Method for determining specific primary ions of traditional Chinese medicinal materials based on high-resolution liquid chromatography and application

Abstract

The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a method for determining specific primary ions of traditional Chinese medicinal materials based on high-resolution liquid chromatography and application. The method for determining the specific first-grade ions of the traditional Chinese medicinal materials based on the high-resolution liquid chromatography comprises the following steps: performing extraction and ultra-high performance liquid chromatography-high-resolution mass spectrometry analysis on different traditional Chinese medicinal materials by using an alcohol-containing solvent; the method comprises the following steps: automatically screening special ultra-high performance liquid chromatography-high resolution mass spectrometry first-level ions of each traditional Chinese medicinal material from ultra-high performance liquid chromatography-high resolution mass spectrometry data of all traditional Chinese medicinal materials, and constructing a special ion database of each traditional Chinese medicinal material; the specific ion database of the traditional Chinese medicinal materials can be used for identifying the traditional Chinese medicinal materials and products thereof. According to the method, the ion specificity is high, the ultra-high performance liquid chromatography-high resolution mass spectrum primary ions, obtained through one-time screening, of all traditional Chinese medicinal materials can be applied to all products containing the traditional Chinese medicinal materials, analysis conditions do not need to be developed again, and the method is rapid, simple, more comprehensive in identification, good in durability and high in universality.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine identification, and specifically relates to a method and application for determining primary ions specific to each traditional Chinese medicine based on high-resolution liquid-mass spectrometry. Background Art

[0002] In recent years, Chinese medicine has become increasingly popular and recognized worldwide for its unique therapeutic effects, and is an important or complementary part of the health care system in many countries. Using the correct varieties of Chinese medicinal materials and ensuring that their products are truly put into production is the key to ensuring the efficacy of Chinese medicine. Therefore, accurate identification of Chinese medicinal materials and their products is an important part of ensuring the quality of Chinese medicine and protecting people's lives and health.

[0003] At present, the identification methods of Chinese herbal medicines and their products (such as Chinese patent medicines, Chinese herbal formula granules, Chinese herbal medicine extracts, etc.) can be divided into four categories: property identification, microscopic identification, physical and chemical identification, and molecular biological identification (such as DNA barcode identification). The above methods have the disadvantages of narrow applicability, poor specificity, cumbersome operation, long time consumption, and the use of a large number of toxic and controlled reagents (Table 1). More importantly, the quality control level of some pharmacopoeial varieties and non-pharmacopoeial variety standards is relatively low. A study published by the China Food and Drug Inspection Institute in 2023 found that when the inspection agency inspected the quality of varieties with lower implementation standards according to higher standards, it often found that no or less materials were added (Nie Lixing, Wu Yanpei, Liu Jing, et al. Reflections on issues related to the study of quality standards for Chinese patent medicines [J]. Acta Pharmaceutica Sinica, 2023, 58(08): 2260-2270). Therefore, it is urgent to develop a simple, fast, accurate, comprehensive and durable identification method for Chinese herbal medicines and their products.

[0004] Table 1 Characteristics of existing identification methods for Chinese medicinal materials and their products

[0005]

[0006]

[0007] Liquid chromatography-mass spectrometry (LC-MS) technology has high sensitivity and excellent qualitative ability. In recent years, this technology has been widely used in the identification of traditional Chinese medicine and its products. For example, in the "Chinese Pharmacopoeia" (2020 edition), donkey-hide gelatin, tortoise shell glue and deer antler glue all use the characteristic MRM ion pairs of tandem mass spectrometry LC-MS / MS to identify characteristic peptides. This technology has also been applied to the identification of animal medicinal materials in many other Chinese patent medicines, such as Fukezhidai Tablets and Fukangbao Oral Liquid, indicating that the identification method based on liquid-mass ions has been recognized. The method using characteristic peptides is mainly based on the differences in protein sequences of different animals, and is identified by different enzyme cleavage sites. Therefore, it is mostly used for the identification of animal drugs. However, when judging whether these peptides are truly characteristic peptides, only a small amount of mixed and counterfeit products are used for comparative studies, and there are reports that the method is considered to have poor specificity (Pan Zhiwen, Huang Wanfeng, Qian Xin, et al. Identification of donkey-hide gelatin and inspection of 6 animal heterologous components based on characteristic peptide detection technology using ultra-performance liquid chromatography-tandem mass spectrometry [J]. China Pharmaceuticals, 2024, 33(11): 79-83).

