Detection method for identifying overground stem doping of rhizoma et radix baphicacanthis cusiae in compound Ganmaoling tablet

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparation detection, and particularly relates to a detection method for identifying adulteration of baphicacanthus cusia overground stems in compound Ganmaoling tablets. According to the detection method, indirubin and 2-benzoxazolinone are used as detection indexes, HPLC-MS is adopted for detection, and the detection accuracy is high. Analyzing and judging according to the ratio of the content of indirubin to the content of 2-benzoxazolinone. According to the present invention, indirubin and 2-benzoxazolinone are adopted as detection indexes, and the contents of the overground stems and the medicinal parts in the rhizoma et radix baphicacanthis cusiae medicinal material are analyzed and compared to establish the HPLC-MS detection method of the rhizoma et radix baphicacanthis cusiae doping feeding in the compound Ganmaoling tablet, and the method can be synchronously used for identifying the rhizoma et radix baphicacanthis cusiae, and a basis can be provided for quality monitoring of the compound Ganmaoling tablets.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation detection, and particularly relates to a detection method for identifying adulteration of southern isatis root aerial stems in compound Ganmaoling tablets. Background Art

[0002] Isatis indigotica Baphicacanthus cusia (Nees) Bremek. The dried rhizomes and roots of Isatis indigotica have the effects of clearing away heat and detoxifying, cooling blood and removing spots. They can be used for fever and sore throat, fever and erysipelas, and are commonly used antiviral medicines for the treatment of respiratory diseases. Isatis indigotica has a variety of biological activities, mainly antibacterial, antiviral, antitumor, anti-inflammatory and antioxidant. The chemical components mainly include alkaloids, lignans, phenylethanol glycosides, sterols, terpenes, etc. Among them, the chemical components with biological activity that have been reported are mainly alkaloids. Alkaloids are the main chemical components of Isatis indigotica with antiviral, anti-inflammatory and anti-tumor activities. Representative components of alkaloids include 2-benzoxazolinone, indigo and indirubin.

[0003] The current quality standard for southern isatis root is Part 1 of the 2020 edition of the "Chinese Pharmacopoeia". The testing items in the standard are relatively simple, with only identification items and routine inspection items. There is a lack of content determination of indicator components, making it difficult to effectively control the quality of southern isatis root. Therefore, the quality of southern isatis root on the market is uneven, which has also brought certain troubles to related research on clinical medication and preparations.

[0004] Malan is mainly produced in Guangdong and Guangxi, Guizhou and Yunnan. It is a perennial herb. The leaves and young stems at the top are often processed into dyes (commonly known as indigo) for sale. The roots and rhizomes of the underground parts are used as southern isatis root for medicinal purposes, and the aboveground stems in the middle are discarded as non-medicinal parts. Due to the scarcity of wild resources of Malan and the small amount of artificial planting, its roots and rhizomes are not well developed, and the yield is low, which is far from meeting market demand. The aboveground stems are non-medicinal parts, and it is difficult to distinguish them from the roots and rhizomes after chopped, resulting in the situation in the circulation of medicinal materials that non-medicinal parts (aboveground stems) and medicinal parts (roots and rhizomes) are mixed and sold together; and southern isatis root, as the minister drug in the compound cold medicine tablets, has the effect of clearing heat and detoxifying, and plays a greater role in exerting the efficacy of the medicine. If non-medicinal parts are used as raw materials, it will bring uncertainty risks to the effectiveness of the preparation. In addition, the current standard for compound cold medicine tablets does not conduct quality control on the southern isatis root in the prescription, so some companies mix the aboveground stems into the medicinal parts for raw materials to reduce costs.

[0005] In view of this, it is necessary to develop a detection method that can identify the adulteration of southern isatis root aboveground stems in Fufang Ganmaoling tablets. Summary of the invention

[0006] In the early stage of the research on Radix Isatidis indigofera, the inventor found that the chemical components contained in the aboveground stems of the non-medicinal parts and the medicinal parts (roots and rhizomes) are very similar, except that the content of each component is different. Therefore, the inventor analyzed and compared multiple batches of aboveground stems and underground parts from multiple sources, and found that there was a large difference in the content ratio of indigo red and 2-benzoxazolinone, which can be used to screen whether the non-medicinal parts and medicinal parts are mixed. Therefore, the present invention provides a detection method for identifying the adulteration of the aboveground stems of Radix Isatidis in Compound Ganmaoling Tablets, using indigo red and 2-benzoxazolinone as detection indicators, analyzing and comparing the aboveground stems and medicinal parts in Radix Isatidis indigofera medicinal materials, and using these two components as indicators to establish an HPLC-MS detection method for the adulteration of the aboveground stems of Radix Isatidis in Compound Ganmaoling Tablets, which can provide a basis for the quality control of Compound Ganmaoling Tablets.

