A quality control method of fangji decoction pieces

Abstract

The application discloses a quality control method of radix stephaniae tetrandrae decoction pieces, adopts liquid chromatography-mass spectrometry to detect the efficacy components in the radix stephaniae tetrandrae decoction pieces, and the efficacy components include any one or several of tetrandrine, fangchinoline, cycilanol, stepholidine, oxystepholidine, stephaoxime, corynoxeine, miliradin and oxymatrine. The specific detection condition can detect multiple efficacy components in the radix stephaniae tetrandrae decoction pieces, which is helpful for comprehensively controlling the quality of the radix stephaniae tetrandrae decoction pieces, and finding the possibility of replacing wild radix stephaniae tetrandrae through efficacy evaluation. In addition, the thin layer chromatography is used to identify the radix stephaniae tetrandrae decoction pieces before detection, and the identification sensitivity can be improved by adjusting the thin layer chromatography condition.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for traditional Chinese medicine decoction pieces, and particularly relates to a quality control method for Fangji decoction pieces. Background Technology

[0002] Fangji is a plant of the Menispermaceae family and the genus Stephania tetrandra (also known as Stephania tetrandra). Stephania tetrandra The root of *Stephania tetrandra* (S. Moore) is bitter and cold in nature, and enters the bladder and lung meridians. It has the effects of dispelling wind and relieving pain, promoting diuresis and reducing swelling, and is used to treat rheumatic pain, edema, beriberi, dysuria, eczema, and sores. Modern research shows that *Stephania tetrandra* contains various components including alkaloids, flavonoids, volatile oils, sterols, and organic acids, exhibiting good pharmacological activity in anti-inflammatory, anti-tumor, anti-platelet aggregation, anti-allergic, and cardioprotective effects.

[0003] The current pharmacopoeia for quality control of Fangji (Stephania tetrandra) slices only includes tetrandrine and fangchinorline, requiring a total content of not less than 1.4%, and using high-performance liquid chromatography (HPLC) for content detection. However, Fangji slices also contain many other pharmacologically active ingredients such as rotenone, tetrandrine, oxytetracycline, tetrandrine, corydaline, mildin, and gentianine, which also possess certain biological activities. Detecting these ingredients is of great significance for the quality control of Fangji slices. Currently, the simple HPLC method is insufficient for the simultaneous detection of multiple pharmacologically active ingredients.

[0004] In addition, Fangji includes wild Fangji and artificially cultivated Fangji. Wild Fangji has unique biological activity and medicinal properties. As the demand for Fangji increases, the storage of wild Fangji is gradually decreasing. Therefore, it is necessary to control the quality of existing Fangji slices and find Fangji slices that are closer to the medicinal components of wild Fangji, so that wild Fangji can be replaced. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a quality control method for Stephania tetrandra slices. This method uses liquid chromatography-mass spectrometry (LC-MS) to detect the active ingredients in Stephania tetrandra slices, thereby controlling their quality. Under specific detection conditions, multiple active ingredients can be detected, facilitating comprehensive quality control and allowing for the identification of potential substitutes for wild Stephania tetrandra through efficacy evaluation. Furthermore, this invention employs thin-layer chromatography (TLC) to identify Stephania tetrandra slices before detection; adjusting the TLC conditions improves the identification sensitivity.

[0006] The first objective of this invention is to use liquid chromatography-mass spectrometry to detect multiple medicinal components in Fangji decoction pieces, thereby enabling comprehensive quality control of Fangji decoction pieces.

[0007] To achieve the first objective, the present invention adopts the following technical solution: A quality control method for Stephania tetrandra slices involves using liquid chromatography-mass spectrometry (LC-MS) to detect the active ingredients in Stephania tetrandra slices for quality control. The detection conditions include: Liquid chromatography conditions: detection wavelength 280 nm, water as mobile phase A, acetonitrile as mobile phase B, gradient elution; Mass spectrometry conditions: First-stage mass spectrometry conditions: drying gas temperature 500℃, spray voltage +5500V, declusing voltage +55V, collision energy +10V, cumulative time 0.1 s; Second-stage mass spectrometry conditions: declusing voltage +80V, collision energy +40V, collision energy variation 15V, cumulative time 0.065 s.

[0008] Preferably, the gradient elution conditions for liquid chromatography are as follows: From 0 to 1.0 min, the volume concentrations of mobile phase A and mobile phase B were 90% and 10%, respectively. 1.0~2.0 min, the volume concentrations of mobile phase A and mobile phase B are 90%~75% and 10%~25%, respectively; The volume concentrations of mobile phase A and mobile phase B were 75%–70% and 25%–30%, respectively, over a period of 2.0–5.4 min. 5.4~5.5 min, the volume concentrations of mobile phase A and mobile phase B are 70%~9% and 30%~91%, respectively; The volume concentrations of mobile phase A and mobile phase B were 9%–7% and 91%–93% respectively over a period of 5.5–9.3 min. From 9.3 to 9.4 min, the volume concentrations of mobile phase A and mobile phase B were 7%–90% and 93%–10%, respectively. 9.4~10 min, the volume concentrations of mobile phase A and mobile phase B are 90% and 10%, respectively.

[0009] Preferably, the liquid chromatography conditions also include: using octadecylsilane-bonded silica gel as the column packing material with a particle size of 1.7 μm, a column size of 2.1 × 100 mm, a column temperature of 35~45℃, a flow rate of 0.2~0.5 ml / min, and an injection volume of 1~3 μL.

[0010] Preferably, the mass spectrometry conditions also include: in the first-stage mass spectrometry conditions, a dual ion source is used, with ion source gas 1 at 45 psi and ion source gas 2 at 50 psi, and the molecular weight range is scanned from 100 Da to 1500 Da; in the second-stage mass spectrometry conditions, the molecular weight range is scanned from 100 Da to 1500 Da.

[0011] Preferably, the active ingredients in the Fangji decoction pieces include any two or more of the following: tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine.

[0012] Preferably, the content of each active ingredient in the Fangji decoction pieces is controlled in accordance with the pharmacopoeia, and the sample test results need to meet the content standards in the pharmacopoeia (calculated on a dried basis, the total amount of fangchialkaloid and fangchinorline shall not be less than 1.4%).

[0013] Preferably, the prepared fangji slices include wild varieties and artificially cultivated varieties.