[0008] At present, there are reports on the use of ultra-high performance liquid chromatography-high resolution mass spectrometry for the identification of Chinese medicines and their products, but none of them systematically explored the unique ion groups of each Chinese medicinal material through experiments and computer programming, nor did they propose the idea of ​​identifying Chinese medicinal materials and their products based on the unique ions of each Chinese medicinal material. Instead, the idea was limited to relying on a few liquid mass ions or a few specific compounds for identification. The analysis process is to first manually screen the characteristic ions of each Chinese medicinal material in the Chinese medicinal product. These ions are non-specific ions, that is, the same characteristic ions may exist in multiple Chinese medicinal materials, and use these characteristic ions for the identification of Chinese medicinal products. More specifically, it is to find the relatively unique ions between the Chinese medicinal materials in a Chinese patent medicine formula, rather than trying to find the relatively larger range of unique ions of this Chinese medicine. This type of processing flow is often only for a specific variety of Chinese medicinal products. If it needs to be applied to other Chinese medicinal products, it is often necessary to find ions again or even redevelop methods, which is time-consuming and has poor universality. It is difficult to use it for the identification of complex Chinese medicinal products. In addition, the specificity of the characteristic ions based on manual screening for identifying each Chinese medicinal material is poor, and there is no systematic mining and discovery of the unique ion groups of many Chinese medicinal materials. For example, the invention patent CN 112903890 (a method for identifying Chinese medicinal material ingredients in food based on high-resolution mass spectrometry) manually specifies the characteristic components of 70 Chinese medicines (about 5 to 10 non-specific ions of the characteristic components of each Chinese medicine). When all the characteristic components of a certain Chinese medicine are detected in the sample to be tested, it is considered that the Chinese medicine may exist in the sample to be tested. The characteristic components are determined by reviewing the literature in the fields of Chinese medicine chemistry and Chinese medicine analysis, rather than by systematic experimental research. The specificity or specificity of the components lacks specific experimental evidence. In addition, the invention is only applicable to the identification of the 70 Chinese medicines proposed. If you want to add other Chinese medicine varieties, you need to reconstruct the characteristic components of the variety, which is cumbersome and time-consuming. In addition, it is important that the characteristic ingredients selected by the patent have poor specificity. For example, there are daucine in both Cornus officinalis and Cyathula officinalis, and the ingredients such as calycosin and formononetin in Astragalus membranaceus are also present in medicinal materials such as Astragalus. Therefore, for samples with complex formulas, false positive results may occur. In addition, for traditional Chinese medicine products with more complicated production processes, false negative results may occur because some characteristic components of traditional Chinese medicine cannot be transferred to the final product.

[0009] How to screen out the ions that are unique to each Chinese herbal medicine and can be used for identification through systematic and real experimental research is the key to restricting the use of ultra-high performance liquid chromatography-high resolution mass spectrometry technology for the identification method of Chinese herbal medicines and their products. This is also an important reason why only a few Chinese herbal medicines and Chinese patent medicines have been identified by this method so far. Although the invention patent CN108152434 (a method for searching for specific components of Chinese herbal medicines based on visual mass spectrometry information) proposes a method for searching for specific components of Chinese herbal medicines based on visual mass spectrometry information, this method can only search for differential components for two groups (two types) of samples, and it is difficult to use it for searching for differential components of multiple Chinese medicines (dozens or more), and it has not been studied whether the differential components found can be used for the identification of Chinese herbal medicine products. Therefore, it is necessary to develop a method that can automatically and quickly identify the specific ions of each Chinese herbal medicine, and use it for the identification of Chinese herbal medicines and their products. Summary of the invention

