Method for detecting chemical components in ganoderma lucidum extract by UPC2 / Qtof combination

Abstract

The invention discloses a method for detecting chemical components in a ganoderma lucidum extract by UPC2 / Qtof combination, which comprises the following steps: adding methanol into ganoderma lucidum sporocarp coarse powder for ultrasonic extraction, and filtering and centrifuging an extracting solution to obtain a test solution; and analyzing and detecting the chemical components in the test solution by adopting ultra performance convergence chromatography tandem quadrupole time-of-flight mass spectrometry. By optimizing the chromatographic conditions, the method can realize separation and detection of 112 chemical components in the lucid ganoderma extracting solution within 24 minutes. Compared with a traditional UPLC method, the UPC2 is remarkably improved in the aspects of peak capacity and separation degree, a complex ganoderma lucidum extracting solution sample can be rapidly separated within 24 min, isomeride of various triterpenoids in ganoderma lucidum can be rapidly read, and the method has the advantages of being rapid, high in sensitivity, environmentally friendly and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a UPC. 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods. Background Technology

[0002] Reishi mushroom (Ganoderma lucidum), also known as red reishi, wood reishi, and reishi grass, is a traditional and precious fungus with a long history of medicinal use. In addition to ancient texts such as the *Shennong Bencao Jing* and *Compendium of Materia Medica* recording its effects of "treating chest congestion" and "tonifying the middle and replenishing qi," modern pharmacological studies have shown that reishi mushroom has significant effects on protecting the liver, fighting tumors, improving cognitive impairment, anti-aging, and lowering blood sugar.

[0003] With the development and application of modern separation and purification techniques, approximately 600 compounds have been isolated from different varieties and medicinal parts of Ganoderma lucidum, mainly including triterpenoids and polysaccharides. In addition, alkaloids, fatty acids, proteins, vitamins, steroids, and some mineral elements are also present. Modern research suggests that different components act on different targets in the body; changes in component compatibility and dosage relationships can alter the efficacy of the drug, even producing new effects, and expanding the therapeutic scope accordingly—that is, "each medicine has its own strengths, but only when combined can they achieve their full potential."

[0004] Currently, the commonly used methods for identifying the chemical components of Ganoderma lucidum and its related products are liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS). However, Ganoderma lucidum has a wide variety of chemical components with complex and similar structures, and existing analytical methods still face some challenges, including long detection times (usually greater than 100 min), poor separation of structurally similar compounds, high consumption of organic solvents, and high analytical costs.

[0005] Therefore, developing a green, environmentally friendly, high-throughput, and rapid detection method for Ganoderma lucidum is of urgent practical significance. Summary of the Invention

[0006] Therefore, the object of the present invention is to provide a UPC. 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupling is proposed. This method can separate and detect 112 chemical components in Ganoderma lucidum extract within 24 minutes, and has the advantages of being fast, highly sensitive and environmentally friendly.

[0007] Ultra-high performance convergence chromatography (UPC) 2 Based on traditional liquid chromatography, it uses compressed liquid CO2 as the main mobile phase. By precisely changing the intensity, pressure and temperature of the mobile phase, it can finely control the separation capability and selectivity of the system. It has a faster separation speed and is more suitable for analyzing components such as structural analogs, isomers and diastereomeric mixtures that are difficult to handle by traditional liquid chromatography.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A type of UPC 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof-based assays includes the following steps:

[0010] (1) Add methanol to extract the crude powder of Ganoderma lucidum fruiting body by ultrasonic extraction, filter the extract, centrifuge to obtain the test solution;

[0011] (2) The chemical components in the test solution were analyzed and detected by ultra-high performance phase chromatography in tandem quadrupole time-of-flight mass spectrometry;

[0012] The ultra-high performance phase chromatographic conditions are as follows:

[0013] Column: Torus Diol 150mm×3mm, 1.7μm;

[0014] System back pressure: 1950-2050 psi;

[0015] Compensation solution: methanol containing 0.1% ammonia in negative ion mode, methanol containing 0.1% formic acid in positive ion mode, with a flow rate of 0.1-0.3 mL / min;

[0016] Mobile phases: Mobile phase A is CO2, and mobile phase B is methanol;

[0017] Elution program: 0–0.5 min, 5% B; 0.5–15 min, 15% B; 15–19 min, 25% B; 19–22.5 min, 35% B; 22.5–24 min, 5% B.

