Method for simultaneously detecting 13 triterpenoids in ganoderma sinense sporocarp

Abstract

The invention provides a method for simultaneously detecting 13 triterpenoids in ganoderma sinense sporocarp. The method comprises the following steps: (1) pretreating ganoderma sinense sporocarp; (2) an ultra-high performance liquid chromatography detection step; (3) obtaining mass spectrum information of the compound; (4) establishing a dynamic multi-reaction monitoring DMRM method; and (5) a data analysis step. According to the method for simultaneously detecting the 13 triterpenoids in the ganoderma sinense sporocarp, provided by the invention, quantitative analysis of the 13 characteristic triterpenoids in the ganoderma sinense sporocarp is realized, and an accurate and reliable detection means is provided for related research and development of the ganoderma sinense sporocarp.

Description

Technical Field

[0001] This invention relates to the field of food and drug analysis, specifically providing a method for simultaneously detecting 13 triterpenoid compounds in the fruiting body of Ganoderma lucidum. Background Technology

[0002] Ganoderma sinense, a fungus belonging to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Ganodermataceae, and genus Ganoderma P. Karst, is listed as a type of "Lingzhi" medicinal material in the Chinese Pharmacopoeia and can be used in the development of pharmaceuticals, health foods, and food-medicine homologous functional foods. For a long time, people have confused the functions of another medicinal material in the Pharmacopoeia, Ganoderma lucidum (also known as Ganodermalingzhi), with Ganoderma sinense, and the same chemical method has been used to determine the triterpenoids in both. In reality, the fruiting body of Ganoderma sinense contains various types of terpenoids, including triterpenoids, sesquiterpenoids, and heteroterpenoids. Compared with Ganoderma lucidum or other species in the genus Ganoderma, the triterpenoid content in Ganoderma sinense is generally lower, and known detection methods are often difficult to use, especially when detecting it simultaneously with other triterpenoid compounds, which presents significant challenges. Therefore, in order to evaluate Ganoderma lucidum raw materials and develop its products in a targeted manner, it is necessary to establish a dedicated method for the simultaneous detection of several Ganoderma lucidum triterpenoid compounds. Summary of the Invention

[0003] This invention provides a method for simultaneously detecting 13 triterpenoid compounds in the fruiting body of Ganoderma lucidum, the method comprising the following steps:

[0004] (1) Pretreatment steps of Ganoderma lucidum fruiting body: Add organic solvent to extract Ganoderma lucidum fruiting body sample, take supernatant, filter, dilute to obtain sample solution to be tested; prepare mixed standard solution of 13 triterpenoid compounds;

[0005] (2) Ultra-high performance liquid chromatography separation steps: The sample solution to be tested and the mixed standard solution of 13 triterpenoids were separated by ultra-high performance liquid chromatography to obtain a better resolution;

[0006] (3) Steps for obtaining mass spectrometry information of triterpenoids: Using mass spectrometry optimization software (Agilent Optimizer), the parent ion was scanned, daughter ion pairs were detected, and the optimal collision energy was found for 13 triterpenoids.

[0007] (4) Steps for establishing the dynamic multiple reaction monitoring (DMRM) analysis method: In the data acquisition software (Agilent MassHunter Data Acquisition), under the conditions of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry, retention time, parent ion, and daughter ion pairs were imported to establish a dynamic multiple reaction monitoring (DMRM) analysis method for 13 triterpenoid compounds; at the same time, mixed standard solutions and test sample solutions of different concentrations were loaded and measured.

[0008] (5) Data analysis steps: Using quantitative analysis software (Agilent MassHunterQuantitativeAnalysis), standard curves for 13 triterpenoid compounds were created to validate the established method and to perform quantitative analysis on the test sample solution of Ganoderma lucidum fruiting body.