[0010] In view of the shortcomings of existing identification methods, the present invention provides a method for determining the primary ions unique to each Chinese medicinal material based on high-resolution liquid-mass spectrometry and applies it to the identification of Chinese medicinal materials and their products. Compared with traditional identification methods, the sample to be tested in the present invention only needs ultrasonic extraction, liquid-mass analysis and result export to obtain the identification result, without the need for complicated pre-treatment, and only about 50 mL of methanol, 0.1% water and acetonitrile are used in the identification process. Compared with the existing identification methods based on ultra-high performance liquid chromatography-high-resolution mass spectrometry technology, the present invention can automatically screen out the primary ions unique to each Chinese medicinal material and use them for identification. These unique primary ions have stronger specificity, and the primary ions unique to each Chinese medicinal material obtained by one screening can be applied to all products containing the Chinese medicine, without the need to redevelop the analysis conditions, and have good durability.

[0011] The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry of the present invention comprises the following steps:

[0012] (1-1) taking each reference Chinese medicinal material, respectively adding a solvent for extraction, and obtaining each reference Chinese medicinal material solution;

[0013] (1-2) analyzing each control Chinese medicinal material solution obtained in step (1-1) using ultra-high performance liquid chromatography-high resolution mass spectrometry;

[0014] (1-3) exporting all raw data obtained from ultra-high performance liquid chromatography-high resolution mass spectrometry analysis into a peak table containing the mass-to-charge ratios and retention times of all ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions;

[0015] (1-4) Using the peak table data of each reference Chinese medicinal material, the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions unique to each Chinese medicinal material were screened by mass-to-charge ratio and retention time.

[0016] The solvent in step (1-1) is alcohol or a mixture of alcohol and water, preferably methanol or methanol-water or water or ethanol or ethanol-water.

[0017] The extraction method described in step (1-1) is ultrasonic extraction, reflux extraction, Soxhlet extraction or decoction.

[0018] The method for screening the primary ions of each Chinese medicinal material by ultra-high performance liquid chromatography-high resolution mass spectrometry described in step (1-4) comprises:

[0019] (1-4-1) Select a peak table of a control Chinese medicinal material, and merge the peak tables of the remaining control Chinese medicinal materials; compare all the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions in the peak table of a single Chinese medicinal material with all the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ion data in the merged peak table; if the difference in mass-to-charge ratio between the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions in the peak table of a single Chinese medicinal material and the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions in the merged peak table is less than a threshold value, and the difference in retention time is less than a threshold value, it indicates that the ion is not an ultra-high performance liquid chromatography-high resolution mass spectrometry primary ion unique to the Chinese medicinal material, and it is excluded, and the remaining ultra-high performance liquid chromatography-high resolution mass spectrometry ions are ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions unique to the Chinese medicinal material;

[0020] (1-4-2) Perform step (1-4-1) on the remaining control Chinese medicinal materials in sequence.

[0021] The threshold value of the difference in mass-to-charge ratio of the primary ions in step (1-4-1) is less than or equal to ±0.02Da, and the threshold value of the difference in retention time is less than or equal to ±0.5min.

[0022] Ultra-high performance liquid chromatography analysis conditions: ultra-high performance liquid chromatography column, column temperature of 30°C; mobile phase A phase is 0.1% formic acid aqueous solution, phase B is acetonitrile, gradient elution program is: 0-5min, 0% B; 5-55min, 0% B-100% B; 55-65min, 100% B; flow rate is 0.3ml / min; injection volume is 3.0μL;

[0023] High-resolution mass spectrometry analysis conditions: positive or negative ion detection mode, nebulizer temperature 350°C, nebulizer flow rate 10 L / min, nebulizer pressure 35 psi, capillary voltage 3500 V, sheath gas temperature 350°C, sheath gas flow rate 12 L / min, capillary voltage 4500 V, cone voltage 500 V.

[0024] The ultra-high performance liquid chromatography (UHPLC) instruments used include ultra-fast liquid chromatography (UFLC), ultra-high pressure liquid chromatography (UPLC), etc., and the high-resolution mass spectrometry (HRMS) instruments used include quadrupole-time-of-flight mass spectrometer (Q-TOF), ion trap-analysis time-of-flight mass spectrometer (IT-TOF), orbitrap mass spectrometer (Orbitrap), etc.

