CN112707944B - Compound, preparation method thereof and application thereof in ganoderma lucidum quality traceability detection - Google Patents
Compound, preparation method thereof and application thereof in ganoderma lucidum quality traceability detection Download PDFInfo
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Abstract
The invention relates to the technical field of drug analysis, in particular to a compound, a preparation method thereof and application thereof in ganoderma lucidum quality traceability detection. The invention has the beneficial effects that: experimental results show that the compound of the formula I can be used for comparison quality detection in a whole-process planting sample of ganoderma lucidum louse and ganoderma lucidum No. 1, and can also be used for tracing chemical components of a ganoderma lucidum quality tracing system.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to a compound, a preparation method thereof and application thereof in ganoderma lucidum quality traceability detection.
Background
The traditional Chinese medicine has high requirements on the growth environment, long growth period, multiple processing links and market circulation, so that the whole industrial chain of the traditional Chinese medicine is very complex, and the quality of the traditional Chinese medicine is uneven. Unreasonable operations in the links of planting, harvesting, processing, extracting, packaging, storing and the like can directly or indirectly cause the quality and curative effect of the traditional Chinese medicine to be reduced. Although traditional Chinese medicine detection methods are more and more abundant and include obvious improvement in primitive identification, appearance shape determination, physicochemical identification, chromatography, spectrum technology, molecular identification technology, one-test-multiple-evaluation technology, chemical characteristic spectrum or fingerprint spectrum technology and the like, the traditional quality control of traditional Chinese medicinal materials is generally only aimed at the detection of finished medicinal materials, so that the integrity of front and back connection among all links of the whole industry chain of the traditional Chinese medicinal materials is ignored, quality traceability information is lacked, traceability cannot be realized, and the traditional Chinese medicine detection method is only subjected to the 'threshold' detection of pharmacopeia standards.
In 2019, the organization of the national drug administration formulates and releases 3 informationized standards of 'basic technical requirements of drug tracing system' (basic data set for vaccine tracing back) 'basic technical requirements for exchanging vaccine tracing back data', and further defines the acquisition requirements of the drug tracing back data. In the special examination meeting of the group standard of the Chinese traditional medicine Association in 2019, three group standards of 'implementation guideline for traditional Chinese medicine tracing system', 'requirement for planting traditional Chinese medicine for tracing information' and 'requirement for producing decoction pieces of traditional Chinese medicine for tracing information' pass expert review, the preparation and release of the three group standards provide clear guidance for implementation of tracing of traditional Chinese medicine enterprises, and new requirements for standardization of traditional Chinese medicine tracing process, standardization of tracing information and networking of tracing information service are provided from the group standard level. Therefore, the construction of a traditional Chinese medicine traceability system with traceability of the whole chain quality is particularly important, and the traditional Chinese medicine traceability system is especially based on the traditional Chinese medicine article traceability and characteristic component traceability in the whole traditional Chinese medicine planting production process.
The Fujian ganoderma lucidum is one of the Fujian Chinese medicinal materials of the Fujian province, is one of high-quality genuine Chinese medicinal materials of the Fujian province, and the construction of a special ganoderma lucidum quality traceability system has important significance for improving the brand and quality traceability of the high-quality ganoderma lucidum.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a compound for tracing the quality of Fujian ganoderma lucidum, a preparation method thereof and application thereof in tracing and detecting the quality of the ganoderma lucidum.
In order to solve the technical problems, the invention adopts the technical scheme that: providing a compound having the formula of formula I:
the other technical scheme provided by the invention is as follows: provides the application of the compound shown as the formula I in the quality traceability detection of the ganoderma lucidum.
Preferably, in the above-mentioned use, the compound represented by formula I is used in the form of a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a stereoisomer thereof, or a solvate thereof.
