Method for detecting contents of multiple lipid components in ganoderma spore oil by quantitative analysis of multi-components by single marker

Abstract

The application belongs to the field of quality standards and detection technology of health food, and discloses a detection method for detecting the content of multiple lipid components in ganoderma lucidum spore oil by a multi-evaluation method. In the application, high performance liquid chromatography is used, glyceryl trioleate is used as an internal reference, correction factors of glyceryl trioleate and other lipid components are established, and the correction factors are used to calculate the concentrations of multiple to-be-measured components in the lipid. In the application, acetonitrile-acetone-isooctane is used as a mobile phase, and HPLC-UV is used in combination, and the specificity is strong.

Description

Technical Field

[0001] This invention belongs to the field of quality standards and testing technology for health food, specifically relating to a method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil. Background Technology

[0002] Reishi spore oil is obtained from the spores of the polypore fungus Ganoderma lucidum (Leyss. ex Fr.) Karst. through cell wall disruption and supercritical fluid extraction technology. Studies have shown that reishi spore oil has good anti-tumor effects, enhances immune regulation, improves memory, and lowers blood lipids. Therefore, with modern people's increasing emphasis on health and pursuit of quality of life, health products containing reishi spore oil have gradually entered the market.

[0003] Ganoderma lucidum spore oil contains various chemically active components such as glycerides, sterols, and triterpenoids, with lipids being the main component. Currently, the detection of lipid content in Ganoderma lucidum spore oil is mostly performed using high-performance liquid chromatography-evaporative optical emission spectrometry (HPLC-ELSD). This method relies on linear fitting using the logarithm of concentration and the logarithm of peak area, which is complex, cumbersome, and has poor reproducibility. While Ganoderma lucidum spore oil contains a wide variety of lipid components, there are few studies on their content detection. The only known report is the determination of eight triglycerides in Ganoderma lucidum spore oil using HPLC-ELSD, indicating that the experimental procedures for detecting lipid content in Ganoderma lucidum spore oil are tedious, and the calculations are computationally intensive. Furthermore, triglycerides are composed of different fatty acids bonded to a glycerol backbone. Due to differences in carbon chain length and saturation, the variety of fatty acids is vast. Different types of sterols and triterpenoids may also be bound to the backbone, resulting in different geometric configurations in the triglycerides, enriching the variety of lipid components. Different lipid structures exhibit different physical and chemical properties, increasing the difficulty of separation. Currently, the detection of lipid components in oils and fats often uses isopropanol-acetonitrile in different proportions as the mobile phase, employs an ELSD detector, and utilizes a linear fitting method based on the logarithm of concentration and the logarithm of peak area for quantitative calculation.

[0004] The Quality Assay Method (QAMS) allows for the simultaneous determination of multiple components by establishing an internal control and setting correction factors for other components relative to the internal control. This simplifies the detection process and saves experimental time and costs. In 2015, the Chinese Pharmacopoeia reported that QAMS could be used for the content determination of traditional Chinese medicinal materials, leading to increased research on QAMS in traditional Chinese medicinal materials and prepared Chinese medicines. While there are reports of using QAMS to determine triterpenoids in Ganoderma lucidum (Reishi mushroom), other components such as lipids have not yet been determined using this method. The five most abundant lipid compounds disclosed in Ganoderma lucidum spore oil are triglyceride (OOO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), cis-1-palmitoyl-2-oleic-3-linoleic acid glyceride (POL), cis-1,2-dioleic-3-linoleic acid glyceride (OOL), and 1,2-dilinoleic-3-oleic acid glyceride (LLO). However, due to the limited number of domestic manufacturers producing lipid-related reference standards, these standards are often ordered from abroad, resulting in long lead times and high prices. Summary of the Invention

[0005] In view of the fact that lipid reference standards are relatively rare and expensive, and the external standard method is cumbersome to calculate, this invention provides for the first time a method for detecting the lipid content in Ganoderma lucidum spore oil by one test and multiple evaluations, which can accurately detect several localizable lipid components.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-measurement-multiple-evaluation method is characterized by employing high-performance liquid chromatography (HPLC) with triglyceride as an internal reference, using HPLC-UV coupling, establishing a correction factor between triglyceride and other lipid components, and using the correction factor to calculate the concentration of multiple analytes in Ganoderma lucidum spore oil. The chromatographic conditions of the HPLC include using acetonitrile-acetone-isooctane as the mobile phase.

