Method for accurately evaluating ganoderma lucidum spore powder triterpenes

Abstract

The invention provides a method for accurately evaluating ganoderma lucidum spore powder triterpenes. The method comprises the following steps: (1) pretreating ganoderma lucidum spore powder; (2) an ultra-high performance liquid chromatography detection step; (3) obtaining mass spectrum information of the compound; (4) establishing an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis method; and (5) a data analysis step. According to the method for accurately evaluating the content of the triterpenes in the ganoderma lucidum spore powder, provided by the invention, accurate analysis of the triterpenes in the ganoderma lucidum spore powder is realized, and a reliable detection technology is provided for evaluation of ganoderma lucidum spore powder raw materials and development of related products.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical, health product, and functional food analysis, and more specifically to a method for accurately evaluating triterpenes in Ganoderma lucidum spore powder. Background Technology

[0002] Reishi mushroom belongs to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Ganodermataceae, and genus Ganoderma. Ganoderma Fungi are a general term encompassing multiple species within a genus, such as Ganoderma lucidum (Ganoderma lucidum). Ganoderma linzhi Also known as Ganoderma lucidum ), Ganoderma lucidum ( Ganoderma sinense ), Pine and Fir Ganoderma ( Ganoderma tsugae Ganoderma lucidum spore powder can be developed as a drug, health food, or a dual-use food and medicine. Ganoderma lucidum spore powder is the common name for its reproductive cells, basidiospores. Its chemical components include triterpenes, sterols, fatty acids, nucleosides, polysaccharides, and proteins. Triterpenes have long been considered one of the main active substances in Ganoderma lucidum spore powder, but detection methods often use oleanolic acid or ursolic acid, which are not present in Ganoderma lucidum basidiospores, as reference standards, and employ chemical reagents such as vanillin-glacial acetic acid and perchloric acid for reaction determination. In reality, fatty acids and sterols in Ganoderma lucidum basidiospores can react with these chemical reagents, greatly interfering with the measurement results. The widespread use of high-performance liquid chromatography (HPLC) has provided a more accurate method for the detection of triterpenes in Ganoderma lucidum spore powder. However, because the content of triterpenes in Ganoderma lucidum spore powder is extremely low, when using HPLC for detection, the spore powder needs to be extracted and then concentrated tens or even hundreds of times to detect the triterpenes. However, the concentrated sample often causes column contamination and increased system pressure, and the overly cumbersome pretreatment can also lead to significant errors in content determination.

[0003] Therefore, a precise detection method is urgently needed for the qualitative and quantitative analysis of triterpenes in Ganoderma lucidum spore powder. Summary of the Invention

[0004] This invention provides a method for accurately evaluating triterpenes in Ganoderma lucidum spore powder, the method comprising the following steps: (1) Pretreatment steps of Ganoderma lucidum spore powder: Add Ganoderma lucidum spore powder sample to organic solvent for extraction, take the supernatant, filter, dilute to obtain the sample solution to be tested; prepare a mixed reference solution of 22 triterpenes; (2) Ultra-high performance liquid chromatography detection steps: The sample solution to be tested and the triterpenoid mixed reference solution are separated by ultra-high performance liquid chromatography to obtain a better resolution; (3) Steps for obtaining compound mass spectrometry information: Using mass spectrometry optimization software (Agilent Optimizer), the parent ion is scanned, daughter ion pairs are detected, and the optimal collision energy is found for 22 triterpenes; (4) Steps for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) method: In the data acquisition software (Agilent MassHunter Data Acquisition), under the conditions of UHPLC and triple quadrupole mass spectrometry, the retention time, precursor ion, and daughter ion pairs were imported to establish the dynamic multiple reaction monitoring (DMRM) method for the determination of triterpenes; at the same time, mixed reference solutions and test sample solutions of different concentrations were loaded and measured. (5) Data analysis steps: Using quantitative analysis software (Agilent MassHunter Quantitative Analysis), standard curves for 22 triterpenes were created to validate the established method and to detect and analyze different Ganoderma lucidum spore powder samples; The 22 Ganoderma lucidum spore powder triterpenes mentioned above are: Compound 1: Ganoderic acid I, Compound 2: Ganoderic acid ε, Compound 3: Ganoderenic acid C, Compound 4: Ganoderic acid C2, Compound 5: Lucidenic acid N, Compound 6: Ganoderic acid C6, Compound 7: Ganoderic acid G, Compound 8: Ganoderenic acid B, Compound 9: Ganoderic acid N, Compound 10: Ganoderic acid B, Compound 11: Lucidenic acid E2, Compound 12: Ganoderenic acid K, Compound 13: Ganoderic acid LM2, Compound 14: Ganoderenic acid A, Compound 15: Ganoderic acid K Compound K), Compound 16: Ganoderenic acid E, Compound 17: Ganoderic acid A, Compound 18: Ganoderic acid H, Compound 19: Lucidenic acid A, Compound 20: Ganoderenic acid D, Compound 21: Ganoderic acid D, Compound 22: Ganoderic acid F.