The invention provides another technical scheme as follows: provides a preparation method of the compound, which comprises the following steps:
(1) drying Ganoderma lucidum of Ganoderma lucidum Lou-Z.K. No. 1, pulverizing, extracting with ethyl acetate, and concentrating the extractive solution under reduced pressure to obtain total extract;
(2) the total extractum is subjected to gradient elution by adopting a silica gel column, and the volume ratio of an eluent is (100-0): 1, a mixed solution of dichloromethane and methanol, with a retention volume ratio of 12.5: 1, namely a liquid eluted from a mixed solution of dichloromethane and methanol, which is named as Fr.4;
(3) taking Fr.4 obtained in the step (2), and performing gradient elution by adopting a silica gel column, wherein the volume ratio of an eluent is (20-0): 1, a mixed solution of dichloromethane and methanol, with a retention volume ratio of 10:1, namely a liquid eluted from a mixed solution of dichloromethane and methanol, which is named as Fr.4-3;
(4) separating and purifying Fr.4-3 obtained in the step (3) by adopting an ODS column, wherein an eluent is acetonitrile aqueous solution with the volume concentration of 50%, and is named as Fr.4-3-3;
(5) and (4) taking Fr.4-3-3 obtained in the step (4), and performing chromatographic preparation by adopting a mass spectrum guided automatic purification preparation liquid phase system, wherein the chromatographic column parameters are as follows: ultimate XB-C18, 10 μm, 20X 250mm, mobile phase 35% acetonitrile in water, and fractions with molecular weight m/z 485 were collected to obtain the compound.
Preferably, in the above preparation method, in the step (1), the extraction specifically comprises: heating and reflux extracting at 80 deg.C for 3 times, each time for 2 hr.
The invention provides another technical scheme as follows: provides a method for detecting the ganoderma lucidum quality tracing by the compounds, which adopts a liquid chromatography-mass spectrometry combined method to detect the ganoderma lucidum quality tracing.
Preferably, the method of detection comprises the preparation of a control solution comprising: adding 50% methanol into the compound to prepare a single reference substance solution with the concentration of 200 ng/mL;
preferably, the method of detection comprises the preparation of a test solution comprising: crushing a ganoderma lucidum sample to be tested, uniformly mixing, weighing 10g of dry powder, placing the dry powder into a triangular flask with a plug, adding 500mL of ethanol, carrying out ultrasonic extraction for 45min, filtering to obtain filtrate, concentrating the filtrate to be dry, carrying out ultrasonic dissolution by using 3mL of 50% acetonitrile, mixing the filtrate with 3mL of water, loading the mixture onto a C18 SPE small column, removing impurities by using 20mL of 25% acetonitrile, eluting by using 18mL of 50% acetonitrile for enrichment, fixing the volume to 25mL by using 50% acetonitrile, shaking up, filtering by using a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the ganoderma lucidum sample.
Preferably, in the method for combining liquid chromatography and mass spectrometry, the chromatographic conditions are as follows: performing gradient elution by taking acetonitrile with the volume concentration of 100% as a mobile phase A and taking a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase B, wherein the total sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100% in percentage by volume; the conditions of the gradient elution are as follows: the proportion of the mobile phase A keeps 20% isocratic elution in 0-0.5 min, the proportion of the mobile phase A changes from 20% to 35% in 0.5-2.5 min, the proportion of the mobile phase A keeps 35% isocratic elution in 2.5-3.0 min, the proportion of the mobile phase A changes from 35% to 65% in 3.0-5.0 min, the proportion of the mobile phase A keeps 65% isocratic elution in 5.0-6.0 min, the proportion of the mobile phase A changes from 65% to 20% in 6.0-6.1 min, the proportion of the mobile phase A keeps 20% in 6.1-8 min, isocratic elution, and the proportion of the mobile phase A is 20%;
in the method of the liquid chromatography-mass spectrometry, the mass spectrometry conditions are as follows: electrospray ion source, negative ion mode ES-, mass spectrum multiple reaction monitoring mode, parent ion m/z 485, daughter ion m/z 455, collision energy: 35eV, capillary voltage negative ion mode-2.5 kV; desolventizing gas flow: nitrogen 800L/. h; desolventizing temperature: 500 ℃; taper hole airflow: 150L/h of nitrogen; ion source temperature: 150 ℃; collision gas: and argon gas.