[0014] Preferably, in order to further evaluate the similarity between the nine medicinal components in artificially cultivated Stephania tetrandra and wild Stephania tetrandra, liquid chromatography-mass spectrometry was used to detect and compare the processed Stephania tetrandra slices of artificially cultivated and wild varieties, and at least one of the following conditions must be met: (1) The total content of tetrandrine and tebufenozide is ≥1.4%; (2) Calculate the ratio of the peak area of ​​the medicinal components of the artificially cultivated variety to that of the wild variety of Stephania tetrandra slices. The sum of the ratios of the peak areas of tetrandrine and tetrandrine is ≥1.0. (3) Sort the active ingredients in the order of: rotenone, tetrandrine, tetrandrine, tetrandrine, tetrandrine, corydaline, mirendine, and peperomialine, in order from 1 to 7. Then, perform polynomial fitting on the peak area of ​​the active ingredients of the cultivated and wild varieties of Stephania tetrandra slices and the corresponding active ingredient number to obtain the polynomial fitting function. The coefficients of the fitting functions of the cultivated and wild varieties must have the same sign and the degree of fit must be ≥0.800. (4) The content of aristolochic acid I or aristolochic acid II is ≤0.001%.

[0015] The second objective of this invention is to identify Fangji slices using thin-layer chromatography, thereby further enhancing the quality control of Fangji slices.

[0016] To achieve the second objective, the present invention adopts the following technical solution: Thin-layer chromatography was used to identify Stephania tetrandra slices, and the identification conditions included at least one of the following: A1, GF254 board; A2. Developing solvent: ethyl acetate-acetone-methanol-5% concentrated ammonia, volume ratio 5:2:0.7:0.3; A3. Colorimetric reagent: dilute potassium bismuth iodide solution; A4. Examine under daylight or ultraviolet light at 254nm; A5. The sample volume is 6~10μL; A6. Spots of the same color appear at the corresponding positions on the chromatogram of the reference standard.

[0017] Preferably, the steps of detection by liquid chromatography-mass spectrometry and identification by thin-layer chromatography further include the preparation of a test solution, wherein the preparation conditions include at least one of the following: B1. Ultrasonic extraction or reflux extraction; B2. Grind the Stephania tetrandra medicinal material to 40-60 mesh before extraction; B3. The extraction solvent is methanol or an aqueous methanol solution, with a volume concentration of 60-80% for the aqueous methanol solution. B4. The volume-to-mass ratio of the extraction solvent to the *Stephania tetrandra* herb is 20-50 mL: 1 g; B5. Extraction time is 15~60min; B6. After extraction, the supernatant is evaporated to dryness to obtain an extract. Methanol is added to prepare a solution of 0.1-0.3 mg / mL, which is then diluted 100-200 times to prepare the test solution.

[0018] More preferably, the preparation conditions for the test solution include at least one of the following: B1. Ultrasonic extraction, power 150~250W; B2. Grind the Stephania tetrandra medicinal material to 60 mesh before extraction; B3. The extraction solvent is an 80% (v / v) methanol aqueous solution; B4. The volume-to-mass ratio of the extraction solvent to the *Stephania tetrandra* herb is 50 mL: 1 g; B5. Extraction time is 20 minutes.

[0019] Preferably, the steps of detection by liquid chromatography-mass spectrometry and identification by thin-layer chromatography further include preparing a reference solution, wherein the reference standard includes tetrandrine and fangchinorline, the solvent for preparing the reference solution is methanol, and the concentration of the reference solution is 0.1~1 mg / mL. More preferably, the concentration of the tetrandrine reference solution is 0.5~1 mg / mL, and the concentration of the fangchinorline reference solution is 0.1~0.5 mg / mL.

[0020] The beneficial effects of this invention are: 1. This invention employs liquid chromatography-mass spectrometry to detect nine pharmacologically active components in Fangji (including tetrandrine, fangchinorinine, rotenone, fangchinensis, oxytetracycline, fangchinone, corydaline, mirendine, and gentianine) in Fangji decoction pieces for quality control. By using specific detection conditions, multiple pharmacologically active components in Fangji decoction pieces can be detected, which helps to conduct comprehensive quality control of Fangji decoction pieces, rather than being limited to the conventional tetrandrine and fangchinorinine.

[0021] 2. This invention further evaluates the similarity between nine medicinal components in artificially cultivated Stephania tetrandra and wild Stephania tetrandra through the above-mentioned detection method. By classifying the medicinal components, fangchinoline, fangchinoline and the other seven medicinal components are evaluated separately, which can more accurately determine the relationship between the different medicinal contents of artificially cultivated Stephania tetrandra and wild Stephania tetrandra, and find the possibility of replacing wild Stephania tetrandra.

[0022] 3. Before detection, the present invention also uses thin-layer chromatography to identify the Fangji medicinal slices. By examining the various conditions of thin-layer chromatography, the identification sensitivity can be improved.

[0023] 4. Furthermore, the liquid chromatography-mass spectrometry (LC-MS) detection method of this invention can also improve the detection sensitivity of harmful components (aristolochic acid I and aristolochic acid II) in medicinal slices. Due to the limitations of previous methods for detecting the active ingredients of Stephania tetrandra, aristolochic acid I or aristolochic acid II may be present in some medicinal slices (low content, difficult to identify). The detection method of this invention uses specific gradient elution and specific detection conditions to accurately identify and obtain the characteristic peaks of aristolochic acid in medicinal slices, thereby accurately detecting aristolochic acid I and aristolochic acid II in Stephania tetrandra and further improving the safety of medicinal slices. Attached Figure Description

