CN112326854B - Method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body - Google Patents
Method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body Download PDFInfo
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Abstract
The invention provides a method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body, which comprises the following steps: (1) pretreating Ganoderma Applanatum fruiting body; (2) detecting by ultra-high performance liquid chromatography; (3) obtaining mass spectrum information of the compound; (4) establishing an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis method; (5) and (5) data analysis. The method for simultaneously detecting six triterpenoids in the Ganoderma applanatum fruiting body establishes a method capable of detecting 6 triterpenoids in the Ganoderma applanatum fruiting body in a trace manner by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and realizes accurate determination of specific triterpenoids in the Ganoderma applanatum fruiting body.
Description
Technical Field
The invention relates to the field of food and drug analysis, in particular to a method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body.
Background
Ganoderma Applanatum is a general name of multiple species of Ganoderma Applanatum of Polyporaceae (Polyporaceae) Ganoderma (Ganoderma sp.), and common Ganoderma Applanatum includes Ganoderma oboditum, Ganoderma Applanatum, Ganoderma australe, etc. Many species of Ganoderma Applanatum have medicinal history in folk, and at present, a small amount of artificial cultivation is available, but scientific and systematic research is still lacking.
Researches show that triterpenes in Ganoderma applanatum comprise applanate epoxy acid H (applanate acid H), applanate epoxy acid A (applanate acid A), applanate epoxy acid G (applanate acid G), applanate epoxy acid C (applanate acid C), applanate epoxy acid E (applanate acid E), applanate epoxy acid F (applanate acid F) and the like, and the applanate epoxy acid compounds are few in types but high in content and have strong capacity of inhibiting tumor cell proliferation.
In the past, the determination of the types and the content of triterpenoids in ganoderma applanatum is mainly carried out by adopting a high performance liquid chromatography-ultraviolet detection method for preliminary analysis. Under the same conditions, the epoxy acid triterpenoids in Ganoderma applanatum have the same retention time as the ganoderic acid triterpenoids and ganoderic acid triterpenoids in Ganoderma lucidum, so that different triterpenoids in Ganoderma applanatum and Ganoderma lucidum cannot be accurately distinguished. Therefore, a more accurate analysis method for detecting the hyoscyamus epoxy acid triterpenoid in the hyoscyamus is needed to be established.
Disclosure of Invention
The invention provides a method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body, which comprises the following steps:
(1) pretreatment of Ganoderma Applanatum fruiting body: adding Ganoderma Applanatum fruiting body into organic solvent, extracting with ultrasound, collecting supernatant, and filtering to obtain filtrate as sample solution to be tested; preparing mixed standard substance solutions of six Ganoderma applanatum epoxy acid compounds;
(2) ultra-high performance liquid chromatography detection: performing ultra-high performance liquid chromatography separation on the sample solution to be detected and the mixed standard substance solution of six Ganoderma applanatum epoxy acid compounds to obtain better separation degree;
(3) obtaining mass spectrum information of the compound: carrying out parent ion scanning, detection of a daughter ion pair and search of optimal collision energy on six tongue epoxy acid compounds by using 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: in Data Acquisition software (Agilent MassHunter Data Acquisition), under the conditions of ultra-high performance liquid chromatography analysis and triple quadrupole mass spectrometry, retention time and parent ion and daughter ion pairs are introduced, a DMRM analysis and determination method of six Ganoderma applanatum epoxy acid compounds is established, and meanwhile, mixed standard solution and sample solution with different concentrations are subjected to sample loading determination;
(5) and (3) data analysis step: and (3) creating a standard curve of six Ganoderma applanatum epoxy acid compounds by using Quantitative Analysis software (Agilent MassHunter Quantitative Analysis), carrying out methodology verification on the created method, and carrying out Quantitative Analysis on Ganoderma applanatum fruiting body sample solution.
Preferably, the step (1) of pretreating Ganoderma Applanatum fruiting body comprises the following steps: completely drying Ganoderma Applanatum fruiting body, and extracting under the following conditions:
the leaching liquor is one of methanol and ethanol, preferably methanol;
the extraction method comprises ultrasonic extraction or heating extraction at 60 deg.C, preferably ultrasonic extraction;
the extraction time is 10-90min, preferably 60 min;
the ratio of the feed to the liquid is 1: 20-1: 40 (weight g: volume ml), preferably 1: 30;
the leaching times comprise one-time leaching or repeated leaching, and preferably one time;
filtering the solution with organic phase microporous filter membrane with pore diameter of 0.20 μm;
and filtering the solution, and diluting the solution by 10 times by mass spectrum methanol to obtain a sample solution to be detected.
