A UPLC-Q / TOF-MS method for identifying Pinellia ternata and Pinellia ternata

Abstract

The present invention relates to the technical field of traditional Chinese medicine detection, and specifically to a UPLC-Q / TOF-MS identification method for pinellia and water pinellia. The present invention is based on the information of pinellia, ginger pinellia, water pinellia and ginger water pinellia collected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, combines multivariate statistical methods to screen characteristic substances, performs OPLS-DA analysis on pinellia and water pinellia, finds differential compounds between the two, combines multiple analysis methods such as box plot analysis, heat map analysis and correlation analysis, determines 5 key compounds for the identification of pinellia, water pinellia and their processed products, and then determines the relative contents of the 5 key compounds, uses OPLS-DA analysis to establish a pinellia and water pinellia identification model, and can realize the detection of water pinellia mixed in pinellia and its processed products.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a UPLC-Q / TOF-MS identification method for pinellia tuber and water pinellia tuber. Background Art

[0002] Pinellia ternata is the dried tuber of the plant Pinellia ternata (Thunb.) Breit. of the Araceae family. It has the effects of drying dampness and resolving phlegm, relieving adverse reactions and stopping vomiting, and eliminating lumps and dispersing nodules. The active ingredients of Pinellia ternata mainly include alkaloids, organic acids, volatile oils, sterols, Pinellia protein, amino acids, inorganic elements, etc. Pinellia ternata is the raw material for the preparation of many Chinese patent medicines. Water Pinellia ternata is the dried tuber of the plant Typhonium flagelliforme (Lodd.) Blume of the Araceae family. Water Pinellia ternata has a mild flavor and a pungent taste, which can numb the tongue and sting the throat. It is mostly used for internal use to treat respiratory diseases, such as cough and phlegm. Due to the price difference between Pinellia ternata and Water Pinellia ternata, some medicinal material markets use Water Pinellia ternata to replace Pinellia ternata, and Pinellia ternata and Water Pinellia ternata are seriously mixed in the market. Common medicinal forms of Pinellia include not only raw materials, but also processed products of Pinellia ternata with ginger, Pinellia ternata with clear water and Pinellia ternata with French method. Among them, lime water and licorice juice are added during the processing of Pinellia ternata with French method, and alum is added during the processing of Pinellia ternata with clear water. These two processed products can be clearly distinguished from the first two in properties, while there is no obvious difference in properties between Pinellia ternata with ginger and Pinellia ternata medicinal materials. Research on the identification of Pinellia ternata and Pinellia ternata with water and the corresponding processed products of Pinellia ternata with ginger is of great significance for the quality control of Pinellia ternata.

[0003] At present, most of the identification methods of Pinellia ternata and Pinellia ternata water are to identify the two by measuring a single component. This method cannot solve the problem of partial adulteration of Pinellia ternata, and the method of identifying a single component is easily disturbed by the deviation sample, which affects the accuracy of the identification result. CN117368340A discloses an identification method of adulterated Pinellia ternata in ginger Pinellia ternata based on LC-Q-TOF. This method is suitable for the identification of Pinellia ternata and Pinellia ternata water, but cannot be used for the identification of Pinellia ternata and Pinellia ternata water raw materials.

[0004] Based on this, establishing a comprehensive and effective identification method to distinguish between Pinellia ternata, hydrated Pinellia ternata and their processed products is of great significance for comprehensively evaluating the quality of the adulterated Pinellia ternata and its processed products on the market and providing a reference for their quality control. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a UPLC-Q / TOF-MS identification method for Pinellia ternata and water Pinellia ternata, which can realize the identification of Pinellia ternata and water Pinellia ternata, and is also suitable for the identification of ginger Pinellia ternata and ginger water Pinellia ternata, and can be used to identify partial adulteration of Pinellia ternata or ginger Pinellia ternata.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides a UPLC-Q / TOF-MS identification method for Pinellia ternata and Pinellia ternata, comprising the following steps:

[0008] S1. Prepare test solution by taking Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water respectively; prepare mixed reference solution by taking α-linolenic acid, rosin acid, hesperidin, atractylodesin, arginine, adenosine, guanosine, uridine, trigonelline, diisobutyl phthalate and dioctyl phthalate;

[0009] S2, the test solutions of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water prepared in S1 were separated by ultra-high performance liquid chromatography and detected by high-resolution mass spectrometry, respectively. E Scanning mode, obtaining mass spectrometry data of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water under positive ion mode, and finding out the differential components of Pinellia ternata and Pinellia ternata with water based on the mass spectrometry data;