[0018] The quadrupole time-of-flight mass spectrometry conditions are as follows:

[0019] Ionization method: ESI+ / -; Acquisition mode: MS E The mass number range was 100–1200 Da, the cone voltage was 30 V, the capillary voltage was 2.5 kV, the desolvation gas temperature was 500 °C, and the desolvation gas flow rate was 1000 L / Hr. (MS) ECollision energy: 15–45 eV.

[0020] Preferably, in step (1), the ratio of the crude powder of Ganoderma lucidum fruiting body to methanol is 1:35-1:40 (g / mL).

[0021] Preferably, in step (1), the ultrasonic temperature is 50-55℃ and the ultrasonic time is 25-35min.

[0022] Preferably, in step (2), the column temperature is 40℃, the flow rate is 0.8-1.2mL / min, and the injection volume is 1-3μL.

[0023] Preferably, in step (2), the analysis and detection are performed using LockSpray and MS. E The technology obtains mass spectrometry data in positive and negative ion modes, and confirms the structure by combining mass number, isotope distribution, and secondary fragment information.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a UPC 2 A method for detecting chemical components in Ganoderma lucidum extract using HPLC-Qtof coupling was developed. By optimizing chromatographic conditions, this method can separate and detect 112 chemical components in Ganoderma lucidum extract within 24 minutes. Compared with the traditional UPLC method, UPC... 2 It has significant improvements in peak capacity and resolution, and can rapidly separate complex Ganoderma lucidum extract samples within 24 minutes. It can also rapidly read isomers of various triterpenoid compounds in Ganoderma lucidum (such as isomers of compound 40 ganoderic acid F and compound 45 ganoderic acid F, and isomers of compound 44 ganoderic acid A and compound 74 ganoderic acid A, etc.), and has the advantages of being fast, highly sensitive and environmentally friendly. Attached Figure Description

[0026] Figure 1 The separation effects of different chromatographic columns on eight ganoderic acids were investigated (A. Viridis HSS C18; B. Viridis BEH; C. Torus 2-PIC; D. Torus Diol).

[0027] Figure 2 The separation effects of different compensation solutions on eight ganoderic acids were investigated (A. methanol + 0.1% NH3; B. methanol).

[0028] Figure 3 The effects of different elution programs on the separation of eight ganoderic acids were investigated (A. First gradient elution program; B. Second elution program).

[0029] Figure 4Ganoderma lucidum extract ACQUITY UPC 2 Total ion flow map of / Xevo G3 QTof in negative and positive ion modes. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments.

[0031] 1. Materials

[0032] 1.1 Instruments

[0033] Xevo G3 QTof quadrupole time-of-flight mass spectrometer (Waters Corporation, USA), ACQUITY UPC 2 Ultra-high performance phase chromatography (Waters Corporation, USA); 0.0001 g electronic balance (Sartorius Scientific Instruments Co., Ltd., Beijing, China); Cascada ultrapure water system (PALL Corporation, USA); YL-060S ultrasonic cleaner (Bark Ultrasonic Technology Co., Ltd., Jinan, China); 800Y traditional Chinese medicine pulverizer (Yongkang Boou Hardware Products Co., Ltd.).

[0034] 1.2 Materials and Reagents

[0035] The Ganoderma lucidum sourced from Jingde County, Anhui Province, was provided by Anhui Huangshan Yunle Ganoderma Co., Ltd., and identified by Professor Yang Qingshan of Anhui University of Traditional Chinese Medicine as the dried fruiting body of the Polyporaceae fungus Ganoderma luicdum. Reference standards for ganoderic acid A (batch number: PS010388), ganoderic acid B (batch number: PS011342), ganoderic acid C2 (batch number: PS010837), ganoderic acid D (batch number: PS000598), ganoderic acid F (batch number: PS210628-12), ganoderic acid B (batch number: PS010839), ganoderic acid C (batch number: PS010840), and ganoderic acid A (batch number: PS010891), with a purity ≥98%, were purchased from Chengdu Pusi Biotechnology Co., Ltd.; methanol (mass spectrometry grade) and acetonitrile (mass spectrometry grade) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0036] 2. Methods

[0037] 2.1 ACQUITY UPC 2 / Xevo G3 QTof detection method

[0038] 2.1.1 Preparation of test solution

[0039] Accurately weigh 5.00 g of crude Ganoderma lucidum fruiting body powder. Extract the Ganoderma lucidum using a Ganoderma lucidum powder:methanol ratio of 1:38 (g / mL), ultrasonic treatment for 30 min, and ultrasonic temperature of 53℃. Collect the extract by membrane filtration. Centrifuge at 12000 r / min for 10 min, dilute to an appropriate concentration, and then inject.