[0024] Figure 1 The results of thin-layer chromatography analysis of Stephania tetrandra in Example 1 of this invention; Figure 2 The results of thin-layer chromatography analysis using methanol aqueous solution of different concentrations and ethanol under heating and reflux extraction were obtained during the investigation of different extraction solvents and extraction methods in Example 1 of this invention. Figure 3 The thin-layer chromatographic analysis results are obtained by ultrasonic extraction with 60% methanol aqueous solution and 80% methanol aqueous solution during the investigation of different extraction solvents and extraction methods in Example 1 of the present invention. Figure 4 The results of thin-layer chromatography analysis of developing solvent 1 during the investigation of different developing solvents in Example 1 of the present invention; Figure 5 The results of thin-layer chromatography analysis of developing solvent 2 during the investigation of different developing solvents in Example 1 of this invention; Figure 6 The results of thin-layer chromatography analysis of developing solvent 3 during the investigation of different developing solvents in Example 1 of this invention; Figure 7 The results of thin-layer chromatography analysis of developing solvent 4 during the investigation of different developing solvents in Example 1 of this invention; Figure 8 The results of thin-layer chromatography analysis were investigated for different sample amounts in Example 1 of this invention. Figure 9The results of thin-layer chromatography analysis using ultraviolet lamp (254+365nm) and 10% sulfuric acid ethanol solution for colorimetric detection during the examination of different detection methods in Example 1 of the present invention; Figure 10 The results of thin-layer chromatography analysis using ultraviolet lamp (254+365nm) and potassium bismuth iodide solution for colorimetric detection during the examination of different detection methods in Example 1 of this invention; Figure 11 The results of thin-layer chromatography analysis of silica gel H thin-layer plate during the investigation of different types of thin-layer plates in Example 1 of the present invention; Figure 12 The results of thin-layer chromatography analysis of silica gel G thin-layer plates during the investigation of different types of thin-layer plates in Example 1 of the present invention; Figure 13 The results of thin-layer chromatography analysis of silica gel GF254 thin-layer plate during the investigation of different types of thin-layer plates in Example 1 of the present invention; Figure 14 The results of thin-layer chromatography analysis of the Merck GF254 thin-layer plate from Germany during the evaluation of thin-layer plates from different manufacturers in Example 1 of this invention; Figure 15 The results of thin-layer chromatography analysis of Qingdao Dingkang GF254 thin-layer plate during the investigation of thin-layer plates from different manufacturers in Example 1 of this invention; Figure 16 The results of thin-layer chromatography analysis of Yantai Jiangyou GF254 thin-layer plate during the investigation of thin-layer plates from different manufacturers in Example 1 of this invention; Figure 17 The results of thin-layer chromatography analysis of Qingdao Ocean GF254 thin-layer plate during the investigation of thin-layer plates from different manufacturers in Example 1 of this invention; Figure 18 This is the liquid chromatography-mass extraction spectrum of the active ingredient of artificially cultivated Stephania tetrandra 5# in Example 2 of the present invention; Figure 19 This is the liquid chromatography-mass extraction spectrum of the active ingredient of artificially cultivated Stephania tetrandra 6# in Example 2 of the present invention; Figure 20 This is the liquid chromatography-mass extraction spectrum of the active ingredient of artificially cultivated Stephania tetrandra 7# in Example 2 of the present invention; Figure 21 This is the fitting result of the peak areas of the other seven medicinal components in artificially cultivated Stephania tetrandra 5# in Example 2 of the present invention; Figure 22 This is the fitting result of the peak areas of the other seven medicinal components in artificially cultivated Stephania tetrandra 6# in Example 2 of the present invention; Figure 23 This is the fitting result of the peak areas of the other seven medicinal components in artificially cultivated Stephania tetrandra 7# in Example 2 of the present invention; Figure 24 This is the fitting result of the peak areas of the other seven medicinal components in wild Stephania tetrandra 8# in Example 2 of the present invention.

[0025] Figure 1 , Figure 2 , Figures 4-8 , Figures 10-17 In the middle: the left images are all under 254nm ultraviolet light, and the right images are all under sunlight after spraying with potassium bismuth iodide; Figure 3 In the middle: the left side shows the color development under sunlight after spraying with potassium bismuth iodide, and the right side shows the color development under a 254nm ultraviolet lamp; Figure 9 In the middle: left is examined under a 254nm UV lamp, middle is examined under a 365nm UV lamp, and right is examined under sunlight after color development with 10% sulfuric acid ethanol solution. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0028] Example 1: Thin-layer chromatographic identification of Stephania tetrandra slices 1. Reagents and Materials Fangji (Stephania tetrandra) slices (randomly collected raw slices from Ruichang, Jiangxi). Fangjinorline reference standard (China National Institutes for Food and Drug Control, catalog number: 110711-201810), fangjinorline reference standard (China National Institutes for Food and Drug Control, batch number: 110793-202108). Ethanol, methanol, ethyl acetate, acetone, ammonia, sulfuric acid, etc., were all analytical grade; water was ultrapure water.

[0029] Thin-layer plates: Silicone GF254 thin-layer plate 20×10cm (Merck, Germany); Silicone GF254 thin-layer plate 20×10cm, Silicone G thin-layer plate 20×10cm, Silicone H thin-layer plate 20×10cm (Qingdao Haiyang Silicone Chemical Plant); Silicone GF254 thin-layer plate 20×10cm (Qingdao Dingkang), Silicone G thin-layer plate 20×10cm (Yantai Jiangyou).

[0030] 2. Preparation of the test solution Take 1g of the herbal powder, pulverize it, and pass it through a 60-mesh sieve. Take 1g of the herbal powder, add 20mL of 80% methanol aqueous solution, and extract by ultrasonication at 200W for 20 min. After centrifugation, collect the supernatant and evaporate it to dryness using a rotary evaporator. Take the extract, add chromatographic methanol to prepare a 0.1mg / ml solution, and then dilute it 100 times to obtain the final product.

[0031] 3. Preparation of reference solution Accurately weigh the tetrandrine and fangchinorline reference standards, and add methanol to prepare reference solutions containing 1.0 mg of tetrandrine and 0.5 mg of fangchinorline per 1 mL.

[0032] 4. Thin-layer chromatography analysis The test was conducted according to the Thin Layer Chromatography Method (General Rule 0502) of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0033] Thin-layer board: German Merck GF254 board or Qingdao Dingkang GF254 board; Sample volume: 8 μL; Developing solvent: ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3). Colorimetric reagent: Spray with potassium bismuth iodide test solution; Inspection: Examine under sunlight and ultraviolet light (254nm) respectively; Result: See Figure 1 (Note: 1. Tetrandrine reference standard; 2. Stephania tetrandra test sample; 3. Tetrandrine powder reference standard) Spots of the same color appear at the corresponding positions on the chromatogram of the reference standard. Through multiple experiments, the development effect is good and the repeatability is good, which can be used as a quality control indicator to control the quality of Stephania tetrandra slices.

[0034] 5. Investigation of different extraction solvents and extraction methods Methanol aqueous solutions with volume concentrations of 15%, 30%, 45%, 60%, 80%, and 100%, as well as 100% ethanol, were selected as extraction solvents. Seven portions of Stephania tetrandra slices were weighed, and each was extracted by heating and reflux for 1 hour using a different extraction solvent. The remaining steps were the same as in step 2 above, resulting in different test solutions. The preparation of the reference solution was the same as in step 3 above.