[0010] S3, the test solution and the mixed reference solution prepared in S1 were separated by ultra-high performance liquid chromatography and detected by high-resolution mass spectrometry, using positive ion MS E Scan mode, obtain the mass spectrometry base peak chromatogram ion current diagram in positive ion mode, and use the collected UPLC-Q / TOF-MS data to extract compound information using UNIFI Portal software. According to the retention time, accurate molecular ion peak and secondary mass spectrum information of the compound, analyze and identify the components of the test solution;

[0011] S4. Progenesis QI software combined with multivariate statistical methods was used to identify the differential components found in S2. It was determined that the differential components between Pinellia ternata and Pinellia ternata were arginine, trigonelline, uridine, trans-p-hydroxycinnamic acid, phenylalanine, valerian proline, galactamine B, galactamine A, linoleic acid ethanolamide, linoleic acid-1-monoglyceride, linoleic acid and α-palmitin.

[0012] S5. The differential components determined in S4 were compared by box plot analysis, heat map analysis and correlation analysis to determine the key compounds for identification of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water, wherein the key compounds are guanidine A, guanidine B, arginine, valerian proline and trigonelline. The semi-quantitative data of the above key compounds detected in S3 were used to establish the OPLS-DA analysis model of Pinellia ternata and Pinellia ternata with water using SIMCA-P 14.1 software;

[0013] S6. Take the sample to be identified, prepare the test solution according to the method of S1, use ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection to obtain the mass spectrometry base peak chromatogram ion flow diagram in positive ion mode, and collect the mass spectrum information of the key compounds in S5;

[0014] S7. Import the mass spectrum information of the key compounds collected in S6 into the OPLS-DA analysis model in S5 for identification.

[0015] Furthermore, in S1, the preparation method of the test solution is to take a sample and bake it at 60°C for 10 hours, crush it, pass it through a No. 4 sieve, accurately weigh the sample powder and place it in a stoppered conical flask, accurately add 50% methanol, weigh it, ultrasonically extract it for 30 minutes, take it out and cool it, make up the lost weight with 50% methanol, shake it well, centrifuge it at 4000rpm for 10 minutes, take the supernatant and pass it through a 0.22μm microporous filter membrane to obtain it.

[0016] Furthermore, the preparation method of the mixed reference substance described in S1 is to take α-linolenic acid, rosin acid, hesperidin, atractylodesin, arginine, adenosine, guanosine, uridine, trigonelline, diisobutyl phthalate, and dioctyl phthalate, respectively, and place them in 10mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and prepare reference substance mother solutions respectively; accurately pipette 100 μL of each of the above reference substance mother solutions into the same 10mL volumetric flask, shake well, add 50% methanol solution to dilute to the scale, shake well, prepare a mixed reference substance solution, and filter through a 0.22 μm microporous filter membrane to obtain.

[0017] Further, in S2, the specific method for finding the differential components between Pinellia ternata and Pinellia ternata includes:

[0018] S21, importing the mass spectrometry data of Pinellia ternata, Pinellia ternata in water, Pinellia ternata in ginger and Pinellia ternata in water collected in positive ion mode into Progenesis QI software respectively, performing peak standardization, peak extraction, peak alignment and peak matching correction on the collected spectra to obtain information including compound retention time and mass-to-charge ratio;

[0019] S22, import all compound data into Ezinfo 3.0 software to obtain a .txt file;

[0020] S23. Import the obtained .txt file into SIMCA-P 14.1 software for multivariate statistical analysis, including principal component analysis and orthogonal partial least squares-discriminant analysis to distinguish the samples of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water, and Pinellia ternata with ginger and water, and use VIP>1 and P<0.05 as the screening conditions for differential components to screen out compounds with significant differences.

[0021] Further, in S3, the specific method for analyzing and identifying the components of the test solution detected includes:

[0022] S31. Ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection were used to perform positive ion MS on the mixed reference solution and the test solutions of Pinellia ternata and Pinellia ternata in water. E Scan to obtain the ion current diagram of the mass spectrometer base peak chromatogram in the positive ion mode;

[0023] S32. The Traditionl TCM database was used for screening to obtain the mass spectrometry data analysis results. The compound list given by the software was identified based on the compound fragmentation rules and the mass spectrometry data of related compounds. Finally, a total of 41 compounds were identified.

[0024] Furthermore, the chromatographic conditions used for ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection in S2 and S3 are:

[0025] Liquid chromatography column: Waters ACQUITY UPLC BEH C18, 2.1 mm × 100 mm × 1.7 μm;

[0026] Mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, the gradient elution program used is: 0-1min, 5%A; 1-8min, 5%-24%A; 8-16min, 24%-48%A; 16-25min, 48%-78%A; 25-30min, 78%-100%A;

[0027] Column temperature: 40°C;

[0028] Flow rate: 0.3 mL / min;

[0029] Injection volume: 1 μL.