[0040] 2.1.2 Preparation of reference solution

[0041] Accurately weigh 1.0 mg of each of the eight Ganoderma lucidum triterpenoid reference standards, place them in a 10 mL volumetric flask, add an appropriate amount of methanol to dilute to volume, and obtain a 0.1 mg / mL stock solution. Store the stock solution at 4 °C for later use.

[0042] 2.1.3 ACQUITY UPC 2 / Xevo G3 QTof conditions

[0043] Xevo G3 QTof mass spectrometry conditions: ESI (+ / -) ionization mode, MS acquisition mode. E The mass number range was 100–1200 Da, the cone voltage was 30 V, the capillary voltage was 2.5 kV, the desolvation gas temperature was 500 °C, and the desolvation gas flow rate was 1000 L / Hr; MS E Collision energy: 15-45 eV.

[0044] ACQUITY UPC 2 Chromatographic conditions: Torus Diol column. (150mm×3mm, 1.7μm), column temperature 40℃, flow rate 1.0mL / min, injection volume 2μL, system back pressure 2000psi, compensation solution is 0.1% ammonia in methanol solution (0.3mL / min, negative ion mode) and 0.1% formic acid in methanol (0.3mL / min, positive ion mode), mobile phase is CO2(A)-methanol(B), elution program is: 0~0.5min, 5% B; 0.5~15min, 15% B; 15~19min, 25% B; 19~22.5min, 35% B; 22.5~24min, 5% B.

[0045] 2.1.4 Data Processing

[0046] Collect MS E The raw data is imported into Waters Connect's automatic data processing system. A workflow template for identifying unknown compounds in traditional Chinese medicine is established, and peak extraction, alignment, and correction are performed on the data to fit possible molecular formulas of unknown compounds.

[0047] 3. Results

[0048] 3.1 UPC 2 Investigation of chromatographic conditions

[0049] 3.1.1 Selection of chromatographic column

[0050] This experiment investigated the effects of four chromatographic columns—Viridis HSS C18 (3.0×100mm, 1.7μm), Viridis BEH (2.1×100mm, 1.7μm), Torus 2-PIC (3.0×100mm, 1.7μm), and Torus Diol (150mm×3mm, 1.7μm)—on the separation of eight ganoderic acids under the following chromatographic conditions: 0–0.5 min, 5% B; 0.5–6 min, 25% B; 6–9 min, 25% B; and 9–10 min, 5% B. Figure 1 It was found that the Viridis HSS C18 column had a low peak height and severe tailing; the Viridis BEH column had poor resolution and an uneven baseline; and the Torus 2-PIC column had a late peak elution time and poor resolution. In contrast, the Torus Diol column (diol-based column) achieved near-baseline separation of the eight ganoderic acid standards within 10 minutes, with good peak shape. Therefore, Torus Diol was selected as the column for this experiment.

[0051] 3.1.2 Selection of Compensation Solution

[0052] This experiment investigated the effects of two compensating solutions (methanol and methanol + 0.1% NH3) on the separation of eight Ganoderma lucidum acid compounds under negative ion mode. Figure 2 It is known that adding ammonia to the compensating solution can significantly reduce background noise and improve signal-to-noise ratio. Therefore, methanol + 0.1% NH3 is chosen as the compensating solution.

[0053] 3.1.3 Optimization of chromatographic elution program

[0054] This experiment investigated the effects of two elution programs on the separation of eight Ganoderma lucidum acid compounds: 0–0.5 min, 5% B; 0.5–6 min, 25% B; 6–9 min, 25% B; and 9–10 min, 5% B; and 0–0.5 min, 5% B; 0.5–15 min, 15% B; 15–19 min, 25% B; 19–22.5 min, 35% B; and 22.5–24 min, 5% B. Figure 3It can be seen that, compared with the second elution program, the first elution program has a poorer ability to separate ganoderic acids, the chromatographic peaks cannot be completely separated and the background noise is high; while the second elution program can quickly achieve good separation of the eight ganoderic acids. Considering the diverse and complex composition of Ganoderma lucidum extract, the second elution program was selected for separation in this experiment.

[0055] Therefore, this experiment selected Torus Diol as the chromatographic column, methanol + 0.1% NH3 as the compensation solution, and performed gradient elution under the following conditions: 0–0.5 min, 5% B; 0.5–15 min, 15% B; 15–19 min, 25% B; 19–22.5 min, 35% B; 22.5–24 min, 5% B.