[0009] In step (1), the pretreatment step of Ganoderma lucidum fruiting bodies includes:

[0010] The organic solvent is one of methanol and ethanol, preferably methanol;

[0011] The extraction method is ultrasonic extraction or heating extraction at 60℃, with ultrasonic extraction being preferred;

[0012] The extraction time is 10-90 min, preferably 60 min; the material-to-liquid ratio is 1:20-1:40, preferably 1:30 (g weight: mL volume);

[0013] The number of extractions can include a single extraction or multiple extractions, with one extraction being preferred;

[0014] Take the supernatant of the extraction, filter it through an organic phase microporous membrane with a pore size of 0.20 μm, and then dilute it 20 times with mass spectrometry grade methanol to obtain the sample solution to be tested for injection.

[0015] The preparation method for the 13 triterpenoid compound standard solutions in step (1) is as follows: accurately weigh the following amounts: Ganoderic acid I (C 30 H 44 O8 compound 1), Ganoderenic acid B (C 30 H 42 O7, compound 2), Ganoderic acid B (C 30 H 44 O7, compound 3), Ganoderic acid K(C) 32 H 46 O9, compound 4), Ganoderic acid H(C 32 H 44O9, compound 5), Lucidenic acid A (C 27 H 38 O6 compound 6), Ganoderic acid D(C 30 H 42 O7, compound 7), Ganoderic acid A (C 30 H 44 O7 compound 8), Ganoderic acid F(C 32 H 42 O9, compound 9), Ganoderic acid GS-3 (C 32 H 46 O8, compound 10), Ganoderic acid GS-1 (C 30 H 42 O6, compound 11), Ganoderic acid GS-2 (C 30 H 44 O6, compound 12), Ganodemanontriol (C 30 H 48 O4 (compound 13), 13 compounds, were prepared into a mixed standard solution with a concentration of 100 μg / mL using mass spectrometry grade methanol (each compound had a concentration of 100 μg / mL), and then serially diluted to prepare mixed standard working solutions of 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL.

[0016] Preferably, the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: An Agilent EclipsePlus C18 column (1.8 μm, 2.1 × 50 mm) is used; the detection wavelength is 254 nm; the column temperature is 35℃; the sample loading volume is 2 μL; the flow rate is 0.4 mL / min; the mobile phase is 0.01% (volume percentage, the same below) acetic acid aqueous solution (A) - acetonitrile (B); the elution program is: 0 min, 62% A, 38% B; 16 min, 52% A, 48% B; 23 min, 20% A, 80% B; 26 min, 20% A, 80% B; 28 min, 0% A, 100% B.

[0017] 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:

[0018] Ganoderic acid I, retention time: 5.98 min; precursor ion: 531.0; quantitative ion: 401.2; collision energy: 13;

[0019] Qualitative ion: 128.9, collision energy 33;

[0020] Ganoderenic acid B, retention time: 9.95 min; precursor ion: 513.0; quantitative ion: 495.2, collision energy:

[0021] 17; Qualitative ion: 451.1, collision energy 33;

[0022] Ganoderic acid B, retention time: 10.90 min; precursor ion: 515.0; quantitative ion: 497.2, collision energy:

[0023] 13; Qualitative ion: 303.1, collision energy 37;

[0024] Ganoderic acid K, retention time: 11.76 min; precursor ion: 573.0; quantitative ion: 555.1, collision energy:

[0025] 13; Qualitative ion: 469.2, Collision energy 37;

[0026] Ganoderic acid H, retention time: 13.18 min; precursor ion: 571.0; quantitative ion: 553.2, collision energy:

[0027] 13; Qualitative ion: 511.2, Collision energy 29;

[0028] Lucidenic acid A, retention time: 14.21 min; precursor ion: 457.0; quantitative ion: 209.1, collision energy:

[0029] 33; Qualitative ion: 149.0, collision energy 37;

[0030] Ganoderic acid D, retention time: 14.71 min; precursor ion: 513.0; quantitative ion: 495.2, collision energy:

[0031] 17; Qualitative ion: 149.0, collision energy 45;

[0032] Ganoderic acid A, retention time: 15.33 min; precursor ion: 515.0; quantitative ion: 497.1, collision energy:

[0033] 25; Qualitative ion: 285.2, Collision energy 41;

[0034] Ganoderic acid F, retention time: 16.06 min; precursor ion: 569.0; quantitative ion: 551.1, collision energy:

[0035] 21; Qualitative ion: 509.2, collision energy 33;