[0030] Furthermore, the mass spectrometry conditions used for ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection in S2 and S3 are:

[0031] MS was performed in positive ion mode E scanning;

[0032] Cone voltage: 40V;

[0033] Source offset: 80V;

[0034] Ion source temperature: 100°C;

[0035] Desolvation temperature: 500°C;

[0036] Cone hole gas volume flow rate: 50L / h;

[0037] Desolvation gas volume flow rate: 800L / h;

[0038] Sampling cone voltage: 40V;

[0039] Low collision voltage: 6V;

[0040] High collision voltage: 40~60V.

[0041] Furthermore, the multivariate statistical method described in S4 includes principal component analysis and orthogonal partial least squares-discriminant analysis.

[0042] Compared with the prior art, the invention has the following beneficial effects: the present invention is based on the component information collected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, combined with multivariate statistical methods to screen characteristic substances, and OPLS-DA analysis is performed on pinellia and water pinellia to find the characteristic compounds of the two, and box plot analysis, heat map analysis and correlation analysis are combined to determine the key compounds of the identification method of pinellia and water pinellia and its processed products, 5, pinelliamine A, pinelliamine B, arginine, valerian proline and trigonelline. By determining the relative content of these 5 key compounds, the pinellia and water pinellia identification model is established by OPLS-DA analysis, and the detection of pinellia and its processed products doped with water pinellia can be realized. The identification method has the advantages of high detection credibility and strong predictive ability, and can effectively realize the identification of pinellia, water pinellia and their processed products at different doping concentrations, and comprehensively and accurately realize the identification and analysis of pinellia and water pinellia medicinal materials and their processed products on the market, and provide a reference for their quality control and further research on drug efficacy related mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the base peak chromatogram of the mixed reference solution in positive ion mode;

[0044] Among them, peak 1 (t R =0.72min) is arginine; Peak 2 (t R =0.80min) is trigonelline; Peak 3 (t R =0.87min) is uridine; Peak 4 (t R =0.87min) is adenosine; Peak 5 (t R =0.89min) is guanosine; Peak 6 (t R =8.17min) is hesperidin; Peak 7 (t R =14.48min) is atractylodesin; Peak 8 (t R =18.57min) is diisobutyl phthalate; Peak 9 (t R =20.85min) is linolenic acid; Peak 10 (t R =22.17min) is abietic acid; Peak 11 (t R =26.23min) is dioctyl phthalate;

[0045] Figure 2 is the base peak chromatogram of the test solution in positive ion mode;

[0046] Among them, the upper picture represents Pinellia ternata, and the lower picture represents Water Pinellia ternata;

[0047] Figure 3 This is a multivariate statistical analysis chart of Pinellia ternata and Pinellia ternata polyphylla;

[0048] Among them, A is the PCA analysis diagram; B is the OPLS-DA analysis diagram; C is the S-plot diagram (the components with VIP>1.0 are red); D is the OPLS-DA model cross-validation diagram;

[0049] Figure 4 This is the multivariate statistical analysis chart of Pinellia ternata with ginger and Pinellia ternata with ginger water;

[0050] Among them, A is the PCA analysis diagram; B is the OPLS-DA analysis diagram; C is the S-plot diagram (the components with VIP>1.0 are red); D is the OPLS-DA model cross-validation diagram;

[0051] Figure 5 This is the box plot of the differential components of Pinellia ternata and Pinellia ternata;

[0052] Figure 6 This is the clustering heat map of the differential components of Pinellia ternata and Pinellia ternata;

[0053] Figure 7 This is the correlation analysis diagram of the differential components of Pinellia ternata and Pinellia ternata;

[0054] Figure 8 OPLS-DA analysis diagram of Pinellia ternata, Pinellia ternata in water and their adulterated samples;

[0055] Fig. 9 This is the OPLS-DA analysis chart of ginger pinellia, ginger water pinellia and their adulterated samples. DETAILED DESCRIPTION

[0056] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0058] Example 1: Establishment of UPLC-Q / TOF-MS Identification Method for Pinellia ternata and Pinellia ternata

[0059] 1. Instruments and Materials

[0060] The ultra-high performance liquid phase system included Waters Acquity high performance liquid chromatograph, Sample Manager FTN autosampler, Binary Solvent Manager binary pump, and WAT-S4LV1467 column oven (Waters, USA); XevoG2-XS Q / TOF mass spectrometer: equipped with Masslynx V4.2 analysis software, UNIFI Portal software, Progenesis QI software, and TCM Chinese database (Waters, USA); KQ-500E CNC ultrasonic extractor (Kunshan Ultrasonic Instrument Co., Ltd., power: 40kHz); MS205DU electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); and low-speed desktop large-capacity multi-tube centrifuge (Shanghai Anting Scientific Instrument Factory).

