CN113917033B - Method for detecting neutral triterpene of ganoderma fungus - Google Patents
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Abstract
The invention provides a method for detecting neutral triterpene of ganoderma fungus, which comprises the following steps: (1) a step of preprocessing ganoderma lucidum fruiting bodies; (2) an ultra performance liquid chromatography detection step; (3) a compound mass spectrometry information obtaining step; (4) 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 neutral triterpene compounds in ganoderma fungi provided by the invention realizes simultaneous qualitative and quantitative analysis of 12 neutral triterpene compounds in ganoderma fungi, and provides accurate and reliable detection analysis for related research of ganoderma fungi.
Description
Technical Field
The invention relates to the field of food and drug analysis, in particular to a method for detecting neutral triterpenes of ganoderma fungi.
Background
Ganoderma (Ganoderma P. Karst) belongs to Basidiomycota, agaricus (Agrocomomyces), polyporales (Polyporales), ganodermataceae (Ganodermataceae), which is an important group of higher Basidiomycetes, and has 137 species in total. Wherein, the Ganoderma lucidum (Ganoderma lucidum) and the Ganoderma lucidum (Ganoderma sinense) can be used as medicinal materials by Chinese pharmacopoeia, and can be used as raw materials for developing health-care foods together with Ganoderma lucidum, ganoderma lucidum and Ganoderma tsugae. The ganoderma fungus contains various triterpene compounds, is one of main active ingredients, and has various physiological activities. These triterpene compounds can be classified into two classes, acidic triterpenes and neutral triterpenes, by means of acid-base extraction. The acidic triterpenes are mainly Ganoderic acid, ganoderic acid and ganoderma lucidum acid compounds, such as Ganoderic acid Ganoderic acid A, B, C, D, E, F, ganoderic acid Ganoderenic acid A, B, D, ganoderma lucidum acid lucidific acid A, B and the like, the structures of the substances are relatively consistent, and most of the substances have a carbonyl group at the C11 position and all have delta 8 (9) double bonds, and the ultraviolet absorption is concentrated at about 253nm, so the substances are relatively suitable for quantitative analysis by high-performance liquid phase-ultraviolet combination. Neutral triterpenes are various, and include various compounds such as ganoderic ketone, ganoderic aldehyde, ganoderic alcohol, ganoderic ester, ganoderic lactone, etc., for example, ganoderic alcohol, ganoderol B, ganoderone A, ganoderol A, lucialdyide A, etc. The neutral triterpenes may also contain some less polar ganoderic acid compounds such as 7-Oxo-ganoderic acid Z, 23S-hydroxy-11, 15-diox-ganoderic acid DM, etc. Neutral triterpenes have been reported to be a ubiquitous class of triterpenes in a wide variety of Ganoderma species, such as Ganoderma lucidum (Ganoderma leucocontextum), ganoderma lucidum (Ganoderma resinaceum), ganoderma sinense, ganoderma tsugae, and the like.
Neutral triterpene is used as an important component of ganoderma triterpene, and has great potential in various aspects such as anti-tumor, antivirus, anti-inflammatory, liver protection, blood sugar reduction, blood lipid reduction, etc. Because the neutral triterpene has a complex and various structure, the maximum ultraviolet absorption of each compound is inconsistent, and accurate detection is difficult to carry out through ultraviolet absorption, a comprehensive neutral triterpene detection method has not been available. Due to the lack of the neutral triterpene detection method, the result is often not comprehensive and objective enough when the quality of ganoderma lucidum and related products is evaluated, and the research and development of ganoderma lucidum triterpene are greatly hindered.
Neutral triterpenes are a class of less polar triterpenes that can be ionized using atmospheric pressure chemical ionization and can therefore be detected by using mass spectrometry. Therefore, it is necessary to establish an accurate analysis method for determining neutral triterpene compounds in fruiting bodies, extracts or related products of Ganoderma genus.