The invention has the beneficial effects that: provides a compound for tracing the quality of Fujian ganoderma lucidum, a preparation method thereof and application thereof in tracing and detecting the quality of the ganoderma lucidum. Experimental results show that the compound of the formula I can be detected in a whole-process planting sample of ganoderma lucidum, namely ganoderma lucidum louse and ganoderma lucidum No. 1, and can also be used for tracing chemical components of a ganoderma lucidum quality tracing system.
Drawings
FIG. 1 is a UV spectrum of Compound I of example 1, an embodiment of the present invention;
FIG. 2 is a high resolution mass spectrum of Compound I of example 1 in accordance with an embodiment of the present invention;
FIG. 3 is a drawing of Compound I of example 1, an embodiment of the present invention1H-NMR(500MHz,CDCl3) A drawing;
FIG. 4 is a drawing of Compound I of example 1, an embodiment of the present invention13C-NMR(125MHz,CDCl3) A drawing;
FIG. 5 shows HSQC (500MHz, CDCl) of Compound I of example 1 according to an embodiment of the present invention3) A drawing;
FIG. 6 shows HMBC (500MHz, CDCl) of Compound I of example 1 in accordance with an embodiment of the present invention3) A drawing;
FIG. 7 shows NOESY (500MHz, CDCl) of Compound I of example 1 in accordance with an embodiment of the present invention3) A drawing;
FIG. 8 is a graph showing the results of detection of each sample in example 2 according to the embodiment of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The raw materials and equipment used in the examples of the present invention were all known products, and were obtained by purchasing commercially available products. The method comprises the following steps:
1. experimental reagent
Column chromatography silica gel (100-200 mesh, 200-300 mesh, Qingdao ocean chemical plant); ODS filler (50 μm, Yuehasa science and technology (Shanghai) Co., Ltd.); thin layer chromatography plate (GF254, 0.20-0.25 mm, Qingdao oceanic chemical plant); dichloromethane, ethyl acetate (analytical grade, chemical reagents of national drug group, ltd.); acetonitrile, methanol (chromatographically pure, MERCK, germany); Milli-Q ultrapure water (Millipore, USA).
2. Laboratory apparatus
High-resolution time-of-flight mass spectrometers (waters, usa); triple quadrupole LC MS (Waters, USA); 2545Autopurification Mass Spectrometry guided Autopurification System (waters, USA); multifunctional extraction and concentration equipment (Shanghai Shunji science and technology Co., Ltd.); one in ten thousand electronic balance (sidoris scientific instruments ltd); KQ-500E desk ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.).
3. Experimental medicinal material
Ganoderma medicinal material is provided by Xianzhi science and technology (Fujian) GmbH, organic Ganoderma lucidum planting whole process samples are collected for 5 stages (fruiting period, parachute-opening period, growth period, maturation period and processing finished product), and identified as dry fruiting body of Ganoderma lucidum (Leys. ex Fr.) Karst in Polyporaceae by professor yellow Jersey in crude drug professor of Fujian university of traditional Chinese medicine, voucher sample is stored in sample room of Fujian university of medicine institute, and 5 conventional Ganoderma lucidum samples in market are collected as reference (S1-S5: CZ20200628-1-1, CZ20200628-2-1, CZ20200628-3-1, CZ20200628-4-1, CZ 20201022-5-1).