[0025] The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry described in the present invention can be applied to the identification of Chinese medicinal materials and their products, and the specific steps are as follows:

[0026] (2-1) taking a sample to be tested and preparing it according to step (1-1) to obtain a sample solution to be tested;

[0027] (2-1) Analyzing the sample solution by ultra-high performance liquid chromatography-high resolution mass spectrometry under the same analytical conditions as step (1-2);

[0028] (2-3) exporting all raw data obtained from ultra-high performance liquid chromatography-high resolution mass spectrometry analysis into a peak table containing mass-to-charge ratios and retention times of all ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions;

[0029] (2-4) Calculate the matching number of ions in the peak table of the sample to be tested and the primary ions unique to each reference medicinal material, and determine whether each Chinese medicine exists in the sample to be tested based on the matching number threshold.

[0030] If the number of specific primary ions of a certain Chinese medicinal material detected in the sample to be tested is greater than or equal to 10, it is considered that the sample to be tested contains the Chinese medicinal material.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Fast and simple. Compared with the identification method of the pharmacopoeia which requires complex pre-treatment, the identification method proposed in the present invention only needs to perform ultrasonic extraction, liquid-to-mass analysis and data processing on the sample to be tested to obtain the identification result. Taking the Xuanmai Ganju Granules used in Example 3 as an example, the identification method described in the pharmacopoeia has a total of 45 operating steps and takes at least 200 minutes. The identification method proposed in the present invention only needs 6 operating steps and takes a maximum of 137 minutes (if multiple samples are processed at the same time, an average of only 77 minutes), which is shortened by 31.50%. And only ultrasonic extraction and liquid-to-mass analysis operations are required, and the identification results are automatically given by a computer program subsequently, without the need for expert experience, so it has good potential for automated inspection.

[0033] (2) Identification is more comprehensive. Compared with the identification methods included in the pharmacopoeia, the identification method proposed in the present invention can identify more Chinese medicines, which is helpful to improve the quality control level of Chinese patent medicines. Taking the Xuanmai Ganju Granules used in Example 3 as an example, the current pharmacopoeia only identifies 2 of the 4 Chinese medicines (Ophiopogon japonicus and Platycodon grandiflorum are not identified), while the identification method proposed in the present invention can identify 4 Chinese medicines at the same time. In addition, the safflower and hawthorn in Lemai Pills in Example 4 cannot be identified by the pharmacopoeia method, but this method can identify them; the scutellaria, mint and safflower in Huayu Quban Capsule cannot be identified by the pharmacopoeia method, but this method can identify them.

[0034] (3) Green, environmentally friendly and safe. Compared with the identification method in the pharmacopoeia that requires the use of a large amount of organic and toxic reagents and produces a large amount of waste liquid, the identification method proposed in the present invention only needs to use methanol, 0.1% formic acid water and acetonitrile totaling about 50ml (26mL of mobile phase waste liquid, 7-14mL of sample extraction waste liquid). Taking Xuanmai Ganju in Example 3 as an example, the pharmacopoeia method uses methanol (40mL), ether (Class II controlled reagent, 90mL), water-saturated n-butanol (180mL), ethanol (3mL), water (60mL), chloroform (Class II controlled reagent, toxic reagent, 20mL), sulfuric acid (prepared with 1% vanillin sulfuric acid solution, prepared on demand, 5-10mL), totaling about 398mL. The present invention saves about 87.44% of reagents.