Disclosure of Invention
The invention provides a method for detecting neutral triterpene of ganoderma fungus, which comprises the following steps:
(1) The pretreatment step of the ganoderma lucidum fruiting body: extracting Ganoderma fruiting body sample with organic solvent, collecting supernatant, filtering, and diluting to obtain sample solution to be tested; preparing mixed standard solution of 12 neutral triterpene compounds;
(2) Ultra-high performance liquid chromatography detection: carrying out ultra-high performance liquid chromatography separation on a sample solution to be detected and mixed standard solution of 12 neutral triterpenoids to obtain a better separation degree;
(3) Compound mass spectrum information obtaining step: carrying out parent ion scanning, detection of child ion pairs and searching of optimal collision energy on 12 triterpene compounds by utilizing mass spectrum optimization software (Agilent Optimizer);
(4) The method for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis method comprises the following steps: introducing retention time, parent ion and ion pairs in data acquisition software (Agilent MassHunter Data Acquisition) under the conditions of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry, and establishing a dynamic multi-reaction monitoring (DMRM) analysis and determination method of 12 neutral triterpene compounds; meanwhile, loading and measuring mixed standard substance solutions with different concentrations and sample solutions to be measured;
(5) And a data analysis step: using quantitative analysis software (Agilent MassHunter QuantitativeAnalysis), creating 12 standard curves of neutral triterpene compounds, performing methodological verification on the established method,
and quantitatively analyzing the solution of the ganoderma lucidum fruiting body sample to be detected.
Wherein in the pretreatment step of the ganoderma lucidum fruiting body in the step (1):
the organic solvent is one of methanol and ethanol, preferably methanol;
the extraction method is ultrasonic extraction or 60 ℃ heating extraction, preferably ultrasonic extraction;
the extraction time is 10-90min, preferably 60min; the feed liquid ratio is 1:20-1:40, preferably 1:30 (weight g: volume mL);
the extraction times comprise 1-3 times of extraction, preferably 1 time;
filtering the extracted supernatant with an organic phase microporous filter membrane with the aperture of 0.20 mu m, and diluting with mass spectrum grade methanol for 20 times to obtain a sample solution to be detected for sample injection;
the preparation method of the 12 neutral triterpenoid standard substance solutions in the step (1) comprises the following steps of precisely weighing: ganodermannriol (C) 30 H 48 O 4 Compound 1), 23S-hydroxy-11, 15-dio-ganoderic acid DM (C) 30 H 40 O 7 Compound 2), 7-Oxo-ganoderic acid Z (C) 30 H 46 O 4 Compound 3), ganoderiol F (C) 30 H 46 O 3 Compound 4), ganodermannodiol (C) 30 H 48 O 3 Compound 5), lucialdehyde B (C) 30 H 44 O 3 Compound 6), lucidal (C) 30 H 46 O 3 Compound 7), ganoderol B (C 30 H 48 O 2 Compound 8), ganoderol a (C 30 H 46 O 2 Compound 9), lucialdehyde a (C) 30 H 46 O 2 Compound 10), ganoderal a (C 30 H 44 O 2 Compound 11), ganoderiol A (C) 30 H 50 O 4 Compound 12), 12 compounds, and a mixed standard solution mother solution (concentration of compound 1 is 200 μg/mL; the concentration of the compounds 2, 3, 4, 5, 7 and 8 is 100 mug/mL; the concentration of Compound 6 was 50. Mu.g/mL; the concentration of the compounds 9, 10 and 12 is 25 mug/mL, the concentration of the compound 11 is 900 mug/mL), and the mother liquor is diluted 20 timesThe mixed standard working solution with the highest concentration is obtained, and then the mixed standard working solution with different concentrations is obtained by stepwise dilution by 2.5 times, 2 times, 2.5 times, 2 times and 2.5 times.
