Method for analyzing chemical component difference of aconitum pendulum and children's excrement product based on UPLC-Q-TOF-MS-MS method

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis and traditional Chinese medicine quality control, in particular to a method for analyzing the chemical component difference of aconitum pendulum and a child excrement product thereof based on a UPLC-Q-TOF-MS-MS (Ultra Performance Liquid Chromatography-Quality Time of Flight Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass analyzing chemical components in the sample; performing principal component analysis and orthogonal partial least squares discriminant analysis on the mass spectrum data by using SPSS software, and comparing the difference of chemical components of the aconitum pendulum and the processed products of the defecate of the aconitum pendulum from different producing areas; according to the invention, through comprehensive analysis of chemical components of the crude aconitum pendulum and the processed children's excrement product, a scientific research on a material basis is further laid for revealing the medicine effect enhancement of the processing of the aconitum pendulum prepared from children's excrement, and meanwhile, the research result shows that the Tibetan medicine strictly complies with the processing procedure in clinical application of the aconitum pendulum; meanwhile, an important basis needs to be provided for achieving the purpose of reducing toxicity by paying attention to reasonable compatibility of medicinal materials.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis and traditional Chinese medicine quality control, and specifically to a method for analyzing the difference in chemical components of an iron hammer and its children's urine products based on a UPLC-Q-TOF-MS-MS method. Background Art

[0002] Aconitum pendulum Busch. and Aconitum flavum Hand.-Mazz. are dried tuberous roots of the Ranunculaceae plants. It is often used to treat rheumatoid arthritis, traumatic injuries and other diseases.

[0003] However, the toxic components of the Chinese medicine Rhizoma Aconitifolia, such as aconitine, deoxyaconitine and 3-acetylaconitine, make the clinical treatment dose of the Chinese medicine Rhizoma Aconitifolia close to the poisoning dose. Due to the frequent reports of poisoning and even death caused by taking too much Chinese medicine Rhizoma Aconitifolia, the clinical use of the Chinese medicine Rhizoma Aconitifolia has been greatly limited. Therefore, the research on the reduction of toxicity and enhancement of efficacy of Chinese medicine Rhizoma Aconitifolia has become a research hotspot in recent years. In clinical practice, Chinese medicine Rhizoma Aconitifolia is generally not used in its raw form, but needs to be processed before use as medicine.

[0004] Due to the different origins of the iron hammer, comparing the differences in the chemical composition of the iron hammers from different origins and their child urine preparations can lay a material foundation for revealing that the preparation of child urine-made iron hammers can enhance the efficacy. At the same time, the research results provide an important basis for Tibetan medicine to pay attention to the rational compatibility of medicinal materials in the clinical application of iron hammers in addition to strictly adhering to the preparation procedures to achieve the purpose of detoxification. Summary of the invention

[0005] In order to clarify the differences in chemical components of an iron hammer and its products made from children's feces, the present invention provides a method for analyzing the differences in chemical components of an iron hammer and its products made from children's feces based on a UPLC-Q-TOF-MS / MS method.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method for analyzing the difference in chemical composition of an iron hammer and its children's urine products based on UPLC-Q-TOF-MS / MS method is characterized by comprising the following steps:

[0008] Step 1, preparation of test solution: pulverize the raw iron hammer products and their corresponding processed products from 4 different origins, pass through a No. 3 sieve, weigh about 0.2 g of each sample, accurately weigh, place in a 50 mL stoppered conical flask, add 10 mL of 95% methanol, shake well, ultrasonically treat (power 300 W, frequency 40 KHz) for 60 min, take out, cool, shake well, take 2 mL of liquid in a centrifuge tube, high-speed centrifuge (12000 rpm / min) for 5 min, take the supernatant, and obtain;

