A method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium

Abstract

The present invention provides a method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium, and the method comprises the following steps: (1) a pretreatment step of Ganoderma sinense mycelium; (2) an ultra-high performance liquid chromatography detection step; (3) a step of obtaining the mass spectrometry information of the compounds; (4) a step of establishing an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis method; (5) a data analysis step. The method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium provided by the present invention can simultaneously perform accurate qualitative and quantitative analysis on 12 triterpenoid compounds metabolized in Ganoderma sinense fermented mycelium, has high sensitivity and strong specificity, and thus provides a rapid and accurate detection and analysis method for the research of triterpenoid compounds in Ganoderma sinense mycelium and the development of related products.

Description

Technical Field

[0001] The present invention relates to the field of detection and analysis of active ingredients in edible and medicinal fungi, and specifically to a method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium. Background Art

[0002] Ganoderma sinense J.D.Zhao, L.W.Hsu & X.Q.Zhan is a fungus of the genus Ganoderma P.Karst., and is a well-known edible and medicinal fungus in China. Since 2000, it has been included in the Pharmacopoeia of the People's Republic of China (National Pharmacopoeia Commission, 2000, Beijing: Chemical Industry Press: 147 - 148) as one of the legally used Ganoderma medicinal materials.

[0003] Compared with Ganoderma lucidum, the artificial cultivation of Ganoderma sinense still has problems such as more pests and diseases and unstable biomass. Therefore, the yield of Ganoderma sinense fruiting bodies is far lower than that of Ganoderma lucidum. With the progress of fermentation technology and processes, many medicinal fungi that are difficult to cultivate artificially can obtain stable and high-yield mycelium materials through liquid or solid fermentation, and are not restricted by seasons and geography. Previous studies have isolated multiple triterpenoid compounds from the fermented mycelium of Ganoderma sinense. These compounds are specifically metabolized at the mycelium stage and have good anti-tumor and anti-inflammatory activities. To further develop the fermented mycelium of Ganoderma sinense, it is necessary to establish a precise quantitative detection method for these active triterpenoid compounds to provide technical support for the screening of high-yield active triterpenoid strains and the optimization of directional fermentation processes in the future. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium, and the method comprises the following steps:

[0005] (1) Pretreatment step of Ganoderma sinense mycelium: Add Ganoderma sinense mycelium to methanol for ultrasonic extraction or heat extraction at 60°C, take the supernatant, filter, and the obtained filtrate is the sample solution to be detected; Prepare a mixed reference solution of 12 triterpenoid compounds;

[0006] (2) Ultra-high performance liquid chromatography detection step: Perform ultra-high performance liquid chromatography separation on the sample solution to be detected and the mixed reference solution of 12 triterpenoid compounds to obtain better separation;

[0007] (3) Step of obtaining compound mass spectrometry information: Through the mass spectrometry optimization software Agilent Optimizer, perform parent ion scanning, detection of daughter ion pairs, and search for the best collision energy for 12 triterpenoid compounds;

[0008] (4) Steps for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analysis method: In the data acquisition software Agilent MassHunter Data Acquisition, under the ultra-high performance liquid chromatography analysis conditions and triple quadrupole mass spectrometry detection conditions, import the retention time, parent ion, daughter ion pairs, and collision energy data to establish the dynamic multiple reaction (DMRM) monitoring analysis and determination method for 12 triterpenoids; meanwhile, inject the mixed reference solution and sample solution of the 12 triterpenoids for determination.

[0009] (5) Data analysis steps: Use the quantitative analysis software Agilent MassHunter QuantitativeAnalysis to create the standard curves for 12 triterpenoids, conduct method validation on the established method, and perform quantitative analysis on the Ganoderma lucidum mycelium sample solution.