[0035] Take 8 μL each of the different test solutions and the reference solutions of tetrandrine and tebufenozide, and spot them separately onto the same silica gel GF254 thin-layer plate. Develop the plate using ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3). Remove the plate, air dry, and examine it under ultraviolet light (254 nm). Spots of the same color should appear at the corresponding positions as those of the reference solutions. Spray with dilute potassium bismuth iodide reagent and examine under sunlight. Spots of the same color should appear at the corresponding positions as those of the reference solutions. See the results below. Figure 2(Note: 1. Tetrandrine reference standard; 2. Test sample prepared from 15% methanol aqueous solution; 3. Test sample prepared from 30% methanol aqueous solution; 4. Test sample prepared from 45% methanol aqueous solution; 5. Test sample prepared from 60% methanol aqueous solution; 6. Test sample prepared from 80% methanol aqueous solution; 7. Test sample prepared from 100% methanol aqueous solution; 8. Test sample prepared from 100% ethanol aqueous solution; 9. Tetrandrine reference standard).

[0036] like Figure 2 As shown, the test solutions prepared with different extraction solvents all exhibited the same color or fluorescent spots at the same positions as the reference standard chromatograms. However, the spots of the test solutions prepared with 60%, 80%, and 100% methanol aqueous solutions were more obvious than those prepared with other solvent gradients, and the number of spots after alkaloid color development of the test solutions prepared with 60% and 80% methanol aqueous solutions was greater than that of the 100% methanol aqueous solution.

[0037] However, both the 60% and 80% methanol aqueous solutions yielded a small amount of precipitate, which may be due to the extraction method. Therefore, two additional 60% and 80% methanol aqueous solutions were used for ultrasonic extraction (200W) for 20 min, with the remaining steps the same as in step 2 above, to prepare two test solutions. Both solutions were clear and showed no precipitate. The two test solutions were spotted together again, and the development was evaluated using ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 2:3:5:0.2). The remaining conditions for thin-layer chromatography were the same as above, and the results are as follows. Figure 3 (Note: 1. Test sample extracted by ultrasonic extraction with 60% methanol aqueous solution; 2. Test sample extracted by ultrasonic extraction with 80% methanol aqueous solution) as shown.

[0038] Depend on Figure 3 It can be seen that the color spots of the test sample solution obtained by ultrasonic extraction with 80% methanol aqueous solution are more concentrated, and the color development of potassium bismuth iodide is more obvious. Therefore, this experiment prefers 80% methanol aqueous solution as the extraction solvent, and suggests that the ultrasonic extraction method is more effective.

[0039] 6. Investigation of different developing solvents The developing effects of the following four developing solvents (developing solvent 1 to developing solvent 4) were examined. 8 μL each of the test solutions prepared in steps 2 and 3 above, the tetrandrine reference solution, and the tebufenozide reference solution were spotted onto the same silica gel GF254 thin-layer plate. Each plate was developed using a different developing solvent, removed, dried, and examined under ultraviolet light (254 nm) and sprayed with dilute potassium bismuth iodide reagent. The results are shown below. Figures 4-7 (Note: 1. Tetrandrine reference standard; 2. Tetrandrine test sample; 3. Tetrandrine reference standard).

[0040] Developing solvent 1: ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3). Developing solvent 2: chloroform-acetone-methanol-5% concentrated ammonia (volume ratio 6:1:1:0.1). Developing solvent 3: Cyclohexane-acetone-ethyl acetate-5% concentrated ammonia (volume ratio 5:2:1:0.3). Developing solvent 4: ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 2:3:5:0.2).

[0041] like Figures 4-7 As shown, the TLC chromatogram of developing solvent 1 has a moderate Rf value compared to the two reference standards using other developing solvents, and the resolution is better. Developing solvents 2 and 3 have lower polarity, and the spot clarity of developing solvent 4 is similar to that of developing solvent 1, but the Rf values ​​of the two reference standards are larger, and the resolution is slightly inferior to that of developing solvent 1. Taking all factors into consideration, developing solvent 1 is preferred, which is ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3).

[0042] 7. Investigation of different sample sizes Take the test solutions, tetrandrine reference solutions, and tebufenozide reference solutions prepared in steps 2 and 3 above, and spot 3 μL, 5 μL, 8 μL, and 10 μL respectively onto the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3) as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (254 nm) and under sunlight after spraying with dilute potassium bismuth iodide reagent. See below for results. Figure 8 (Note: 1. Sample volume 3 μL; 2. Sample volume 5 μL; 3. Sample volume 8 μL; 4. Sample volume 10 μL).

[0043] like Figure 8 As shown, when the sample volume was 3 μL, 5 μL, 8 μL, and 10 μL, the test solution showed the same spots at the positions opposite to the reference chromatogram under both sunlight and fluorescence. However, at 3 μL and 5 μL, the clarity of the test solution after color development with potassium bismuth iodide solution was insufficient, while at 10 μL, each spot showed varying degrees of tailing under 254 nm fluorescence, indicating that the sample volume was too large. Considering all factors, 8 μL was the preferred sample volume.

[0044] 8. Examination of different inspection methods Two inspection methods were investigated: ultraviolet light (254 nm and 365 nm) and spraying with colorimetric reagents (bismuth iodide potassium solution for alkaloids and 10% sulfuric acid ethanol solution for general colorimetric reagents). 8 μL each of the test solutions prepared in steps 2 and 3 above, the tetrandrine reference solution, and the tebufenozide reference solution were spotted onto the same silica gel GF254 thin-layer plate (produced by Qingdao Dingkang). The plate was developed using ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 2:3:5:0.2). The plates were then removed, dried, and examined under ultraviolet light at 254 nm and 365 nm, respectively. After spraying with 10% sulfuric acid ethanol solution for general colorimetric reagents and heating until color development was observed, the plates were examined again. Results are shown below. Figure 9 (Note: 1. Tetrandrine reference standard; 2. Tetrandrine test sample; 3. Tetrandrine reference standard).

[0045] like Figure 9 As shown, only tetrandrine and tebufenozide are visible under 254nm ultraviolet light, while more spots are visible under 365nm ultraviolet light, but the clarity is poor. After spraying with 10% sulfuric acid ethanol solution and heating for color development, tetrandrine and tebufenozide are no longer visible.

[0046] Therefore, examination was performed under 254 nm ultraviolet light. Separately, 8 μL each of the test solutions prepared in steps 2 and 3 above, the tetrandrine reference solution, and the tebufenozide reference solution were spotted onto the same silica gel GF254 thin-layer plate (produced by Yantai Jiangyou). The plate was developed using ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3). The plates were then removed, dried, and examined under a 254 nm ultraviolet lamp, followed by examination with bismuth potassium iodide reagent, a special colorimetric reagent for alkaloids. The results are shown below. Figure 10 (Note: 1. Tetrandrine reference standard; 2. Tetrandrine test sample; 3. Tetrandrine reference standard).