Preferably, the preparation method of six Ganoderma applanatum epoxy acid compounds in Ganoderma applanatum fruiting body in step (1) comprises precisely weighing six compounds of Ganoderma applanatum epoxy acid H, Ganoderma applanatum epoxy acid A, Ganoderma applanatum epoxy acid G, Ganoderma applanatum epoxy acid C, Ganoderma applanatum epoxy acid E and Ganoderma applanatum epoxy acid F, preparing into 200ng/mL mixed standard solution with mass spectrum methanol, and diluting with mass spectrum methanol solution step by step into a series of mixed standard solutions of 100ng/mL, 50ng/mL, 20ng/mL and 10 ng/mL.
Preferably, the chromatographic conditions for the ultra-high performance liquid chromatographic separation in the step (2) are as follows: agilent ZORBAX SB-Aq C-18(2.1mm x 100mm, 1.8 μm) is selected; detection wavelength: 252 nm; column temperature: 30 ℃; the sample loading amount is 5 uL; flow rate: 0.3 mL/min; mobile phase: acetonitrile (a) -0.01% acetic acid water (B). Elution procedure: 0-1min, A: 20% -20%; 1-3min, A: 20% -40%; 3-12min, A: 40% -60%; 12-13min, A: 60 to 100 percent.
Preferably, the retention time, parent ion, daughter ion pair and collision energy information of each compound required for analysis in step (3) are obtained by mass spectrometry optimization software (Agilent Optimizer) as follows:
and (3) the epoxy acid of the Ganoderma applanatum H: retention time: 4.83 min; parent ion: 529.3, respectively; and (3) quantifying ions: 128.9, collision energy: 21; and (3) qualitative ion: 399.2, collision energy 21;
ganoderma applanatum epoxy acid A: retention time: 5.40 min; parent ion: 511.3, respectively; and (3) quantifying ions: 467.2, collision energy: 29; and (3) qualitative ion: 257.0, collision energy 37;
ganoderma applanatum epoxy acid G: retention time: 6.27 min; parent ion: 527.3, respectively; and (3) quantifying ions: 129.0, collision energy: 33; and (3) qualitative ion: 397.1, collision energy 17;
c, Ganoderma applanatum epoxy acid: retention time: 6.89 min; parent ion: 525.2, respectively; and (3) quantifying ions: 128.9, collision energy: 13; and (3) qualitative ion: 395.2, collision energy 17;
and E, Ganoderma applanatum epoxy acid: retention time: 7.21 min; parent ion: 511.0; and (3) quantifying ions: 257.0, collision energy: 37; and (3) qualitative ion: 467.2, collision energy 29;
and (3) Ganoderma applanatum epoxy acid F: retention time: 7.62 min; parent ion: 509.2; and (3) quantifying ions: 491.2, collision energy: 17; and (3) qualitative ion: 447.1, collision energy 25.
Preferably, in the step (4), the ultra performance liquid chromatography-triple quadrupole mass spectrometry is used as an analysis and detection instrument, wherein the chromatographic conditions of the ultra performance liquid chromatography separation are as follows: agilent ZORBAX SB-Aq C-18(2.1mm x 100mm, 1.8 μm) is selected; detection wavelength: 252 nm; column temperature: 30 ℃; the sample loading amount is 5 uL; flow rate: 0.3 mL/min; mobile phase: acetonitrile (a) -0.01% acetic acid water (B). Elution procedure: 0-1min, A: 20% -20%; 1-3min, A: 20% -40%; 3-12min, A: 40% -60%; 12-13min, A: 60% -100%; the mass spectrum conditions are as follows: electrospray ionization source (AJS ESI) is as the ion source, and detection is carried out under the negative ion mode, and dynamic multi-reaction monitoring (DMRM) is selected, capillary voltage: 3000V, capillary exit voltage: 380V. Flow rate of drying gas: 14L/min, drying gas temperature: 200 ℃, sheath gas temperature: 300 ℃, sheath gas flow rate: 11L/min, nozzle voltage: 1500V.