EXAMPLE 1 preparation of Compounds of the invention
A method of preparing a compound comprising: pulverizing Ganoderma (23kg), extracting with ethyl acetate under reflux at 80 deg.C for 3 times (each time for 2 hr). Concentrating to obtain extract, and gradient eluting with silica gel column (100-200 mesh, 120 cm. times.15 cm) and dichloromethane/methanol (100: 1-0: 1). Comprehensive analysis and judgment are carried out according to the result of a TLC development system, and the collected liquids are combined to finally obtain 12 subfractions (Fr.1-12). Separating Fr.4(94.3g, silica gel column dichloromethane/methanol (12.5: 1 elution part) by using a silica gel column (200-300 meshes), selecting dichloromethane/methanol (20-0): 1, gradient elution is carried out to obtain 9 sub-components (Fr.4-1-Fr.4-9), Fr.4-3(12.5g, 10:1 elution part of dichloromethane/methanol of silica gel column) is continuously and finely separated through an ODS column, 10%, 30%, 50%, 70% and 100% acetonitrile/water system are selected for gradient elution, 50% acetonitrile water solution eluent is collected to obtain Fr.4-3-3, the chromatographic preparation is carried out through a mass spectrum guided automatic purification preparation liquid phase system, a chromatographic column (Ultimate XB-C18, 10 mu m, 20 x 250mm) is used as a mobile phase, 35% acetonitrile water solution is used as a mobile phase, and a component with the molecular weight of m/z 485 (retention time 20.2-23.7 min) is collected to obtain the compound I (9.80 mg).
Fr. in this example denotes fraction, meaning component. The proportions of the mixed solvents used in the present invention are volume ratios.
The structure of the above compound I of the present invention was confirmed:
a compound I: a white powder; UV λ maxMeOH (nm) 254 (. epsilon.2.34); HR-ESI-MS m/z: 485.2532[ M-H]-Theoretical value of 485.2545, calculated to obtain formula C28H37O7Error 2.67 ppm. Combined 1H-NMR (500MHz, CDCl)3) And13C-NMR(500MHz,CDCl3) Spectrum, presuming the molecular formula of C28H38O7。13C-NMR(500MHz，CDCl3) The spectrum shows 28 carbon signals, and combined with DEPT spectrum, the structure of the spectrum is known to contain 7 methyl groups, 6 methylene groups, 4 methine groups and 11 quaternary carbons.13The low field region of C-NMR showed 4 ketocarbonyl groups [ chemical shifts delta 214.86(C-3), 206.51(C-15), 201.81(C-11), 198.58(C-7)]1 carboxyl group [174.08(C-24)]And one intra-ring double bond [ chemical shifts δ 148.67(C-8) and 147.47(C-9)]. Delta H3.658 (-OCH) in HMBC spectra3) And delta 174.08(C-24) indicating that the methoxy group is linked at position 24. The spectral data of the fluorescence spectrum of D-Ganoderma lucidum are compared with those reported in the literature (Kikuchi Tohru et al, Chem Pharm Bull, 1986; 34(10):4018-4029)2Methyl ester (Methyl-lucidenate D)2) And performing nuclear magnetic data attribution on the compound I according to the C spectrum, the H spectrum and the two-dimensional correlation spectrum by combining two similar compound nuclear magnetic data, wherein the compound I and the D-ganoderic acid are subjected to nuclear magnetic data attribution2Methyl ester (Methyl-lucidenate D)2) The differences in nuclear magnetic data are shown in Table 1, suggesting that Compound I is Ganoderma lucidum acid D2Methyl ester (Methyl-lucidenate D)2) The position of the greatest difference between the H spectrum data of the deacetylated product of (1) is the 12-position hydrogen signal, wherein the D-ganoderma lucidum acid2Methyl ester (Methyl-lucidenate D)2) Is delta 5.67(H-12, alpha configuration), while compound I is delta 4.509 (H-12); the 12-position H configurations of the two compounds are likely to be similar, and the 12-position hydrogen signal of the compound I is further determined to be alpha configuration according to a two-dimensional spectrum NOESY, wherein the 12-position hydrogen signal is related to the 14-position methyl. Further searching the isomer of the same isomer of the compound I, namely the ganoderma lucidum acidK Methyl ester (Methyl-lucidenate K, from T.Nishitoba et al, Pyrochemirry, 1987; 26(6):1777-1784), in which the absolute configuration of the 12-position hydrogen is beta configuration, and the H spectral data under the same nuclear magnetic solvent is highly similar to that of the compound I, but the chemical shift delta 3.94(H-12) of the 12-position hydrogen is greatly different from that delta 4.509(H-12) of the 12-position hydrogen of the compound I obtained by the separation, and further confirms that the compound obtained by the separation is different from K Methyl erythronate (Methyl-lucidenate K).