[0047] like Figure 10 As shown, under 254 nm ultraviolet light, the test solution showed spots at the same positions as the reference chromatograms. After spraying with dilute potassium bismuth iodide solution and examining under sunlight, both tetrandrine and tebufenozide reference standards showed orange-red spots, and the test solution showed spots of the same color at the same positions as the reference chromatograms. Taking all factors into consideration, fluorescence examination at 254 nm under ultraviolet light and examination under sunlight after spraying with potassium bismuth iodide solution (a special colorimetric reagent for alkaloids) were selected.

[0048] 9. Examination of different types of thin-layer boards Take 8 μL each of the test solutions, tetrandrine reference solutions, and tebufenozide reference solutions prepared in steps 2 and 3 above, and spot them onto silica gel H, silica gel G, and silica gel GF254 thin-layer plates produced by Qingdao Marine Silica Gel Chemical Plant, respectively. Develop the plates using ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3), remove them, air dry, and examine them under ultraviolet light (254 nm) and after spraying with dilute potassium bismuth iodide solution and then under sunlight. The results are shown in the table below. Figures 11-13 (Note: 1. Tetrandrine reference standard; 2. Tetrandrine test sample; 3. Tetrandrine reference standard).

[0049] like Figures 11-13 As shown, both silica gel G and silica gel H thin-layer plates showed no color development at 254 nm. However, after spraying with potassium bismuth iodide as a colorimetric reagent, the spots were either unclear or appeared as bands due to tailing. In contrast, the GF254 thin-layer plate showed corresponding spots at the reference standard after both 254 nm fluorescence development and development under sunlight with potassium bismuth iodide. Therefore, the GF254 thin-layer plate was selected.

[0050] 10. Comparison of thin-layer boards from different manufacturers Take 8 μL each of the test solutions, tetrandrine reference solutions, and tebufenozide reference solutions prepared in steps 2 and 3 above, and spot them onto silica gel GF254 thin-layer plates produced by different manufacturers (Merck, Qingdao Dingkang, Yantai Jiangyou, and Qingdao Haiyang). Use ethyl acetate-acetone-methanol-5% concentrated ammonia (volume ratio 5:2:0.7:0.3) as the developing solvent. Develop, remove, and air-dry. Examine under ultraviolet light (254 nm) and under sunlight after spraying with dilute potassium bismuth iodide reagent, respectively, to investigate the thin-layer plates from different manufacturers. Results are shown below. Figures 14-17 (Note: 1. Tetrandrine reference standard; 2. Tetrandrine test sample; 3. Tetrandrine reference standard).

[0051] like Figures 14-17 As shown, under 254nm UV light examination, thin-layer chromatography (TLC) plates from different manufacturers could separate tetrandrine and fangchinorline reference standards. However, after development with potassium bismuth iodide solution, no spots were visible on the Yantai Jiangyou GF254 TLC plate, while the Qingdao Haiyang GF254 TLC plate still showed alkaloid spots, but with a low Rf value. The German Merck silica gel GF254 and Qingdao Dingkang GF254 TLC plates showed good separation of tetrandrine and fangchinorline reference standards, with moderate Rf values. Clear spots were visible under both UV fluorescence and sunlight development with potassium bismuth iodide solution. However, considering the higher price of the imported German Merck GF254 plate, the Qingdao Dingkang GF254 plate was preferred for TLC identification.

[0052] Example 2: Detection of medicinal components in Stephania tetrandra slices by liquid chromatography-mass spectrometry 1. Instruments, reagents and materials Eight batches of samples were randomly collected, including Stephania tetrandra slices #1-#4 (Ruichang, Jiangxi), Stephania tetrandra slices #5-#7 (Shanggao, Jiangxi), and wild Stephania tetrandra #8 (Shanggao, Jiangxi). Tetrandrine reference standard (China National Institutes for Food and Drug Control, catalog number: 110711-201810) and tetrandrine reference standard (China National Institutes for Food and Drug Control, batch number: 110793-202108). Methanol, acetonitrile (analytical grade, Tianjin Baishi; chromatographic grade, Fisher), water was ultrapure water, and other reagents were analytical grade.

[0053] Instrument: Liquid chromatography-mass spectrometry (SCIEX X500R UPLC-QTOF-MS).

[0054] 2. Preparation of the test solution In Example 1, different extraction solvents and methods for the test sample were investigated during thin-layer chromatography identification. The color of the thin-layer chromatographic spots after development can preliminarily determine the content of the corresponding tetrandrine and fangchinorline. Using the test sample solution preparation method determined in Example 1, the obtained thin-layer chromatographic spots showed good results, indicating that the content of tetrandrine and fangchinorline extracted from the test sample was also high. Therefore, the same test sample solution preparation steps as in Example 1 were selected for this liquid chromatography-mass spectrometry (LC-MS) detection.

[0055] Take the fangji (Stephania tetrandra) slices, pulverize them, and pass them through a 60-mesh sieve. Take 1g of the powder, add 20mL of 80% methanol aqueous solution, and extract by sonication for 20min. Centrifuge, collect the supernatant, and evaporate it to dryness using a rotary evaporator. Take the extract, add chromatographic methanol to prepare a 0.1mg / ml solution, and then dilute it 100 times to obtain the final product.

[0056] 3. Preparation of reference solution Accurately weigh the tetrandrine and fangchinorline reference standards, and add methanol to prepare reference solutions containing 1.0 mg of tetrandrine and 0.5 mg of fangchinorline per 1 mL.

[0057] 4. Detection by liquid chromatography-mass spectrometry (1) Liquid chromatography conditions: The chromatographic column was ACQUITY UPLC C18 1.7μm, 2.1×100 mm, the mobile phase was acetonitrile (B)-water (A), the flow rate was 0.3mL / min, the detection wavelength was 280nm, the column temperature was 40℃, and the injection volume was 2 μL. The gradient elution conditions are shown in Table 1.

[0058] Table 1 Gradient elution conditions for liquid chromatography

[0059] (2) Mass spectrometry conditions: First-order mass spectrometry conditions: Ion source gas 1, 45 PSI; ion source gas 2, 50 PSI; drying gas temperature 500 ℃; spray voltage +5500V; scanning molecular weight range 100 Da~1500 Da; declustering voltage +55 V; collision energy +10 V; accumulation time 0.1 s.

[0060] Secondary mass spectrometry conditions: molecular weight range 100 Da~1500 Da; declustering voltage +80 V; collision energy +40 V; collision energy variation 15 V; cumulative time 0.065 s.