Preferably, the chromatographic conditions of the ultra performance liquid chromatography in step (2) are: selecting Agilent ZORBAX SB-Aq chromatographic column with a diameter of 1.8 μm and a diameter of 2.1X106 mm; detection wavelength: 240nm. Column temperature: 35 ℃; the loading amount is 2 mu L; flow rate: 0.4mL/min; mobile phase: 0.01% acetic acid water (a) -methanol (B); elution procedure: 0min,20% A,80% B;8min,20% A,80% B;10min,10% A,90% B;15min,0% A,100% B;18min,0% A,100% B.
Preferably, the retention time, parent ion, daughter ion pair and collision energy information for each compound required for analysis in step (3) is obtained by mass spectrometry optimization software (Agilent Optimizer) as follows:
ganodermantiol, retention time: 3.79min; parent ion: 455.0; quantitative ion: 105.1, collision energy:
80; qualitative ion: 107.0, collision energy: 37, respectively;
23S-hydroxy-11, 15-dio-ganoderic acid DM, retention time: 3.98min; parent ion: 495.1;
quantitative ion: 435.3, collision energy: 17; qualitative ion: 353.1, collision energy: 29;
7-Oxo-ganoderic acid Z, retention time: 4.34min; parent ion: 471.2; quantitative ion: 107.1, collision energy: 61; qualitative ion: 135.1, collision energy: 45;
ganoderiol F, retention time: 5.94min; parent ion: 437.2; quantitative ion: 67.2, collision energy: 73;
qualitative ion: 95.0, collision energy: 41;
ganodermannodiol, retention time: 6.40min; parent ion: 439.2; quantitative ion: 105.1, collision energy:
73; qualitative ion: 109.1, collision energy: 41;
lucialdehyde B, retention time: 6.79min; parent ion: 453.1; quantitative ion: 327.1, collision energy:
17; qualitative ion: 121.1, collision energy: 49;
lucidal, retention time: 7.89min; parent ion: 437.1; quantitative ion: 201.1, collision energy: 29; qualitative ion: 119.1, collision energy: 37, respectively;
ganodrol B, retention time: 10.80min; parent ion: 423.2; quantitative ion: 67.0, collision energy: 61;
qualitative ion: 95.1, collision energy: 37, respectively;
ganoderol A, retention time: 11.25min; parent ion: 421.1; quantitative ion: 67.1, collision energy: 61;
qualitative ion: 95.0, collision energy: 45;
lucialdehyde a, retention time: 11.25min; parent ion: 421.1; quantitative ion: 109.1, collision energy:
29; qualitative ion: 69.1, collision energy: 45;
ganoderal A, retention time: 12.21min; parent ion: 437.0; quantitative ion: 91.0, collision energy: 77;
qualitative ion: 81.0, collision energy: 49;
ganoderiol A, retention time: 13.99min; parent ion: 475.0; quantitative ion: 177.0, collision energy:
21, a step of; qualitative ion: 145.1, collision energy: 53;
preferably, in the step (4), the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as an analysis and detection instrument, wherein the ultra-high performance liquid condition is as follows: selecting Agilent ZORBAX SB-Aq chromatographic column with a diameter of 1.8 μm and a diameter of 2.1X106 mm; detection wavelength: 240nm; column temperature: 35 ℃; the loading amount is 2 mu L; flow rate: 0.4mL/min; mobile phase: 0.01% acetic acid water (a) -methanol (B); elution procedure: 0min,20% A,80% B;8min,20% A,80% B;10min,10% A,90% B;15min,0% A,100% B;18min,0% A,100% B; the mass spectrum conditions are as follows: atmospheric pressure chemical ionization source (APCI) is used as ion source, detection is carried out in positive ion mode, dynamic multi-reaction monitoring (DMRM) is selected, and capillary voltage is 3500V; capillary outlet voltage: 380V; corona needle current: 8 μA; the temperature of the drying gas is 290 ℃; the dry air flow is 13L/min; gasification chamber temperature: 350 ℃; atomization gas pressure: 30psi.