[0005] Specifically, using the following structural formulas A, B, C, and D as basic general formulas, the structural formulas of 22 Ganoderma lucidum spore powder triterpenes are shown in Table 1 below:

[0006] Formula A Formula B

[0007] Formula C Formula D Table 1. Structural formulas of 22 triterpenoid compounds

[0008] In step (1), the pretreatment step of Ganoderma lucidum fruiting bodies includes: The extractant used is either methanol or ethanol, with ethanol being preferred; The extraction method is ultrasonic extraction and / or heating extraction, with ultrasonic heating extraction being preferred; The extraction time is 10-90 min, preferably 60 min; the heating temperature is 30℃-60℃, preferably 40℃; the material-liquid ratio is 1:20-1:40, preferably 1:20 (weight g: volume mL). The number of extractions can include a single extraction or repeated extractions, with one extraction being preferred; Take the extracted supernatant and filter it through an organic phase microporous membrane with a pore size of 0.20 μm to obtain the sample solution to be tested for injection. The preparation method for the 22 triterpenoid reference solutions in step (1) is as follows: Accurately weigh the following: Ganoderic acid I (C 30 H 44 O8, compound 1), Ganoderic acid ε(C 30 H 44 O7, compound 2), Ganoderenic acid C(C 30 H 44 O7, compound 3), Ganoderic acid C2 (C 30 H 46 O7, compound 4), Lucidenic acid N(C 27 H 40 O6, compound 5), Ganoderic acid C6 (C 30 H 42 O8, compound 6), Ganoderic acid G(C 30 H 44 O8, compound 7), Ganoderenic acid B (C 30 H 42 O7, compound 8), Ganoderic acid N(C 30 H 42 O8, compound 9), Ganoderic acid B (C 30 H 44 O7, compound 10), Lucidenic acid E2 (C 29 H 40 O8, compound 11), Ganoderenic acid K (C 32 H 44 O9, compound 12), Ganoderic acid LM2 (C 30 H 42O7, compound 13), Ganoderenic acid A (C 30 H 42 O7, compound 14), Ganoderic acid K(C 32 H 46 O9, compound 15), Ganoderenic acid E (C 30 H 40 O8, compound 16), Ganoderic acid A (C 30 H 44 O7, compound 17), Ganoderic acid H(C 32 H 44 O9, compound 18), Lucidenic acid A (C 27 H 38 O6 compound 19), Ganoderenic acid D(C 30 H 40 O7, compound 20), Ganoderic acid D(C 30 H 42 O7, compound 21), Ganoderic acid F(C 32 H 42 O9, compound 22), were prepared into a mixed reference solution with a concentration of 10 ppm using mass spectrometry grade methanol (where the concentration of each compound was 10 ppm), and then serially diluted to prepare mixed reference working solutions of 5 ppm, 2 ppm, 1 ppm, 500 ppb, 200 ppb, 100 ppb, 50 ppb, and 20 ppb.