[0008] As a preferred embodiment of the method of the present invention, the other lipid components include one or more of 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dioleic acid-3-linoleic acid glyceride, cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride, and 1,2-dioleoyl-3-palmitoyl-rac-glycerol.

[0009] As a preferred embodiment of the method of the present invention, the chromatographic conditions of the high performance liquid chromatography include: the mobile phase is acetonitrile-acetone-isooctane with a volume ratio of 53-57.7:32-29.1:15-13.2; the stationary phase is a C18 column with a column temperature of 35-40℃.

[0010] As a preferred embodiment of the method of the present invention, the mobile phase is acetonitrile-acetone-isooctane with a volume ratio of 57.7:29.1:13.2; the stationary phase is a YMC-Triart-C18 chromatographic column with specifications of 250*4.6mm DS-3μm 12nm and a column temperature of 38℃.

[0011] As a preferred embodiment of the method of the present invention, the method includes the following steps:

[0012] A. Preparation of the test solution: Accurately weigh the Ganoderma lucidum spore oil sample, dissolve and dilute it in a solvent to obtain the test solution;

[0013] B. Preparation of reference standard stock solution:

[0014] B1) Accurately weigh the standards of 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dioleic acid-3-linoleic acid glyceride, and cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride, dissolve them in isopropanol, and dilute them to obtain the reference stock solutions of 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dioleic acid-3-linoleic acid glyceride, and cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride, with a concentration of approximately 3 mg / mL, for later use;

[0015] B2) Accurately weigh the standards of glycerol trioleate and 1,2-dioleoyl-3-palmitoyl-rac-glycerol, dissolve them in acetonitrile-isopropanol at a volume ratio of 53:47, and dilute them to obtain glycerol trioleate reference stock solution and 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference stock solution, with a concentration of about 2 mg / mL, for later use;

[0016] C. Preparation of mixed standard solution: Accurately take the 1,2-dilinoleic acid-3-oleic acid glyceride reference standard stock solution, the cis-1,2-dilinoleic acid-3-oleic acid glyceride reference standard stock solution, the cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride reference standard stock solution, the trioleic acid ester, and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol standard, mix them to prepare a mixed standard solution, which will be used for detection and calculation of the correction factor;

[0017] D. Preparation of standard curve solutions: Accurately pipette the stock solution of triglyceride reference standard described in step B, and dissolve it to prepare triglyceride standard curve solutions of different concentrations;

[0018] E. Detection: Accurately pipette the standard curve solutions of triglycerides of different concentrations described in step D and perform high performance liquid chromatography (HPLC) to detect them, and prepare an internal reference standard curve; accurately pipette the test solution described in step A and perform HPLC to detect it, and obtain the chromatogram of the test solution;

[0019] F. Calculation: The chromatogram of the test sample solution shows characteristic peaks of triglycerides and the other lipid components. Using the internal standard method, the concentration of a certain analyte is calculated using the correction factor and the standard curve of the internal reference in step E. The calculation method is as follows:

[0020] F1) Take the test solution and perform high-performance liquid chromatography (HPLC) to obtain the chromatogram of the test solution.

[0021] F2) Based on the spectrum of the test sample solution, the concentration of triglyceride in the test sample solution is obtained through the standard curve of the internal reference in step E;

[0022] F3) The triglyceride ester in the test solution is used as an internal reference, designated 4x. The analyte in the test solution is designated ax. Based on the chromatogram of the test solution in step F1) and the triglyceride ester concentration in F2), the concentration of the analyte is calculated using the correction factor. The formula for the concentration of the analyte is as follows:

[0023]

[0024] f is a correction factor for a certain analyte.

[0025] A 4x The peak area of ​​the internal reference in the test solution.

[0026] W 4x The concentration of the internal reference in the test solution.

[0027] A ax The peak area of ​​a certain analyte in the test solution.

[0028] W ax The concentration of a certain analyte in the test solution.

[0029] Furthermore, the correction factor is obtained in the following manner:

[0030] a) Inject the mixed standard solution into a high-performance liquid chromatograph to obtain the chromatogram of the mixed standard solution;

[0031] b) Based on the chromatogram of the mixed standard solution, triglyceride ester in the mixed standard solution is used as an internal reference and is numbered 4. The number of the analyte in the mixed standard solution is denoted as a. The formula for the relative correction factor between triglyceride ester and the analyte is as follows:

[0032]

[0033] f 4a This is the relative correction factor between triglyceride and a certain analyte.

[0034] A4 represents the peak area of ​​the internal reference standard.

[0035] W4 represents the concentration of the internal reference standard.