The identification basis of the detection method of six Ganoderma applanatum epoxy acid compounds in Ganoderma applanatum fruiting body of the invention is as follows: when the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined detection is adopted, the six Ganoderma applanatum epoxy acid compounds in the Ganoderma applanatum fruiting body not only need to meet the condition that the retention time is consistent, but also need to meet the condition that the primary and secondary ion pairs meet the detection parameters under the specific mass spectrometry condition, and then the compounds can be accurately and quantitatively analyzed.
The invention has the beneficial effects that: the technical scheme of the invention can carry out accurate qualitative and quantitative analysis on six specific Ganoderma applanatum epoxy acid compounds in Ganoderma applanatum fruiting body, has high accuracy, high sensitivity and strong specificity, and overcomes the defects of the conventional chemical method and high performance liquid chromatography.
The invention establishes a liquid chromatography-mass spectrometry DMRM analysis method by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry on the basis of preparing six Ganoderma applanatum epoxy acid compounds from Ganoderma applanatum in the early stage. The method not only depends on retention time, but also carries out accurate qualitative and quantitative determination on the Ganoderma Applanatum epoxy acid compounds in Ganoderma Applanatum. Compared with the conventional high performance liquid chromatography-ultraviolet detection method, the method has higher accuracy, sensitivity and resolution, and is more suitable for the detection and analysis of the Ganoderma applanatum epoxy acid compounds in Ganoderma applanatum.
Drawings
FIG. 1 shows the structural formula of epoxy acid compounds of six Ganoderma Applanatum in Ganoderma Applanatum fruiting body
FIG. 2 shows the total ion flow diagram of six Ganoderma applanatum epoxy acid compounds in Ganoderma applanatum fruiting body
Wherein, the first and the second end of the pipe are connected with each other,
compound 1: applanatic acid H (applanatic acid H),
compound 2: applanatic acid A (applanatic acid A),
compound 3: applanatic acid G (applanatic acid G),
compound 4: ganoderma applanatum epoxy acid C (applanoxic acid C),
compound 5: applanatic acid E (applanatic acid E),
compound 6: ganoderma applanatum Oxycoside F (applanoxic acid F)
Detailed Description
The technical solutions of the present invention are further described below by specific examples, but the present invention is not limited thereto, and the scope of the present invention should not be limited thereby, and all equivalent changes and modifications made in the claims of the present invention should be covered by the present invention.
Instruments and materials:
ultra high performance liquid chromatography (Agilent LC1290 definition II, Agilent Inc. of Agilent, USA),
triple quadrupole mass spectrometer (Agilent 6495, Agilent, USA),
a freeze dryer (LGJ-18C, Shanghai Bilang instruments manufacturing Co., Ltd.),
an ultrasonic cleaner (KQ2200E, ultrasonic instruments Co., Ltd., Kunshan city),
an electronic balance (FA2004A, Shanghai precision scientific instruments Co., Ltd.),
organic filter membranes (0.20 μm, Agilent, USA),
mass spec grade methanol, acetonitrile and water were all purchased from America merk.
6 standard substances of the Ganoderma applanatum epoxy acid compounds: compound 1: applanatic acid h, compound 2: applanatic acid a, compound 3: applanatic acid g, compound 4: ganoderma applanatum epoxidic acid C (applanatoic acid C), Compound 5: applanatic acid e, compound 6: ganoderma applanatum epoxidic acid F (applanatoic acid F). The above compounds are prepared according to the literature (Chairul, Tokuyama T, Hayashi Y, et al, applied botanical acids A, B, C and D, biological active tetracyclic tripeptides from Garoderma applanatum [ J ]. Phytochemistry, 1991, 30 (12): 4105-4109.Chairul S M, Hayashi Y.Lanostand tripeptides from Garoderma applanatum [ J ]. Phytochemistry, 1994, 35 (5): 1308.Mothana R A, Awadh Ali N A, Jansen R, et al, antibacterial triterpenes from Garoderma applanate [ J ]. Phytochemistry ], 1994, 35 (5): 1305-1308.Mothana R A, Awadh Alni N A, Jansen R, et al, Antiviral polysaccharides from Garoderma [ J ]. 2003, 74, 92. xanthecotype J.76, No. 99, Leybox, 92. 11. Labiatric acids J.251, Leybox, M.251, Leybox.7. 21. 100. environmental active extracts, and D. environmental active extracts from Garoderma.