[0010] The 12 triterpenoids are as follows:

[0011] (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid (C 30 H 44 O6, Compound 1);

[0012] 3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid (C 30 H 46 O5, Compound 2);

[0013] (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid (C 34 H 44 O8, Compound 3);

[0014] (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid (C 30 H 44 O5, Compound 4);

[0015] (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid (C 32 H 46 O6, Compound 5);

[0016] (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid (C 32 H 48 O6, Compound 6);

[0017] (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 48 O7, Compound 7);

[0018] (22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 50 O7, Compound 8);

[0019] (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 50 O7, Compound 9);

[0020] lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid (C 36 H 52 O8, Compound 10);

[0021] (24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 50 O6, Compound 11);

[0022] (22S,24E)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C34 H 50 O6, Compound 12);

[0023] Preferably, the pretreatment step of Ganoderma sinense mycelium in step (1) is to vacuum freeze-dry the mycelium to be tested, add methanol at a solid-liquid ratio of 1:20 - 40 (weight g: volume ml) (preferably 1:20), extract by ultrasonic wave or heat extraction at 60 °C for 10 - 90 min (preferably: ultrasonic wave for 60 min), filter the extract with an organic phase microporous filter membrane with a pore size of 0.20 μm, and then dilute it 100 times with mass spectrometry grade methanol to obtain the sample solution for loading.

[0024] Preferably, the preparation method of the mixed reference solution of 12 triterpenoid compounds in step (1) is: accurately weigh 12 triterpenoid compounds respectively, prepare a mixed standard solution with a concentration of 5 μg / mL with mass spectrometry grade methanol (the concentration of each triterpenoid compound is 5 μg / mL), and then serially dilute to obtain mixed standard working solutions with concentrations of 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, and 10 ng / mL.

[0025] Preferably, the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are: select an Agilent ZORBAX Eclipse Plus C18 chromatographic column, 1.8 μm, 2.1×150 mm, detection wavelength: 240 nm; column temperature: 35 °C; room temperature: 20 °C; sample loading volume: 2 μL; flow rate: 0.4 mL / min; pressure: 800 bar; mobile phase A: 0.01% glacial acetic acid; mobile phase B: acetonitrile; elution program: 0 min, 55% A, 45% B; 5 min, 55% A, 45% B; 10 min, 25% A, 75% B; 15 min, 15% A, 85% B; 17 min, 0% A, 100% B; 20 min, 0% A, 100% B;

[0026] Preferably, through the mass spectrometry optimization software (Agilent Optimizer), the retention time, parent ion, daughter ion pairs, and collision energy information of each compound required for analysis in step (3) are obtained as follows:

[0027] (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid (C 30 H 44 O6, Compound 1): Retention time: 3.79 min; Parent ion: 499; Quantitative ion: 399.2, Collision energy: 29; Qualitative ion: 99.0, Collision energy 29;

[0028] 3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid (C 30 H 46 O5, Compound 2): Retention time: 5.37 min; Parent ion: 485; Quantitative ion: 99.0, Collision energy: 25; Qualitative ion: 141.0, Collision energy 17;

[0029] (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid (C 34 H 44 O8, Compound 3): Retention time: 7.12 min; Parent ion: 555; Quantitative ion: 495.1, Collision energy: 25; Qualitative ion: 301.2, Collision energy 33;

[0030] (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid (C 30 H 44 O5, Compound 4): Retention time: 8.28 min; Parent ion: 483; Quantitative ion: 99.0, Collision energy: 21; Qualitative ion: 425.1, Collision energy 21;

[0031] (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid (C 32 H 46 O6, Compound 5): Retention time: 10.59 min; Parent ion: 525; Quantitative ion: 465.4, Collision energy: 29; Qualitative ion: 99.0, Collision energy 41;

[0032] (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oicacid (C 32 H 48 O6, Compound 6): Retention time: 11.03 min; Parent ion: 527; Quantitative ion: 99.1, Collision energy: 25; Qualitative ion: 117.1, Collision energy 29;

[0033] (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 48 O7, compound 7): Retention time: 12.02 min; Parent ion: 567; Quantification ion: 507.3, Collision energy: 33; Qualitative ion: 99.1, Collision energy 41;

[0034] (22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 50 O7, compound 8): Retention time: 12.46 min; Parent ion: 569; Quantification ion: 99.0, Collision energy: 25; Qualitative ion: 434.0, Collision energy 61;