[0007] The invention provides a detection method for identifying adulteration of southern isatis root aerial stems in compound Ganmaoling tablets. The detection method uses indigo carmine and 2-benzoxazolinone as detection indicators, adopts HPLC-MS for detection, and analyzes and judges according to the ratio of indigo carmine to 2-benzoxazolinone content. The method specifically comprises the following steps: (1) preparing a test solution: taking the compound Ganmaoling tablets, removing the coating, grinding, taking an appropriate amount, accurately weighing, moistening with 4 mL of concentrated ammonia test solution, adding chloroform, extracting at 80°C, filtering, evaporating to dryness, adding chloroform-methanol mixed solution to dissolve the residue and transferring it to a 5 mL volumetric flask, adding chloroform-methanol mixed solution to dilute to scale, shaking well, filtering, and taking a filtrate to obtain the solution; (2) Preparation of mixed reference solution: Take appropriate amount of 2-benzoxazolinone reference substance and indirubin reference substance, weigh accurately, add methanol to make a solution containing 0.1 mg per 1 mL, then accurately measure an appropriate amount and dilute with chloroform-methanol mixed solution to make a mixed reference solution containing 100 ng of 2-benzoxazolinone and indirubin per 1 mL. (3) Determination: The test solution and the reference solution are tested by HPLC-MS and then analyzed and judged.

[0008] Furthermore, in the above technical scheme, the analysis and judgment principles are: a. In the extracted ion flow chromatogram of the test solution, if the chromatographic peak corresponding to the chromatogram of the mixed reference solution does not appear simultaneously, it is regarded as that the southern isatis root is not detected; b. In the extracted ion flow chromatogram of the test solution, if the chromatographic peak corresponding to the chromatogram of the mixed reference solution appears simultaneously, and the content ratio of indigo red to 2-benzoxazolinone is not greater than 0.15, it is regarded as that the southern isatis root is not adulterated; c. In the extracted ion flow chromatogram of the test solution, if the chromatographic peak corresponding to the chromatogram of the mixed reference solution appears simultaneously, and the content ratio of indigo red to 2-benzoxazolinone is greater than 0.15, it is regarded as that the southern isatis root is adulterated.

[0009] Furthermore, in the above technical solution, the HPLC detection conditions are: the chromatographic column uses octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, 0.1% formic acid aqueous solution as a mobile phase B, gradient elution, a flow rate of 0.3 mL / min, and a column temperature of 25° C. Specifically, the chromatographic column is Thermo Hypersil GOLD aQ C18.

[0010] Furthermore, in the above technical scheme, the gradient elution program is: from 0min to 15.0min, the mobile phase A increases from 10% to 60%, and the mobile phase B decreases from 90% to 40%; from 15.0min to 15.5min, the mobile phase A increases from 60% to 90%, and the mobile phase B decreases from 40% to 10%; from 15.5min to 18.0min, the mobile phase A is 90%, and the mobile phase B is 10%.

[0011] Furthermore, in the above technical solution, the specifications of the chromatographic column are 100 mm×2.1 mm×1.7 μm.

[0012] Furthermore, in the above technical scheme, the mass spectrometry conditions of MS are: using a triple quadrupole mass spectrometer detector, electrospray ionization positive and negative ion mode multiple reaction monitoring, and the parameters used are as follows: the nebulizer gas, sheath gas, dry gas, and collision gas are all high-purity nitrogen; the nebulizer pressure is 45psi; the dry gas flow rate is 6L / min; the dry gas temperature is 350℃; the sheath gas flow rate is 11L / min; the sheath gas temperature is 350℃; the spray voltage is 500V; and the capillary voltage is 3500V.

[0013] Furthermore, in the above technical solution, in step (1), the extraction method is to extract by reflux for 60 minutes.

[0014] Furthermore, in the above technical solution, in step (1), the volume mass ratio (mL / g) of chloroform to sample is 40-45:1, preferably 43.5:1.

[0015] Furthermore, in the above technical solution, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is 1:1.

[0016] Advantages compared to the prior art: 1. The present invention uses indigo carmine and 2-benzoxazolinone as detection indicators, analyzes and compares the contents of the aerial stems and medicinal parts in the Radix Isatidis indigotica medicinal materials, and uses these two components as indicators at the same time. According to the different contents of different parts, an HPLC-MS detection method for the adulteration of the aerial stems of Radix Isatidis in Compound Ganmaoling Tablets is established, which can be used for screening random samples.

[0017] 2. The present invention analyzes the rules of the contents of indirubin and 2-benzoxazolinone in the aerial stems and medicinal parts of the southern isatis root medicinal material, and establishes a detection method for judging whether southern isatis root is added to the compound cold medicine tablets, whether the feeding is standardized, and whether the aerial stems are mixed. The method can be used simultaneously for the identification of southern isatis root, and can provide a basis for the quality control of the compound cold medicine tablets.