The identification basis of the method for detecting neutral triterpene of ganoderma fungus is as follows: when ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used for detection, 12 neutral triterpene compounds in ganoderma fungi not only can meet the requirement of consistent retention time, but also can meet the requirement that both primary parent-secondary ion pairs and secondary ion pairs meet detection parameters under specific mass spectrometry conditions, so that the compounds can be accurately and quantitatively analyzed.
The method for detecting the neutral triterpene of the ganoderma fungus not only depends on the retention time, but also can accurately, qualitatively and quantitatively detect up to 12 neutral triterpene compounds in the ganoderma fungus through the parent ion and the child ion pairs. Compared with the previous chemical method and high performance liquid-ultraviolet detection method, the method has higher accuracy, sensitivity and resolution, and is more suitable for detecting and analyzing triterpene compounds in fruiting bodies, extracts and related products of ganoderma lucidum.
Drawings
FIG. 1 shows the structural formula of 12 triterpene compounds in Ganoderma fungus
FIG. 2 is a mass spectrum total ion flow diagram of 12 triterpene compounds in ganoderma fungus
Wherein, the liquid crystal display device comprises a liquid crystal display device,
compound 1: ganodermannriol,
compound 2:23S-hydroxy-11, 15-dio-ganoderic acid DM,
compound 3:7-Oxo-ganoderic acid Z,
compound 4: ganoderiol F is used as a carrier,
compound 5: ganodermanndiol the process comprises,
compound 6: the Lucialdehyde B is used to prepare a pharmaceutical composition,
compound 7: the expression of Lucidal (r) in the form of,
compound 8: ganodrol B is used to control the rate of the gas,
compound 9: ganodrol A is used to control the rate of change of the gas,
compound 10: the Lucialdehyde a is used to prepare,
compound 11: ganoderal A,
compound 12: ganoderiol A.
Detailed Description
The following embodiments further illustrate the technical solution of the present invention, but not limit the scope of the present invention, and all equivalent changes and modifications according to the claims of the present invention shall fall within the scope of the present invention.
Instrument and material:
ultra-high performance liquid chromatography (Agilent LC1290 in definition II, agilent, usa);
triple quadrupole mass spectrometer (Agilent 6495, agilent, usa);
ultrasonic cleaners (KQ 2200E, kunshan ultrasonic instruments Co., ltd.);
electronic balances (FA 2004A, shanghai precision scientific instruments limited);
organic filters (0.20 μm, agilent, USA);
mass spectrometry grade methanol, acetonitrile and water were all purchased from merk corporation, usa;
agilent ZORBAX SB-Aq chromatography column (1.8 μm, 2.1X106 mm, agilent, USA).
Standard of 12 triterpene compounds: compound 1: ganodermantiol, compound 2:23S-hydroxy-11, 15-dio-ganoderic acid DM, compound 3:7-Oxo-ganoderic acid Z, compound 4: ganoderiol F, compound 5: ganodermannodiol, compound 6: lucialdehyde B, compound 7: lucidal, compound 8: ganoderol B, compound 9: ganoderol A, compound 10: lucialdehyde a, compound 11: ganodiral A, compound 12: ganoderiol A. The above compounds are purchased from the Wuhan Qiong Biotechnology Co., ltd, and the purity is more than or equal to 98%.
Fruiting body of Ganoderma fungus: fruiting body 1: ganoderma lucidum Ganoderma lucidum from Shanghai; fruiting body 2: ganoderma lucidum Ganoderma lucidum from Shanghai; fruiting body 3: ganoderma lucidum Ganoderma lucidum from Shanghai; fruiting body 4: ganoderma lucidum Ganoderma lucidum from Shanghai; fruiting body 5: ganoderma lucidum Ganoderma lucidum from Shanghai; fruiting body 6: ganoderma sinensis Ganoderma sinense from Zhejiang, fruiting body 7: ganoderma tsugae, from Fujian; fruiting body 8: ganoderma lucidum Ganoderma leucocontextum from Tibetan; fruiting body 9: ganoderma sessiliflorum Ganoderma resinaceum is from Beijing.