[0035] (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 50 O7, compound 9): Retention time: 12.89 min; Parent ion: 569; Quantification ion: 509.2, Collision energy: 33; Qualitative ion: 99.0, Collision energy 37;

[0036] lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid (C 36 H 52 O8, compound 10): Retention time: 14.46 min; Parent ion: 611; Quantification ion: 551.3, Collision energy: 37; Qualitative ion: 99.0, Collision energy 41;

[0037] (24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C 34 H 50 O6, compound 11): Retention time: 16.49 min; Parent ion: 553; Quantification ion: 511.3, Collision energy: 33; Qualitative ion: 493.4, Collision energy 37;

[0038] (22S,24E)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid (C34 H 50 O6, Compound 12): Retention time: 16.74 min; Parent ion: 553; Quantification ion: 493.3, Collision energy: 33; Qualitative ion: 98.9, Collision energy 37;

[0039] Preferably, in step (4), an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical and detection instrument. The chromatographic conditions for ultra-high performance liquid chromatography separation (the same as the chromatographic conditions in step (2)) are as follows: An Agilent ZORBAX Eclipse Plus C18 chromatographic column, 1.8 μm, 2.1×150 mm is selected, the detection wavelength: 240 nm; Column temperature: 35 °C; Room temperature: 20 °C; Sample injection volume: 2 μL; Flow rate: 0.4 mL / min; Pressure: 800 bar; Mobile phase A: 0.01% glacial acetic acid; Mobile phase B: acetonitrile; Elution program: 0 min, 55% A, 45% B; 5 min, 55% A, 45% B; 10 min, 25% A, 75% B; 15 min, 15% A, 85% B; 17 min, 0% A, 100% B; 20 min, 0% A, 100% B; The mass spectrometry conditions are as follows: Electrospray ionization source (AJS ESI) is used as the ion source, the detection is carried out in the negative ion mode, dynamic multiple reaction monitoring (DMRM) is selected, capillary voltage: 3000 V, capillary outlet voltage: 380 V; Dry gas flow rate: 14 L / min, dry gas temperature: 200 °C, sheath gas temperature: 250 °C, sheath gas flow rate: 11 L / min, nozzle voltage: 1500 V.

[0040] The identification basis of a method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium provided by the present invention is: Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used for detection. On the basis of qualitatively identifying compounds by retention time, further qualitative and quantitative analysis is carried out according to the molecular ion information of the compounds to accurately target and detect the compounds.

[0041] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technology can not only accurately qualitatively and quantitatively detect triterpenoid compounds through retention time, but also from mass spectrometry information such as parent ions and daughter ions. This technology is especially suitable for quantitative analysis in biological materials with a lot of impurity interference and when the target compound structures are similar. Therefore, the present invention uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and takes 12 triterpenoid compounds obtained from Ganoderma sinense fermented mycelium as reference substances to establish a method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium.

[0042] The beneficial effects of the present invention are as follows: The technical solution of the present invention combines the high separation ability of liquid chromatography and the function of mass spectrometry to provide structural information, can obtain rich and effective compound structural information, has the advantages of high sensitivity and strong selectivity, and can effectively overcome the disadvantages of chemical methods or liquid chromatography methods, thereby providing an accurate and reliable detection and analysis method for the related research and products of Ganoderma sinense mycelium. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Structural formula diagrams of 12 triterpenoid compounds in Ganoderma sinense mycelium

[0044] Figure 2 Ultra-high performance liquid chromatography diagrams of 12 triterpenoid compounds in Ganoderma sinense mycelium

[0045] Figure 3 Total ion current diagrams of 12 triterpenoid compounds in Ganoderma sinense mycelium

[0046] Among them, Compound 1: (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid;

[0047] Compound 2: 3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid;

[0048] Compound 3: (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid;

[0049] Compound 4: (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid;

[0050] Compound 5: (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid;

[0051] Compound 6: (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid;

[0052] Compound 7: (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid;

[0053] Compound 8: (22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid;

[0054] Compound 9: (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid;

[0055] Compound 10: lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid;

[0056] Compound 11: (24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid;

[0057] Compound 12: (22S,24E)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid Detailed implementation mode

[0058] The technical solutions of the present invention are further described below through specific embodiments, but it is not used to limit the solutions of the present invention, and the protection scope of the present invention cannot be limited thereby. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

[0059] Instruments and materials:

[0060] Ultra-high performance liquid chromatograph (Agilent LC1290 infinity II, Agilent Technologies, USA);

[0061] Triple quadrupole mass spectrometer (Agilent 6495, Agilent Technologies, USA)

[0062] Ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instruments Co., Ltd.)