[0018] 3. The detection method established by the present invention is simple, has high precision, good stability and repeatability, low detection limit and accurate judgment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a chromatogram of the 2-benzoxazolinone reference substance solution in Example 1 of the present invention; Figure 2 The chromatogram of the indigo red reference substance solution in Example 1 of the present invention; Figure 3 This is a chromatogram of the test solution of the aboveground stem of Radix Isatidis in Example 1 of the present invention; Figure 4 This is a chromatogram of the test solution of the medicinal part of Radix Isatidis indigotica in Example 1 of the present invention; Figure 5 This is the MRM ion flow chart of the specificity test of the mixed reference solution in Example 2 of the present invention, wherein a is 2-benzoxazolinone; b is indirubin; Figure 6 This is the MRM ion flow chart of the specificity test of the test solution in Example 2 of the present invention, wherein a is 2-benzoxazolinone; b is indirubin; Figure 7 This is the MRM ion flow chart of the specificity test of the negative control solution in Example 2 of the present invention; Figure 8 The MRM extracted ion chromatogram and mass spectrum of the 2-benzoxazolinone specificity test in Example 2 of the present invention, wherein A-1 is the extracted ion chromatogram and A-2 is the mass spectrum; Fig. 9 The MRM extracted ion chromatogram and mass spectrum of the indigo carmine specificity test in Example 2 of the present invention, wherein B-1 is the extracted ion chromatogram and B-2 is the mass spectrum; Fig.10 The standard curve diagram of 2-benzoxazolinone prepared by the blank solvent in Example 2 of the present invention; Fig.11 The standard curve diagram of indigo red prepared by the blank solvent in Example 2 of the present invention; Fig.12 The standard curve of 2-benzoxazolinone prepared for the blank matrix in Example 2 of the present invention; Fig.13 This is a standard curve diagram of indigo red prepared with the blank matrix in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0022] The present invention is further described in detail below in conjunction with the drawings and embodiments: Experimental instruments and reagents: 1. Instrument Agilent 1290II-6470 ultra high performance liquid chromatography-mass spectrometry, Masshunter chromatography workstation; Ultimate 3000 Dionex high performance liquid chromatograph, Chromeleon7 chromatography workstation; Sartorius BSA 124S-CW electronic balance, Sartorius BT 25S electronic balance.

[0023] 2. Drug test reagents Southern Isatis Root: See Table 2 for sample information.

[0024] Negative control: Take other raw and auxiliary materials lacking southern isatis root, and prepare a negative sample lacking southern isatis root according to the prescription and process of this product.

[0025] Indigo carmine reference substance (China Food and Drug Inspection Institute, batch number: 110717-202407, 98.9%), 2-benzoxazolinone reference substance (STANDARD CHEMICAL, batch number: BE231022-13).