[0061] 5. Results of the content of tetrandrine and fangchinorline in Fangji decoction pieces and the pharmacopoeia regulations. Under the detection conditions determined above, liquid chromatography-mass spectrometry was used to detect the contents of Fangji slices 1#~4# (Ruichang, Jiangxi Province). The results of the content of fangji alkaloid and fangjinorline are shown in Table 2.

[0062] Table 2. Content of tetrandrine and fangchinorline in Stephania tetrandra slices 1#~4#

[0063] As shown in Table 2, the total content of tetrandrine and fangchinorline in the Fangji slices 1#~4# is greater than 1.4%, which meets the relevant requirements in the pharmacopoeia.

[0064] 6. Detection of nine medicinal components in Fangji decoction pieces Under the aforementioned detection conditions, liquid chromatography-mass spectrometry (LC-MS) was used to detect the active ingredients in cultivated Stephania tetrandra 5#~7# (Shanggao, Jiangxi) and wild Stephania tetrandra 8# (Shanggao, Jiangxi). The LC-MS extraction chromatograms of the active ingredients in samples 5#~7# are shown below. Figures 18-20 Nine medicinal components were detected in the prepared slices of Stephania tetrandra, including tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, and tetrandrine. The relative contents (peak areas) of the nine medicinal components are shown in Table 3.

[0065] Table 3. Relative contents (peak area) of nine medicinal components in artificially cultivated Stephania tetrandra 5#~7# and wild Stephania tetrandra 8#.

[0066] As shown in Table 3, the contents of the nine medicinal components in different Stephania tetrandra samples varied considerably. Overall, the contents of medicinal components in artificially cultivated Stephania tetrandra were slightly lower than those in wild Stephania tetrandra (especially tetrandrine and tetrandrine), showing the advantage of wild Stephania tetrandra in terms of medicinal components.

[0067] To further evaluate the similarity between the nine medicinal components in artificially cultivated Stephania tetrandra 5#~7# and wild Stephania tetrandra 8#, the following evaluation experiment was conducted.

[0068] 7. Evaluation of the substitution of wild Stephania tetrandra by artificial cultivation (1) Assessment of the content of tetrandrine and tebufenozide Tetrandrine and fangchinorline are key pharmacological components in Stephania tetrandra decoction pieces and are also components subject to quality control in the pharmacopoeia. Based on the results in Table 3, the ratios of the peak areas of tetrandrine and fangchinorline in artificially cultivated Stephania tetrandra 5#~7# to the peak areas of the corresponding components in wild Stephania tetrandra 8# were calculated. The trend of the content of tetrandrine and fangchinorline in artificially cultivated Stephania tetrandra 5#~7# approaching that in wild Stephania tetrandra 8# was assessed based on whether the sum of the ratios of the peak areas of tetrandrine and fangchinorline was ≥1.0. The results are shown in Table 4.

[0069] Table 4. Ratios of peak area of ​​tetrandrine and peak area of ​​tetrandrine in artificially cultivated Stephania tetrandra 5#~7# and wild Stephania tetrandra 8#

[0070] Table 4 shows that the peak area ratios of tetrandrine and fangchinorline in artificially cultivated Stephania tetrandra 5# and 6# and wild Stephania tetrandra 8# are relatively small, with a total ratio of less than 0.1, indicating that the contents of tetrandrine and fangchinorline in artificially cultivated Stephania tetrandra 5# and 6# are not similar to those in wild Stephania tetrandra 8#. However, the total ratio of the peak area ratios of tetrandrine and fangchinorline in artificially cultivated Stephania tetrandra 7# and wild Stephania tetrandra 8# is relatively high at 1.285, indicating that the contents of tetrandrine and fangchinorline in artificially cultivated Stephania tetrandra 7# are relatively similar to those in wild Stephania tetrandra 8#.

[0071] (2) Evaluation of the content of the remaining seven active ingredients The active ingredients in Stephania tetrandra include rotenone, tetrandrine, tetrandrine, tetrandrine, corydaline, mirendine, and peperomialine. As shown in Table 3, these seven active ingredients account for a large proportion and can be used as quality indicators for Stephania tetrandra slices.

[0072] The active ingredients were ordered as follows: Stephania tetrandra alkaloids, Stephania tetrandra alkaloids, Stephania tetrandra alkaloids, Stephania tetrandra alkaloids, Corydaline alkaloids, Milidinyl benzoate, and Paederia tetrandra alkaloids, numbered 1 to 7 respectively. Then, polynomial fitting was performed on the peak areas of the active ingredients in cultivated Stephania tetrandra 5#~7# and wild Stephania tetrandra 8#, respectively, to obtain polynomial fitting functions. Based on whether the coefficients of the fitting functions of cultivated Stephania tetrandra 5#~7# and wild Stephania tetrandra 8# were all identical and the goodness of fit was ≥0.800, the similarity between the contents of the other seven active ingredients in cultivated Stephania tetrandra 5#~7# and wild Stephania tetrandra 8# was evaluated. The results are shown in the table. Figures 21-24 .

[0073] The fitting functions are as follows: Artificial cultivation of Stephania tetrandra 5#: y = 1.26E+04x 3 - 6.47E+04x 2 - 1.82E+05x + 8.79E+05, R² =0.939; Artificially cultivated Stephania tetrandra 6#: y = -1.87E+04x 3 + 7.89E+05x 2 -5.45E+06x + 9.58E+06, R² =0.880; Artificial cultivation of Stephania tetrandra 7#: y = -6.39E+04x 3 + 1.45E+06x 2 - 8.47E+06x + 1.37E+07, R² =0.887; Wild Stephania tetrandra #8: y = -2.05E+05x 3 + 3.03E+06x 2 - 1.39E+07x + 1.95E+07, R² =0.944.

[0074] It can be seen that the coefficients of the fitting functions of artificially cultivated Fangji 6#, 7# and wild Fangji 8# are all the same and the fitting degree is ≥0.800, indicating that the contents of the other seven medicinal components of artificially cultivated Fangji 6#, 7# and wild Fangji 8# are relatively similar.

[0075] Based on the above assessment of the contents of tetrandrine, fangchinorline, and the other seven medicinal components, it can be concluded that the contents of artificially cultivated Fangji 7# and wild Fangji 8# are quite similar in terms of the contents of the nine medicinal components, and it is expected to replace wild Fangji.

[0076] Example 3 Methodological Investigation 1. Stability test A 1.0g sample of Stephania tetrandra slices (1#, Ruichang, Jiangxi) was tested for stability under the liquid chromatography-mass spectrometry conditions described in Example 2, at 0h, 2h, 4h, 8h, 12h, and 24h. The RSD of the peak areas of nine active ingredients—tetrandrine, fangchinorinine, fangchiphenine, oxytetrandrine, fangchiquinone, corydaline, mirendine, and gentianine—was calculated. The results showed that the RSD of all eight batches of samples was less than 3.0%, indicating good stability of the test solution within 24 hours.