[0036] Ganoderic acid GS-3, retention time: 19.81 min; precursor ion: 557.3; quantitative ion: 539.2, collision energy:

[0037] 17; Qualitative ion: 497.1, Collision energy 21;

[0038] Ganoderic acid GS-1, retention time: 20.77 min; precursor ion: 497.3; quantitative ion: 423.2, collision energy:

[0039] 21; Qualitative ion: 149.0, collision energy 45;

[0040] Ganoderic acid GS-2, retention time: 21.12 min; precursor ion: 499.3; quantitative ion: 299.8, collision energy:

[0041] 37; Qualitative ion: 285.0, collision energy 45;

[0042] Ganodermanontriol, retention time: 23.95 min; precursor ion: 531.4; quantitative ion: 471.2, collision energy:

[0043] 13; Qualitative ion: 439.2, Collision energy 33;

[0044] Preferably, in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) is used as the analytical instrument. The UHPLC conditions are as follows: Column: Agilent Eclipse Plus C18 column (1.8 μm, 2.1 × 50 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, 62% A, 38% B; 16 min, 52% A, 48% B; 23 min, 20% A, 80% B; 26 min, 20% A, 80% B; 28 min, 0% A, 100% B; Mass spectrometry conditions: Electrospray ionization source (AJS ESI) as the ion source, detection in negative ion mode, dynamic multiple reaction monitoring (DMRM) selected, capillary voltage: 3500 V, capillary outlet voltage: 380 V. Drying gas flow rate: 16 L / min, drying gas temperature: 200 °C, sheath gas temperature: 320 °C, sheath gas flow rate: 12 L / min, nebulization pressure: 30 psi, nozzle voltage: 2000 V.

[0045] The basis for this invention's ability to simultaneously detect 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies is that, under ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS), these 13 triterpenoid compounds not only achieved baseline separation but also conformed to primary precursor ion and secondary daughter ion pair parameter information under specific mass spectrometry conditions. This technique achieves both accurate qualitative analysis of triterpenoid compounds in Ganoderma lucidum through retention time and characteristic ion information, and precise quantification of trace amounts of triterpenoids. Therefore, this method is currently the most accurate triterpenoid detection technique in Ganoderma lucidum.

[0046] The detection method of this invention relies not only on retention time but also on parent and daughter ion pairs to accurately qualitatively and quantitatively identify up to 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies. In particular, Ganoderic acid GS-1, Ganoderic acid GS-2, and Ganoderic acid GS-3 are difficult to detect using previous methods due to their low concentrations in Ganoderma lucidum fruiting body extracts. Compared to previous chemical methods, the detection method of this invention offers higher accuracy, sensitivity, and resolution, making it more suitable for the simultaneous detection and analysis of multiple triterpenoid compounds in Ganoderma lucidum fruiting bodies, extracts, and related products.

[0047] This invention provides a method for simultaneously detecting 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies. By using liquid chromatography-mass spectrometry (LC-MS), it can effectively qualitatively identify and accurately detect triterpenoids at ppb levels in Ganoderma lucidum fruiting bodies. This method is suitable for developing a detection method for evaluating triterpenoid compounds in Ganoderma lucidum raw materials and products, providing technical support for the quality evaluation and targeted development of Ganoderma lucidum fruiting bodies. Attached Figure Description

[0048] Figure 1 Structural diagrams of 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies

[0049] Figure 2 Total ion chromatogram of 13 triterpenoid compounds in Ganoderma lucidum fruiting body

[0050] in,

[0051] Compound 1: Ganoderic acid I,

[0052] Compound 2: Ganoderenic acid B,

[0053] Compound 3: Ganoderic acid B,

[0054] Compound 4: Ganoderic acid K,

[0055] Compound 5: Ganoderic acid H,

[0056] Compound 6: Lucidenic acid A

[0057] Compound 7: Ganoderic acid D,

[0058] Compound 8: Ganoderic acid A,

[0059] Compound 9: Ganoderic acid F,

[0060] Compound 10: Ganoderic acid GS-3,

[0061] Compound 11: Ganoderic acid GS-1,

[0062] Compound 12: Ganoderic acid GS-2,

[0063] Compound 13: Ganodermanontriol. Detailed Implementation

[0064] The technical solution of the present invention is further illustrated below through specific embodiments, but the scope of protection of the present invention is not limited to the content 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.