[0026] Test product information: Yili Pharmaceutical Co., Ltd., batch number 3231009, 3231243, 3231202, 3230718, 3231209, 3231113, 3230807, 3230201, 3230208, 3231221-1, 3220712, 3231225, 3240106, 3230723, 3231213, 3231114, 3230715, 3231140, 3231208, 3231229, 3240102, 3231233, 3231151, 3231232, 323113 0, 3231006, 3231221-2, 3230901, 3221115, 3231210, 3240511, 3231150, 3231102; Guangxi Jinye Pharmaceutical Co., Ltd., batch number 230702-1, 230603, 231101-1, 231101-2, 230402, 230401, 240302, 240201, 240503, 240502, 240202-1, 230702-2, 240202-2; Guangxi Jimintang Pharmaceutical Co., Ltd., batch number 230701-1, 230402, 230303 -1, 230801-1, 230801-2, 231001-1, 230701-2, 231001-2, 230301, 230501, 230701-3, 231001-3, 231001-4, 231001-5, 230303-2, 231001-6, 230801-3, 231001-7, 230801-4, 240101, 231102, 240102-1, 230101-1, 230101-2, 240303, 240102-2; Guangzhou Nuojin Pharmaceutical Co., Ltd., batch number 3507001 -1, 350A002-1, 3509003, 3505009, 3509006, 3507002, 3509007, 3507001-2, 350A002-2, 350A002-3, 3507002, 3502001, 3507001-3, 350A002-4, 3505010, 4501004, 3502006, 4501002, 350A001; Guangzhou Baiyunshan Huacheng Pharmaceutical Co., Ltd., batch number 20221220, 20230915, 20230903, 20221219, 20221210;Hainan Huluwa Pharmaceutical Group Co., Ltd., batch numbers 221215, 221207, 231110-1, 221211-1, 231003, 231005-1, 231008, 231004, 231109, 221223-1, 231006, 221223-2, 231005-2, 221223-3, 221211-2, 231001, 231107, 221213, 231108, 231110-2, 240101, 240102; Guangdong Luofushan White Crane Pharmaceutical Factory, batch numbers 2312016-1, 2302004, 2210010, 2310015, 2304007, 230 9012, 2302004, 2212015, 2401002-1, 2402003, 2312016-2, 2403005, 2403006, 2401002-2, 2401001; Jiangxi Jiulianshan Pharmaceutical Co., Ltd., batch number 202312143, 202311071, 2023120 93-1, 202301311, 202309143, 202307031-1, 202302031-1, 202312093-2, 202302031-2, 202302031-3, 202302063-1, 202212202, 202307031-2, 202307 031-3, 202401133, 202401251, 202312253-1, 202311251, 202302063-2, 202312253-2, 202301112; Guangdong Huizhou Traditional Chinese Medicine Factory Co., Ltd., batch number 231125, 240101-1, 231228-1, 231228-2, 240102, 231228-3, 240205-1, 240205-2, 221223, 240101-2, 240207, 240412, 240411, 240206, 240205-3; Guangxi Beibu Gulf Pharmaceutical Co., Ltd., batch number 231001, 231201- 1, 231201-2, 230202, 221101, 230601, 230201, 240201, 231201-3; Guangdong Zaitian Pharmaceutical Co., Ltd., batch number 230601, 230201, 231203-1, 221204-1, 231002-1, 231202, 221204-2, 231201, 231203-2, 231001, 231204, 231002-2; Guangdong Kangqili Pharmaceutical Co., Ltd., batch number 230101, 231101-1, 231110, 231109, 231101-2, 240402; Guangzhou Xiaoqiling Pharmaceutical Co., Ltd., batch number 240301. ;

[0027] Reagents: Acetonitrile was of chromatographic grade, formic acid was of mass spectrometry grade, water was ultrapure water, and the other reagents were of analytical grade.

[0028] Example 1: Establishment of HPLC Detection Method for the Aboveground Stems and Medicinal Parts of Radix Isatidis 1. Chromatographic conditions and system suitability test The chromatographic column was filled with octadecylsilane bonded silica gel (250 mm × 4.6 mm, 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions of Table 1; the detection wavelength was 280 nm; the column temperature was 30 °C; the flow rate was 1.0 mL / min. The theoretical plate number calculated based on the 2-benzoxazolinone peak should not be less than 4000.

[0029] Table 1 Mobile phase gradient elution table

[0030] 2. Preparation of reference solution Take an appropriate amount of 2-benzoxazolinone reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg of 2-benzoxazolinone per 1 mL; take an appropriate amount of indigo red reference substance, accurately weigh it, and add methanol (first dissolve it in chloroform) to make a solution containing 2 μg of indigo red per 1 mL.

[0031] 3. Preparation of the test solution of Isatis indigotica aboveground stem Take about 1g of the powder of this product (passed through No. 2 sieve), weigh it accurately, put it in a stoppered conical flask, add 50mL of ethyl acetate accurately, weigh it, heat and reflux at 80℃ for 1h, cool it, weigh it again, make up the lost weight with ethyl acetate, filter it, evaporate it to dryness, add a mixed solution of chloroform-methanol (1:1) to dissolve the residue and transfer it to a 10mL volumetric flask, add a mixed solution of chloroform-methanol (1:1) to dilute it to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0032] 4. Preparation of test solution of medicinal parts of Radix Isatidis The same method for preparing the test solution of the aerial stem of Isatis indigotica.

[0033] 5. Determination method The 2-benzoxazolinone reference solution, indigo red reference solution, southern isatis root aerial stem test solution, southern isatis root medicinal part test solution were taken separately and the test was carried out according to the above conditions. The results are as follows Figures 1 to 4 The results showed that the test solution of the aboveground stem of Radix Isatidis and the test solution of the medicinal part of Radix Isatidis both presented chromatographic peaks with the same retention time as the two reference solutions.

[0034] 6. Results Analysis According to the above method, 14 batches of southern isatis root aboveground stems and 26 batches of southern isatis root medicinal parts (roots and rhizomes) collected in this experiment were tested, and the test results are shown in Table 2. From the data in Table 2, it can be seen that the content of 2-benzoxazolinone in the southern isatis root aboveground stems is lower than that of indigo red, and the ratio of indigo red content to 2-benzoxazolinone content (F=W indigo red / W2-benzoxazolinone) ranges from 1.12 to 10.22; while the content of 2-benzoxazolinone in the medicinal parts of southern isatis root is higher than that of indigo red, and the ratio of indigo red content to 2-benzoxazolinone content ranges from 0.10 to 0.50. It can be seen that the ratio of indigo red content to 2-benzoxazolinone content can be used to screen whether the southern isatis root aboveground stems are mixed into the southern isatis root medicinal parts.