Ganoderma Applanatum fruiting body: sample 1: ganoderma Applanatum fruiting body, sample 2: ganoderma Applanatum fruiting body, sample 3: ganoderma Applanatum fruiting body, sample 4: ganoderma Applanatum fruiting body, sample 5: ganoderma australe fruiting body, sample 6: ganoderma australe fruiting body, sample 7: ganoderma australe fruiting body, sample 8: ganoderma australe fruiting body, sample 9: the fruiting body of Ganoderma lobatum, samples 1-8 from Beijing university of forestry, and sample 9 from Kunming edible fungus institute of China national supply and marketing Cooperation headquarters.
EXAMPLE 1 establishment of detection method
1. Sample pretreatment: accurately weighing 0.50g of sample in a test tube with a plug, adding 15mL of methanol according to the material-liquid ratio of 1: 30 (weight-volume ratio), ultrasonically extracting for 60min, taking supernatant, filtering with a 0.20 mu m organic filter membrane, and diluting with mass spectrum methanol by 10 times.
Preparation of mixed standard solution: taking six Ganoderma applanatum epoxy acid compounds, preparing a200 ng/mL mixed standard solution (wherein the concentration of each compound is 200ng/mL) by using mass spectrum methanol, diluting the mixed standard solution into a series of mixed standard solutions of 100ng/mL, 50ng/mL, 20ng/mL and 10ng/mL step by using the mass spectrum methanol solution, and storing the mixed standard solutions in a refrigerator at 4 ℃.
2. Ultra-high performance liquid chromatography detection: performing ultra-high performance liquid chromatography separation on the sample solution to be detected and 6 Ganoderma applanatum epoxy acid compounds to obtain better separation degree;
wherein, the ultra-high performance liquid phase condition is as follows: a chromatographic column: agilent ZORBAX SB-Aq C-18(2.1 mm. times.100 mm, 1.8 μm); detection wavelength: 252 nm; column temperature: 30 ℃; the sample loading amount is 5 uL; flow rate: 0.3 mL/min; mobile phase: acetonitrile (a) -0.01% acetic acid water (B). Elution procedure: 0-1min, A: 20% -20%; 1-3min, A: 20% -40%; 3-12min, A: 40% -60%; 12-13min, A: 60 to 100 percent.
3. Obtaining mass spectrum information of the compound: preparing 6 standard substances of the curse epoxy acid compounds into 5ppm methanol solutions respectively, confirming parent ion information in a negative ion mode, and automatically optimizing the daughter ions and the collision energy under the condition of determining the parent ions through Agilent Optimizer software.
And (3) the epoxy acid of the Ganoderma applanatum H: retention time: 4.83 min; parent ion: 529.3, respectively; and (3) quantifying ions: 128.9, collision energy: 21; and (3) qualitative ion: 399.2, collision energy 21;
ganoderma applanatum epoxy acid A: retention time: 5.40 min; parent ion: 511.3, respectively; and (3) quantifying ions: 467.2, collision energy: 29; and (3) qualitative ion: 257.0, collision energy 37;
ganoderma applanatum epoxy acid G: retention time: 6.27 min; parent ion: 527.3, respectively; and (3) quantifying ions: 129.0, collision energy: 33; and (3) qualitative ion: 397.1, collision energy 17;
c, Ganoderma applanatum epoxy acid: retention time: 6.89 min; parent ion: 525.2, respectively; and (3) quantifying ions: 128.9, collision energy: 13; and (3) qualitative ion: 395.2, collision energy 17;
and E, Ganoderma applanatum epoxy acid: retention time: 7.21 min; parent ion: 511.0; and (3) quantifying ions: 257.0, collision energy: 37; and (3) qualitative ion: 467.2, collision energy 29;
and (3) Ganoderma applanatum epoxy acid F: retention time: 7.62 min; parent ion: 509.2; and (3) quantifying ions: 491.2, collision energy: 17; and (3) qualitative ion: 447.1, collision energy 25.