[0077] 2. Precision test 1.0 g of Stephania tetrandra slices (1#, Ruichang, Jiangxi) was accurately weighed and analyzed under the liquid chromatography-mass spectrometry conditions described in Example 2. Six consecutive injections of 2 μL each were performed to assess precision. The RSD of the peak areas of nine active ingredients—tetrandrine, fangchinorline, fangchiphenine, oxytetrandrine, fangchiquinone, corydaline, mirtinoline, and gentianine—was calculated. The results showed that the RSD of all six samples was less than 2.0%, indicating good instrument precision.

[0078] 3. Repeatability test Six samples of Stephania tetrandra slices (1#, Ruichang, Jiangxi Province) were taken, each accurately weighed to 1.0 g. The samples were analyzed under the liquid chromatography and mass spectrometry conditions described in Example 2 to assess repeatability. The RSD of the peak areas of nine active ingredients—stephania tetrandra alkaloids, stephania tetrandra alkaloids, stephania tetrandra alkaloids, stephania tetrandra alkaloids, stephania tetrandra alkaloids, corydaline alkaloids, milidine, and gentianin—were calculated. The results showed that the RSD of all six samples was less than 4.0%, indicating good repeatability of the method.

[0079] Example 4: Investigation of harmful components in Stephania tetrandra In addition, ten batches of Fangji medicinal slices (9#~18#) from different production areas across the country were randomly collected in this invention. During the determination process using the liquid chromatography and mass spectrometry conditions of Example 2, it was found that aristolochic acid I and aristolochic acid II were detected in three batches of samples. The results of the aristolochic acid I and aristolochic acid II content are shown in Table 5.

[0080] Table 5. Content (%) of aristolochic acid I and aristolochic acid II in three batches of samples.

[0081] As shown in Table 5, a certain amount of aristolochic acid I and aristolochic acid II were detected in samples 10#, 13# and 17#. Although the content was not very high, aristolochic acid I and aristolochic acid II are nephrotoxic and carcinogenic, and their content needs to be strictly controlled.

[0082] Modern research has found that *Stephania tetrandra* contains aristolochic acid, which can cause liver and kidney poisoning, and therefore it has been banned by the state. The *Stephania tetrandra* currently circulating in the market is *Stephania tetrandra* var. *pinnatifida*, which is a plant in the Menispermaceae family. Stephania tetrandra The dried root of *Stephania tetrandra* (S. Moore). However, this invention detected aristolochic acid I and aristolochic acid II in three batches of *Stephania tetrandra* samples, indicating the limitations of previous methods for detecting the active ingredients of *Stephania tetrandra*. This leads to the concealment of aristolochic acid I and aristolochic acid II in some samples, or problems with the source of some medicinal slices, which poses a challenge to the safety control of *Stephania tetrandra* medicinal slices. How to accurately detect aristolochic acid I and aristolochic acid II in *Stephania tetrandra* is the key to quality control.

[0083] The 2020 edition of the Chinese Pharmacopoeia, Part I, stipulates the limit for aristolochic acid I in Asarum medicinal materials and processed slices as follows: The content of aristolochic acid I (C...) 17 H 11 NO7) must not exceed 0.001%. Therefore, the limits for aristolochic acid I and aristolochic acid II in the Fangji herbal slices of this invention also refer to this standard. Samples 10#, 13#, and 17#, which tested positive for aristolochic acid I and aristolochic acid II in this study, were evaluated according to this standard. It was concluded that samples 10# and 13# exceeded the limits for aristolochic acid I and aristolochic acid II, and were therefore unqualified. Although the levels of aristolochic acid I and aristolochic acid II in sample 17# were within the limits, the dosage should still be strictly controlled in practical applications.

[0084] Next, the detection method for Fangji decoction pieces was changed, and Comparative Example 1 was set up to compare and verify the accuracy of the liquid chromatography-mass spectrometry method of the present invention in detecting aristolochic acid I in Fangji decoction pieces.

[0085] Comparative Example 1 1. Instruments, reagents and materials Ten batches of samples, numbered 9# to 18#, were collected from Fangji (Stephania tetrandra) slices. Reference standards for tetrandrine (China National Institutes for Food and Drug Control, catalog number: 110711-201810) and fangchinorline (China National Institutes for Food and Drug Control, batch number: 110793-202108) were used. Mass spectrometry-grade methanol and acetonitrile were from ACS Encor Chemicals, Inc. (USA); mass spectrometry-grade formic acid was from Fisher Scientific, Inc. (USA); water was ultrapure water; other reagents were of analytical grade.

[0086] Instruments: TripleTOF 5600 high-resolution mass spectrometer (SCIEX Corporation, USA); LC-30AD ultra-high performance liquid chromatograph (Shimadzu Corporation, Japan).

[0087] 2. Preparation of the test solution Take the fangji slices, pulverize them, and pass them through a 60-mesh sieve. Take 1.0g of the medicinal powder, add 25ml of 80% methanol aqueous solution, heat under reflux for 1h, filter, concentrate the filtrate to dryness, and add 80% methanol aqueous solution to make up to 4mL as the test solution.

[0088] 3. Preparation of reference solution Accurately weigh the tetrandrine and fangchinorline reference standards, and add methanol to prepare reference solutions containing 1.0 mg of tetrandrine and 0.5 mg of fangchinorline per 1 mL.

[0089] 4. Detection by liquid chromatography-mass spectrometry (1) Liquid chromatography conditions: An ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) was used. The mobile phase was 0.1% formic acid aqueous solution (A) - acetonitrile (B), with gradient elution. The flow rate was 0.6 mL / min, the detection wavelength was 280 nm, the column temperature was 40 °C, and the injection volume was 5 μL. The gradient elution conditions are shown in Table 6.

[0090] Table 6 Gradient elution conditions for liquid chromatography

[0091] (2) Mass spectrometry conditions: An ESI electrospray ionization source was used for detection in positive ion mode. The mass scan range was 100–1500 m / z, the spray voltage (ISVF) was 5.5 kV, the ion source temperature (TEM) was 500 °C, the declustering voltage (DP) was 100 V, the collision energy (CE) was 45 V, the collision energy superposition (CES) was 15 V, the curtain gas pressure (CUR) was 40 psi, and the pressures of both the nebulizer gas (GS1) and the auxiliary gas (GS2) were 50 psi. The data acquisition time was 55 min, and data were acquired using the TOF-MS-IDA-MS / MS method. The IDA was set to perform secondary mass spectrometry scans on the six peaks with response values ​​exceeding 100 cps. The daughter ion scan range was 50–1250 m / z, and those meeting this condition were prioritized for secondary scanning. Dynamic background subtraction (DBS) was enabled.