[0063] Electronic balance (FA2004A, Shanghai Precision Scientific Instruments Co., Ltd.)

[0064] Disposable syringe (1 mL, Taizhou Baike Medical Instrument Co., Ltd.)

[0065] Organic phase microporous filter membrane (0.20 μm, Agilent Technologies, USA)

[0066] Mass spectrometry grade methanol and acetonitrile were both purchased from Merk, USA.

[0067] Reference substances of 12 triterpenoid compounds were prepared by the method disclosed in Chinese Patent Application (Publication No. CN115746076A), and the purity of the compounds was ≥98%.

[0068] Source of Ganoderma sinense mycelium: Strain 1: Ganoderma sinense, strain number: Dai20077, preserved in the College of Ecology and Nature Conservation, Beijing Forestry University; Strain 2: Ganoderma sinense, strain number: 6066, preserved in the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences.

[0069] Establishment of the detection method in Example 1

[0070] 1. Preparation of the mixed reference substance solution: Take reference substances of 12 triterpenoid compounds and prepare a mixed standard solution with a concentration of 5 μg / mL in mass spectrometry grade methanol (the concentration of each reference substance is 5 μg / mL), and then serially dilute it with mass spectrometry grade methanol to obtain mixed reference substance solutions with concentrations of 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, and 10 ng / mL, and store them in a 4°C refrigerator for later use.

[0071] Fermentation of Ganoderma sinense mycelium: Inoculate Strain 1 and Strain 2 into the liquid medium respectively (prepared in distilled water with soluble starch 3%, glucose 1.2%, yeast powder 1%, MgSO4·H2O 0.15%, KH2PO4 0.3%, natural pH, sterilized at 121°C for 15 min), culture at 150 r / min and 25°C in the dark for 7 days to obtain the first-stage seed liquid. Then inoculate the first-stage seed liquid into the second-stage culture medium (with the same formula above) at a ratio of 10%, culture at 150 r / min and 25°C in the dark for 7 days, transfer to static culture for 15 days, collect the mycelium, and freeze-dry it for later use.

[0072] Extraction of Ganoderma sinense mycelium: Weigh 0.50 g each of the dried Ganoderma sinense mycelium 1 and 2, add 10 mL of analytical grade methanol according to the solid-liquid ratio of 1:20 (weight-volume ratio, weight g: volume ml), ultrasonically extract for 60 min, take the supernatant and filter it through an organic phase microporous filter membrane with a pore size of 0.20 μm, and then dilute it 100 times with mass spectrometry grade methanol to obtain the sample solution to be tested;

[0073] 2. Ultra - high performance liquid chromatography detection steps: Optimize the ultra - high performance liquid chromatography elution conditions for a mixed solution of 12 triterpene reference substances to obtain the best resolution. The final liquid chromatography diagram is shown in Figure 2 ;

[0074] Among them, the ultra - high performance liquid conditions are as follows: Agilent ZORBAX Eclipse Plμs C18 chromatographic column, 1.8 μm, 2.1×150 mm, detection wavelength: 240 nm; column temperature: 35 °C; room temperature: 20 °C; sample injection volume: 2 μL; flow rate: 0.4 mL / min; pressure: 800 bar; mobile phase A: 0.01% glacial acetic acid; mobile phase B: acetonitrile; elution program: 0 min, 55% A, 45% B; 5 min, 55% A, 45% B; 10 min, 25% A, 75% B; 15 min, 15% A, 85% B; 17 min, 0% A, 100% B; 20 min, 0% A, 100% B.