[0035] (4) Strong specificity. In the Chinese Pharmacopoeia, Chinese patent medicines containing the Chinese medicinal materials such as Citrus aurantium, Citrus aurantium fructus, and dried orange peel are often identified by "hesperidin" and "naringin". As stipulated in the Pharmacopoeia, in the Children's Qingre Daozhi Pills, hesperidin and naringin reference substances are used to identify Citrus aurantium. However, the specificity of these two substances is not strong, and they are also present in many citrus peels (Zhang Fangfang, Zhang Jinjun, Xia Huili, et al. Simultaneous determination of 12 flavonoid compounds in different tissues of Red Beauty Citrus by high performance liquid chromatography [J]. Journal of Food Safety and Quality, 2022, 13(07): 2267-2273.). The method of the present invention can avoid this problem. This is because Citrus aurantium, Citrus aurantium fructus, and dried orange peel all contain the above substances, so they can all be detected in the liquid mass spectrometry platform. Since they are the same substances, they have the same retention time and mass-to-charge ratio, and will be excluded by the screening algorithm of the present invention, which effectively improves the specificity. Furthermore, no ions related to hesperidin and naringin were observed in the specific ions of dried tangerine peel in Example 1 (judged by mass-to-charge ratio), which indirectly verifies the strong specificity of the present invention.

[0036] (5) Strong universality. Since this method uses the same extraction and analysis conditions for all Chinese medicines, there is no need to develop pretreatment methods for new Chinese patent medicines that need to be identified. Only methanol ultrasonic extraction is required, which has good universality.

[0037] (6) Good durability. Since the method uses primary mass spectrometry ions for analysis, similar primary mass spectrometry ions will be generated even on different LC-MS platforms (as long as they are all ESI sources), which has good durability. As shown in Example 4 of the present invention, we have proved that the present invention is applicable to three different LC-MS platforms, and therefore has good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The schematic diagram of the principle of the method for discovering the unique primary ions of each Chinese medicinal material according to the present invention;

[0039] Figure 2 The present invention is a flow chart of discovering primary ions unique to each Chinese medicinal material based on ultra-high performance liquid chromatography-high resolution mass spectrometry and using them for identification of Chinese medicinal products;

[0040] Figure 3 Schematic diagram of the primary ions specific to each Chinese medicinal material described in this method (taking 4 medicinal materials as examples);

[0041] Figure 4 This is a flow chart and time comparison chart of the present method and the pharmacopoeia method for identifying the medicinal materials in the Xuanmai Ganju Granules formula. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below through specific embodiments.

[0043] Example 1 Determination of primary ions specific to each Chinese herbal medicine

[0044] (1) Instruments and reagents

[0045] The ultra-high performance liquid chromatography-high resolution mass spectrometer was SCIEX triple TOF 6600+, including binary pump, online degasser, autosampler, column oven. BP 211D electronic balance (Sartorius, Germany); KH-250B ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); TGL-16B high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory); Plus-E2TS ultrapure water device (Nanjing Yipu Yida Technology Development Co., Ltd.).

[0046] Acetonitrile (mass spectrometry grade, Merck, Germany), formic acid (mass spectrometry grade, Merck, Germany), and water were homemade ultrapure water; 83 reference medicinal materials were used to construct the unique ions of each medicinal material. All reference Chinese medicinal materials were purchased from the China Food and Drug Administration.

[0047] Table 2 Information of 83 kinds of control Chinese herbal medicine samples

[0048]

[0049]

[0050]

[0051] (2) Methods

[0052] (1-1) Take 1g of the control Chinese medicinal materials in turn, weigh accurately, add 7mL of methanol, perform ultrasonic extraction at room temperature for 45min, centrifuge, take the supernatant, and filter through a 0.22μm filter membrane to obtain the control Chinese medicinal material solution. The remaining samples to be tested are prepared according to the method to obtain the solutions of the samples to be tested;

[0053] (1-2) The control Chinese medicinal material solutions obtained in step (1-1) are analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry; the ultra-high performance liquid chromatography-high resolution mass spectrometry analysis conditions are as follows:

[0054] Ultra-high performance liquid chromatography analysis conditions: chromatographic column is ACQUITYUPLC BEH C 18 Column (2.1×150 mm, 1.7 μm, Waters, USA), column temperature 30°C; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, gradient elution method was as shown in Table 3, flow rate was 0.3 ml / min; injection volume was 3.0 μL.