[0009] Step 2, chromatographic conditions: Agilent Poroshell 120Aq-C 18 (2.1×100mm, 2.7μm) chromatographic column; mobile phase acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-3min, 5% A, 95% B; 3-15min, 5%-25% A, 95%-75% B; 15-28min, 25%-35% A, 75%-65% B; 28-33min, 35%-72% A, 65%-28 % B; 33-43min, 72%-95% A, 28%-5% B; 43-47min, 95% A, 5% B; 47-47.1min, 95%-5% A, 5%-95% B; 49.1-50min, 5% A, 95% B); column temperature 30°C; flow rate 0.3mL / min; injection volume 2μL; detection wavelength range 190~400nm;

[0010] Step 3, mass spectrometry conditions: detection in ESI-Negative / Positive ion mode; TOF mass range 50-1700; spray gas pressure 50psi; auxiliary heater pressure 50psi; curtain gas pressure 35psi; ionization voltage -4500 / 5000V; ion source temperature 500℃; declustering voltage 100V; collision voltage 10V; MS / MS secondary mass range 50-1250; declustering voltage 100V; collision voltage ±40eV; collision voltage swing 20eV; ion release delay 30ms; ion beam width 15ms;

[0011] Step 4: Data processing and analysis, specifically including the following steps:

[0012] Step (1), using UPLC-Q-TOF-MS / MS to collect mass spectrometry data of the raw iron rod hammer products and processed products thereof from different locations according to the above-mentioned chromatographic and mass spectrometric conditions, with the mass spectrometry deviation being less than or equal to 5.7 ppm, and obtaining a UPLC-HRMS base peak ion chromatogram (BPC) of full scan in positive and negative ion modes;

[0013] Step (2), analyzing the chemical components in the sample in combination with literature reports and the Stende natural product high-resolution mass spectrometry database to obtain structural information such as the actual and theoretical values ​​of the compound name and molecular formula m / z, and secondary mass spectrometry fragmentation data;

[0014] Step (3), selecting the mass spectrometry data of the chemical components of the iron club and its child urine processed product under the positive ion mode with a strong response, a total of 50 diterpene alkaloids were identified, including 13 non-ester types, 11 monoester types, 15 diester types, 2 multiester types and 9 long-chain ester types, as shown in Tables 1 and 2;

[0015] Table 1 Identification results of chemical components of iron hammers from different origins and their processed urine products in positive ion mode

[0016]

[0017]

[0018]

[0019] As shown in Table 1, the compounds detected in the positive ion mode with higher response were subjected to accurate mass qualitative analysis and secondary fragment structure analysis, and TOF-MS matched the molecular formula. A total of 50 diterpene alkaloids were identified through comparative analysis combined with Scifinder database and HR-MS / MS Spectral Library 1.1 mass spectrum database matching, including 13 non-ester types, 11 monoester types, 15 diester types, 2 polyester types and 9 long-chain ester types.

[0020] Table 2 Peak areas of iron hammer ions from different origins and their change indexes before and after processing under positive ion mode

[0021]

[0022]

[0023]

[0024] The processing technology is as follows: wrap it with wet paper, then simmer it with wood ash, remove the paper, immerse the iron hammer in children's urine for 24 hours, take it out, rinse it with running water and dry it, and you will get the processed product.

[0025] As shown in Table 2, the chemical component types and contents of raw iron rods from different collection points were different. The component types after processing did not change, the contents of MDAs and ADAs decreased slightly, the contents of LDAs and PDAs increased slightly, and the content of DDAs did not change significantly. Among them, the content of Dehydrolucidusculine increased significantly after processing, Polyschistine D disappeared after processing, the contents of Delpinine and Chasmaconitine decreased significantly after processing, and Falconeridine did not exist in the three production areas and their processed products in this study.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: through a comprehensive analysis of the chemical composition of the raw iron club hammer and the wine-processed product, the present invention further lays a material foundation for scientific research to reveal that the processing of the iron club hammer made from highland barley wine can enhance the efficacy. At the same time, the research results provide an important basis for Tibetan medicine to pay attention to the rational compatibility of medicinal materials in addition to strictly abiding by the processing procedures in the clinical application of the iron club hammer to achieve the purpose of detoxification. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the MS chart of compound 11 in Example 1 of the present invention;