[0009] Preferably, the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: InfinityLabPoroshell 120 EC-C18 column (1.9 μm, 2.1 × 150 mm), detection wavelength: 254 nm; column temperature: 35 ℃; sample loading volume: 2 μL; flow rate: 0.4 mL / min. Mobile phase: 0.01% (v / v) acetic acid aqueous solution (A) - acetonitrile (B); elution program: 0 min, 75% A, 25% B; 33 min, 75% A, 25% B; 37 min, 65% A, 35% B; 41 min, 40% A, 60% B; 43 min, 0% A, 100% B. Preferably, the retention time, parent ion, daughter ion pair, and collision energy of each compound required for analysis in step (3) are obtained using mass spectrometry optimization software (Agilent Optimizer) as follows: Ganoderic acid I, retention time: 7.13 min; precursor ion: 531.2; quantitative ion: 401.2, collision energy: 13; qualitative ion: 128.9, collision energy: 33; Ganoderic acid ε, retention time: 8.31 min; precursor ion: 515.1; quantitative ion: 441.2, collision energy: 25; qualitative ion: 249.0, collision energy: 38; Ganoderenic acid C, retention time: 9.62 min; precursor ion: 515.1; quantitative ion: 193.0, collision energy: 21; qualitative ion: 79.0, collision energy: 33; Ganoderic acid C2, retention time: 11.41 min; precursor ion: 517.1; quantitative ion: 499.2, collision energy: 33; qualitative ion: 287.2, collision energy: 37; Lucidenic acid N, retention time: 12.83 min; precursor ion: 459.1; quantitative ion: 249.0, collision energy: 37; qualitative ion: 209.1, collision energy: 37; Ganoderic acid C6, retention time: 13.47 min; precursor ion: 529.1; quantitative ion: 511.2, collision energy: 13; qualitative ion: 467.3, collision energy: 37; Ganoderic acid G, retention time: 15.76 min; precursor ion: 531.1; quantitative ion: 513.1, collision energy: 17; qualitative ion: 265.1, collision energy: 41; Ganoderenic acid B, retention time: 16.88 min; precursor ion: 513.1; quantitative ion: 495.2, collision energy: 17; qualitative ion: 451.1, collision energy: 33; Ganoderic acid N, retention time: 17.53 min; precursor ion: 529.0; quantitative ion: 511.3, collision energy: 13; qualitative ion: 129.0, collision energy: 17; Ganoderic acid B, retention time: 18.57 min; precursor ion: 515.2; quantitative ion: 497.2, collision energy: 13; qualitative ion: 303.1, collision energy: 37; Lucidenic acid E2, retention time: 20.41 min; precursor ion: 515.1; quantitative ion: 473.2, collision energy: 29; qualitative ion: 443.3, collision energy: 45; Ganoderenic acid K, retention time: 22.85 min; precursor ion: 571.0; quantitative ion: 553.2, collision energy: 9; qualitative ion: 467.3, collision energy: 37; Ganoderic acid LM2, retention time: 23.29 min; precursor ion: 513.1; quantitative ion: 439.2, collision energy: 25; qualitative ion: 149.2, collision energy: 49; Ganoderenic acid A, retention time: 23.84 min; precursor ion: 513.1; quantitative ion: 193.1, collision energy: 21; qualitative ion: 79.1, collision energy: 33; Ganoderic acid K, retention time: 23.96 min; precursor ion: 573.1; quantitative ion: 555.1, collision energy: 13; qualitative ion: 469.2, collision energy: 37; Ganoderenic acid E, retention time: 25.96 min; precursor ion: 527.1; quantitative ion: 509.1, collision energy: 17; qualitative ion: 491.1, collision energy: 21; Ganoderic acid A, retention time: 29.96 min; precursor ion: 515.1; quantitative ion: 497.1, collision energy: 25; qualitative ion: 285.2, collision energy: 41; Ganoderic acid H, retention time: 30.45 min; precursor ion: 571.1; quantitative ion: 553.2, collision energy: 13; qualitative ion: 511.2, collision energy: 29; Lucidenic acid A, retention time: 33.21 min; precursor ion: 457.2; quantitative ion: 209.1, collision energy: 33; qualitative ion: 149, collision energy: 37; Ganoderenic acid D, retention time: 36.72 min; precursor ion: 511.0; quantitative ion: 493.2, collision energy: 17; qualitative ion: 149, collision energy: 53; Ganoderic acid D, retention time: 38.08 min; precursor ion: 513.1; quantitative ion: 495.2, collision energy: 17; qualitative ion: 149, collision energy: 45; Ganoderic acid F, retention time: 39.80 min; precursor ion: 569.1; quantitative ion: 551.1, collision energy: 21; qualitative ion: 509.2, collision energy: 33; Preferably, in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical instrument, wherein the ultra-high performance liquid chromatography conditions are: column: InfinityLab Poroshell 120 EC-C18 column (1.9μm, 2.1×150mm). Detection wavelength: 254nm. Column temperature: 35℃; sample loading volume: 2uL; flow rate: 0.4mL / min; mobile phase: 0.01% v / v acetic acid aqueous solution (A) - acetonitrile (B); elution program: 0 min, 75%A, 25%B; 33 min, 75%A, 25%B; 37 min, 65%A, 35%B; 41 min, 40%A, 60%B; 43 min, 0%A, 100%B; mass spectrometry conditions: electrospray ionization source (AJS ESI) as ion source, detection in negative ion mode, dynamic multiple reaction monitoring (DMRM) selected, capillary voltage: 3500V, capillary outlet voltage: 380V, drying gas flow rate: 16L / min, drying gas temperature: 200℃, sheath gas temperature: 320℃, sheath gas flow rate: 12L / min, nozzle voltage: 2000V.

[0010] The basis of the method for accurately evaluating triterpenes in Ganoderma lucidum spore powder of the present invention is that when using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection, the 22 triterpenes in Ganoderma lucidum spore powder must not only meet the requirement of consistent retention time, but also meet the requirement that the primary mother-daughter and secondary daughter ion pairs meet the detection parameters under specific mass spectrometry conditions in order to accurately quantify the triterpenoid compounds in Ganoderma lucidum spore powder.