[0035] Table 2 Test results of Radix Isatidis indigotica

[0036] Example 2: Establishment of HPLC-MS Detection Method for Radix Isatidis in Fufang Ganmaoling Tablets Because indigo carmine has poor solubility in water and ethanol, while 2-benzoxazolinone has relatively good solubility, the indigo carmine content in the preparation of Isatis indigotica root is very low after being treated with water decoction and alcohol precipitation process, and it is difficult to detect it by conventional liquid phase. Therefore, the determination of the contents of the two components in the preparation must be detected by liquid spectrometry.

[0037] 1. Chromatographic, mass spectrometry conditions and system suitability test Thermo Hypersil GOLD aQ C18 (100 mm × 2.1 mm × 1.7 μm) was used as the chromatographic column, acetonitrile was used as the mobile phase A, 0.1% formic acid aqueous solution was used as the mobile phase B, the flow rate was 0.3 mL / min; the column temperature was 25 °C, and gradient elution was performed according to the regulations in Table 3.

[0038] Table 3 Gradient elution table

[0039] A triple quadrupole mass spectrometer was used for multiple reaction monitoring (MRM) in positive and negative ion modes using electrospray ionization (ESI). The monitored ion pairs are shown in Table 4. The nebulizer gas, sheath gas, drying gas, and collision gas were all high-purity nitrogen; the nebulizer pressure was 45 psi; the drying gas flow rate was 6 L / min; the drying gas temperature was 350 °C; the sheath gas flow rate was 11 L / min; the sheath gas temperature was 350 °C; the spray voltage was 500 V; and the capillary voltage was 3500 V.

[0040] Table 4 Mass spectrometry detection parameters

[0041] 2. Solution preparation Preparation of reference solution: Take appropriate amount of 2-benzoxazolinone reference substance and indirubin reference substance, weigh accurately, and add methanol to make a solution containing 0.1 mg per 1 mL. Accurately measure appropriate amount and dilute with chloroform-methanol (1:1) mixed solution to make a mixed reference solution containing 100 ng of 2-benzoxazolinone and indirubin per 1 mL.

[0042] Preparation of matrix reference solution: Take an appropriate amount of blank matrix sample and treat it in the same way as the preparation method of the test solution until it is "evaporated to dryness", accurately add 5 mL of the above mixed reference solution to dissolve it, filter it, and take the filtrate to obtain the solution.

[0043] Preparation of test solution: Take 20 tablets of Fufang Ganmaoling tablets, remove the coating, grind them into powder, take an appropriate amount (approximately equivalent to 7.81g of southern isatis root), accurately weigh, moisten with 4mL of concentrated ammonia test solution, add 100mL of chloroform, reflux at 80℃ for 1h, filter, evaporate to dryness, add chloroform-methanol (1:1) mixed solution to dissolve the residue and transfer it to a 5mL volumetric flask, add chloroform-methanol (1:1) mixed solution to dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0044] Preparation of negative control solution: Take an appropriate amount of negative control preparation, grind it into powder, and prepare it in the same way as the test sample solution.

[0045] 3. System suitability test Take the reference solution, test solution and negative control solution separately and measure according to the above conditions. Figures 5 to 9 The results showed that the test solution presented a chromatographic peak with the same retention time as the reference solution; while it was not detected in the negative control and had good chromatographic separation, indicating that the experiment has certain specificity.

[0046] 4. Methodological investigation (1) Investigation and optimization of extraction methods Extraction method: Compound Ganmaoling Tablets have many medicinal flavors and complex matrix. Both 2-benzoxazolinone and indigo red are alkaloid components. Therefore, concentrated ammonia test solution is used for alkalization and then chloroform extraction. The ultrasonic and reflux extraction methods are compared: two portions (Yili Pharmaceutical Co., Ltd., batch number: 3231009) are taken respectively, the coating is removed, and the powder is ground. About 2.3g is taken, accurately weighed, moistened with 4mL of concentrated ammonia test solution, 100mL of chloroform is added, ultrasonic and refluxed for 1h, filtered, evaporated, and the residue is dissolved with chloroform-methanol (1:1) mixed solution and transferred to a 5mL volumetric flask, diluted to the scale with chloroform-methanol (1:1) mixed solution, shaken, filtered, and the filtrate is taken. The above chromatographic conditions are used for determination, and the results are shown in Table 5. The results show that the extraction efficiency of the reflux extraction method is higher, so the reflux extraction method is finally selected.