[0028] Figure 2 It is the MS chart of compound 13 in Example 1 of the present invention;

[0029] Figure 3 is the MS chart of compound 30 in Example 2 of the present invention;

[0030] Figure 4 is the MS chart of compound 29 in Example 2 of the present invention;

[0031] Figure 5 is the MS chart of compound 27 in Example 3 of the present invention;

[0032] Figure 6 is the MS chart of compound 16 in Example 3 of the present invention;

[0033] Figure 7 It is the MS chart of compound 44 in Example 4 of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, 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.

[0035] Instrument: Waters H-Class ultra-high performance liquid chromatograph (Waters Technology Co., Ltd.); AB SciexTriple 4600 high-resolution mass spectrometer (SCIEX); ME104 electronic balance (Mettler-Toledo International Trading (Shanghai) Co., Ltd.); KQ-300BD ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); SIGMA 3K15 high-speed centrifuge (SIGMA).

[0036] Materials: The samples of the iron club were collected in early October 2023 from Zhiduo County, Yushu Prefecture, Qinghai Province (numbered QHZD), Qumalai County, Yushu Prefecture, Qinghai Province (numbered QHQML), and Guide County, Hainan Prefecture, Qinghai Province (numbered QHgNgMy). They were identified as dried tubers of the iron club by Jiawa, chief physician of the Qinghai Institute of Tibetan Medicine, and the specimens were stored in the sample room of the Qinghai Institute of Tibetan Medicine. The processed barley wine products were provided by the Qinghai Institute of Tibetan Medicine, and the numbers were QHLD-1, QHQML-1, and QHgNgMy-1. Acetonitrile was mass spectrometry grade (I1133829105, Merck), methanol was mass spectrometry grade (I1139035113, Merck), water was purified water (20230911C, Guangzhou Watsons Food and Beverage Co., Ltd.), and formic acid was mass spectrometry grade (K9050188, CNW).

[0037] The most toxic C19-DAs are the main components of the iron club, which are classified according to the ester bond into alcohol amine type, monoester type, diester type, multiester type, and long-chain ester type. Among them, diester type diterpene alkaloids are the most toxic, and alcohol amine type diterpene alkaloids are the least toxic. In this experiment, full scan mass spectrometry was used to obtain the relevant mass spectrometry data of iron club and its children's urine preparations in positive and negative ion modes.

[0038] Example 1 Alcoholamine diterpenes (C19-ADAs)

[0039] Common substituent positions of C19-ADAs are C1, C6 and C16. The types of substituents include α-type oxygen-containing substitutions at C-1 and C-6, and β-type -CH3OH and -OH substitutions at C-16. There are no ester groups in the molecular structure. Common mass loss fragments during the cleavage of C19-ADAs include neutral molecules such as CH3OH (32Da), H2O (18Da) and CO (28Da), which then produce [M+H-32] + , [M+H-18] + , [M+H-28] + The corresponding characteristic fragment ion peaks are as follows. Figure 1 As shown, the quasi-ion peak of compound 11 is m / z 400.2505 [M+H] + , fragment ion peak m / z 382.2378 [M+H-H2O] + 、m / z 340.2268[M+H-CH3COOH] + , m / z322.2159[M+H-CH3COOH-H2O] + Combined with the prior art, compound 11 was identified as Dehydrolucidusculine.

[0040] like Figure 2As shown, compound 13 is a diterpenoid with a quasi-ion peak of m / z 466.3144 [M+H] + , fragment ion peak m / z 434.2893[M+H-CH3OH] + 、m / z402.2635[M+H-CH3OH] + Combined with the prior art, compound 13 was identified as Homochasmanine.