[0065] Thirteen triterpenoid compounds were used as standards: Compound 1: Ganoderic acid I, Compound 2: Ganoderenic acid B, Compound 3: Ganoderic acid B, Compound 4: Ganoderic acid K, Compound 5: Ganoderic acid H, Compound 6: Lucidenic acid A, Compound 7: Ganoderic acid D, Compound 8: Ganoderic acid A, Compound 9: Ganoderic acid F, Compound 10: Ganoderic acid GS-3, Compound 11: Ganoderic acid GS-1, Compound 12: Ganoderic acid GS-2, and Compound 13: Ganodermanontriol. All compounds were purchased from Wuhan Tianzhi Biotechnology Co., Ltd., with a purity ≥98%.

[0066] Fruiting bodies of Ganoderma sinense: Sample 1: collected from Guanxian County, Shandong Province; Sample 2: collected from Tengchong, Yunnan Province; Sample 3: collected from Xunwu, Jiangxi Province; Sample 4: collected from Sandu, Guizhou Province; Sample 5: collected from Fengkai, Guangdong Province; Sample 6: collected from Longquan, Zhejiang Province; Sample 7: collected from Rongshui, Guangxi Province; Sample 8: collected from Wuping, Fujian Province.

[0067] Example 1: Establishment of the detection method

[0068] 1. Pretreatment steps of Ganoderma lucidum fruiting bodies: Accurately weigh 0.50g of dried Ganoderma lucidum fruiting body samples (fruiting bodies 1, 2, 3, 4, 5, 6, 7, 8) into a stoppered test tube, add 15mL of methanol at a material-to-liquid ratio of 1:20 (weight-to-volume ratio, weight g: volume ml), extract by ultrasonication for 60min, take the supernatant, filter it through a 0.20μm organic filter membrane, and then dilute it 20 times (volume factor) with mass spectrometry grade methanol to obtain the sample solution to be tested for loading;

[0069] Preparation of mixed standard solutions: Take 13 triterpenoid compound standards and prepare a mixed standard solution with a concentration of 100 μg / mL using mass spectrometry grade methanol (where the concentration of each compound is 100 μg / mL). Then, dilute stepwise to prepare mixed standard working solutions of 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL.

[0070] 2. Ultra-high performance liquid chromatography (UHPLC) detection procedure: The sample solution to be tested and the standard solutions of 13 triterpenoid compounds were separated by UHPLC to obtain better resolution;

[0071] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: Column: Agilent Eclipse Plus C18 column (1.8 μm, 2.1 × 50 mm). Detection wavelength: 254 nm. Column temperature: 35℃. Sample loading volume: 2 μL. Flow rate: 0.4 mL / min. Mobile phase: 0.01% aqueous acetic acid (A) - acetonitrile (B). Elution program: 0 min, 62% A, 38% B; 16 min, 52% A, 48% B; 23 min, 20% A, 80% B; 26 min, 20% A, 80% B; 28 min, 0% A, 100% B.

[0072] 3. Procedure for obtaining compound mass spectrometry information: Thirteen triterpenoid compound standards were prepared into 5 ppm solutions with methanol. The parent ion information of the 13 triterpenoid compounds was confirmed in negative ion mode. Then, the daughter ions and collision energies were automatically optimized using Agilent Optimizer software after the parent ions were determined.