[0061] Acetonitrile was of mass spectrometry grade (Merck, Germany); formic acid was of mass spectrometry grade (Thermo Fisher Scientific (China) Co., Ltd.); purified water was (Hangzhou Wahaha Drinking Water Co., Ltd.).

[0062] α-linolenic acid (batch number: 11631-200502), abietic acid (batch number: 111938-201201), hesperidin (batch number: 110721-201316), atractylodesin (batch number: 111924-201404) and uridine (batch number: 887-200202) were purchased from the China Food and Drug Administration; arginine (Shanghai Yuanye Biotechnology Co., Ltd., batch number: T01J11H11 7320), adenosine (batch number: WP24032911), guanosine (batch number: WP24060505) and trigonelline (batch number: WP23101009) reference substances were purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; diisobutyl phthalate (batch number: 1003651670) and dioctyl phthalate (batch number: 102656455) reference substances were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0063] The Pinellia ternata and Pinellia ternata used in the experiment were purchased from various production areas and identified as Pinellia ternata of the Araceae family by the Chinese Medicine Identification Teaching and Research Office of the School of Pharmacy of Jiangxi University of Traditional Chinese Medicine Pinellia Rhizoma The dried tuber of Thunb. Breit. or the Araceae plant Typhonium flagelliforme Dried tubers of (Lodd.) Blume. The processed products of Pinellia ternata and Pinellia ternata in water are processed according to the provisions of "Pinellia ternata" in Volume 1 of the Chinese Pharmacopoeia. The specimens are kept in the research room of Jiangxi Provincial Institute of Drug Inspection and Testing. The information of Pinellia ternata and Pinellia ternata samples is shown in Table 1.

[0064] Table 1: Information on the medicinal materials and decoction pieces of Pinellia ternata and Pinellia ternata aquat

[0065]

[0066] 2. Test conditions

[0067] 2.1 Chromatographic conditions: Waters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm × 1.7 μm) liquid chromatography column; mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B); gradient elution: 0-1 min, 5% A; 1-8 min, 5%-24% A; 8-16 min, 24%-48% A; 16-25 min, 48%-78% A; 25-30 min, 78%-100% A. Column temperature: 40 °C; flow rate: 0.3 mL / min; injection volume: 1 μL.

[0068] 2.2 Mass spectrometry conditions: Electrospray ionization source (ESI): MS was performed in positive ion mode. E Scanning; cone voltage: 40V; source offset: 80V; ion source temperature: 100℃; desolvation gas temperature: 500℃; cone gas volume flow rate (N2) is 50L / h; desolvation gas volume flow rate (N2) is 800 L / h; sampling cone voltage: 40V; low collision voltage is 6V; high collision voltage is 40~60V. Real-time calibration of mass number of leucine enkephalin. UNIFI Chinese medicine chemical component database and self-built Pinellia component database.

[0069] 3. Preparation of reference solution

[0070] Accurately weigh about 10 mg each of α-linolenic acid, abietic acid, hesperidin, atractylodesin, arginine, adenosine, guanosine, uridine, trigonelline, diisobutyl phthalate, and dioctyl phthalate reference substances, respectively, and place them in 10 mL volumetric flasks, add a small amount of methanol to dissolve and dilute to the scale, shake well, and prepare reference substance mother solutions with a mass concentration of about 1.00 mg / mL; accurately pipette 100 μL of the above reference substance mother solutions respectively, place them in the same 10 mL volumetric flask, shake well, add 50% methanol solution to dilute to the scale, shake well, and prepare a mixed reference substance solution with a mass concentration of about 10.0 μg / mL, and filter through a 0.22 μm microporous filter membrane to obtain.

[0071] 4. Preparation of test solution

[0072] Take samples of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water, and Pinellia ternata with ginger water and dry at 60℃ for 10h, crush, and pass through a No. 4 sieve. Accurately weigh about 0.5g of sample powder and place it in a stoppered conical bottle, accurately add 20mL of 50% methanol, weigh, and ultrasonically extract (power: 40kHz) for 30min, take out and cool, make up the lost weight with 50% methanol, shake well, centrifuge at 4000rpm for 10min, and take the supernatant to pass through a 0.22μm microporous filter membrane to obtain the product.