[0055] High-resolution mass spectrometry analysis conditions: nebulizer temperature 350°C, nebulizer flow rate 10 L / min, nebulizer pressure 35 psi, capillary voltage 3500 V, sheath gas temperature 350°C, sheath gas flow rate 12 L / min, capillary voltage 4500 V, cone voltage 500 V; acquisition was performed in negative ion mode with a mass-to-charge ratio range of m / z 100-1500.

[0056] Mobile phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile, and the gradient elution method is shown in the following table:

[0057] Table 3 Liquid phase conditions

[0058]

[0059]

[0060] (1-3) All raw data obtained from ultra-high performance liquid chromatography-high resolution mass spectrometry analysis were exported using MS-DIAL software as a peak table containing the mass-to-charge ratio and retention time of all ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions, and the unique ions of each Chinese medicinal material were screened according to the mass-to-charge ratio threshold of ±0.02 Da and the retention time threshold of ±0.5 min.

[0061] (3) Results

[0062] More than 100 unique primary ions were detected for 83 kinds of Chinese medicinal materials in both ion modes. The details are shown in Table 4.

[0063] Table 4 The number of primary mass spectrometry ions and unique primary ions of 83 Chinese medicinal materials in positive and negative ion modes

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] Example 2 Application of the method for determining the primary ions specific to each Chinese herbal medicine in the identification of various Chinese herbal medicine pieces

[0070] (1) Instruments and reagents

[0071] The instruments and reagents are the same as those in Example 1.

[0072] (2) Methods

[0073] 9 kinds of Chinese herbal medicine pieces (Qinjiao, Qianghuo, Huangqi, Dahuang, Baizhi, Gancao, Danshen, Muxiang, Chuanxiong) were purchased from the market, and after being ground into powder and passed through a No. 4 sieve, 9 kinds of test sample powders (test samples) were obtained to verify the identification effect of this method. The specific steps are:

[0074] (2-1) taking a sample to be tested and preparing it according to step (1-1) to obtain a sample solution to be tested;

[0075] (2-1) Accurately weigh 1 g of the sample to be tested, add 7 mL of methanol, and perform ultrasonic extraction at room temperature for 45 min. Centrifuge, take the supernatant, and filter through a 0.22 μm filter membrane to obtain the sample solution to be tested.

[0076] (2-2) The sample solution obtained in step (2-1) is analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry; the ultra-high performance liquid chromatography-high resolution mass spectrometry analysis conditions are the same as those in step (1-2) of Example 1:

[0077] (2-3) exporting all raw data obtained from ultra-high performance liquid chromatography-high resolution mass spectrometry analysis into a peak table containing mass-to-charge ratios and retention times of all ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions;

[0078] (2-4) Calculate the matching number of ions in the peak table of the sample to be tested and the unique ions of each reference medicinal material, and determine whether each Chinese medicine exists in the sample to be tested based on the matching number threshold.

[0079] Then, the unique primary ions of each Chinese herbal medicine are searched in the peak table of the nine Chinese herbal medicines to be tested, and the matching number of ions in the peak table of the nine Chinese herbal medicines and the unique ions of each control herbal medicine is calculated. If the number of unique ions of a Chinese herbal medicine detected in the sample to be tested is greater than or equal to 10, it is considered that the sample to be tested contains the Chinese herbal medicine.

[0080] (3) Identification results of 9 kinds of Chinese herbal medicine pieces

[0081] Nine kinds of Chinese herbal medicines were successfully and accurately identified without any false positive results. The specific test results are shown in Table 5.

[0082] Table 5 Identification results of 9 kinds of Chinese herbal medicine pieces

[0083]

[0084]

[0085] Example 3 Using the unique primary ions of each Chinese herbal medicine to distinguish Xuanmai Ganju granules from counterfeit Xuanmai Ganju granules

[0086] (1) Instruments and reagents

[0087] The apparatus and reagents are the same as those in Example 1. Xuanmai Ganju Granules are a Chinese patent medicine prepared by extracting and concentrating four Chinese medicinal herbs, namely, Scrophularia, Radix Ophiopogonis, Radix Glycyrrhizae and Platycodon grandiflorum, and adding auxiliary materials.