[0011] Based on the technical advantages of ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS), this invention, through literature review, selected triterpenoid compounds reported in Ganoderma lucidum spore powder as reference standards. By optimizing chromatographic and mass spectrometric parameters and conditions, a method for detecting triterpenoids in Ganoderma lucidum spore powder was established. Compared with previous chemical and high performance liquid chromatography methods, this method has higher accuracy, convenience, and sensitivity, and is more suitable for the detection and analysis of triterpenoids in Ganoderma lucidum spore powder and related products.

[0012] This invention not only enables preliminary qualitative analysis of analytes using retention time, but also allows for precise identification and quantification of mixtures that are difficult to completely separate chromatographically based on the characteristic information of the parent and daughter ions. It can accurately quantify trace substances in samples at concentrations as low as ppb (parts per billion). Attached Figure Description

[0013] Figure 1 Total ion chromatogram of 22 triterpenoids in, Compound 1: Ganoderic acid I. Compound 2: Ganoderic acid ε. Compound 3: Ganoderenic acid C, Compound 4: Ganoderic acid C2. Compound 5: Lucidenic acid N. Compound 6: Ganoderic acid C6. Compound 7: Ganoderic acid G. Compound 8: Ganoderenic acid B. Compound 9: Ganoderic acid N. Compound 10: Ganoderic acid B. Compound 11: Lucidenic acid E2. Compound 12: Ganoderenic acid K. Compound 13: Ganoderic acid LM2. Compound 14: Ganoderenic acid A. Compound 15: Ganoderic acid K. Compound 16: Ganoderenic acid E. Compound 17: Ganoderic acid A. Compound 18: Ganoderic acid H. Compound 19: Lucidenic acid A. Compound 20: Ganoderenic acid D. Compound 21: Ganoderic acid D. Compound 22: Ganoderic acid F. Detailed Implementation

[0014] The present invention will be further described in detail below through embodiments, but the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0015] Ganoderma lucidum spore powder: Sample 1: Ganoderma lucidum Ganoderma lingzhi (Former name) Ganoderma lucidum ), Collection location: Shandong (Taishan Ganoderma); Sample 2: Ganoderma lucidum Ganoderma lingzhi (Former name) Ganoderma lucidum ), Collection location: Guizhou (Hu Nong Lingzhi No. 1); Sample 3: Purple Ganoderma ( Ganoderma sinense ), Collection location: Jiangxi (Zizhi No. 8); Sample 4: Zizhi ( Ganoderma sinense ), Collection location: Fujian (Wuzhi No. 2); Sample 5: Ganoderma lucidum without stalk ( Ganoderma sessile ), Collection location: Shandong (Meizhi); Sample 6: Resin Ganoderma ( Ganoderma resinaceum ), Collection location: Slovakia (wild species); Sample 7: White-fleshed Ganoderma lucidum ( Ganoderma leucocontextum ), Source: Yunnan (Yunbai Lingzhi No. 1); Sample 8: Tropical Lingzhi ( Ganoderma tropicum ), Source: Guangxi (wild species).

[0016] The 22 triterpenoid reference standards were sourced from Yunnan Xili Biotechnology Co., Ltd., with a purity ≥98%. Organic phase microporous filter membrane with 0.20 μm pore size: Agilent Technologies, USA; InfinityLab Poroshell 120 EC-C18 column (1.9 μm, 2.1 × 150 mm): Agilent Technologies, USA.

[0017] Example 1 Establishment of detection methods: (1). Pretreatment steps of Ganoderma lucidum spore powder: Accurately weigh 1g of dried Ganoderma lucidum spore powder sample (samples 1, 2, 3, 4, 5, 6, 7, 8) into a stoppered test tube, add 20mL of ethanol at a material-to-liquid ratio of 1:20 (weight-to-volume ratio, weight g: volume ml), extract by ultrasonication at 40℃ for 60min, take the supernatant, filter it through a 0.20μm organic filter membrane to obtain the sample solution to be tested for loading; Preparation of mixed reference solutions: Take 22 triterpenoid reference standards and prepare a mixed standard solution with a concentration of 10 ppm using mass spectrometry grade methanol (where the concentration of each compound is 10 ppm). Then, dilute stepwise to prepare mixed reference solutions of 5 ppm, 2 ppm, 1 ppm, 500 ppb, 200 ppb, 100 ppb, 50 ppb, and 20 ppb.

[0018] (2). Ultra-high performance liquid chromatography detection steps: The sample solution to be tested and the mixed reference solution of 22 triterpenes were separated by ultra-high performance liquid chromatography to obtain a better resolution; The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: Column: InfinityLab Poroshell 120 EC-C18 column (1.9 μm, 2.1 × 150 mm). Detection wavelength: 254 nm. Column temperature: 35℃. Sample loading volume: 2 μL. Flow rate: 0.4 mL / min. Mobile phase: 0.01% acetic acid aqueous solution (A) - acetonitrile (B). Elution program: 0 min, 75% A, 25% B; 33 min, 75% A, 25% B; 37 min, 65% A, 35% B; 41 min, 40% A, 60% B; 43 min, 0% A, 100% B.