[0073] 5. Data Collection and Processing

[0074] Take the samples of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water, and Pinellia ternata with ginger water in Table 1, and prepare the test solutions of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water, and Pinellia ternata with ginger water respectively according to the method under "4. Preparation of test solution", and use UPLC-Q-TOF-MS instrument for positive ion MS E Scanning, Masslynx V4.2 was used to collect data on Pinellia, Pinellia radix ginger, Pinellia ternata and Pinellia ternata in ginger water in positive ion mode, and the mass spectrometry data were imported into Progenesis QI software, and the acquired spectra were subjected to peak standardization, peak extraction, peak alignment, peak matching correction, etc. to obtain the compound retention time and mass-to-charge ratio information, and all the compound data were imported into EZinfo 3.0 software to obtain a .txt file; the obtained file was imported into SIMCA-P 14.1 software, and PCA and OPLS-DA data processing methods were used to distinguish Pinellia, Pinellia radix ginger, Pinellia ternata and Pinellia ternata in ginger water samples, and VIP>1 and P<0.05 were used as the screening conditions for differential components to find out the differential components between Pinellia ternata and Pinellia ternata in ginger water.

[0075] 6. Qualitative analysis of chemical components of Pinellia ternata by UPLC-Q-TOF-MSE

[0076] The mixed reference solution, Pinellia ternata and water Pinellia ternata test solution were scanned by positive ion MSE using UPLC-Q-TOF-MS to obtain the mass spectrometry base peak chromatogram in positive ion mode, see Figure 1 and Figure 2 The Traditionl TCM database provided by UNIFI Portal software and the Pinellia database built by the research group (the Pinellia database built by the research group was established using UNIFI Portal software based on the molecular formula, structural formula and compound category of the chemical components of Pinellia reported in the literature) were used as the basis for screening, and preliminary mass spectrometry data analysis results were obtained. The list of possible compounds given by the software was manually identified based on the compound fragmentation rules and the mass spectrometry data of related compounds. Finally, a total of 41 compounds were identified. The retention time, quasi-molecular ion and fragment ion information of the 41 compounds are detailed in Table 2 below.

[0077] Table 2: UPLC-Q-TOF-MS of Pinellia ternata E Chemical composition identification table

[0078]

[0079]

[0080]

[0081] Note: The * is verified by the reference substance. The reference substance, trigonelline, is detected in Pinellia ternata, so it is not shown in this table.

[0082] 7. Identification of Differential Components in Pinellia ternata and Pinellia ternata

[0083] The mass spectrometry data of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water collected in the positive ion mode under "5. Data acquisition and processing" were imported into Progenesis QI software respectively, and all compound data were imported into Ezinfo 3.0 software to obtain .txt files; the obtained files were imported into SIMCA-P 14.1 software for multivariate statistical analysis, including PCA (principal component analysis) analysis and OPLS-DA (orthogonal partial least squares-discriminant) analysis.

[0084] Five randomly selected Pinellia samples and Pinellia QC samples (Pinellia QC samples are mixed samples of all Pinellia) were divided into one group, and five water Pinellia samples and water Pinellia QC samples (water Pinellia QC samples are mixed samples of all water Pinellia) were divided into another group. Principal component PCA analysis and supervised OPLS-DA analysis were performed. The results are shown in Figure 3 Among them, the PCA diagram in positive ion mode is shown in Figure 3 As shown in A, R²=0.547, Q²=0.215, it can be seen from the figure that the two groups of Pinellia ternata and water Pinellia ternata are significantly different; supervised OPLS-DA analysis is performed on them, and the results are shown in Figure 3 As shown in B, the difference between the two groups of Pinellia ternata and Pinellia ternata is obvious. 2 X=0.516, R 2 Y=0.969, Q 2 =0.867, indicating that the model has high credibility and strong predictive ability; scatter score graph (S-plot) see Figure 3 As shown in C in the figure, the S-plot can represent the difference between groups. The farther the data point is from the origin, the greater the contribution of the variable to the difference in sample grouping. The S-plot can be used to screen out compounds with significant differences (VIP>1). Figure 3 As shown in D, it can be seen that the R 2and Q 2 The slopes of the regression line Q 2 The intercept with the Y axis is less than 0, indicating that the OPLS-DA model is reliable.