The triterpenoids in Ganoderma are lanoline type tetracyclic triterpenes, which belong to lanoline highly oxidized derivatives, wherein the methyl groups at 10, 13 and 14 positions are respectively beta, beta and alpha configuration, C-20 is R configuration, and A/B, B/C, C/D rings are trans.
Through the spectroscopy and the biogenic characteristics of the lanoline tetracyclic triterpene, the structure of the compound I is determined and shown in the formula I. Naming: methyl 12 beta-hydroxy-4, 4,14 alpha-trimethy-3, 7,11, 15-tetroxy-5 alpha-chol-8-en-24-oate,
the data for compound 1 above are shown in table 1. Table 1 shows hydrogen (500MHz) and carbon (500, 125 MHz; CDCl) spectra data of the above-mentioned compound I of the present invention3)。
TABLE 1
The ultraviolet spectrum of the compound I is shown in figure 1;
the high resolution mass spectrum of compound I of the present invention is shown in fig. 2;
of the Compounds I of the invention1H-NMR(500MHz,CDCl3) As shown in fig. 3;
of the Compounds I of the invention13C-NMR(125MHz,CDCl3) As shown in fig. 4;
HSQC (500MHz, CDCl) of Compound I of the present invention3) As shown in fig. 5;
HMBC (500MHz, CDCl) of Compound I of the present invention3) As shown in fig. 6;
NOESY (500MHz, CDCl) of Compound I of the invention3) As shown in fig. 7;
the new lanostane type triterpene compound with an unusual structure is prepared by adopting the chromatographic separation combined with the modern spectroscopic technology (NMR) for identification.
Experimental example 2 application of the compound of the invention in ganoderma lucidum quality tracing
1. Laboratory apparatus
Triple quadrupole LC MS (Waters, USA); one-tenth-ten-thousandth electronic balance (sidoris scientific instruments ltd); KQ-500E desk ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.).
2. Test reagents and drugs
Acetonitrile, methanol (chromatographically pure, MERCK, germany); Milli-Q ultrapure water (Millipore, USA); the other reagents are analytically pure.
Ganoderma medicinal material is provided by Xianzhi science and technology (Fujian) GmbH, organic Ganoderma lucidum planting whole process samples are collected for 5 stages (fruiting period, parachute opening period, growth period, maturation period and processing finished product), identified as dry fruiting body of Ganoderma lucidum (Leys. ex Fr.) Karst of Polyporaceae by professor yellow Jersey in crude drug research room of Fujian Chinese medicine university, and 5 conventional Ganoderma lucidum samples in market are collected as reference (S1-S5: CZ20200628-1-1, CZ20200628-2-1, CZ20200628-3-1, CZ20200628-4-1, CZ 20201022-5-1). The voucher specimen is stored in the specimen room of the college of medicine of Fujian Chinese medicine university.
3. Preparation of solutions
Blank sample: 50% acetonitrile solution (solvent to dissolve the sample).