[0092] 5. Test Results The results of the detection of aristolochic acid I and aristolochic acid II in the samples are shown in Table 7. Aristolochic acid I and aristolochic acid II were not detected in any of the ten batches of Stephania tetrandra samples, which indicates the limitations of Comparative Example 1 in quality control.

[0093] Table 7. Content (%) of aristolochic acid I and aristolochic acid II in ten batches of samples

[0094] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A quality control method for Stephania tetrandra slices, characterized in that: The active ingredients in Stephania tetrandra slices were detected by liquid chromatography-mass spectrometry (LC-MS) for quality control. The detection conditions included: Liquid chromatography conditions: detection wavelength 280 nm, water as mobile phase A, acetonitrile as mobile phase B, gradient elution; Mass spectrometry conditions: First-stage mass spectrometry conditions: drying gas temperature 500℃, spray voltage +5500V, declusing voltage +55V, collision energy +10V, cumulative time 0.1 s; Second-stage mass spectrometry conditions: declusing voltage +80V, collision energy +40V, collision energy variation 15V, cumulative time 0.065 s.

2. The quality control method according to claim 1, characterized in that: The gradient elution conditions for liquid chromatography are as follows: From 0 to 1.0 min, the volume concentrations of mobile phase A and mobile phase B were 90% and 10%, respectively. 1.0~2.0 min, the volume concentrations of mobile phase A and mobile phase B are 90%~75% and 10%~25%, respectively; The volume concentrations of mobile phase A and mobile phase B were 75%–70% and 25%–30%, respectively, over a period of 2.0–5.4 min. 5.4~5.5 min, the volume concentrations of mobile phase A and mobile phase B are 70%~9% and 30%~91%, respectively; The volume concentrations of mobile phase A and mobile phase B were 9%–7% and 91%–93% respectively over a period of 5.5–9.3 min. From 9.3 to 9.4 min, the volume concentrations of mobile phase A and mobile phase B were 7%–90% and 93%–10%, respectively. 9.4~10 min, the volume concentrations of mobile phase A and mobile phase B are 90% and 10%, respectively.

3. The quality control method according to claim 1, characterized in that: The liquid chromatography conditions also include: using octadecylsilane-bonded silica gel as the column packing material with a particle size of 1.7 μm, a column size of 2.1 × 100 mm, a column temperature of 35–45 °C, a flow rate of 0.2–0.5 ml / min, and an injection volume of 1–3 μL.

4. The quality control method according to claim 1, characterized in that: The mass spectrometry conditions also include: in the first-stage mass spectrometry conditions, dual ion sources are used, with ion source gas 1 at 45 psi and ion source gas 2 at 50 psi, and the molecular weight range is 100 Da to 1500 Da; in the second-stage mass spectrometry conditions, the molecular weight range is 100 Da to 1500 Da.

5. The quality control method according to claim 1, characterized in that: The medicinal components in Fangji decoction pieces include any two or more of the following: tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, tetrandrine, and tetrandrine.

6. The quality control method according to claim 1, characterized in that: The prepared slices of Fangji include wild and cultivated varieties. The cultivated and wild varieties of Fangji slices are detected and compared using liquid chromatography-mass spectrometry, and must meet at least one of the following conditions: (1) The total content of tetrandrine and tebufenozide is ≥1.4%; (2) Calculate the ratio of the peak area of ​​the medicinal components of the artificially cultivated variety to that of the wild variety of Stephania tetrandra slices. The sum of the ratios of the peak areas of tetrandrine and tetrandrine is ≥1.

0. (3) Sort the active ingredients in the order of: rotenone, tetrandrine, tetrandrine, tetrandrine, tetrandrine, corydaline, mirendine, and peperomialine, in order from 1 to 7. Then, perform polynomial fitting on the peak area of ​​the active ingredients of the cultivated and wild varieties of Stephania tetrandra slices and the corresponding active ingredient number to obtain the polynomial fitting function. The coefficients of the fitting functions of the cultivated and wild varieties must have the same sign and the degree of fit must be ≥0.

800. (4) The content of aristolochic acid I or aristolochic acid II is ≤0.001%.

7. The quality control method according to claim 1, characterized in that: It also includes the identification of Stephania tetrandra slices using thin-layer chromatography, with identification conditions including at least one of the following: A1, GF254 board; A2. Developing solvent: ethyl acetate-acetone-methanol-5% concentrated ammonia, volume ratio 5:2:0.7:0.3; A3. Colorimetric reagent: dilute potassium bismuth iodide solution; A4. Examine under daylight or ultraviolet light at 254nm; A5. The sample volume is 6~10μL; A6. Spots of the same color appear at the corresponding positions on the chromatogram of the reference standard.

8. The quality control method according to claim 1 or 7, characterized in that: It also includes the preparation of a test solution, wherein the preparation conditions include at least one of the following: B1. Ultrasonic extraction or reflux extraction; B2. Grind the Stephania tetrandra medicinal material to 40-60 mesh before extraction; B3. The extraction solvent is methanol or an aqueous methanol solution, with a volume concentration of 60-80% for the aqueous methanol solution. B4. The volume-to-mass ratio of the extraction solvent to the *Stephania tetrandra* herb is 20-50 mL: 1 g; B5. Extraction time is 15~60min; B6. After extraction, the supernatant is evaporated to dryness to obtain an extract. Methanol is added to prepare a solution of 0.1-0.3 mg / mL, which is then diluted 100-200 times to prepare the test solution.

9. The quality control method according to claim 1 or 7, characterized in that: It also includes the preparation of a reference solution, wherein the reference standard includes tetrandrine and tebufenozide, the solvent for preparing the reference solution is methanol, and the concentration of the reference solution is 0.1~1 mg / ml.

10. The quality control method according to claim 8, characterized in that: The preparation conditions for the test solution include at least one of the following: B1. Ultrasonic extraction, power 150~250W; B2. Grind the Stephania tetrandra medicinal material to 60 mesh before extraction; B3. The extraction solvent is an 80% (v / v) methanol aqueous solution; B4. The volume-to-mass ratio of the extraction solvent to the *Stephania tetrandra* herb is 50 mL: 1 g; B5. Extraction time is 20 minutes.

Images