[0085] All samples of Pinellia ternata and Pinellia ternata in ginger water were divided into one group and PCA and OPLS-DA were performed. The results are shown in Figure 4 PCA diagram in positive ion mode Figure 4 As shown in A, R²=0.739, Q²=0.386. It can be seen from the figure that the two groups of samples of ginger pinellia and ginger water pinellia are significantly different. A supervised OPLS-DA analysis was performed on them; the results are shown in Figure 4 As shown in B, the difference between the two groups of samples of ginger pinellia and ginger water pinellia is obvious. 2 X=0.793, R 2 Y=0.986, Q 2 =0.885, indicating that the model has high credibility and strong predictive ability; scatter score graph (S-plot) see Figure 4 As shown in C in the figure, the S-plot can represent the difference between groups. The farther the data point is from the origin, the greater the contribution of the variable to the difference in sample grouping. The S-plot can be used to screen out compounds with significant differences (VIP>1). Figure 4 As shown in D, the R values ​​of two groups of samples in positive mode 2 and Q 2 The slopes of the regression line Q 2 The intercept with the Y axis is less than 0, indicating that the OPLS-DA model is reliable.

[0086] The screened differential components of Pinellia ternata and water Pinellia ternata were compared with those of Pinellia ternata with ginger and water Pinellia ternata. There were 12 identical differential components, including arginine, menthaamine B, etc. The results of the 12 differential components of Pinellia ternata, water Pinellia ternata and their processed products are shown in Table 3.

[0087] Table 3: Different components of Pinellia ternata, Pinellia ternata in water and their processed products

[0088]

[0089] 8. Determination of characteristic compounds for identification of Pinellia ternata and Pinellia ternata

[0090] Under the item “7. Identification of differential components in the identification of Pinellia ternata and Pinellia ternata”, on the basis of multivariate statistical analysis, a box plot analysis was performed on the 12 differential components in the Pinellia ternata samples in Table 3. The results are as follows Figure 5As shown, it was found that: the content of compound No. 1 arginine in Pinellia ternata and its processed products was higher than that in Pinellia ternata and its processed products, and it can be used as a characteristic compound for identifying Pinellia ternata; the content of compound No. 2 trigonelline in Pinellia ternata and its processed products was much higher than that in Pinellia ternata and its processed products, and it can be used as a characteristic compound for identifying Pinellia ternata; the content of compound No. 6 valproic acid in Pinellia ternata with ginger water was much higher than that in Pinellia ternata and its processed products, and it can be used as a characteristic compound for identifying Pinellia ternata with ginger water; the contents of compound No. 7 guanidine B and compound No. 8 guanidine A in Pinellia ternata were much higher than those in Pinellia ternata with ginger water, and they can be used as characteristic compounds for identifying Pinellia ternata.

[0091] Cluster heat map analysis of samples such as Pinellia ternata was performed using 12 differential components. The results are as follows: Figure 6 As shown, it was found that: the pinellia samples were clustered into one category, and the contents of pinelliamine A, pinelliamine B, linoleic acid ethanolamide, phenylalanine, α-palmitin and arginine in pinellia were higher than those in other samples; the ginger water pinellia was clustered into a group compared with ginger pinellia and water pinellia, and the content of valerian proline in ginger water pinellia was much higher than that in other groups, which was significantly different from other samples; the content of trigonelline in the water pinellia group was much higher than that in other groups, and the clustering was good.

[0092] Correlation analysis was performed on these 12 differential components in samples such as Pinellia ternata. The results are as follows: Figure 7 As shown, it was found that the correlation coefficient between compound No. 7 benzoate B and compound No. 8 benzoate A was 0.8, indicating that the two were strongly positively correlated; the correlation coefficient between trans-p-hydroxycinnamic acid and phenylalanine was 1, and the correlation coefficients of both with linoleic acid-1-monoglyceride were both 0.8, indicating that the three were strongly positively correlated.

[0093] The above analysis screened out five characteristic compounds for identification of Pinellia ternata and Pinellia ternata, including: quinqueline A, quinqueline B, arginine, valerian proline, and trigonelline.

[0094] Example 2: Identification of Pinellia ternata and Pinellia ternata and their processed products using the five identified characteristic compounds

[0095] Take the pinellia and water pinellia samples, and prepare water pinellia adulterated samples with different doping amounts (the doping amounts of water pinellia are 10%, 20%, 80% and 90%, respectively) as the test solution according to the method under "4. Preparation of test solution" in Example 1, and perform positive ion MS on the water pinellia adulterated samples using UPLC-Q-TOF-MS according to the method under "2. Detection conditions" in Example 1. EScanning determination, the relative contents of the five characteristic compounds obtained: quinquercetin A, quinquercetin B, arginine, valerian proline, and trigonelline were brought into the previously established pinellia identification model for OPLS-DA analysis. The pinellia samples mixed with a small amount (10%, 20%) of water pinellia and a large amount (80%, 90%) of water pinellia were analyzed. The results are as follows Figure 8 As shown, it was found that: no matter the samples of Pinellia ternata were adulterated with a small amount of water Pinellia ternata or the samples were adulterated with a large amount of water Pinellia ternata, they could be clearly distinguished from the samples of Pinellia ternata and water Pinellia ternata, and were obviously far away from the Pinellia ternata samples, indicating that the relative contents of the above five characteristic compounds can be used to effectively distinguish the Pinellia ternata samples and the samples of Pinellia ternata adulterated with water Pinellia ternata.