Preparation of a test solution: crushing lucid ganoderma, uniformly mixing, precisely weighing 10g of lucid ganoderma dry powder, placing the powder into a triangular flask with a plug, precisely adding 500mL of ethanol, ultrasonically extracting (with the power of 250W and the frequency of 50kHz) for 45min, filtering to obtain filtrate, concentrating the filtrate to dryness, ultrasonically dissolving the filtrate by using 3mL of 50% acetonitrile, mixing the filtrate with 3mL of water, loading the mixture onto a C18 SPE small column (1000mg and 6mL), removing impurities by using 20mL of 25% acetonitrile, eluting by using 18mL of 50% acetonitrile to enrich, fixing the volume to 25mL by using 50% acetonitrile, shaking uniformly, filtering by using a 0.22 mu m filter membrane, and taking subsequent filtrate to obtain the lucid ganoderma extract.
Preparation of control solutions: compound I from example 1 was taken as a control, precisely weighed, and 50% methanol was added to make a single control solution with a concentration of 200 ng/mL.
4. Liquid quality detection method
The liquid chromatogram tandem mass spectrometer detects according to the following chromatographic conditions: performing gradient elution by taking acetonitrile with the volume concentration of 100% as a mobile phase A and taking a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase B, wherein the total sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100% in percentage by volume; the conditions of the gradient elution are as follows: the proportion of the mobile phase A keeps 20% isocratic elution in 0-0.5 min, the proportion of the mobile phase A changes from 20% to 35% in 0.5-2.5 min, the proportion of the mobile phase A keeps 35% isocratic elution in 2.5-3.0 min, the proportion of the mobile phase A changes from 35% to 65% in 3.0-5.0 min, the proportion of the mobile phase A keeps 65% isocratic elution in 5.0-6.0 min, the proportion of the mobile phase A changes from 65% to 20% in 6.0-6.1 min, the proportion of the mobile phase A keeps 20% in 6.1-8 min, isocratic elution, and the proportion of the mobile phase A is 20%;
the mass spectrometry conditions were set as follows: electrospray ion source, negative ion mode ES-, mass spectrometry multiple reaction monitoring mode (MRM mode), parent ion m/z 485, daughter ion m/z 455, collision energy: 35eV, capillary voltage negative ion mode-2.5 kV; desolventizing gas flow: 800 L.h of nitrogen–1(ii) a Desolventizing temperature: 500 ℃; taper hole airflow: nitrogen 150 L.h–1(ii) a Ion source temperature: 150 ℃; collision gas: and argon gas.
5. The result of the detection
(1) A blank sample;
(2) control (control solution);
(3) ganoderma lucidum sample in Ganoderma lucidum stage;
(4) ganoderma lucidum sample in umbrella opening period;
(5) a growth-stage ganoderma lucidum sample;
(6) a mature-stage ganoderma lucidum sample;
(7) processing finished ganoderma lucidum samples;
(8) market sample S1;
(9) market sample S2;
(10) market sample S3;
(11) market sample S4;
(12) market sample S5
The experimental results are shown in fig. 8, and comparison of (1) to (12) in fig. 8 with the samples (1) to (12) shows that the compound I of the present invention can be detected in the whole process planting sample of ganoderma lucidum, ganoderma lucidum No. 1, and is not detected in the market collecting sample, and the compound I of the present invention can be used for the chemical composition tracing of the ganoderma lucidum quality tracing system.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (9)
1. A compound having the formula I:
2. use of the compound of claim 1 in ganoderma lucidum quality traceability test.
3. Use according to claim 2, characterized in that said compound is used in the form of a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a stereoisomer thereof, or a solvate thereof.