[0075] 3. Steps to obtain the mass spectrometry information of compounds: Prepare methanol solutions of 5 ppm for 12 triterpene compound reference substances respectively, confirm the parent ion information of 12 triterpene compounds in the negative ion mode, and then automatically optimize the daughter ions and collision energies through Agilent Optimizer software when the parent ions are determined.

[0076] Compound 1: Retention time: 3.79 min; parent ion: 499; quantitative ion: 399.2, collision energy: 29; qualitative ion: 99.0, collision energy 29;

[0077] Compound 2: Retention time: 5.37 min; parent ion: 485; quantitative ion: 99.0, collision energy: 25; qualitative ion: 141.0, collision energy 17;

[0078] Compound 3: Retention time: 7.12 min; parent ion: 555; quantitative ion: 495.1, collision energy: 25; qualitative ion: 301.2, collision energy 33;

[0079] Compound 4: Retention time: 8.28 min; parent ion: 483; quantitative ion: 99.0, collision energy: 21; qualitative ion: 425.1, collision energy 21;

[0080] Compound 5: Retention time: 10.59 min; parent ion: 525; quantitative ion: 465.4, collision energy: 29; qualitative ion: 99.0, collision energy 41;

[0081] Compound 6: Retention time: 11.03 min; parent ion: 527; quantitative ion: 99.1, collision energy: 25; qualitative ion: 117.1, collision energy 29;

[0082] Compound 7: Retention time: 12.02 min; Parent ion: 567; Quantification ion: 507.3, Collision energy: 33; Qualification ion: 99.1, Collision energy 41;

[0083] Compound 8: Retention time: 12.46 min; Parent ion: 569; Quantification ion: 99.0, Collision energy: 25; Qualification ion: 434.0, Collision energy 61;

[0084] Compound 9: Retention time: 12.89 min; Parent ion: 569; Quantification ion: 509.2, Collision energy: 33; Qualification ion: 99.0, Collision energy 37;

[0085] Compound 10: Retention time: 14.46 min; Parent ion: 611; Quantification ion: 551.3, Collision energy: 37; Qualification ion: 99.0, Collision energy 41;

[0086] Compound 11: Retention time: 16.49 min; Parent ion: 553; Quantification ion: 511.3, Collision energy: 33; Qualification ion: 493.4, Collision energy 37;

[0087] Compound 12: Retention time: 16.74 min; Parent ion: 553; Quantification ion: 493.3, Collision energy: 33; Qualification ion: 98.9, Collision energy 37.

[0088] 4. Establishment of a quantitative analysis method by ultra - performance liquid chromatography - triple quadrupole mass spectrometry: Under the conditions of setting the parameters of ultra - performance liquid chromatography and triple quadrupole mass spectrometry in the data acquisition software (Agilent MassHunter Data Acquisition), change the mass spectrometry acquisition mode to multiple reaction monitoring (MRM), then import the information of the parent ion of the compound, the information of the quantification and qualification daughter ion pairs, the collision energy, etc. into the detection method. Inject a mixed reference solution of 12 triterpenoid compounds, and after running the program, update the acquisition mode to dynamic multiple reaction monitoring (DMRM). The mass spectrometry diagram is shown in Figure 3 .

[0089] Among them, the chromatographic conditions for ultra-high performance liquid chromatography separation are as follows: An Agilent ZORBAX Eclipse Plμs C18 chromatographic column, 1.8 μm, 2.1×150 mm, detection wavelength: 240 nm; column temperature: 35 °C; room temperature: 20 °C; sample injection volume: 2 μL; flow rate: 0.4 mL / min; pressure: 800 bar; mobile phase A: 0.01% glacial acetic acid; mobile phase B: acetonitrile; elution program: 0 min, 55% A, 45% B; 5 min, 55% A, 45% B; 10 min, 25% A, 75% B; 15 min, 15% A, 85% B; 17 min, 0% A, 100% B; 20 min, 0% A, 100% B.

