the five-fingered peach is specifically as follows: a) extracting the Fuzheng Jiedu granules, the negative test sample of Eleutherodendron chinense and the Eleutherodendron chinense control medicinal material solution with ethyl acetate to obtain a test sample solution, a negative test sample solution of Eleutherodendron chinense and a Eleutherodendron chinense control medicinal material solution, respectively; Extracting psoralen with ethyl acetate to obtain a psoralen control medicinal material solution; b) performing thin layer chromatography on the test solution, the negative test sample of Psoralea corylifolia, the psoralen reference solution and the Psoralea corylifolia reference medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is n-hexane-butyl acetate, and the volume ratio of n-hexane-butyl acetate is 4:1; c) Inspection: If the test sample, the reference medicinal material of Eleutherodactylus quinquefolius, and psoralen have fluorescent spots of the same color at the same Rf value, and the Eleutherodactylus quinquefolius negative test sample has no spots, then it is Eleutherodactylus quinquefolius; The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 1 μL.

13. The method according to claim 4, characterized in that The method of using thin layer chromatography to identify dried orange peel is specifically as follows: a) extracting the Fuzheng Jiedu granules and the tangerine peel negative test solution with 0.05% sodium hydroxide, adding acid to adjust the pH value to 4-5, adding water to the precipitate and centrifuging it, dissolving the precipitate after centrifugation with methanol, evaporating it to dryness and dissolving it with methanol to obtain a test solution and a tangerine peel negative test solution, respectively; Extract the tangerine peel control medicinal material with methanol to obtain a tangerine peel control medicinal material solution; Hesperidin was dissolved in methanol to obtain a hesperidin reference solution; b) performing thin layer chromatography on the test solution, the tangerine peel negative test solution, the hesperidin reference solution and the tangerine peel reference medicinal material solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is ethyl acetate-methanol-water, and the volume ratio of ethyl acetate-methanol-water is 100:17:13; c) Inspection: If the test sample, the tangerine peel control medicinal material, and hesperidin have fluorescent spots of the same color at the same Rf value, and the tangerine peel negative test sample has no spots, then it is tangerine peel; The inspection is specifically as follows: spraying with 1% aluminum chloride solution and placing under ultraviolet light for inspection; the wavelength of the ultraviolet light is 365nm; and the sample spotting volume is 4 to 6 μL.

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