[0036] A a The peak area of ​​a reference standard for a certain analyte is given.

[0037] W a This represents the concentration of a reference standard for a specific analyte.

[0038] c) Inject 10 μL, 20 μL, 25 μL and 30 μL of the mixed standard solution respectively, calculate the relative correction factor between the triglyceride ester and the analyte, and take the average value to obtain the correction factor of the analyte.

[0039] Furthermore, the preparation of the test solution in step A of the method of the present invention includes the following steps: accurately weighing the Ganoderma lucidum spore oil sample, dissolving and diluting it with acetonitrile-isopropanol at a volume ratio of 53:47 to about 4.5 mg / mL, to obtain the test solution.

[0040] Further, the preparation of the mixed standard solution in step C of the method of the present invention includes the following steps: accurately pipetting the 1,2-dilinoleic acid-3-oleic acid glyceride reference stock solution, the cis-1,2-dioleic acid-3-linoleic acid glyceride reference stock solution, the cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride reference stock solution, the trioleic acid ester reference stock solution, and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference stock solution in step B, at a volume ratio of 53:4. Dilute with acetonitrile-isopropanol to prepare a mixed standard solution with the following concentrations: 1,2-dilinoleic acid-3-oleic acid glyceride at approximately 0.12 mg / mL, cis-1,2-dioleic acid-3-linoleic acid glyceride at approximately 0.12 mg / mL, cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride at approximately 0.12 mg / mL, trioleic acid trioleate at approximately 0.4 mg / mL, and 1,2-dioleoyl-3-palmitoyl-rac-glycerol at approximately 0.4 mg / mL.

[0041] Further, the preparation of the standard curve solution in step D of the method of the present invention includes the following steps: accurately pipetting the stock solution of the triglyceride reference standard in step B into a 5 mL volumetric flask, diluting it with acetonitrile-isopropanol at a volume ratio of 53:47 to prepare the triglyceride standard curve solutions with concentrations of approximately 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.2 mg / mL.

[0042] As a preferred embodiment of the method of the present invention, the chromatographic conditions of the high performance liquid chromatography include the following: the detection instrument is a Thermo Fisher U3000 high performance liquid chromatograph with an ultraviolet detector, the ultraviolet detection wavelength is selected as 205-206 nm, the injection volume is 20 μL, the mobile phase constant flow rate is 1.0 mL / min, and the mobile phase is maintained for 60 minutes.

[0043] Furthermore, in addition to its application in Ganoderma lucidum spore oil, the method of the present invention is also applicable to the separation, purification, detection of lipid content, identification of component structure, or qualitative identification of other oils (such as olive oil, soybean oil, medium-chain triglycerides, etc.).

[0044] In their research on the detection of lipid components in Ganoderma lucidum spore oil, the inventors discovered that lipid component determination exhibits greater stability under ultraviolet (UV) detection. Through extensive experimentation, they found that using a specific proportion of mobile phase system for separating lipid components in Ganoderma lucidum spore oil resulted in better separation of structurally similar components, demonstrating improved separation efficiency compared to commonly used isopropanol-acetonitrile mobile phase systems. When using a multi-evaluation method to detect the lipid content in Ganoderma lucidum spore oil, the inventors selected triglyceride (OOO) as an internal reference and successfully established correction factors for LLO, OOL, POL, and OOP for calculation.

[0045] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0046] 1) For the first time, acetonitrile-acetone-isooctane with a volume ratio of (53-57.7):(32-29.1):(15-13.2) was used as the mobile phase, which resulted in better separation effect, enabling the effective separation and quantitative detection of overlapping components, which is beneficial for studying the structure of the prototype components in the product.

[0047] 2) In view of the fact that the logarithmic fitting linear method of HPLC-ELSD is cumbersome and has poor reproducibility, the HPLC-UV coupling method has strong specificity, small detection error, accurate and reliable results, good reproducibility, and a simpler calculation process.