[0047] Table 5 Experimental results of different extraction methods

[0048] Extraction time: Take 3 portions of (Yili Pharmaceutical Co., Ltd., batch number: 3231009), remove the coating, grind, take about 2.3g, accurately weigh, moisten with 4mL of concentrated ammonia test solution, add 100mL of chloroform, reflux for 30min, 60min, 90min, filter, evaporate to dryness, dissolve the residue with chloroform-methanol (1:1) mixed solution and transfer to a 5mL volumetric flask, add chloroform-methanol (1:1) mixed solution to dilute to the scale, shake well, filter, and take the filtrate. The results are shown in Table 6 for determination under the above chromatographic conditions. The results show that the extraction efficiency of 60min is complete, so the final choice is to reflux for 60min.

[0049] Table 6 Experimental results of different extraction times

[0050] Extraction volume: Take 3 portions of (Yili Pharmaceutical Co., Ltd., batch number: 3231009), remove the coating, grind, take about 2.3g, accurately weigh, moisten with 4mL of concentrated ammonia test solution, add 50mL, 100mL, and 150mL of chloroform, reflux for 60min, filter, evaporate to dryness, dissolve the residue with chloroform-methanol (1:1) mixed solution and transfer to a 5mL volumetric flask, add chloroform-methanol (1:1) mixed solution to dilute to scale, shake well, filter, and take the filtrate. The results are shown in Table 7 when the above chromatographic conditions are measured. The results show that the extraction efficiency is complete when 100mL of chloroform is added, so 100mL of chloroform is finally selected for reflux.

[0051] Table 7 Experimental results of different extraction volumes

[0052] (2) Investigation of matrix effect and linear relationship Take appropriate amount of 2-benzoxazolinone reference substance and indigo red reference substance, and prepare a series of mixed reference substance solutions of 10-1000 ng / mL with blank solvent (mixed solution of chloroform-methanol (1:1)) and blank matrix sample, respectively. Determine the peak area according to the chromatographic mass spectrometry conditions in the text, and draw a standard curve with the peak area as the ordinate and the concentration of the reference substance solution as the abscissa. The results are shown in Tables 8 to 11 and Figures 10 to 13 shown.

[0053] Standard curve prepared with blank solvent: The regression equation of 2-benzoxazolinone is y=0.6248X-4.489153, R²=0.9992; the regression equation of indigo carmine is y=1.5782X+75.372, R²=0.9995.

[0054] Standard curve prepared with blank matrix: The regression equation of 2-benzoxazolinone is y=0.4929X+3.4052, R²=0.9989; the regression equation of indigo carmine is y=0.9822+16.247, R²=0.9991.

[0055] Judging from the slope ratio, the matrix effect of 2-benzoxazolinone was 78.98%, and the matrix effect of indirubin was 62.23%, which showed obvious matrix inhibition effect. Therefore, the series of mixed reference solutions were prepared using blank matrix samples.

[0056] Table 8 Linear relationship determination results of 2-benzoxazolinone prepared with blank solvent

[0057] Table 9 Linear relationship determination results of indigo red prepared with blank solvent

[0058] Table 10 Linear relationship determination results of blank matrix preparation 2-benzoxazolinone

[0059] Table 11 Results of linear relationship determination of blank matrix preparation of indigo red

[0060] (3) Precision test Accurately pipette the mixed reference solution, repeat the injection 6 times continuously under the above chromatographic conditions, and take the integral value of the peak area as the evaluation index. The results are shown in Tables 12 and 13.

[0061] Table 12 2-Benzoxazolinone precision test results

[0062] Table 13 Indigo carmine precision test results

[0063] (4) Stability test The test solution (Yili Pharmaceutical Co., Ltd., batch number: 3231009) was precisely aspirated and injected once at regular intervals according to the above chromatographic conditions. The results are shown in Tables 14 and 15. The results showed that there was no significant change in the peak area of ​​the two components of the test solution within 24 hours, and the stability was good.

[0064] Table 14 2-Benzoxazolinone stability test results

[0065] Table 15 Indigo carmine stability test results

[0066] (5) Repeatability test Six samples of the test sample (Yili Pharmaceutical Co., Ltd., batch number: 3231009) were taken and processed according to the above preparation method, and the sample was injected and analyzed according to the above chromatographic conditions. The results are shown in Tables 16 and 17. The results showed that the average content of 2-benzoxazolinone was 2.09 μg / g, with an RSD of 1.0%; the average content of indirubin was 0.29 μg / g, with an RSD of 1.9%, and the repeatability was good.