[0041] Example 2 C19-MDAs

[0042] The common substituent position of monoester C19-MDAs is only at C14, and the substituent types include benzoyl, acetyl, and cinnamoyl. The β-OH at C8 is not esterified. Therefore, the common mass loss fragments in the cleavage law of monoester diterpene alkaloids include neutral molecules such as C6H5COOH (Da211) or AcOH (Da60), H2O (Da18), and CH3OH (Da32), forming [M+H-122] + ([M+H-106] + ) or [M+H-60] + , [M+H-18] + , [M+H-32] + , [M+H-50] + 、[M+H-64] + , [M+H-82] + etc. fragment ions.

[0043] like Figure 3 As shown, the quasi-ion peak of compound 30 is m / z 510.3050 [M+H] + , fragment ion peak m / z450.2845[M+H-AcOH] + , m / z 418.2579[M+H-AcOH-CH3OH] + , m / z 390.2632[M+H-AcOH-CH3OH-CO] + Combined with the prior art, compound 30 was identified as 14-Acetylbrowniine.

[0044] like Figure 4 As shown, the quasi-ion peak of compound 29 is m / z 496.2917 [M+H] + , fragment ion peak m / z478.2818[M+H-H2O] + 、m / z 436.2703[M+H-AcOH] + 、m / z 418.2600[M+H-AcOH-H2O] +、m / z376.2492[M+H-2AcOH] + , m / z 358.2381[M+H-2AcOH-H2O] + , m / z 326.2112[M+H-2AcOH-H2O-CH3OH] + Combined with the existing technology, it is speculated that compound 29 is identified as Taurenine.

[0045] Example 3 C19-DDAs

[0046] Common substituent positions of C19-diester diterpene alkaloids are at C8 and C14, and the substituent types include -COCH3, -C7H5O2, -OH, and -OCH3. Common mass loss fragments of this type of compound during the secondary mass spectrometry fragmentation process include H2O (18Da), CH3OH (32Da), AcOH (60Da), and BzOH (122Da). In the positive ion mode, the quasi-molecular ion peak in the secondary mass spectrum of compound 16 with a retention time of 17.34min is m / z 646.3210 [M+H] + , fragment ion peak m / z586.3007[M+H-CH3COOH] + , m / z 554.2740[M+H-CH3COOH-CH3OH] + , m / z 522.2489[M+H-CH3COOH-2CH3OH] + .like Figure 5 As shown, the possible molecular formula C matched by TOF-MS 34 H 47 NO 11 According to the mass spectrometry data combined with the prior art, compound 16 was identified as Polyschistine D. The fragment ion peak in the secondary mass spectrum of compound 27 with a retention time of 26.93 min was observed to be m / z 614.3660 [M+H] + ,m / z 554.3143[M+H-AcOH] + , m / z522.2879[M+H-AcOH-CH3OH] + , m / z 462.2660[M+H-2AcOH-CH3OH] + . Combined with the possible molecular formula C matched by TOF-MS 34 H 47 NO9, combined with the prior art, compound 27 was identified as Chasmaconitine.

[0047] Example 4 C19-LDAs

[0048] The structural characteristics of long-chain ester-type LDAs are that the acetyl group at the C8 position of diester-type diterpene alkaloids is replaced by a fatty acyl group. Therefore, the long-chain fatty acid at the C8 position is first removed during the cleavage process, which is consistent with the cleavage pathway of diester-type diterpene alkaloids. Common mass loss fragments in the cleavage pattern of long-chain ester-type LDAs include C 18 H 32 Neutral molecules such as O2 (280Da), CH3OH (32Da), H2O (18Da) and CO (28Da) produce corresponding characteristic fragment ions, forming [M+H-280] + , [M+H-32] + , [M+H-18] + , [M+H-28] + etc. fragment ions.

[0049] like Figure 6 As shown, compound 44 is a long lipid diterpenoid organism, and its quasi-quasi-ion peak is m / z850.5484[M+H] + , fragment ion peak m / z 570.3065 [M+HC 18 H 32 O2] + 、m / z538.2805[M+HC 18 H 32 O2-CH3OH] + .

[0050] Combined with the prior art, compound 44 was identified as Lipodeoxyaconitine.