4. A process for the preparation of a compound according to claim 1, comprising the steps of:
(1) drying Ganoderma lucidum of Ganoderma lucidum Lou-Z.K. No. 1, pulverizing, extracting with ethyl acetate, and concentrating the extractive solution under reduced pressure to obtain total extract;
(2) the total extractum is subjected to gradient elution by adopting a silica gel column, and the volume ratio of an eluent is (100-0): 1, a mixed solution of dichloromethane and methanol, with a retention volume ratio of 12.5: 1, namely a liquid eluted from a mixed solution of dichloromethane and methanol, which is named as Fr.4;
(3) taking Fr.4 obtained in the step (2), and performing gradient elution by adopting a silica gel column, wherein the volume ratio of an eluent is (20-0): 1, a mixed solution of dichloromethane and methanol, with a retention volume ratio of 10:1, namely a liquid eluted from a mixed solution of dichloromethane and methanol, which is named as Fr.4-3;
(4) separating and purifying Fr.4-3 obtained in the step (3) by adopting an ODS column, wherein an eluent is acetonitrile aqueous solution with the volume concentration of 50%, and is named as Fr.4-3-3;
(5) and (4) taking Fr.4-3-3 obtained in the step (4), and performing chromatographic preparation by adopting a mass spectrum guided automatic purification preparation liquid phase system, wherein the chromatographic column parameters are as follows: ultimate XB-C18, 10 μm, 20X 250mm, mobile phase 35% acetonitrile in water, and fractions with molecular weight m/z 485 were collected to obtain the compound.
5. The method of claim 4, wherein: in the step (1), the extraction specifically comprises: heating and reflux extracting at 80 deg.C for 3 times, each time for 2 hr.
6. A detection method for performing ganoderma lucidum quality traceability detection by adopting the compound of claim 1, wherein the detection method adopts the compound as a reference substance and adopts a liquid chromatography-mass spectrometry combined method to perform ganoderma lucidum quality traceability detection.
7. The assay of claim 6, wherein the assay comprises preparation of a control solution;
the preparation of the control solution comprises: the compound was added to 50% methanol to make a single control solution with a concentration of 200 ng/mL.
8. The assay of claim 6, wherein the assay comprises preparation of a test solution;
the preparation of the test solution comprises the following steps: crushing a ganoderma lucidum sample to be tested, uniformly mixing, weighing 10g of dry powder, placing the dry powder into a triangular flask with a plug, adding 500mL of ethanol, carrying out ultrasonic extraction for 45min, filtering to obtain filtrate, concentrating the filtrate to be dry, carrying out ultrasonic dissolution by using 3mL of 50% acetonitrile, mixing the filtrate with 3mL of water, loading the mixture onto a C18 SPE small column, removing impurities by using 20mL of 25% acetonitrile, eluting by using 18mL of 50% acetonitrile for enrichment, fixing the volume to 25mL by using 50% acetonitrile, shaking up, filtering by using a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the ganoderma lucidum sample.
9. The detection method according to claim 6, wherein in the liquid chromatography-mass spectrometry combined method, the chromatographic conditions are as follows: performing gradient elution by taking acetonitrile with the volume concentration of 100% as a mobile phase A and taking a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase B, wherein the total sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100% in percentage by volume; the conditions of the gradient elution are as follows: the proportion of the mobile phase A keeps 20% isocratic elution in 0-0.5 min, the proportion of the mobile phase A changes from 20% to 35% in 0.5-2.5 min, the proportion of the mobile phase A keeps 35% isocratic elution in 2.5-3.0 min, the proportion of the mobile phase A changes from 35% to 65% in 3.0-5.0 min, the proportion of the mobile phase A keeps 65% isocratic elution in 5.0-6.0 min, the proportion of the mobile phase A changes from 65% to 20% in 6.0-6.1 min, the proportion of the mobile phase A keeps 20% in 6.1-8 min, isocratic elution, and the proportion of the mobile phase A is 20%;
in the method of the liquid chromatography-mass spectrometry, the mass spectrometry conditions are as follows: electrospray ion source, negative ion mode ES-, mass spectrum multiple reaction monitoring mode, parent ion m/z 485, daughter ion m/z 455, collision energy: 35eV, capillary voltage negative ion mode-2.5 kV; desolventizing gas flow: 800L/h of nitrogen; desolventizing temperature: 500 ℃; taper hole airflow: 150L/h of nitrogen; ion source temperature: 150 ℃; collision gas: and argon gas.
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