Example 1 establishment of the detection method
1. The pretreatment step of the ganoderma lucidum fruiting body: accurately weighing 0.50g of dried ganoderma lucidum fruiting body samples ( fruiting bodies 1, 2, 3, 4 and 5) in a test tube with a plug, adding 15mL of methanol according to a feed liquid ratio of 1:30 (weight volume ratio, weight g: volume mL), ultrasonically extracting for 60min, taking supernatant, passing through a 0.20 mu m organic filter membrane, and diluting 20 times (volume multiple) with mass spectrum grade methanol to obtain a sample solution to be tested for loading;
preparation of mixed standard solution: preparing a mixed standard solution mother solution (the concentration of a compound 1 is 200 mug/mL, the concentration of a compound 2, 3, 4, 5, 7 and 8 is 100 mug/mL, the concentration of a compound 6 is 50 mug/mL, the concentration of a compound 9, 10 and 12 is 25 mug/mL, the concentration of a compound 11 is 900 mug/mL) by using mass spectrometry grade methanol, diluting the mother solution by 20 times to obtain a mixed standard working solution with the highest concentration, and then gradually diluting the mixed standard working solution by 2.5 times, 2 times, 2.5 times, 2 times and 2.5 times to obtain mixed standard working solutions with different concentrations.
2. Ultra-high performance liquid chromatography detection: carrying out ultra-high performance liquid chromatography separation on the sample solution to be detected and 12 triterpene compound standard substance solutions to obtain a better separation degree;
wherein, ultra-high performance liquid phase condition: chromatographic column: agilent ZORBAX SB-Aq chromatographic column, 1.8 μm, 2.1X106 mm; detection wavelength: 240nm. Column temperature: 35 ℃; the loading amount is 2 mu L; flow rate: 0.4mL/min; mobile phase: 0.01% acetic acid water (a) -methanol (B); elution procedure: 0min,20% A,80% B;8min,20% A,80% B;10min,10% A,90% B;15min,0% A,100% B;18min,0% A,100% B.
3. Compound mass spectrum information obtaining step: preparing the 12 triterpene compound standard substances into 5ppm solutions by using methanol respectively, confirming the parent ion information of the 12 triterpene compounds in a positive ion mode, and automatically optimizing the child ions and collision energy under the condition of confirming the parent ions by Agilent Optimizer software;
the retention time, parent ion, daughter ion pair, and collision energy information for each compound are as follows:
qualitative ion: 107.0, collision energy: 37, respectively;
qualitative ion: 353.1, collision energy: 29;
qualitative ion: 135.1, collision energy: 45;
qualitative ion: 109.1, collision energy: 41;
qualitative ion: 121.1, collision energy: 49;
qualitative ion: 119.1, collision energy: 37, respectively;
qualitative ion: 95.1, collision energy: 37, respectively;
qualitative ion: 95.0, collision energy: 45;
qualitative ion: 69.1, collision energy: 45;
qualitative ion: 81.0, collision energy: 49;
qualitative ion: 145.1, collision energy: 53;
4. establishing an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined quantitative analysis method: under the conditions of ultra-high performance liquid chromatography and triple quadrupole mass spectrum parameter setting in data acquisition software (Agilent MassHunter Data Acquisition), changing a mass spectrum acquisition mode into multi-reaction monitoring (MRM), then leading the parent ion information, quantitative and qualitative sub-ion pair information, collision energy and other information of the compound and the ultra-high performance liquid chromatography method into a detection method, taking mixed standard substance solution for loading, and updating an acquisition mode into dynamic multi-reaction monitoring (DMRM) after running a program.