[0088] Xuanmai Ganju Granules were purchased from Yunnan Baiyao and Shuzhong Pharmaceutical from the market. According to the 2020 edition of the Chinese Pharmacopoeia, Xuanmai Ganju Granules positive drug, negative preparation 1 (lacking Scrophularia ningpoensis), negative preparation 2 (lacking Ophiopogon japonicus), negative preparation 3 (lacking Licorice), and negative preparation 4 (lacking Platycodon grandiflorum) were prepared.

[0089] (2) Methods

[0090] Take 1g of Xuanmai Ganju Granules (Yunnan Baiyao), weigh accurately, add 7ml of methanol, and extract by ultrasonic for 45min. Centrifuge to remove the supernatant, filter through a 0.22μm filter membrane, and obtain the test solution. Take 2g of Xuanmai Ganju Granules (Shuzhong Pharmaceutical) and other homemade samples, prepare in the same way as above, and obtain the sample solutions to be tested;

[0091] Ultra-high performance liquid chromatography-high resolution mass spectrometry analysis conditions and data processing: same as in Example 2.

[0092] The specific ions of each Chinese medicinal material are all from Example 1.

[0093] (3) Results

[0094] Table 6 Identification results of positive preparations, negative preparations and Chinese patent medicines

[0095]

[0096]

[0097] The results showed that the identification method based on the unique primary ions of each Chinese medicinal material can accurately identify the Chinese medicinal materials that make up Chinese patent medicines without producing false positive results.

[0098] Example 4 Durability of the method on different high-resolution liquid-mass spectrometry platforms

[0099] (1) Instruments and reagents

[0100] The reagents were the same as those in Example 1. The high performance liquid chromatography-high resolution mass spectrometers were SCIEX triple TOF 6600+ (Ibrance), Orbitrap Exploris 240 (Thermo Fisher Scientific) and LCMS-9050 (Shimadzu).

[0101] (2) Methods

[0102] Eleven other Chinese medicinal materials and three Chinese patent medicines (Compound Honeysuckle Granules, Lemai Pills and Huayu Quban Capsules) were prepared as samples according to the method in Example 2 and analyzed on different liquid chromatography-mass spectrometry platforms, and matched with the unique primary ions of each medicinal material obtained in Example 1 to obtain identification results.

[0103] Take negative ion mode analysis as an example:

[0104] The SCIEX triple TOF 6600+ high-resolution mass spectrometry analysis conditions were the same as in Example 1.

[0105] Orbitrap Exploris 240 high-resolution mass spectrometry analysis conditions: negative ion mode; capillary ionization voltage of 1.5 kV; ion source temperature of 120 °C; both nebulizer gas and backflush gas were nitrogen (N2); solvent gas flow rate of 650 L / h; mass range of m / z 100 to 1500 (MS) and m / z 100 to 1500 (MS2); temperature of 350 °C.

[0106] LCMS-9050: negative ion mode; interface voltage -3.5 kV. The nebulizer uses nitrogen gas with a flow rate of 3.0 L / min; the drying gas is nitrogen gas with a flow rate of 10.0 L / min. The heating gas uses air with a flow rate of 10.0 L / min; the interface temperature is set to 300°C, the DL temperature is 250°C, and the heating block temperature is 400°C.

[0107] Data processing: The peak table is exported using the data processing software provided with each instrument.

[0108] The specific ions of each Chinese medicinal material are all from Example 1.

[0109] (3) Results

[0110] Table 6 Identification results of 11 Chinese medicinal materials and 2 Chinese patent medicines on different HPLC platforms

[0111]

[0112]

[0113] The results showed that all Chinese medicinal materials and Chinese patent medicine formulas (except for those that are not included in the 83 medicinal materials-specific primary ion database) can be accurately identified on different LC-MS platforms.

Claims

1. A method for determining the primary ions specific to each Chinese herbal medicine based on high-resolution liquid-mass spectrometry, characterized in that: The following steps are involved: (1-1) taking each reference Chinese medicinal material, respectively adding a solvent for extraction, and obtaining each reference Chinese medicinal material solution; (1-2) analyzing each control Chinese medicinal material solution obtained in step (1-1) using ultra-high performance liquid chromatography-high resolution mass spectrometry; (1-3) exporting all raw data obtained from ultra-high performance liquid chromatography-high resolution mass spectrometry analysis into a peak table containing the mass-to-charge ratios and retention times of all ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions; (1-4) Using the peak table data of each reference Chinese medicinal material, the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions unique to each Chinese medicinal material were screened by mass-to-charge ratio and retention time.