[0073] The retention times, parent ions, daughter ion pairs, and collision energies of each compound are as follows:

[0074] Ganoderic acid I, retention time: 5.98 min; precursor ion: 531.0; quantitative ion: 401.2, collision energy:

[0075] 13; Qualitative ion: 128.9, collision energy 33;

[0076] Ganoderenic acid B, retention time: 9.95 min; precursor ion: 513.0; quantitative ion: 495.2, collision energy: 17; qualitative ion: 451.1, collision energy: 33;

[0077] Ganoderic acid B, retention time: 10.90 min; precursor ion: 515.0; quantitative ion: 497.2, collision energy:

[0078] 13; Qualitative ion: 303.1, collision energy 37;

[0079] Ganoderic acid K, retention time: 11.76 min; precursor ion: 573.0; quantitative ion: 555.1, collision energy:

[0080] 13; Qualitative ion: 469.2, Collision energy 37;

[0081] Ganoderic acid H, retention time: 13.18 min; precursor ion: 571.0; quantitative ion: 553.2, collision energy:

[0082] 13; Qualitative ion: 511.2, Collision energy 29;

[0083] Lucidenic acid A, retention time: 14.21 min; precursor ion: 457.0; quantitative ion: 209.1, collision energy:

[0084] 33; Qualitative ion: 149.0, collision energy 37;

[0085] Ganoderic acid D, retention time: 14.71 min; precursor ion: 513.0; quantitative ion: 495.2, collision energy:

[0086] 17; Qualitative ion: 149.0, collision energy 45;

[0087] Ganoderic acid A, retention time: 15.33 min; precursor ion: 515.0; quantitative ion: 497.1, collision energy:

[0088] 25; Qualitative ion: 285.2, Collision energy 41;

[0089] Ganoderic acid F, retention time: 16.06 min; precursor ion: 569.0; quantitative ion: 551.1, collision energy:

[0090] 21; Qualitative ion: 509.2, collision energy 33;

[0091] Ganoderic acid GS-3, retention time: 19.81 min; precursor ion: 557.3; quantitative ion: 539.2, collision energy: 17; qualitative ion: 497.1, collision energy: 21;

[0092] Ganoderic acid GS-1, retention time: 20.77 min; precursor ion: 497.3; quantitative ion: 423.2, collision energy: 21; qualitative ion: 149.0, collision energy: 45;

[0093] Ganoderic acid GS-2, retention time: 21.12 min; precursor ion: 499.3; quantitative ion: 299.8, collision energy: 37; qualitative ion: 285.0, collision energy: 45;

[0094] Ganodermanontriol, retention time: 23.95 min; precursor ion: 531.4; quantitative ion: 471.2, collision energy: 13; qualitative ion: 439.2, collision energy: 33;

[0095] 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). Then, the parent ion information of the compound, the quantitative and qualitative daughter ion pair information, collision energy, and the ultra-high performance liquid chromatography method were all imported into the detection method. A mixed standard solution was loaded, and after running the program, the acquisition mode was updated to dynamic multiple reaction monitoring (DMRM).

[0096] The ultra-high performance liquid chromatography (UHPLC) conditions were the same as in step 2: Column: Agilent Eclipse Plus C18 column (1.8 μm, 2.1 × 50 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, 62% A, 38% B; 16 min, 52% A, 48% B; 23 min, 20% A, 80% B; 26 min, 20% A, 80% B; 28 min, 0% A, 100% B.

[0097] The triple quadrupole 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; dynamic multiple reaction monitoring (DMRM) was used; capillary voltage: 3500 V; capillary outlet voltage: 380 V; drying gas flow rate: 16 L / min; drying gas temperature: 200 °C; sheath gas temperature: 320 °C; sheath gas flow rate: 12 L / min; nebulization pressure: 30 psi; nozzle voltage: 2000 V.

[0098] 5. Limit of Detection and Limit of Quantification: The limit of detection (LOD) and limit of quantification (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.

[0099] 6. Standard curves for 13 triterpenoid compounds were created using Agilent MassHunter Quantitative Analysis software: Mixed standard working solutions of 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL were prepared and loaded under the optimized HPLC and mass spectrometry conditions described above. The concentration of the compound was plotted on the x-axis, and the quantitative ion response value of the compound was plotted on the y-axis. The quantitative standard curves are shown in Table 1.

[0100] Example 2: Methodological Validation and Sample Testing

[0101] Methodological validation was conducted in accordance with laboratory quality control standards, food physicochemical testing standards, and relevant pharmacopoeia regulations.

[0102] 1. Precision: A 5 μg / mL standard mixture solution was injected six times on the same day. The concentrations of the triterpenes obtained from the six experiments were calculated based on the standard curve, and the intra-day precision was calculated. The 5 μg / mL 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 13 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 2.