[0096] Take the samples of Pinellia ternata and Pinellia ternata in ginger water, and prepare Pinellia ternata adulterated samples with different doping amounts in ginger water (the doping amounts of Pinellia ternata in ginger water are 10%, 20%, 80% and 90%, respectively) as the test solution according to the method under "4. Preparation of test solution" in Example 1, and perform positive ion MS on the Pinellia ternata adulterated samples in ginger water according to the method under "2. Detection conditions" in Example 1 using UPLC-Q-TOF-MS instrument. E Scanning determination, using the above analytical method to measure the sample, the relative contents of the five characteristic compounds obtained: quinquercetin A, quinquercetin B, arginine, valerian proline, and fenugreek alkaloids were brought into the previously established pinellia identification model for OPLS-DA analysis. OPLS-DA analysis was performed on the samples of ginger pinellia mixed with a small amount (10%, 20%) of ginger water pinellia and a large amount (80%, 90%) of ginger water pinellia. The results are shown in Fig. 9 As shown, it was found that: the samples of ginger pinellia, whether they were mixed with a small amount of ginger pinellia or a large amount of ginger pinellia, could be clearly distinguished from the samples of ginger pinellia and ginger pinellia, and were obviously far away from the ginger pinellia samples, indicating that the relative content of the above five characteristic compounds can be used to effectively distinguish the ginger pinellia samples and the samples of ginger pinellia adulterated with ginger pinellia.

[0097] In summary, the use of UPLC-Q / TOF-MS E The technology combined with multivariate statistical methods, through the analysis of the chemical composition of the samples of Pinellia and Pinellia ternata, found that there are 12 different components between Pinellia and Pinellia ternata. According to the results of correlation analysis, combined with the optimization of the compound content of the box plot and heat map, the following results were obtained: the key compounds for establishing the identification method of Pinellia ternata and Pinellia ternata ternata are: baicalin A, baicalin B, arginine, valerian proline, and fenugreek alkaloids. Then, by measuring the relative content of these five key compounds, the OPLS-DA analysis was used to establish the identification model of Pinellia ternata and Pinellia ternata ternata, which can realize the rapid and effective identification of Pinellia ternata and its processed products mixed with Pinellia ternata ternata.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solution requested for protection in this application.