2. The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 1, characterized in that: The solvent in step (1-1) is alcohol or a mixture of alcohol and water.

3. The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 1, characterized in that: The extraction method described in step (1-1) is ultrasonic extraction, reflux extraction, Soxhlet extraction or decoction.

4. The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 1, characterized in that: The method for screening the primary ions of each Chinese medicinal material by ultra-high performance liquid chromatography-high resolution mass spectrometry described in step (1-4) comprises: (1-4-1) Select a peak table of a control Chinese medicinal material, and merge the peak tables of the remaining control Chinese medicinal materials; compare all the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions in the peak table of a single Chinese medicinal material with all the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ion data in the merged peak table; if the difference in mass-to-charge ratio between the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions in the peak table of a single Chinese medicinal material and the ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions in the merged peak table is less than a threshold value, and the difference in retention time is less than a threshold value, it indicates that the ion is not an ultra-high performance liquid chromatography-high resolution mass spectrometry primary ion unique to the Chinese medicinal material, and it is excluded, and the remaining ultra-high performance liquid chromatography-high resolution mass spectrometry ions are ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions unique to the Chinese medicinal material; (1-4-2) Perform step (1-4-1) on the remaining control Chinese medicinal materials in sequence.

5. The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 4, characterized in that: The threshold value of the difference in mass-to-charge ratio of the primary ions in step (1-4-1) is less than or equal to ±0.02Da, and the threshold value of the difference in retention time is less than ±0.5min.

6. The method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 1, characterized in that: Ultra-high performance liquid chromatography-high resolution mass spectrometry was selected for analysis; Ultra-high performance liquid chromatography analysis conditions: ultra-high performance liquid chromatography column, column temperature of 30°C; mobile phase A phase is 0.1% formic acid aqueous solution, phase B is acetonitrile, gradient elution program is: 0-5min, 0% B; 5-55min, 0% B-100% B; 55-65min, 100% B; flow rate is 0.3ml / min; injection volume is 3.0μL; High-resolution mass spectrometry analysis conditions: positive or negative ion detection mode, nebulizer temperature 350°C, nebulizer flow rate 10 L / min, nebulizer pressure 35 psi, capillary voltage 3500 V, sheath gas temperature 350°C, sheath gas flow rate 12 L / min, capillary voltage 4500 V, cone voltage 500 V.

7. Use of the method for determining the unique primary ions of each Chinese medicinal material based on high-resolution liquid-mass spectrometry as described in any one of claims 1 to 6 in identifying Chinese medicinal materials and their products.

8. Application of the method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 7 in identifying Chinese medicinal materials and their products, characterized in that: (2-1) taking a sample to be tested and preparing it according to step (1-1) to obtain a sample solution to be tested; (2-1) Analyzing the sample solution by ultra-high performance liquid chromatography-high resolution mass spectrometry under the same analytical conditions as step (1-2); (2-3) exporting all raw data obtained from ultra-high performance liquid chromatography-high resolution mass spectrometry analysis into a peak table containing mass-to-charge ratios and retention times of all ultra-high performance liquid chromatography-high resolution mass spectrometry primary ions; (2-4) Calculate the matching number of ions in the peak table of the sample to be tested and the primary ions unique to each reference Chinese medicinal material, and determine whether each Chinese medicinal material exists in the sample to be tested based on the matching number threshold.

9. Application of the method for determining the primary ions specific to each Chinese medicinal material based on high-resolution liquid-mass spectrometry according to claim 8 in identifying Chinese medicinal materials and their products, characterized in that: If the number of unique primary ions of a certain Chinese medicinal material detected in the sample to be tested is greater than or equal to 10, it is considered that the sample to be tested contains the Chinese medicinal material.

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