4. The establishment of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry quantitative analysis method comprises the following steps: under the conditions of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry parameter setting in Data Acquisition software (Agilent mass hunter Data Acquisition), a mass spectrometry Acquisition mode is changed into multi-reaction monitoring (MRM), then parent ion information, quantitative and qualitative daughter ion pair information, collision energy and other information of a compound and an ultra-high performance liquid chromatography method are all introduced into a detection method, a mixed standard sample is sampled, and an Acquisition mode is updated to dynamic multi-reaction monitoring (DMRM) after a program is operated.
Wherein, the ultra-high performance liquid phase condition is as follows: a chromatographic column: agilent ZORBAX SB-Aq C-18(2.1 mm. times.100 mm, 1.8 μm); detection wavelength: 252 nm; column temperature: 30 ℃; the sample loading amount is 5 uL; flow rate: 0.3 mL/min; mobile phase: acetonitrile (a) -0.01% acetic acid water (B). Elution procedure: 0-1min, A: 20% -20%; 1-3min, A: 20% -40%; 3-12min, A: 40% -60%; 12-13min, A: 60 to 100 percent.
Wherein, the mass spectrum conditions of the triple quadrupole are as follows: electrospray ionization source (AJS ESI) is as the ion source, and detection is carried out under the negative ion mode, and dynamic multi-reaction monitoring (DMRM) is selected, capillary voltage: 3000V, capillary exit voltage: 380V. Flow rate of drying gas: 14L/min, drying gas temperature: 200 ℃, sheath gas temperature: 300 ℃, sheath gas flow rate: 11L/min, nozzle voltage: 1500V.
5. Detection limit and quantitation limit: the limit of detection (LOD) and limit of quantitation (LOQ) are calculated based on the standard deviation of the response values and the slope of the standard curve. Wherein: LOD 3 σ/S, LOQ 10 σ/S, σ: standard deviation of response value, S: the slope of the standard curve, the standard deviation of the response values is the remaining standard deviation of the standard curve.
6. And (3) standard curve formulation: and (3) sampling the prepared mixed standard working solution of 200ng/mL, 100ng/mL, 50ng/mL, 20ng/mL and 10ng/mL according to the optimized conditions of the liquid phase and the mass spectrum, and preparing a quantitative standard curve by taking the abscissa as the concentration of the compound and the ordinate as the quantitative ion response value of the compound.
Example 2
Methodology validation and result detection: the methodology verification refers to the laboratory quality control standard food physicochemical detection standard and the related regulations of pharmacopoeia.
Precision within 1 day: and (3) repeatedly injecting the mixed standard substance solution for 6 times in the same day, calculating the concentration of the Ganoderma applanatum epoxy acid compound obtained in 6 experiments according to the standard curve, and calculating the precision in the day. The results show that the RSD of the results of the in-day precision measurement of the six Ganoderma applanatum epoxy acid compounds is less than 15.00 percent, which indicates that the in-day precision of the method is good. The specific results are shown in Table 2.
Precision between 2 days: and (3) sampling the mixed standard substance solution for 2 times in three consecutive days, respectively, calculating the concentration of the epoxy acid compound of the tongue obtained in 6 experiments according to a standard curve, and calculating the daytime precision. The result shows that the RSD of the daytime precision measurement results of the six Ganoderma applanatum epoxy acid compounds is less than 15.00 percent, which indicates that the daytime precision of the method is good. The specific results are shown in Table 2.
3, stability: after a sample to be detected (sample 9) is taken and treated according to the extraction step of the embodiment 1, sample solutions to be detected are injected for 0h, 2h, 4h, 6h, 8h, 12h and 24h respectively, and the stability of the sample is calculated according to the results of 7 experiments. The results confirmed that the RSD of the six applanate epoxies were less than 15.00% indicating that the samples were stable in performance over 24 hours. The specific results are shown in Table 2.
4, repeatability: 6 parts of the sample to be tested (sample 9) are weighed in parallel, extracted and processed respectively according to the embodiment 1, then sample injection analysis is carried out, and the repeatability of the sample is calculated according to the experimental results of 6 times. The results confirm that the RSD of the six applanate epoxy acid compounds is less than 15.00 percent, which indicates that the sample has good repeatability. The specific results are shown in Table 2.