Wherein, the ultra-high performance liquid chromatography conditions are the same as in step 2: chromatographic column: agilent ZORBAX SB-Aq chromatographic column, 1.8 μm, 2.1X106 mm; detection wavelength: 240nm. Column temperature: 35 ℃; the loading amount is 2 mu L; flow rate: 0.4mL/min; mobile phase: 0.01% acetic acid water (a) -methanol (B); elution procedure: 0min,20% A,80% B;8min,20% A,80% B;10min,10% A,90% B;15min,0% A,100% B;18min,0% A,100% B.
Wherein, triple quadrupole mass spectrometry conditions: atmospheric pressure chemical ionization source (APCI) is used as ion source, detection is carried out in positive ion mode, dynamic multi-reaction monitoring (DMRM) is selected, and capillary voltage is 3500V; capillary outlet voltage: 380V; corona needle current: 8 μA; the temperature of the drying gas is 290 ℃; the dry air flow is 13L/min; gasification chamber temperature: 350 ℃; atomization gas pressure: 30psi.
5. Detection limit and quantification limit: a detection Limit (LOD) and a quantification Limit (LOQ) are calculated based on the standard deviation of the response values and the slope of the standard curve. Wherein: lod=3σ/S, loq=10σ/S, σ: standard deviation of response values, S: slope of the standard curve.
6. Standard curves for 12 triterpene compounds were created using quantitative analysis software (Agilent MassHunter Quantitative Analysis): and loading the prepared mixed standard working solution according to the optimized liquid phase and mass spectrum conditions, taking the abscissa as the concentration of the compound and the ordinate as the quantitative ion response value of the compound, and preparing a quantitative standard curve shown in Table 1.
Example 2
Methodological verification and outcome detection:
1. precision: and (3) taking the mixed standard solution with the highest concentration, diluting the mixed standard solution for 4 times step by step, repeatedly sampling the mixed standard solution for 6 times in the same day, calculating the concentration of triterpene obtained by 6 experiments according to a standard curve, and calculating the daily precision. Taking the mixed standard solution with the highest concentration, gradually diluting the mixed standard solution for 4 times, continuously injecting the mixed standard solution for three days twice a day, and calculating the daytime precision according to the results of 6 experiments. The results show that the RSD of the measurement results of the daily precision and the daytime precision of 12 triterpene compounds are less than 15.00 percent, which indicates that the method has good daily precision and daytime precision. The specific results are shown in Table 2.
2. Stability: and taking the fruiting body 5, preparing one part of sample solution to be tested after extracting according to the sample pretreatment step in the embodiment 1, and respectively sampling at 0h, 2h, 4h, 6h, 8h, 12h and 24h, and calculating the sample stability according to the results of 7 experiments. The results demonstrate that the RSD of all 12 triterpene compounds is less than 15.00%, indicating that the samples are stable over 24 hours. The specific results are shown in Table 2.
3. Repeatability: and weighing 6 parts of fruiting body 5 in parallel, respectively extracting according to the sample pretreatment step in the embodiment 1, preparing a sample solution to be detected, carrying out sample injection measurement, and calculating the sample repeatability according to the experimental result of 6 times. As a result, it was confirmed that the RSDs of the 12 triterpene compounds were all less than 15.00%, indicating good sample reproducibility. The specific results are shown in Table 2.
4. Sample recovery rate: and taking a sample with a known concentration, adding 12 triterpene compound standard samples, uniformly mixing, repeating sample loading for three times, and calculating the sample recovery rate. Specific results of% recovery = (measured value-measured component amount contained in test sample)/amount of added control×100% recovery are shown in table 3. The result proves that the recovery rate RSD of 12 triterpene compounds in the sample is within 15.00%, which meets the method requirement.