[0051] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A method for analyzing the chemical composition differences of an iron hammer and its feces products based on UPLC-Q-TOF-MS-MS, characterized in that: UPLC-Q-TOF-MS / MS technology was used, Agilent Poroshell 120Aq-C18 2.1×100mm, 2.7μm chromatographic column was selected, gradient elution was performed, and ESI electrospray ion source was used to perform positive and negative ion scanning in the mass range of m / z 50-1700 to analyze the chemical components in the samples; SPSS software was used to perform principal component analysis and orthogonal partial least squares discriminant analysis on the mass spectrometry data to compare the differences in the chemical components of iron hammers and children's urine products from different origins; the specific steps included: Step 1, preparation of test solution: crush the raw iron hammer products and their corresponding processed products from 4 different origins, pass through a No. 3 sieve, weigh about 0.2g of each sample, accurately weigh, put into a 50mL stoppered conical flask, add 10mL of 95% methanol, shake well, ultrasonically treat for 60min, take out, cool, shake well, take 2mL of liquid into a centrifuge tube, centrifuge at high speed for 5min, and take the supernatant; Step 2, chromatographic conditions: Agilent Poroshell 120Aq-C 18 Chromatographic column, 2.1×100 mm, 2.7 μm; mobile phase: acetonitrile A-0.1% formic acid aqueous solution B, gradient elution program: 0-3 min, 5% A, 95% B; 3-15 min, 5%-25% A, 95%-75% B; 15-28 min, 25%-35% A, 75%-65% B; 28-33 min, 35%-72% A, 65%-28 %B; 33-43min, 72%-95%A, 28%-5%B; 43-47min, 95%A, 5%B; 47-47.1min, 95%-5%A, 5%-95%B; 49.1-50min, 5%A, 95%B; column temperature 30°C; flow rate 0.3mL / min; injection volume 2μL; detection wavelength range 190~400nm; Step 3, mass spectrometry conditions: detection under ESI-Negative / Positive ion mode; TOF mass range 50-1700; spray gas pressure 50psi; auxiliary heater pressure 50psi; curtain gas pressure 35psi; ionization voltage -4500 / 5000V; ion source temperature 500℃; declustering voltage 100V; collision voltage 10V; MS / MS secondary mass range 50-1250; declustering voltage 100V; collision voltage ±40eV; collision voltage swing 20eV; ion release delay 30ms; ion beam width 15ms; Step 4: Data processing and analysis, specifically including the following steps: Step (1), using UPLC-Q-TOF-MS / MS to collect mass spectrometry data of the raw iron rod hammer products and processed products thereof from different locations according to the above-mentioned chromatographic and mass spectrometric conditions, with the mass spectrometry deviation being less than or equal to 5.7 ppm, and obtaining a UPLC-HRMS base peak ion chromatogram in full scan in positive and negative ion modes; Step (2), analyzing the chemical components in the sample to obtain structural information such as compound name, molecular formula, m / z actual value and theoretical value, secondary mass spectrometry fragmentation data; Step (3), selecting the mass spectrometry data of the chemical components of the iron club and its child urine processed product under the positive ion mode with a strong response, a total of 50 diterpene alkaloids were identified, including 13 non-ester types, 11 monoester types, 15 diester types, 2 multiester types and 9 long-chain ester types, as shown in Tables 1 and 2; Table 1 Identification results of chemical components of iron hammers from different origins and their processed urine products in positive ion mode Table 2 Peak areas of iron hammer ions from different origins under positive ion mode and their change index before and after processing 2. The method for analyzing the difference in chemical composition of the iron hammer and its children's urine products based on UPLC-Q-TOF-MS-MS method according to claim 1, characterized in that: In step 1, the parameters of the ultrasonic treatment are power 300W and frequency 40KHz.

3. The method for analyzing the chemical composition differences of the iron hammer and its children's urine products based on UPLC-Q-TOF-MS-MS method according to claim 1, characterized in that: In step 1, the rotation speed of the high-speed centrifugation is 12000 rpm / min.

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