[0067] Table 16 2-Benzoxazolinone repeatability test results

[0068] Table 17 Indigo red repeatability test results

[0069] (6) Recovery test A sample addition recovery test was performed. Six portions of the product (Yili Pharmaceutical Co., Ltd., batch number: 3231009) with a known content were taken. About 1.15 g was taken and accurately weighed. Then, 1.2 mL of 2-benzoxazolinone reference solution (concentration: 2.092 μg / mL) and 0.2 mL of indigo red reference solution (concentration: 1.904 μg / mL) were accurately added in a ratio relative to 100% of the content of the product. The test solution was prepared according to the above method, and the recovery rate was calculated according to the above chromatographic conditions and the method. The results are shown in Tables 18 and 19.

[0070] Table 18 2-Benzoxazolinone recovery test results

[0071] Table 19 Indigo red recovery test results

[0072] (7) Detection limit Accurately measure an appropriate amount of the mixed reference solution, add it to the blank matrix sample, and process it according to the test solution preparation method. Accurately pipette 5 μL and inject it into the LC-MS / MS instrument. Record the chromatogram. The detection limit is when the signal-to-noise ratio is about 3. The detection limit of 2-benzoxazolinone is 0.012 μg / g, and the detection limit of indirubin is 0.009 μg / g.

[0073] 5. Setting limits (1) Investigation of the ratio of the two components in the simulated preparation The present embodiment selects 6 batches of Radix Isatidis Radix medicinal parts and 6 batches of Radix Isatidis Radix aerial stems respectively, simulates the prescription process, and makes a simulated preparation with other raw materials and auxiliary materials in the prescription, to investigate the rule of indirubin and 2-benzoxazolinone content ratio in the preparation obtained by feeding with Radix Isatidis Radix different positions. The measured result is shown in Table 20, and the corresponding medicinal material content data in the data of Table 20 and Table 2 show that 2-benzoxazolinone is less affected by the decoction and alcohol precipitation process, and indirubin is due to poor solubility in water and ethanol, and is affected by the decoction and alcohol precipitation process, and content is obviously reduced. But indirubin and 2-benzoxazolinone content ratio still have large differences in Radix Isatidis Radix medicinal parts and Radix Isatidis Radix aerial stems, and there is a certain rule. The preparation obtained by normal use of the roots and rhizomes of southern isatis root has a ratio of indirubin to 2-benzoxazolinone in the range of 0.01-0.05; the preparation obtained by using the above-ground stems of southern isatis root has a ratio of indirubin to 2-benzoxazolinone in the range of 0.12-0.73.

[0074] Table 20 Determination results of the ratio of indirubin to 2-benzoxazolinone in simulated preparations for different medicinal parts

[0075] (2) Study on the ratio of two components in positive preparations The general rules of the fourth volume of the 2020 edition of the "Chinese Pharmacopoeia" stipulate that drug scraps and impurities should generally not exceed 3%. At the same time, considering that the above-ground stems at the lower end of the southern isatis root are connected to the medicinal parts (roots and rhizomes) and it is difficult to cut and separate them completely and accurately, the adulteration limit is proposed to be 10%, that is, 10% of the above-ground stems are allowed to be mixed into the roots and rhizomes for feeding.

[0076] Three batches of southern isatis root medicinal parts were taken, and two southern isatis root aerial stems with large F values ​​were added at a ratio of 10% respectively as the southern isatis root for feeding, and then the positive preparation doped with 10% southern isatis root aerial stems was prepared according to the prescription process with other raw materials and auxiliary materials in the prescription, and the test sample solution was prepared according to the test sample preparation method, and the sample was injected and analyzed according to the above chromatographic conditions. The results are shown in Table 21. The results showed that the maximum value of the ratio of indirubin to 2-benzoxazolinone in the prepared 6 positive preparations was 0.14.

[0077] Table 21 Investigation of 10% doped positive preparations

[0078] Based on the above-mentioned simulation test results and doping test results, and taking into account the influence of multiple factors in actual large-scale production, it is proposed that when the ratio of indigo carmine to 2-benzoxazolinone in the preparation is greater than 0.15, there is doping of the aboveground stems of Isatis indigotica in the feed.

[0079] 6. Judgment principles (1) If the chromatographic peaks corresponding to those in the mixed reference solution do not appear in the extracted ion flow chromatogram of the test sample, it is deemed that southern isatis root is not detected; (2) If the chromatographic peaks corresponding to those in the mixed reference solution appear in the extracted ion flow chromatogram of the test sample, and the ratio of indirubin to 2-benzoxazolinone is not greater than 0.15, it is deemed that the southern isatis root feed is not adulterated; (3) If the chromatographic peaks corresponding to those in the mixed reference solution appear in the extracted ion flow chromatogram of the test sample, and the ratio of indirubin to 2-benzoxazolinone is greater than 0.15, it is deemed that southern isatis root feed is adulterated.