[0103] 2. Stability: Five fruiting bodies were taken, and after extracting fruiting body sample 4 according to the sample pretreatment steps in Example 1, one sample solution was prepared. The sample was injected at 0h, 2h, 4h, 6h, 8h, 12h, and 24h, respectively. The sample stability was calculated based on the results of the seven experiments. The results confirmed that the RSD of all 13 triterpenoid compounds was less than 15.00%, indicating that the samples were stable within 24 hours. Specific results are shown in Table 2.

[0104] 3. Repeatability: Four fruiting body samples were taken, and six parallel samples were weighed. Each sample was extracted according to the sample pretreatment steps in Example 1, and the resulting sample solutions were prepared and injected for analysis. The repeatability of the samples was calculated based on the results of the six experiments. The results confirmed that the RSDs of all 13 triterpenoid compounds were less than 15.00%, indicating good sample repeatability. Specific results are shown in Table 2.

[0105] 4. Recovery Rate: Take a sample of known concentration, add 13 triterpenoid standard samples, mix well, and repeat the loading three times. Calculate the sample recovery rate. Recovery rate % = (Measured value - Amount of analyte in the test sample) / Amount of added reference standard × 100%

[0106] The specific recovery results are shown in Table 3. The results confirm that the RSD of the recoveries of the 13 triterpenoid compounds in the samples were all within 15.00%, which meets the method requirements.

[0107] 5. Results Detection: Eight Ganoderma lucidum fruiting bodies were collected (Sample 1: collected from Guanxian County, Shandong Province; Sample 2: collected from Tengchong, Yunnan Province; Sample 3: collected from Xunwu, Jiangxi Province; Sample 4: collected from Sandu, Guizhou Province; Sample 5: collected from Fengkai, Guangdong Province; Sample 6: collected from Longquan, Zhejiang Province; Sample 7: collected from Rongshui, Guangxi Province; Sample 8: collected from Wuping, Fujian Province), and were tested according to the detection method in Example 1. The specific test results are shown in Table 4.

[0108] Table 1. Standard curves and parameters for quantitative analysis of 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies

[0109]

[0110] Table 2. Intra-day precision, inter-day precision, repeatability, and stability.

[0111]

[0112]

[0113] Table 3. Recovery Rate Results

[0114]

[0115]

[0116] Table 4. Sample Measurement Results

[0117]

Claims

1. A method for simultaneously detecting 13 triterpenoid compounds in the fruiting body of Ganoderma lucidum, the method comprising the following steps: (1) Pretreatment steps for Ganoderma lucidum fruiting bodies: Add organic solvent to the Ganoderma lucidum fruiting body sample for extraction, and take the supernatant. Filter and dilute to obtain the sample solution to be tested; prepare a mixed standard solution of 13 triterpenoid compounds; (2) Ultra-high performance liquid chromatography separation steps: The sample solution to be tested and the mixed standard solution of 13 triterpenoids were separated by ultra-high performance liquid chromatography to obtain a better resolution; (3) Steps for obtaining mass spectrometry information of triterpenoids: Using the mass spectrometry optimization software Agilent Optimizer, the parent ion was scanned, daughter ion pairs were detected, and the optimal collision energy was found for 13 triterpenoids. (4) Steps for establishing dynamic multiple reaction monitoring (DMRM) analysis method: In the data acquisition software Agilent MassHunterData Acquisition, under the conditions of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry, retention time, parent ion, and daughter ion pairs were imported to establish a dynamic multiple reaction monitoring (DMRM) analysis method for 13 triterpenoid compounds; at the same time, mixed standard solutions and test sample solutions of different concentrations were loaded and measured. (5) Data analysis steps: Using the quantitative analysis software Agilent MassHunterQuantitative Analysis, standard curves for 13 triterpenoid compounds were created to validate the established method and to perform quantitative analysis on the test sample solution of Ganoderma lucidum fruiting body. The 13 triterpenoid compounds mentioned are: Ganoderic acid I, Ganoderenic acid B, Ganoderic acid B, Ganoderic acid K, Ganoderic acid H, Lucidenic acid A, Ganoderic acid D, Ganoderic acid A, Ganoderic acid F, Ganoderic acid GS-3, Ganoderic acid GS-1, Ganoderic acid GS-2, Ganoderic acid GS-2, Ganoderic acid GS-2.