Claims

1. A UPLC-Q / TOF-MS identification method for Pinellia ternata and Pinellia ternata, characterized in that: The following steps are involved: S1. Prepare test solution by taking Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water respectively; prepare mixed reference solution by taking α-linolenic acid, rosin acid, hesperidin, atractylodesin, arginine, adenosine, guanosine, uridine, trigonelline, diisobutyl phthalate and dioctyl phthalate; S2, the test solutions of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water prepared in S1 were separated by ultra-high performance liquid chromatography and detected by high-resolution mass spectrometry, respectively. E Scanning mode, obtaining mass spectrometry data of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water under positive ion mode, and finding out the differential components of Pinellia ternata and Pinellia ternata with water based on the mass spectrometry data; S3, the test solution and the mixed reference solution prepared in S1 were separated by ultra-high performance liquid chromatography and detected by high-resolution mass spectrometry, using positive ion MS E Scan mode, obtain the mass spectrometry base peak chromatogram ion current diagram in positive ion mode, and use the collected UPLC-Q / TOF-MS data to extract compound information using UNIFI Portal software. According to the retention time, accurate molecular ion peak and secondary mass spectrum information of the compound, analyze and identify the components of the test solution; S4. Progenesis QI software combined with multivariate statistical methods was used to identify the differential components found in S2. It was determined that the differential components between Pinellia ternata and Pinellia ternata were arginine, trigonelline, uridine, trans-p-hydroxycinnamic acid, phenylalanine, valerian proline, galactamine B, galactamine A, linoleic acid ethanolamide, linoleic acid-1-monoglyceride, linoleic acid and α-palmitin. S5. The differential components determined in S4 were compared by box plot analysis, heat map analysis and correlation analysis to determine the key compounds for identification of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water and Pinellia ternata with ginger water, wherein the key compounds are guanidine A, guanidine B, arginine, valerian proline and trigonelline. The semi-quantitative data of the above key compounds detected in S3 were used to establish the OPLS-DA analysis model of Pinellia ternata and Pinellia ternata with water using SIMCA-P 14.1 software; S6. Take the sample to be identified, prepare the test solution according to the method of S1, use ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection to obtain the mass spectrometry base peak chromatogram ion flow diagram in positive ion mode, and collect the mass spectrum information of the key compounds in S5; S7, importing the mass spectrum information of the key compounds collected in S6 into the OPLS-DA analysis model in S5 for identification; In S2, the specific method for finding the differential components of Pinellia ternata and Pinellia ternata includes: S21, importing the mass spectrometry data of Pinellia ternata, Pinellia ternata in water, Pinellia ternata in ginger and Pinellia ternata in water collected in positive ion mode into Progenesis QI software respectively, performing peak standardization, peak extraction, peak alignment and peak matching correction on the collected spectra to obtain information including compound retention time and mass-to-charge ratio; S22, import all compound data into Ezinfo 3.0 software to obtain a .txt file; S23, importing the obtained .txt file into SIMCA-P 14.1 software for multivariate statistical analysis, including principal component analysis and orthogonal partial least squares-discriminant analysis to distinguish the samples of Pinellia ternata, Pinellia ternata with ginger, Pinellia ternata with water, and Pinellia ternata with ginger water, and using VIP>1 and P<0.05 as the screening conditions for differential components to screen out compounds with significant differences; The chromatographic conditions used for ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection in S2 and S3 were: Liquid chromatography column: Waters ACQUITY UPLC BEH C18, 2.1 mm × 100 mm × 1.7 μm; Mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, the gradient elution program used is: 0-1min, 5%A; 1-8min, 5%-24%A; 8-16min, 24%-48%A; 16-25min, 48%-78%A; 25-30min, 78%-100%A; Column temperature: 40°C; Flow rate: 0.3 mL / min; Injection volume: 1 μL; The mass spectrometry conditions used for ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection in S2 and S3 were: MS was performed in positive ion mode E scanning; Cone voltage: 40V; Source offset: 80V; Ion source temperature: 100°C; Desolvation temperature: 500°C; Cone hole gas volume flow rate: 50L / h; Desolvation gas volume flow rate: 800L / h; Sampling cone voltage: 40V; Low collision voltage: 6V; High collision voltage: 40~60V.

2. The UPLC-Q / TOF-MS identification method of Pinellia ternata and Pinellia ternata according to claim 1, characterized in that: In S1, the preparation method of the test solution is to take a sample and dry it at 60°C for 10 hours, crush it, pass it through a No. 4 sieve, accurately weigh the sample powder and place it in a stoppered conical flask, accurately add 50% methanol, weigh it, ultrasonically extract it for 30 minutes, take it out and cool it, make up the lost weight with 50% methanol, shake it well, centrifuge it at 4000rpm for 10 minutes, take the supernatant and pass it through a 0.22μm microporous filter membrane to obtain it.

3. The UPLC-Q / TOF-MS identification method of Pinellia ternata and Rhizoma Pinelliae Rhizoma according to claim 1, characterized in that: The preparation method of the mixed reference substance described in S1 is as follows: take α-linolenic acid, rosin acid, hesperidin, atractylodesin, arginine, adenosine, guanosine, uridine, fenugreek alkaloids, diisobutyl phthalate, and dioctyl phthalate, respectively, put them in 10mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and prepare reference substance mother solutions respectively; accurately pipette 100μL of each of the above reference substance mother solutions into the same 10mL volumetric flask, shake well, add 50% methanol solution to dilute to the scale, shake well, prepare a mixed reference substance solution, and filter through a 0.22μm microporous filter membrane to obtain.

4. The UPLC-Q / TOF-MS identification method of Pinellia ternata and Rhizoma Pinelliae Rhizoma according to claim 1, characterized in that: In S3, the specific methods for analyzing and identifying the components of the test solution include: S31. Ultra-high performance liquid chromatography separation and high-resolution mass spectrometry detection were used to perform positive ion MS on the mixed reference solution and the test solutions of Pinellia ternata and Pinellia ternata in water. E Scan to obtain the ion current diagram of the mass spectrometer base peak chromatogram in the positive ion mode; S32. The Traditionl TCM database was used for screening to obtain mass spectrometry data analysis results. The compound list given by the software was identified based on the compound fragmentation rules and mass spectrometry data of related compounds. Finally, a total of 41 compounds were identified.

5. The UPLC-Q / TOF-MS identification method of Pinellia ternata and Pinellia ternata according to claim 1, characterized in that: The multivariate statistical methods described in S4 include principal component analysis and orthogonal partial least squares-discriminant analysis.

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