[0080] 7. Result determination In the extracted ion flow chromatogram of the test sample, the chromatographic peak corresponding to the chromatogram of the mixed reference solution should be detected simultaneously, and the content ratio of indirubin to 2-benzoxazolinone should not be greater than 0.15.

[0081] Example 3: Determination of Radix Isatidis in the Sample Compound Ganmaoling Tablets According to the test method established in Example 2, 197 samples of Fufang Ganmaoling Tablets from 13 production companies were screened. The results showed that 54 samples (31 batches) from 5 companies did not contain southern isatis root, and the unqualified rate was 27.4%; although southern isatis root was detected in 37 samples (33 batches) from 5 companies, there was contamination with aerial stems, and the positive detection rate (unqualified rate) was 18.8%.

[0082] In summary, the detection method established by the present invention can not only perform qualitative identification of southern isatis root, but also detect whether there is southern isatis root aerial stem adulteration in the preparation. The detection method has strong specificity, good stability and repeatability, and high accuracy, and can provide a basis for quality monitoring of compound Ganmaoling tablets.

[0083] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets, characterized in that: The detection method uses indigo carmine and 2-benzoxazolinone as detection indicators, adopts HPLC-MS for detection, and analyzes and judges according to the ratio of the content of indigo carmine and 2-benzoxazolinone; specifically comprises the following steps: (1) preparation of the test solution: take compound Ganmaoling tablets, remove the coating, grind them into powder, take an appropriate amount, accurately weigh, moisten with 4 mL of concentrated ammonia test solution, add chloroform, extract at 80°C, filter, evaporate to dryness, add chloroform-methanol mixed solution to dissolve the residue and transfer it to a 5 mL volumetric flask, add chloroform-methanol mixed solution to dilute to the scale, shake well, filter, and take the filtrate to obtain; (2) Preparation of mixed reference solution: Take appropriate amount of 2-benzoxazolinone reference substance and indirubin reference substance, weigh accurately, add methanol to make a solution containing 0.1 mg per 1 mL, then accurately measure an appropriate amount and dilute with chloroform-methanol mixed solution to make a mixed reference solution containing 100 ng of 2-benzoxazolinone and indirubin per 1 mL. (3) Determination: The test solution and the reference solution are tested by HPLC-MS and then analyzed and judged.

2. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 1, characterized in that: The analysis and judgment principles are as follows: a. If the chromatographic peaks corresponding to those in the chromatogram of the mixed reference solution do not appear simultaneously in the extracted ion flow chromatogram of the test solution, it is deemed that southern isatis root is not detected; b. If the chromatographic peaks corresponding to those in the chromatogram of the mixed reference solution appear simultaneously in the extracted ion flow chromatogram of the test solution, and the content ratio of indirubin to 2-benzoxazolinone is not greater than 0.15, it is deemed that the southern isatis root feed is not adulterated; c. If the chromatographic peaks corresponding to those in the chromatogram of the mixed reference solution appear simultaneously in the extracted ion flow chromatogram of the test solution, and the content ratio of indirubin to 2-benzoxazolinone is greater than 0.15, it is deemed that southern isatis root is adulterated.

3. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 1, characterized in that: The detection conditions of HPLC were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel, acetonitrile was used as mobile phase A, 0.1% formic acid aqueous solution was used as mobile phase B, gradient elution was performed, the flow rate was 0.3 mL / min, and the column temperature was 25°C.

4. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 3, characterized in that: The gradient elution procedure is: from 0 min to 15.0 min, the mobile phase A increases from 10% to 60%, and the mobile phase B decreases from 90% to 40%; From 15.0min to 15.5min, mobile phase A increased from 60% to 90%, and mobile phase B decreased from 40% to 10%; from 15.5min to 18.0min, mobile phase A was 90% and mobile phase B was 10%.

5. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 3, characterized in that: The specifications of the chromatographic column are 100 mm × 2.1 mm × 1.7 μm.

6. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 1, characterized in that: The mass spectrometry conditions of MS are: using a triple quadrupole mass spectrometer, electrospray ionization positive and negative ion mode multiple reaction monitoring, the parameters used are as follows: nebulizer gas, sheath gas, drying gas, and collision gas are all high-purity nitrogen; nebulizer pressure is 45psi; drying gas flow rate is 6L / min; drying gas temperature is 350℃; sheath gas flow rate is 11L / min; sheath gas temperature is 350℃; spray voltage is 500V; capillary voltage is 3500V.

7. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 1, characterized in that: In step (1), the extraction method is to use reflux extraction for 60 minutes.

8. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 1, characterized in that: In step (1), the volume mass ratio of chloroform to sample is 40-45:

1.

9. A detection method for identifying adulteration of Radix Isatidis Radix in Fufang Ganmaoling Tablets according to claim 1, characterized in that: The volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is 1:1.

Images