5. And (3) result detection: 8 Ganoderma lucidum fruiting bodies (fruiting body 1: ganoderma lucidum Ganoderma lucidum from Shanghai city, fruiting body 2: ganoderma lucidum Ganoderma lucidum from Shanghai city, fruiting body 3: ganoderma lucidum Ganoderma lucidum from Shanghai city, fruiting body 4: ganoderma lucidum Ganoderma lucidum from Shanghai city, fruiting body 6: ganoderma lucidum Ganoderma sinense from Zhejiang, fruiting body 7: ganoderma tsugae from Fujian, fruiting body 8: ganoderma lucidum Ganoderma leucocontextum from Tibet, fruiting body 9: ganoderma lucidum Ganoderma resinaceum from Beijing) were taken and detected separately according to the detection method of example 1, and the specific detection results are shown in Table 4 and Table 5.
TABLE 1 quantitative analysis standard curve, detection limit and quantitative limit for 12 neutral triterpene compounds
TABLE 2 within day precision, daytime precision, repeatability, stability
TABLE 3 sample recovery results
TABLE 4 sample measurement results
TABLE 5 sample measurement results
Claims (3)
1. A method for detecting neutral triterpenes in ganoderma fungi, which is characterized by comprising the following steps:
(1) The pretreatment step of the ganoderma lucidum fruiting body: extracting Ganoderma fruiting body sample with organic solvent,
taking supernatant, filtering and diluting to obtain a sample solution to be detected; preparing mixed standard solution of 12 neutral triterpene compounds;
(2) Ultra-high performance liquid chromatography detection: carrying out ultra-high performance liquid chromatography separation on a sample solution to be detected and a mixed standard solution of 12 triterpene compounds to obtain a better separation degree;
(3) Compound mass spectrum information obtaining step: utilizing mass spectrum optimization software Agilent Optimizer to scan the parent ions, detect the child ion pairs and search the optimal collision energy for 12 neutral triterpenoids;
(4) The method for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis method comprises the following steps: introducing retention time, parent ion and ion pairs in data acquisition software Agilent MassHunter Data Acquisition under the conditions of ultra-high performance liquid chromatography analysis and triple quadrupole mass spectrometry detection, and establishing a dynamic multi-reaction monitoring DMRM analysis and determination method of 12 neutral triterpene compounds; at the same time, the method comprises the steps of,
loading and measuring mixed standard substance solutions with different concentrations and sample solutions to be measured;
(5) And a data analysis step: using quantitative analysis software Agilent MassHunter Quantitative
Analyzing, namely creating 12 standard curves of neutral triterpene compounds, performing methodological verification on the established method, and quantitatively analyzing a sample solution to be detected of the ganoderma lucidum fruiting body;
wherein the chromatographic conditions of the ultra performance liquid chromatography separation in the step (2) are as follows: agilent ZORBAX SB-Aq chromatographic column, 1.8 μm, 2.1X106 mm, detection wavelength: 240nm; column temperature: 35 ℃; the loading amount is 2 mu L; flow rate: 0.4mL/min; mobile phase: a is 0.01% acetic acid water, B is methanol; elution procedure: 0min,20% A,80% B;8min,20% A,80% B;10min,10% A,90% B;15min,0% A,100% B;18min,0% A,100% B;
in the step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as an analysis and detection instrument, wherein the conditions of the ultra-high performance liquid chromatography are as follows: chromatographic column: agilent ZORBAX SB-Aq chromatographic column, 1.8 μm, 2.1X106 mm; detection wavelength: 240nm; column temperature: 35 ℃; the loading amount is 2 mu L; flow rate: 0.4mL/min; mobile phase: a is 0.01% acetic acid water, B is methanol; elution procedure: 0min,20% A,80% B;8min,20% A,80% B;10min,10% A,90% B;15min,0% A,100% B;18min,0% A,100% B;
wherein the mass spectrometry conditions are: the atmospheric pressure chemical ionization source APCI is used as an ion source, detection is carried out in a positive ion mode, dynamic multi-reaction monitoring DMRM is selected, and capillary voltage is 3500V; capillary outlet voltage: 380V; corona needle current: 8 μA; the temperature of the drying gas is 290 ℃; the dry air flow is 13L/min; gasification chamber temperature: 350 ℃; atomization gas pressure: 30psi;
wherein 12 triterpene compounds are as follows:
compound 1: ganodermantiol, compound 2:23S-hydroxy-11, 15-dio-ganoderic acid DM, compound 3:7-Oxo-ganoderic acid Z, compound 4: ganoderiol F, compound 5: ganodermannodiol, compound 6: lucialdehyde B, compound 7: lucidal, compound 8: ganoderol B, compound 9: ganoderol A, compound 10: lucialdehyde a, compound 11: ganodiral A, compound 12: ganoderiol A.