[0090] Among them, the triple quadrupole mass spectrometry analysis conditions: The electrospray ionization source AJS ESI is used as the ion source, the detection is carried out in the negative ion mode, the dynamic multiple reaction monitoring DMRM is selected, capillary voltage: 3000 V, capillary outlet voltage: 380 V; drying gas flow rate: 14 L / min, drying gas temperature: 200 °C, sheath gas temperature: 250 °C, sheath gas flow rate: 11 L / min, nozzle voltage: 1500 V.

[0091] 5. Detection limit and quantification limit: The detection limit (LOD) and quantification limit (LOQ) are calculated based on the standard deviation of the response value and the slope of the standard curve. Among them: LOD = 3σ / S, LOQ = 10σ / S, σ: the standard deviation of the response value, S: the slope of the standard curve, and the standard deviation of the response value is the residual standard deviation of the standard curve.

[0092] 6. Standard curve formulation: Take the prepared mixed reference solution with concentrations of 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, and 10 ng / mL and inject samples according to the chromatographic conditions for the optimized ultra-high performance liquid chromatography separation and the triple quadrupole mass spectrometry analysis conditions in step 4 above. Use the compound concentration as the abscissa and the quantitative ion response value of the compound as the ordinate to make the quantitative standard curve equation as shown in Table 1 below.

[0093] Table 1. Quantitative analysis standard curves and parameters of 12 triterpenoid compounds

[0094]

[0095] Example 2

[0096] Methodology verification and result detection: The methodology verification refers to the laboratory quality control specifications for food physical and chemical testing standards and relevant regulations of the pharmacopoeia.

[0097] 1 Precision: Take the mixed reference solution and inject it repeatedly 6 times on the same day. Calculate the concentrations of the 12 triterpenoid compounds obtained from the 6 experiments according to the standard curve, and calculate the within-day precision. Take the mixed reference solution for three consecutive days, inject it 3 times each day, and calculate the between-day precision according to the results of the 9 experiments. The results show that the RSDs of the within-day precision determination results of the 12 triterpenoid compounds are 1.55%, 1.61%, 1.31%, 0.84%, 1.93%, 1.85%, 2.29%, 3.41%, 1.77%, 1.46%, 2.86%, 2.81% respectively; the RSDs of the between-day precision determination results are 2.05%, 1.88%, 1.67%, 2.75%, 3.43%, 4.23%, 2.37%, 3.84%, 2.54%, 2.11%, 9.20%, 2.94% respectively, all less than 15.00%, indicating that the within-day and between-day precisions of this method are good. The specific results are shown in Table 2 and Table 3.

[0098] 2 Stability: Take the dried mycelium of strain 1, extract it according to the steps in Example 1, and inject the test sample solution at 0h, 2h, 4h, 6h, 8h, 12h, 24h respectively. Calculate the sample stability according to the results of the 7 experiments. The results confirm that the RSDs of the 12 triterpenoid compounds are 1.48%, 4.02%, 2.94%, 2.86%, 4.24%, 3.97%, 5.71%, 9.54%, 6.30%, 5.01%, 13.14%, 10.12% respectively, all less than 15.00%, indicating that the properties of the sample test results are stable within 24 hours. The specific results are shown in Table 4.

[0099] 3 Repeatability: Weigh 6 portions of the dried mycelium of strain 1 in parallel, treat them according to the extraction steps in Example 1 respectively, inject the test sample solution, and calculate the sample repeatability according to the results of the 6 experiments. The results confirm that the RSDs of the 12 triterpenoid compounds are 1.05%, 3.88%, 2.16%, 0.65%, 1.75%, 3.56%, 2.91%, 4.94%, 2.04%, 3.15%, 5.94%, 7.85% respectively, all less than 15.00%, indicating that the sample repeatability is good. The specific results are shown in Table 5.

[0100] 4 Recovery rate: Take the test sample solution with a known concentration (see Table 6), add the triterpenoid reference substance, mix well and load the sample three times repeatedly, and calculate the sample recovery rate.