[0056] 3.2 Identification and Characterization of Chemical Constituents of Ganoderma lucidum

[0057] Ganoderma lucidum active ingredients contain hundreds of coexisting structures with a wide concentration range. The Xevo G3 QTof boasts ultra-high resolution and wide mass range coverage. Its LockSpray technology ensures real-time accuracy of acquired data, combining high-accuracy mass numbers, isotope distributions, and secondary fragment information to obtain accurate molecular formulas. MS E The technology enables the simultaneous acquisition of primary and secondary mass spectrometry information for all compounds with a single injection, facilitating further structural confirmation.

[0058] This invention utilizes LockSpray and MS in high-resolution mass spectrometry with Xevo G3 QTof. E The technology obtained mass spectrometry data of Ganoderma lucidum extract in positive and negative ion modes. The total ion chromatogram (TIC) is shown below. Figure 4 The results showed that Ganoderma lucidum extract exhibited abundant signals in both ESI + / - modes, and ESI - The mode yielded more chromatographic peaks. Simultaneously, the Waters_connect automated data processing system was used to analyze the acquired mass spectrometry data. For unknown compounds, elemental composition analysis and characteristic fragment / neutral loss search were used to automatically search the ChemSpider online database for possible structures, and secondary fragment matching was used to confirm the structure. Ultimately, 112 chemical components were identified or inferred from the Ganoderma lucidum extract (Table 1), including ganoderic acids, ganoderic acids, ganoderic acids, sterols, and fatty acids. Among these, 8 ganoderic acid components were compared with standards. 91 compounds were identified in negative ion mode, and 86 compounds were identified in positive ion mode. A total of 65 compounds responded in both positive and negative ion modes at the same retention time.

[0059] Table 1. Chemical composition of Ganoderma lucidum extract. 2Xevo G3 QTof mass spectrometry information

[0060]

[0061]

[0062]

[0063]

[0064] The specific exemplary embodiments of the present invention described herein are intended to be illustrative and not to limit the invention to the precise forms disclosed. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the invention, and all such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A type of UPC 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods, characterized in that... Includes the following steps: (1) Add methanol to extract the crude powder of Ganoderma lucidum fruiting body by ultrasonic extraction, filter the extract, centrifuge to obtain the test solution; (2) The chemical components in the test solution were analyzed and detected by ultra-high performance phase chromatography in tandem quadrupole time-of-flight mass spectrometry; The ultra-high performance phase chromatographic conditions are as follows: Column: Torus Diol, 130 Å, 150 mm × 3 mm, 1.7 µm; System back pressure: 1950-2050 psi; Compensation solution: methanol containing 0.1% ammonia in negative ion mode, methanol containing 0.1% formic acid in positive ion mode, with a flow rate of 0.1-0.3 mL / min; Mobile phases: Mobile phase A is CO2, and mobile phase B is methanol; Elution program: 0~0.5 min, 5% B; 0.5~15 min, 15% B; 15~19 min, 25% B; 19~22.5 min, 35% B; 22.5~24 min, 5% B.

2. The UPC according to claim 1 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods, characterized in that... The quadrupole time-of-flight mass spectrometry conditions are as follows: Ionization method: ESI + / -; Acquisition mode: MS E The mass number range was 100~1200 Da, the cone voltage was 30 V, the capillary voltage was 2.5 kV, the desolvation gas temperature was 500℃, and the desolvation gas flow rate was 1000 L / Hr. (MS) E Collision energy: 15~45 eV.

3. The UPC according to claim 1 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods, characterized in that... In step (1), the ratio of the crude powder of Ganoderma lucidum fruiting body to methanol is 1:35-1:40 g / mL.

4. The UPC according to claim 1 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods, characterized in that... In step (1), the ultrasonic temperature is 50-55℃ and the ultrasonic time is 25-35 min.

5. The UPC according to claim 1 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods, characterized in that... In step (2), the column temperature is 40℃, the flow rate is 0.8-1.2 mL / min, and the injection volume is 1-3 μL.

6. The UPC according to claim 1 2 A method for detecting chemical components in Ganoderma lucidum extract using Qtof coupled with other methods, characterized in that... In step (2), the analysis and detection are performed using LockSpray and MS. E The technology obtains mass spectrometry data in both positive and negative ion modes. The structure was confirmed by combining mass number, isotope distribution, and secondary fragment information.

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