2. The method for detecting neutral triterpenes from fungi of the genus ganoderma according to claim 1, wherein in the pretreatment step of the fruiting body of ganoderma in step (1):
the organic solvent is one of methanol or ethanol;
the extraction method is ultrasonic extraction or heating extraction at 60 ℃;
the extraction time is 10-90min;
the feed liquid ratio is 1:20-1:40, the weight volume ratio is that g: volume ml;
filtering the extracted supernatant with an organic phase microporous filter membrane with the aperture of 0.20 mu m, and diluting with mass spectrum grade methanol for 20 times to obtain a sample solution to be detected for sample injection.
3. The method for detecting neutral triterpenes according to claim 1, wherein the retention time, parent ion, child ion pair and collision energy information of each compound required for the analysis in the step (3) is obtained by mass spectrometry optimization software Agilent Optimizer as follows:
ganodermantiol, retention time: 3.79min; parent ion: 455.0; quantitative ion: 105.1, collision energy: 80; qualitative ion: 107.0, collision energy: 37, respectively;
23S-hydroxy-11, 15-dio-ganoderic acid DM, retention time: 3.98min; parent ion: 495.1; quantitative ion: 435.3, collision energy: 17; qualitative ion: 353.1, collision energy: 29;
7-Oxo-ganoderic acid Z, retention time: 4.34min; parent ion: 471.2; quantitative ion: 107.1, collision energy: 61; qualitative ion: 135.1, collision energy: 45;
ganoderiol F, retention time: 5.94min; parent ion: 437.2; quantitative ion: 67.2, collision energy: 73; qualitative ion: 95.0, collision energy: 41;
ganodermannodiol, retention time: 6.40min; parent ion: 439.2; quantitative ion: 105.1, collision energy: 73; qualitative ion: 109.1, collision energy: 41;
lucialdehyde B, retention time: 6.79min; parent ion: 453.1; quantitative ion: 327.1, collision energy: 17; qualitative ion: 121.1, collision energy: 49;
lucidal, retention time: 7.89min; parent ion: 437.1; quantitative ion: 201.1, collision energy: 29; qualitative ion: 119.1, collision energy: 37, respectively;
ganodrol B, retention time: 10.80min; parent ion: 423.2; quantitative ion: 67.0, collision energy: 61; qualitative ion: 95.1, collision energy: 37, respectively;
ganoderol A, retention time: 11.25min; parent ion: 421.1; quantitative ion: 67.1, collision energy: 61; qualitative ion: 95.0, collision energy: 45;
lucialdehyde a, retention time: 11.25min; parent ion: 421.1; quantitative ion: 109.1, collision energy: 29; qualitative ion: 69.1, collision energy: 45;
ganoderal A, retention time: 12.21min; parent ion: 437.0; quantitative ion: 91.0, collision energy: 77; qualitative ion: 81.0, collision energy: 49;
ganoderiol A, retention time: 13.99min; parent ion: 475.0; quantitative ion: 177.0, collision energy: 21, a step of; qualitative ion: 145.1, collision energy: 53.
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