5, recovery rate: taking a sample (sample 9) solution with a known concentration to be detected, adding a certain amount of standard solution to prepare a sample solution, mixing uniformly, then repeatedly loading the sample for three times, and calculating the recovery rate of the sample.
Percent recovery%
Specific results of recovery are shown in table 3. The results prove that the recovery rates RSD of the six applanate epoxy acid compounds in the sample are all within 15.00 percent, and the method requirements are met.
6, detecting the result: eight Ganoderma Applanatum species (sample 1: Ganoderma Applanatum sporophore, sample 2: Ganoderma Applanatum sporophore, sample 3: Ganoderma Applanatum sporophore, sample 4: Ganoderma Applanatum sporophore, sample 5: Ganoderma australe sporophore, sample 6: Ganoderma australe sporophore, sample 7: Ganoderma australe sporophore, sample 8: Ganoderma australe sporophore) were extracted and processed, and then sample injection analysis was performed according to the detection method of example 1, and the results are shown in Table 3.
As can be seen from the results, the total content of epoxy acid compounds of six linguistics among the four types of fruiting bodies of Garoderma australia was relatively high. In the four Garoderma apple fruit bodies, except that the content of the epoxy acid G in the Ganoderma Applanatum in the sample 2 is higher, the content of the epoxy acid compounds in the Ganoderma Applanatum is obviously lower than that in the Garoderma apple fruit body. Therefore, the types and the contents of the ganoderma applanatum epoxy acid compounds of the two ganoderma applanatum types are different to a certain extent, and the ganoderma applanatum epoxy acid G is the main ganoderma applanatum epoxy acid component in the two ganoderma applanatum types.
TABLE 1 quantitative analysis standard curve and parameters of 6 Ganoderma applanatum epoxy acid compounds in Ganoderma applanatum fruiting body
TABLE 2 results of in-day precision, reproducibility, stability, and recovery from sample addition
TABLE 3 results of sample measurement
Claims (3)
1. A method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body is characterized by comprising the following steps:
(1) pretreatment of Ganoderma Applanatum fruiting body: adding Ganoderma Applanatum fruiting body into organic solvent, extracting with ultrasound, collecting supernatant, and filtering to obtain filtrate as sample solution; preparing mixed standard substance solutions of six Ganoderma applanatum epoxy acid compounds;
(2) ultra-high performance liquid chromatography detection: performing ultra-high performance liquid chromatography separation on the sample solution to be detected and six Ganoderma applanatum epoxy acid compounds to obtain better separation degree;
(3) obtaining mass spectrum information of the compound: carrying out parent ion scanning, detection of a daughter ion pair and search of optimal collision energy on six tongue epoxy acid compounds by using a mass spectrum optimization software Agilent Optimizer;
(4) the method for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined analysis comprises the following steps: in Data Acquisition software Agilent MassHunter Data Acquisition, retention time, parent ion and daughter ion pairs are introduced under the conditions of ultra-high performance liquid chromatography analysis and triple quadrupole mass spectrometry detection, a DMRM analysis and determination method of six Ganoderma applanatum epoxy acid compounds is established, and meanwhile, mixed standard substance solutions and sample solutions with different concentrations are subjected to sample loading determination;
(5) and (3) data analysis step: establishing six standard curves of the Ganoderma applanatum epoxy acid compounds by using Quantitative Analysis software Agilent MassHunter Quantitative Analysis, carrying out methodology verification on the established method, and carrying out Quantitative Analysis on Ganoderma applanatum fruiting body sample solution;
wherein the six triterpene compounds are as follows: the artificial ganoderma lucidum karst comprises ganoderma lucidum karst epoxy acid H, ganoderma lucidum karst epoxy acid A, ganoderma lucidum karst epoxy acid G, ganoderma lucidum karst epoxy acid C, ganoderma lucidum karst epoxy acid E and ganoderma lucidum karst epoxy acid F;
wherein the chromatographic conditions of the ultra-high performance liquid chromatography in the step (2) are as follows: selecting Agilent ZORBAX SB-AqC-18 with the diameter of 2.1mm multiplied by 100mm and the diameter of 1.8 mu m; detection wavelength: 252 nm; column temperature: 30 ℃; the sample loading amount is 5 uL; flow rate: 0.3 mL/min; mobile phase: acetonitrile-0.01% acetic acid water; elution procedure: 0-1min, acetonitrile: 20% -20%; 1-3min, acetonitrile: 20% -40%; 3-12min, acetonitrile: 40% -60%; 12-13min, acetonitrile: 60 to 100 percent.