[0019] (3). Steps for obtaining compound mass spectrometry information: 22 triterpenoid reference standards were prepared into 5 ppm solutions with methanol. The parent ion information of each triterpenoid was confirmed in negative ion mode. Then, the daughter ion and collision energy were automatically optimized by Agilent Optimizer software after the parent ion was determined. The retention times, parent ions, daughter ion pairs, and collision energies of each compound are as follows: Ganoderic acid I, retention time: 7.13 min; precursor ion: 531.2; quantitative ion: 401.2, collision energy: 13; qualitative ion: 128.9, collision energy: 33; Ganoderic acid ε, retention time: 8.31 min; precursor ion: 515.1; quantitative ion: 441.2, collision energy: 25; qualitative ion: 249.0, collision energy: 38; Ganoderenic acid C, retention time: 9.62 min; precursor ion: 515.1; quantitative ion: 193.0, collision energy: 21; qualitative ion: 79.0, collision energy: 33; Ganoderic acid C2, retention time: 11.41 min; precursor ion: 517.1; quantitative ion: 499.2, collision energy: 33; qualitative ion: 287.2, collision energy: 37; Lucidenic acid N, retention time: 12.83 min; precursor ion: 459.1; quantitative ion: 249.0, collision energy: 37; qualitative ion: 209.1, collision energy: 37; Ganoderic acid C6, retention time: 13.47 min; precursor ion: 529.1; quantitative ion: 511.2, collision energy: 13; qualitative ion: 467.3, collision energy: 37; Ganoderic acid G, retention time: 15.76 min; precursor ion: 531.1; quantitative ion: 513.1, collision energy: 17; qualitative ion: 265.1, collision energy: 41; Ganoderenic acid B, retention time: 16.88 min; precursor ion: 513.1; quantitative ion: 495.2, collision energy: 17; qualitative ion: 451.1, collision energy: 33; Ganoderic acid N, retention time: 17.53 min; precursor ion: 529.0; quantitative ion: 511.3, collision energy: 13; qualitative ion: 129.0, collision energy: 17; Ganoderic acid B, retention time: 18.57 min; precursor ion: 515.2; quantitative ion: 497.2, collision energy: 13; qualitative ion: 303.1, collision energy: 37; Lucidenic acid E2, retention time: 20.41 min; precursor ion: 515.1; quantitative ion: 473.2, collision energy: 29; qualitative ion: 443.3, collision energy: 45; Ganoderenic acid K, retention time: 22.85 min; precursor ion: 571.0; quantitative ion: 553.2, collision energy: 9; qualitative ion: 467.3, collision energy: 37; Ganoderic acid LM2, retention time: 23.29 min; precursor ion: 513.1; quantitative ion: 439.2, collision energy: 25; qualitative ion: 149.2, collision energy: 49; Ganoderenic acid A, retention time: 23.84 min; precursor ion: 513.1; quantitative ion: 193.1, collision energy: 21; qualitative ion: 79.1, collision energy: 33; Ganoderic acid K, retention time: 23.96 min; precursor ion: 573.1; quantitative ion: 555.1, collision energy: 13; qualitative ion: 469.2, collision energy: 37; Ganoderenic acid E, retention time: 25.96 min; precursor ion: 527.1; quantitative ion: 509.1, collision energy: 17; qualitative ion: 491.1, collision energy: 21; Ganoderic acid A, retention time: 29.96 min; precursor ion: 515.1; quantitative ion: 497.1, collision energy: 25; qualitative ion: 285.2, collision energy: 41; Ganoderic acid H, retention time: 30.45 min; precursor ion: 571.1; quantitative ion: 553.2, collision energy: 13; qualitative ion: 511.2, collision energy: 29; Lucidenic acid A, retention time: 33.21 min; precursor ion: 457.2; quantitative ion: 209.1, collision energy: 33; qualitative ion: 149, collision energy: 37; Ganoderenic acid D, retention time: 36.72 min; precursor ion: 511.0; quantitative ion: 493.2, collision energy: 17; qualitative ion: 149, collision energy: 53; Ganoderic acid D, retention time: 38.08 min; precursor ion: 513.1; quantitative ion: 495.2, collision energy: 17; qualitative ion: 149, collision energy: 45; Ganoderic acid F, retention time: 39.80 min; precursor ion: 569.1; quantitative ion: 551.1, collision energy: 21; qualitative ion: 509.2, collision energy: 33.