[0048] 3) This invention is the first to use a single-test-multiple-evaluation method to determine the content of lipid components in Ganoderma lucidum spore oil. Given that lipid component reference standards are relatively expensive and the application of a single-test-multiple-evaluation method is lacking, this invention fills a gap in the field. By using triglyceride ester (OOO) as an internal reference, the use of other expensive reference standards is reduced, which can save costs while ensuring product quality control, and the operation is simple. Attached Figure Description

[0049] Figure 1 High performance liquid chromatography (HPLC) chromatogram of lipid components separated from Ganoderma lucidum spore oil sample using acetonitrile-isopropanol (55:45) as the mobile phase;

[0050] Figure 2 High-performance liquid chromatography (HPLC) chromatogram of lipid components separated from Ganoderma lucidum spore oil sample using acetonitrile-acetone-isooctane (57.7:29.1:13.2) as the mobile phase;

[0051] Figure 3 High-performance liquid chromatography (HPLC) chromatogram of lipid components separated from Ganoderma lucidum spore oil sample using an acetonitrile-acetone-isooctane (57.7:29.1:13.2)-ELSD system;

[0052] Figure 4 High-performance liquid chromatography (HPLC) chromatogram of lipid components separated from Ganoderma lucidum spore oil sample using an acetonitrile-acetone-isooctane (57.7:29.1:13.2)-UV system;

[0053] Figure 5 This is the high-performance liquid chromatogram of the mixed standard solution in Example 5;

[0054] Figure 6 High-performance liquid chromatography (HPLC) chromatogram of lipid components separated from Ganoderma lucidum spore oil sample using an acetonitrile-dichloromethane (65:35)-ELSD system;

[0055] Figure 7 The above are high-performance liquid chromatograms of the detection method of the present invention applied to other oils; wherein the test sample 1 is olive oil, the test sample 2 is soybean oil, the test sample 3 is medium-chain oil, and the test sample 4 is Ganoderma lucidum spore oil. Detailed Implementation

[0056] To enable those skilled in the art to better understand and implement the present invention, the embodiments described are not intended to limit the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from its spirit and essence are within the scope of the invention.

[0057] Tables 1 to 4 provide information on the relevant experimental instruments, standards, reagents, and samples used in the examples:

[0058] Table 1. Information on reagents used in this invention.

[0059] Reagent Name level factory Acetonitrile HPLC Simga acetone HPLC Guangzhou Chemical Reagent Factory Isooctane HPLC McLean Isopropanol HPLC SK Chemicals methanol HPLC Sigma dichloromethane HPLC Sigma n-Hexane HPLC Inter-Lab

[0060] Table 2 Information on instruments used in this invention

[0061] Instrument Name Manufacturer and Model Electronic balance Shimadzu AP225WD High Performance Liquid Chromatography Thermo U3000 UV detector Thermo U3000 accessories ELSD detector AllChrom ELSD 6100

[0062] Table 3 Information on the reference standards used in this invention

[0063] Reference source 1,2-Dilinoleic-3-oleic glyceryl ester (LLO) Larodan cis-1,2-dioleoyl-3-linoleic acid glyceride (OOL) Larodan cis-1-palmitoyl-2-oleic-3-linoleic acid glyceride (POL) Larodan Triglycerides (OOO) Larodan 1,2-Dioleoyl-3-palmitoyl-rac-glycerol (OOP) Larodan

[0064] Table 4 Sample information used in this invention

[0065] sample Preparation method Ganoderma lucidum spore oil Ganoderma lucidum spores prepared via supercritical fluid processing

[0066] Example 1: Preparation of the test solution

[0067] Take 45 mg of Ganoderma lucidum spore oil sample, accurately weigh its mass, dissolve and dilute it to the mark with acetonitrile-isopropanol (53:47) in a 10 mL volumetric flask, shake well, and use it as the test solution.

[0068] Example 2: Preparation of Standard Solution

[0069] (I) Preparation of reference stock solution

[0070] LLO reference stock solution: Accurately dissolve LLO reference standard in a 25 mL volumetric flask, dilute to the mark with isopropanol, and the concentration is 3.0308 mg / mL.

[0071] OOL reference standard stock solution: Accurately dissolve OOL reference standard in a 25 mL volumetric flask, dilute to the mark with isopropanol, and the concentration is 3.1152 mg / mL.

[0072] POL reference standard stock solution: Accurately dissolve POL reference standard in a 25 mL volumetric flask, dilute to the mark with isopropanol, and the concentration is 3.1080 mg / mL.

[0073] OOO reference standard stock solution: Accurately take OOO reference standard, place it in a 25 mL volumetric flask, dissolve it with acetonitrile-isopropanol (53:47), and dilute to the mark to a concentration of 2.1705 mg / mL.

[0074] OOP reference stock solution: Accurately dissolve OOP reference standard in a 25 mL volumetric flask with acetonitrile-isopropanol (53:47) and dilute to the mark to a concentration of 1.6043 mg / mL.