[0101] Recovery rate % = (measured value - amount of the tested component contained in the test sample) / amount of added reference substance × 100%

[0102] The specific results of the recovery rate are shown in Table 6. The results confirmed that the sample recovery rates of the 12 triterpenoid compounds were 97.29%, 88.87%, 93.11%, 105.03%, 100.26%, 102.25%, 102.18%, 98.04%, 102.42%, 102.33%, 108.98%, and 108.02% respectively. The RSDs were all within 7.86%, meeting the method requirements.

[0103] 5 Result detection: Take two extracts of Ganoderma sinense mycelia (strains 1 and 2), and according to the detection method of Example 1, perform pretreatment and detection respectively. The specific detection results are shown in Table 7.

[0104] It can be seen from the results that the compositions of the 12 triterpenoid compounds in the two mycelia of Ganoderma sinense are similar, indicating that Ganoderma sinense of the same species is similar in the composition of triterpenoid compounds. Among the fermented mycelia of Ganoderma sinense, the four compounds with higher contents are lanosta - 7,9(11),24 - trien - 3β,15α,22β - triacetoxy - 26 - oic acid, (22S,24E) - 3β,22β - diacetoxylanosta - 7,9(11),24 - trien - 26 - oic acid, (22S,24E) - 15α - hydroxy - 3β,22β - diacetoxylanosta - 7,9(11),24 - trien - 26 - oic acid, and (24Z) - 3β,15α - diacetoxylanosta - 7,9(11),24 - trien - 26 - oic acid, and these four compounds can be prepared in large quantities.

[0105] Table 2, Results of within - day precision

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[0107] Table 3, Results of between - day precision

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[0110] Table 4, Results of sample stability

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[0112] Table 5, Results of sample repeatability

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[0115] Table 6, Results of Sample Recovery Rate

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[0117] Table 7, Results of Sample Determination

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[0119] Content of Each Compound (mg / kg)

[0120]

Claims

1. A method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium, characterized in that The method comprises the following steps: (1) Pretreatment step of Ganoderma sinense mycelium: adding Ganoderma sinense mycelium into methanol for ultrasonic extraction or heating extraction at 60 °C, taking the supernatant, filtering, and the obtained filtrate is serially diluted to obtain the sample solution to be measured; preparing a mixed reference solution of 12 triterpenoid compounds; (2) Ultra-high performance liquid chromatography detection step: performing ultra-high performance liquid chromatography separation on the sample solution to be measured and the mixed reference solution of 12 triterpenoid compounds; (3) Step of obtaining compound mass spectrometry information: through the mass spectrometry optimization software Agilent Optimizer, performing parent ion scanning, detection of daughter ion pairs, and searching for the optimal collision energy for 12 triterpenoid compounds; (4) Step of establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis method: in the data acquisition software Agilent MassHunter Data Acquisition, under the ultra-high performance liquid chromatography analysis conditions and triple quadrupole mass spectrometry detection conditions, importing retention time, parent ion, daughter ion pair, and collision energy data, and establishing a dynamic multiple reaction monitoring (DMRM) analysis and determination method for 12 triterpenoid compounds; meanwhile, performing sample injection determination on mixed reference solutions and sample solutions with different concentrations; (5) Data analysis step: using the quantitative analysis software Agilent MassHunter Quantitative Analysis to create a standard curve for 12 triterpenoid compounds, performing method validation on the established method, and performing quantitative analysis on the Ganoderma sinense mycelium sample solution; Among them, the 12 triterpenoid compounds are: (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid, Compound 1; 3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid, Compound 2; (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid, Compound 3; (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid, Compound 4; (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid, Compound 5; (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid, Compound 6; (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid, Compound 7; (22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid, Compound 8; (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid, Compound 9; lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid, Compound 10; (24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid, Compound 11; (22S,24E)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid, Compound 12; Among them, the chromatographic conditions for ultra-high performance liquid chromatography separation in steps (2) and (4) are as follows: An Agilent ZORBAX Eclipse Plus C18 chromatographic column, 1.8 μm, 2.1×150 mm, detection wavelength: 240 nm; column temperature: 35 °C; room temperature: 20 °C; sample injection volume: 2 μL; flow rate: 0.4 mL / min; Pressure: 800 bar; mobile phase A: 0.01% glacial acetic acid; mobile phase B: acetonitrile; elution program: 0 min, 55% A, 45% B; 5 min, 55% A, 45% B; 10 min, 25% A, 75% B; 15 min, 15% A, 85% B; 17 min, 0% A, 100% B; 20 min, 0% A, 100% B.