2. The method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body 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) is obtained by Agilent Optimizer:
and (3) the epoxy acid of the Ganoderma applanatum H: retention time: 4.83 min; parent ion: 529.3, respectively; and (3) quantifying ions: 128.9, collision energy: 21; and (3) qualitative ion: 399.2, collision energy 21;
ganoderma applanatum epoxy acid A: retention time: 5.40 min; parent ion: 511.3, respectively; and (3) quantifying ions: 467.2, collision energy: 29; and (3) qualitative ion: 257.0, collision energy 37;
ganoderma applanatum epoxy acid G: retention time: 6.27 min; parent ion: 527.3, respectively; and (3) quantifying ions: 129.0, collision energy: 33; and (3) qualitative ion: 397.1, collision energy 17;
c, Ganoderma applanatum epoxy acid: retention time: 6.89 min; parent ion: 525.2, respectively; and (3) quantifying ions: 128.9, collision energy: 13; and (3) qualitative ion: 395.2, collision energy 17;
and E, Ganoderma applanatum epoxy acid: retention time: 7.21 min; parent ion: 511.0; and (3) quantifying ions: 257.0, collision energy: 37; and (3) qualitative ion: 467.2, collision energy 29;
and (3) Ganoderma applanatum epoxy acid F: retention time: 7.62 min; parent ion: 509.2; and (3) quantifying ions: 491.2, collision energy: 17; and (3) qualitative ion: 447.1, collision energy 25.
3. The method for simultaneously detecting six triterpene compounds in Ganoderma applanatum fruiting body as claimed in claim 1, wherein in step (4), ultra high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analysis and detection instrument, wherein the chromatographic conditions of ultra high performance liquid chromatography separation are as follows: selecting Agilent ZORBAX SB-AqC-18 with the diameter of 2.1mm multiplied by 100mm and the diameter of 1.8 mu m; detection wavelength: 252 nm; column temperature: 30 ℃; the sample loading amount is 5 uL; flow rate: 0.3 mL/min; mobile phase: acetonitrile-0.01% acetic acid water; elution procedure: 0-1min, acetonitrile: 20% -20%; 1-3min, acetonitrile: 20% -40%; 3-12min, acetonitrile: 40% -60%; 12-13min, acetonitrile: 60% -100%; the mass spectrum conditions are as follows: the electrospray ionization source AJS ESI is used as an ion source, detection is carried out in a negative ion mode, and dynamic multi-reaction monitoring DMRM is selected, and capillary voltage is as follows: 3000V, capillary exit voltage: 380V; flow rate of drying gas: 14L/min, drying gas temperature: 200 ℃, sheath gas temperature: 300 ℃, sheath gas flow rate: 11L/min, and the nozzle voltage is 1500V.
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| CN102721764A (en) * | 2012-07-02 | 2012-10-10 | 福建省药品检验所 | Method for controlling quality of ganoderma lucidum alcohol extract |
| CN105878529A (en) * | 2016-05-20 | 2016-08-24 | 于学军 | Medicine aiming at infection after chemotherapy of lymphoma and preparation method thereof |
| CN111307966A (en) * | 2020-01-23 | 2020-06-19 | 上海市农业科学院 | HPLC (high Performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and product thereof |
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| CN102721764A (en) * | 2012-07-02 | 2012-10-10 | 福建省药品检验所 | Method for controlling quality of ganoderma lucidum alcohol extract |
| CN105878529A (en) * | 2016-05-20 | 2016-08-24 | 于学军 | Medicine aiming at infection after chemotherapy of lymphoma and preparation method thereof |
| CN111307966A (en) * | 2020-01-23 | 2020-06-19 | 上海市农业科学院 | HPLC (high Performance liquid chromatography) determination method for triterpenoid components in ganoderma lucidum spore powder and product thereof |
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| Ganoderma applanatum secondary metabolites induced apoptosis through different pathways: In vivo and in vitro anticancer studies;Waill A. Elkhateeb et al.;《Biomedicine & Pharmacotherapy》;20180227;第101卷;264-277 * |
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