[0020] (4). Establishment of a quantitative analysis method using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry: Under the parameter settings of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry in the data acquisition software (Agilent MassHunter Data Acquisition), the mass spectrometry acquisition mode was changed to multiple reaction monitoring (MRM). The parent ion information, quantitative and qualitative daughter ion pair information, collision energy and other information of the compound, as well as the ultra-high performance liquid chromatography method were all imported into the detection method. The mixed reference solution was loaded, and after running the program, the acquisition mode was updated to dynamic multiple reaction monitoring (DMRM).

[0021] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: column: InfinityLab Poroshell 120 EC-C18 column (1.9 μm, 2.1 × 150 mm); detection wavelength: 254 nm; column temperature: 35 ℃; sample loading volume: 2 μL; flow rate: 0.4 mL / min; mobile phase: 0.01% acetic acid aqueous solution (A) - acetonitrile (B); elution program: 0 min, 75% A, 25% B; 33 min, 75% A, 25% B; 37 min, 65% A, 35% B; 41 min, 40% A, 60% B; 43 min, 0% A, 100% B.

[0022] The triple quadrupole mass spectrometry conditions were as follows: an electrospray ionization source (AJS ESI) was used as the ion source; detection was performed in negative ion mode; dynamic multiple reaction monitoring (DMRM) was used; capillary voltage: 3500V; capillary outlet voltage: 380V; drying gas flow rate: 16L / min; drying gas temperature: 200℃; sheath gas temperature: 320℃; sheath gas flow rate: 12L / min; nozzle voltage: 2000V.

[0023] (5) Limit of detection and limit of quantitation: The limit of detection (LOD) and limit of quantitation (LOQ) are calculated based on the standard deviation of the response value and the slope of the standard curve. Where: LOD = 3σ / S, LOQ = 10σ / S, σ: standard deviation of the response value, S: slope of the standard curve, and the standard deviation of the response value is the residual standard deviation of the standard curve.

[0024] (6) Using quantitative analysis software (Agilent MassHunter Quantitative Analysis), standard curves for 22 triterpenoid compounds were created: The prepared mixed reference working solutions of 5ppm, 2ppm, 1ppm, 500ppb, 200ppb, 100ppb, 50ppb, and 20ppb were loaded onto the samples under the optimized liquid chromatography and mass spectrometry conditions described above. The concentration of the compound was used as the abscissa and the quantitative ion response value of the compound was used as the ordinate to create the quantitative standard curves, as shown in Table 2.

[0025] Example 2 Methodological validation and result testing: Methodological validation was conducted in accordance with laboratory quality control standards, food physicochemical testing standards, and relevant pharmacopoeia regulations.

[0026] 1. Precision: A 500 ppb standard mixture solution was injected six times on the same day. The concentrations of triterpenes obtained from the six experiments were calculated based on the standard curve, and the intra-day precision was calculated. The 500 ppb standard mixture solution was injected twice daily for three consecutive days, and the inter-day precision was calculated based on the results of the six experiments. The results showed that the RSDs of the intra-day and inter-day precision determinations of the 22 triterpenoid compounds were all less than 15.00%, indicating that the method has good intra-day and inter-day precision. Specific results are shown in Table 3.

[0027] 2. Stability: Sample 2 was extracted according to the sample pretreatment steps in Example 1, and a sample solution was prepared. The solution was injected at 0h, 2h, 4h, 6h, 8h, 12h, and 24h. The sample stability was calculated based on the results of the seven experiments. The results confirmed that the RSD of all 22 triterpenes was less than 15.00%, indicating that the samples were stable within 24 hours. Specific results are shown in Table 3.

[0028] 3. Repeatability: Sample 2 was taken, and six parallel samples were weighed. The samples were extracted according to the sample pretreatment steps in Example 1, and the test solutions were prepared. The samples were then injected and analyzed. The repeatability of the samples was calculated based on the results of the six experiments. The results confirmed that the RSD of all 22 triterpenes was less than 15.00%, indicating good sample repeatability. Specific results are shown in Table 3.

[0029] 4. Recovery Rate: Take a sample solution with known triterpenoid content, and add 22 triterpenoid reference standards at ratios of 1:0.5, 1:1, and 1:1.5 for each triterpenoid content in the sample. Repeat each addition three times. Calculate the sample recovery rate. Recovery rate % = (Measured value - Amount of analyte in the test sample) / Amount of reference standard added × 100% The specific recovery results are shown in Table 4. The results confirm that the RSD of the recoveries of the 22 triterpenes in the samples were all within 15.00%, which meets the method requirements.

[0030] 5. Sample Testing: Eight Ganoderma lucidum spore powder samples were taken and tested according to the testing method in Example 1. The specific test results are shown in Table 5. The results show that various triterpenoid compounds were detected in all eight Ganoderma lucidum samples. While the triterpenoid composition types were generally similar across the different samples, the triterpenoid content showed significant differences.