2. The method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium according to claim 1, wherein the pretreatment step of Ganoderma sinense mycelium in step (1) is that the mycelium to be tested is frozen until completely dry, and methanol is added according to a solid-liquid ratio of 1:20 - 40, weight g: volume ml, and ultrasonic extraction is carried out for 10 - 90 min or heating extraction is carried out at 60 °C for 10 - 90 min. The extract is filtered through an organic phase microporous filter membrane with a pore size of 0.20 μm and then diluted 100 times with mass spectrometry grade methanol to obtain the sample solution to be tested.

3. The method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium according to claim 1, wherein the preparation method of the mixed reference solution of 12 triterpenoid compounds in step (1) is as follows: Take the reference substances of 12 triterpenoid compounds, and prepare a mixed standard solution with a concentration of 5 μg / mL using mass spectrometry grade methanol, and then serially dilute it to obtain mixed standard working solutions with concentrations of 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, and 10 ng / mL.

4. The method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium according to claim 1, wherein through the mass spectrometry optimization software Agilent Optimizer, the retention time, parent ion, daughter ion pairs, and collision energy information of each compound required for analysis in step (3) are as follows: (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid: Retention time: 3.79 min; Parent ion: 499; Quantitative ion: 399.2, Collision energy: 29; Qualitative ion: 99.0, Collision energy 29; 3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid: Retention time: 5.37 min; Parent ion: 485; Quantitative ion: 99.0, Collision energy: 25; Qualitative ion: 141.0, Collision energy 17; (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid: Retention time: 7.12 min; Parent ion: 555; Quantitative ion: 495.1, Collision energy: 25; Qualitative ion: 301.2, Collision energy 33; (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 8.28 min; Parent ion: 483; Quantitative ion: 99.0, Collision energy: 21; Qualitative ion: 425.1, Collision energy 21; (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 10.59 min; Parent ion: 525; Quantitative ion: 465.4, Collision energy: 29; Qualitative ion: 99.0, Collision energy 41; (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 11.03 min; Parent ion: 527; Quantification ion: 99.1, Collision energy: 25; Qualitative ion: 117.1, Collision energy 29; (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 12.02 min; Parent ion: 567; Quantification ion: 507.3, Collision energy: 33; Qualitative ion: 99.1, Collision energy 41; (22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 12.46 min; Parent ion: 569; Quantification ion: 99.0, Collision energy: 25; Qualitative ion: 434.0, Collision energy 61; (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 12.89 min; Parent ion: 569; Quantification ion: 509.2, Collision energy: 33; Qualitative ion: 99.0, Collision energy 37; lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid: Retention time: 14.46 min; Parent ion: 611; Quantification ion: 551.3, Collision energy: 37; Qualitative ion: 99.0, Collision energy 41; (24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 16.49 min; Parent ion: 553; Quantification ion: 511.3, Collision energy: 33; Qualitative ion: 493.4, Collision energy 37; (22S,24E)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid: Retention time: 16.74 min; Parent ion: 553; Quantification ion: 493.3, Collision energy: 33; Qualitative ion: 98.9, Collision energy 37.

5. The method for simultaneously detecting 12 triterpenoid compounds in Ganoderma sinense mycelium according to claim 1, wherein in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical and detection instrument, and the mass spectrometry conditions are as follows: AJS ESI of electrospray ionization source is used as the ion source, the detection is carried out in the negative ion mode, dynamic multiple reaction monitoring (DMRM) is selected, capillary voltage: 3000 V, capillary outlet voltage: 380 V; drying gas flow rate: 14 L / min, drying gas temperature: 200 °C, sheath gas temperature: 250 °C, sheath gas flow rate: 11 L / min, nozzle voltage: 1500 V.

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