2. The method for simultaneously detecting 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies according to claim 1, wherein in step (1), the pretreatment step of Ganoderma lucidum fruiting bodies includes: The organic solvent is one of methanol and ethanol; The extraction methods are ultrasonic extraction or extraction by heating at 60℃; Extraction time: 10-90 min; material-to-liquid ratio: 1:20-1:40; weight (g): volume (mL). Extract the supernatant, filter it through an organic phase microporous membrane with a pore size of 0.20 μm, and then dilute it 20 times with mass spectrometry grade methanol to obtain the sample solution to be tested for injection.

3. The method for simultaneous detection of 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies according to claim 1, wherein the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: Agilent Eclipse Plus C18 column, 1.8 μm, 2.1 × 50 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 water A-acetonitrile B; elution program: 0 min, 62% A, 38% B; 16 min, 52% A, 48% B; 23 min, 20% A, 80% B; 26 min, 20% A, 80% B; 28 min, 0% A, 100% B.

4. The method for simultaneous detection of 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies 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: 5.98 min; precursor ion: 531.0; quantitative ion: 401.2; collision energy: 13; Qualitative ion: 128.9, collision energy 33; Ganoderenic acid B, retention time: 9.95 min; precursor ion: 513.0; quantitative ion: 495.2, collision energy: 17; Qualitative ion: 451.1, collision energy 33; Ganoderic acid B, retention time: 10.90 min; precursor ion: 515.0; quantitative ion: 497.2, collision energy: 13; Qualitative ion: 303.1, collision energy 37; Ganoderic acid K, retention time: 11.76 min; precursor ion: 573.0; quantitative ion: 555.1, collision energy: 13; Qualitative ion: 469.2, Collision energy 37; Ganoderic acid H, retention time: 13.18 min; precursor ion: 571.0; quantitative ion: 553.2, collision energy: 13; Qualitative ion: 511.2, Collision energy 29; Lucidenic acid A, retention time: 14.21 min; precursor ion: 457.0; quantitative ion: 209.1, collision energy: 33; Qualitative ion: 149.0, collision energy 37; Ganoderic acid D, retention time: 14.71 min; precursor ion: 513.0; quantitative ion: 495.2, collision energy: 17; Qualitative ion: 149.0, collision energy 45; Ganoderic acid A, retention time: 15.33 min; precursor ion: 515.0; quantitative ion: 497.1, collision energy: 25; Qualitative ion: 285.2, Collision energy 41; Ganoderic acid F, retention time: 16.06 min; precursor ion: 569.0; quantitative ion: 551.1, collision energy: 21; Qualitative ion: 509.2, collision energy 33; Ganoderic acid GS-3, retention time: 19.81 min; precursor ion: 557.3; quantitative ion: 539.2, collision energy: 17; Qualitative ion: 497.1, Collision energy 21; Ganoderic acid GS-1, retention time: 20.77 min; precursor ion: 497.3; Quantitative ion count: 423.2, Collision energy: 21; Qualitative ion: 149.0, collision energy 45; Ganoderic acid GS-2, retention time: 21.12 min; precursor ion: 499.3; Quantitative ion count: 299.8, Collision energy: 37; Qualitative ion: 285.0, collision energy 45; Ganodermanontriol, retention time: 23.95 min; precursor ion: 531.4; quantitative ion: 471.2, collision energy: 13; Qualitative ion: 439.2, Collision energy 33.

5. The method for simultaneous detection of 13 triterpenoid compounds in Ganoderma lucidum fruiting bodies 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: Agilent Eclipse Plus C18 column, 1.8 μm, 2.1 × 50 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 (A) - acetonitrile (B); Elution program: 0 min, 62% A, 38% B; 16 min, 52% A, 48% B; 23 min, 20% A, 80% B; 26 min, 20% A, 80% B; 28 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, nebulization pressure: 30psi, and nozzle voltage: 2000V.

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