[0031] Table 2. Standard curves and parameters for quantitative analysis of 22 triterpenes

[0032] Table 3. Intra-day precision, inter-day precision, repeatability, and stability.

[0033] Table 4. Recovery Rate Results

[0034] Table 5. Sample Measurement Results

Claims

1. A method for accurately evaluating triterpenes in Ganoderma lucidum spore powder, characterized in that... The method includes the following steps: (1) Pretreatment steps of Ganoderma lucidum spore powder: Add Ganoderma lucidum spore powder sample to organic solvent for extraction, take the supernatant, filter, dilute to obtain the sample solution to be tested; prepare a mixed reference solution of 22 triterpenes; (2) Ultra-high performance liquid chromatography detection steps: The sample solution to be tested and the mixed reference solution of triterpenes are separated by ultra-high performance liquid chromatography to obtain a better resolution; (3) Steps for obtaining compound mass spectrometry information: Using the mass spectrometry optimization software Agilent Optimizer, the parent ion of triterpenes is scanned, daughter ion pairs are detected, and the optimal collision energy is found. (4) Steps for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) method: In the data acquisition software Agilent MassHunter Data Acquisition, under the conditions of UHPLC and triple quadrupole mass spectrometry, retention time, parent ion, and daughter ion pairs were imported to establish a dynamic multiple reaction monitoring (DMRM) method for the determination of triterpenes; at the same time, mixed reference solutions and test sample solutions of different concentrations were loaded and measured. (5) Data analysis steps: Using the quantitative analysis software Agilent MassHunter Quantitative Analysis, standard curves for 22 triterpenes were created to validate the established method and to detect and analyze different Ganoderma lucidum spore powder samples. The 22 Ganoderma lucidum spore powder triterpenes mentioned above are: Compound 1: Ganoderic acid I, Compound 2: Ganoderic acid ε, Compound 3: Ganoderic acid C, Compound 4: Ganoderic acid C2, Compound 5: Ganoderma lucidum N, Compound 6: Ganoderic acid C6, Compound 7: Ganoderic acid G, Compound 8: Ganoderic acid B, Compound 9: Ganoderic acid N, Compound 10: Ganoderic acid B, Compound 11: Ganoderma lucidum E2, Compound 12: Ganoderic acid K, Compound 13: Ganoderic acid LM2, Compound 14: Ganoderic acid A, Compound 15: Ganoderic acid K, Compound 16: Ganoderic acid E, Compound 17: Ganoderic acid A, Compound 18: Ganoderic acid H, Compound 19: Ganoderma lucidum A, Compound 20: Ganoderic acid D, Compound 21: Ganoderic acid D, Compound 22: Ganoderic acid F.

2. The method for accurately evaluating Ganoderma lucidum spore powder triterpenes according to claim 1, wherein in step (1) the pretreatment step of Ganoderma lucidum spore powder: The extraction solution used is either methanol or ethanol; The extraction methods are ultrasonic extraction and / or heating extraction; Extraction time is 10-90 minutes; The extraction temperature is 30℃-60℃; The material-to-liquid ratio is 1:20 to 1:40, and the weight-to-volume ratio is (g:ml). The supernatant of the extraction was filtered through an organic phase microporous membrane with a pore size of 0.20 μm, and then diluted 20 times with mass spectrometry grade methanol to obtain the sample solution to be tested for injection.

3. The method for accurately evaluating Ganoderma lucidum spore powder triterpenes according to claim 1, wherein the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: InfinityLab Poroshell 120 EC-C18 column, 1.9 μm, 2.1 × 150 mm, detection wavelength: 254 nm; column temperature: 35 °C; sample loading volume: 2 μL; flow rate: 0.4 mL / min; mobile phase: 0.01% acetic acid aqueous solution A-acetonitrile B; elution program: 0 min, 75% A, 25% B; 33 min, 75% A, 25% B; 37 min, 65% A, 35% B; 41 min, 40% A, 60% B; 43 min, 0% A, 100% B.

4. The method for accurately evaluating Ganoderma lucidum spore powder triterpenes according to claim 1, wherein the retention time, parent ion, daughter ion pair and collision energy information of each compound required for analysis in step (3) are obtained by using the mass spectrometry optimization software Agilent Optimizer as follows: Ganoderic acid I, retention time: 7.13 min; precursor ion: 531.2; quantitative ion: 401.2, collision energy: 13; qualitative ion: 128.9, collision energy: 33; Ganoderic acid ε, retention time: 8.31 min; precursor ion: 515.1; quantitative ion: 441.2, collision energy: 25; qualitative ion: 249.0, collision energy: 38; Ganoderenic acid C, retention time: 9.62 min; precursor ion: 515.1; Quantitative ions: 193.0, collision energy: 21; Qualitative ions: 79.0, collision energy: 33; Ganoderic acid C2, retention time: 11.41 min; precursor ion: 517.1; quantitative ion: 499.2, collision energy: 33; qualitative ion: 287.2, collision energy: 37; Lucidenic acid N, retention time: 12.83 min; precursor ion: 459.1; quantitative ion: 249.0, collision energy: 37; qualitative ion: 209.1, collision energy: 37; Ganoderic acid C6, retention time: 13.47 min; precursor ion: 529.1; quantitative ion: 511.2, collision energy: 13; qualitative ion: 467.3, collision energy: 37; Ganoderic acid G, retention time: 15.76 min; precursor ion: 531.1; Quantitative ion: 513.1, collision energy: 17; Qualitative ion: 265.1, collision energy: 41; Ganoderenic acid B, retention time: 16.88 min; precursor ion: 513.1; quantitative ion: 495.2, collision energy: 17; qualitative ion: 451.1, collision energy: 33; Ganoderic acid N, retention time: 17.53 min; precursor ion: 529.0; quantitative ion: 511.3, collision energy: 13; qualitative ion: 129.0, collision energy: 17; Ganoderic acid B, retention time: 18.57 min; precursor ion: 515.2; quantitative ion: 497.2, collision energy: 13; qualitative ion: 303.1, collision energy: 37; Lucidenic acid E2, retention time: 20.41 min; precursor ion: 515.1; quantitative ion: 473.2, collision energy: 29; qualitative ion: 443.3, collision energy: 45; Ganoderenic acid K, retention time: 22.85 min; precursor ion: 571.0; quantitative ion: 553.2, collision energy: 9; qualitative ion: 467.3, collision energy: 37; Ganoderic acid LM2, retention time: 23.29 min; precursor ion: 513.1; quantitative ion: 439.2, collision energy: 25; qualitative ion: 149.2, collision energy: 49; Ganoderenic acid A, retention time: 23.84 min; precursor ion: 513.1; quantitative ion: 193.1, collision energy: 21; qualitative ion: 79.1, collision energy: 33; Ganoderic acid K, retention time: 23.96 min; precursor ion: 573.1; quantitative ion: 555.1, collision energy: 13; qualitative ion: 469.2, collision energy: 37; Ganoderenic acid E, retention time: 25.96 min; precursor ion: 527.1; quantitative ion: 509.1, collision energy: 17; qualitative ion: 491.1, collision energy: 21; Ganoderic acid A, retention time: 29.96 min; precursor ion: 515.1; quantitative ion: 497.1, collision energy: 25; qualitative ion: 285.2, collision energy: 41; Ganoderic acid H, retention time: 30.45 min; precursor ion: 571.1; quantitative ion: 553.2, collision energy: 13; qualitative ion: 511.2, collision energy: 29; Lucidenic acid A, retention time: 33.21 min; precursor ion: 457.2; quantitative ion: 209.1, collision energy: 33; qualitative ion: 149, collision energy: 37; Ganoderenic acid D, retention time: 36.72 min; precursor ion: 511.0; quantitative ion: 493.2, collision energy: 17; qualitative ion: 149, collision energy: 53; Ganoderic acid D, retention time: 38.08 min; precursor ion: 513.1; quantitative ion: 495.2, collision energy: 17; qualitative ion: 149, collision energy: 45; Ganoderic acid F, retention time: 39.80 min; precursor ion: 569.1; quantitative ion: 551.1, collision energy: 21; qualitative ion: 509.2, collision energy:

33.

5. The method for accurately evaluating Ganoderma lucidum spore powder triterpenes according to claim 1, wherein in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical instrument. The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: Column: InfinityLab Poroshell 120 EC-C18 column, 1.9 μm, 2.1 × 150 mm; Detection wavelength: 254 nm; Column temperature: 35℃; Sample loading volume: 2 μL; Flow rate: 0.4 mL / min; Mobile phase: 0.01% acetic acid aqueous solution A-acetonitrile B; Elution program: 0 min, 75% A, 25% B; 33 min, 75% A, 25% B; 37 min, 65% A, 35% B; 41 min, 40% A, 60% B; 43 min, 0% A, 100% B. The mass spectrometry conditions were as follows: an AJS ESI electrospray ionization source was used as the ion source, detection was performed in negative ion mode, a dynamic multiple reaction monitoring (DMRM) system was selected, capillary voltage: 3500V, capillary outlet voltage: 380V; drying gas flow rate: 16L / min, drying gas temperature: 200℃, sheath gas temperature: 320℃, sheath gas flow rate: 12L / min, and nozzle voltage: 2000V.

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