[0075] (II) Preparation of OOO standard curve solution

[0076] Take the OOO reference stock solution from Example (I) as OOO standard curve solution 5. Accurately pipette 4 mL, 3 mL, 2 mL and 1 mL of OOO reference stock solution into 5 mL volumetric flasks, dilute to the mark with acetonitrile-isopropanol (53:47), shake well to obtain OOO standard curve solutions 4-1.

[0077] (III) Preparation of mixed standard solution

[0078] Accurately pipette 0.2 mL of LLO reference stock solution, 0.2 mL of OOL reference stock solution, 0.2 mL of POL reference stock solution, 1.0 mL of OOO reference stock solution, and 1.0 mL of OOP reference stock solution from Example (I) into a 5 mL volumetric flask, dilute to the mark with acetonitrile-isopropanol (53:47), and shake well to obtain a mixed standard solution.

[0079] Example 3 Chromatographic determination and chromatographic conditions

[0080] Acetonitrile-isopropanol (53:47) was used as a blank solution. The test solution prepared according to the method of Example 1, the OOO standard curve solution prepared according to the method of Example 2 (II), and the mixed standard solution prepared according to Example 2 (III) were injected into the chromatograph for determination under the following high-performance liquid chromatography (HPLC) conditions:

[0081] Column: YMC-Triart-C18 250*4.6mm DS-3μm 12nm

[0082] Instrument: Thermo Fisher U3000 with UV detector

[0083] Mobile phase: Acetonitrile-acetone-isooctane (57.7:29.1:13.2)

[0084] Flow rate: constant flow rate 1.0 mL / min

[0085] Elution: Keep the mobile phase for up to 60 minutes.

[0086] Column oven: 38℃

[0087] Injection volume: 20 μL (injection volumes of the mixed standard solution are 10 μL, 20 μL, 25 μL, and 30 μL respectively)

[0088] Detection wavelength: 205–206 nm

[0089] Example 4: Calculation of the content of the analyte in the "one test, multiple evaluations" method

[0090] Using triglyceride (OOO) as an internal reference, 5-1 ml of the OOO standard curve solution prepared in Example 2 (II) was injected into the chromatograph according to the high performance liquid chromatography determination and chromatographic conditions of Example 3. The chromatograms were recorded, and OOO standard curves with concentrations of 0.4341 mg / mL, 0.8682 mg / mL, 1.3023 mg / mL, 1.7364 mg / mL, and 2.1705 mg / mL were established.

[0091] The mixed standard solution was prepared according to the steps in Example 2 (III). The concentrations of LLO, OOL, POL, OOO, and OOP in the mixed standard solution were 0.1212 mg / mL, 0.1246 mg / mL, 0.1243 mg / mL, 0.4341 mg / mL, and 0.3209 mg / mL, respectively. The remaining conditions were determined by high-performance liquid chromatography (HPLC) under the conditions described in Example 3, and the chromatograms of the mixed standard solution were collected. In the mixed standard results, OOO was selected as the internal reference, numbered 4. Other components were numbered as follows: LLO as 1, OOL as 2, POL as 3, and OOP as 5. The relative correction factor was calculated using the following formula:

[0092]

[0093] f 4a This is the relative correction factor between triglyceride and a certain analyte.

[0094] A4 represents the peak area of ​​the internal reference standard.

[0095] W4 represents the concentration of the internal reference standard.

[0096] A a The peak area of ​​a reference standard for a certain analyte;

[0097] W a This represents the concentration of a reference standard for a certain analyte.

[0098] Inject 10 μL, 20 μL, 25 μL, and 30 μL of the mixed standard solution respectively. After obtaining the relative correction factor of each component with different injection volumes, take the average value, which is the correction factor of that component.

[0099] The calculated correction factors are 0.1783 for LLO, 0.3012 for OOL, 0.2996 for POL, and 1.4017 for OOP (Table 9).

[0100] Take the test solution prepared in Example 1, and determine the chromatogram using the same high-performance liquid chromatography (HPLC) conditions as in Example 3. Record the chromatogram. Obtain the actual concentration of OOO in the test solution using the OOO standard curve; calculate the concentrations of the remaining analytes using the relative correction factor, as shown in the following formula:

[0101]

[0102] f is a correction factor for a certain analyte.

[0103] A 4x The peak area of ​​the internal reference in the test solution.

[0104] W 4x The concentration of the internal reference in the test solution.

[0105] A ax The peak area of ​​a certain analyte in the test solution.

[0106] W ax The concentration of a certain analyte in the test solution.

[0107] The concentration to content conversion formula can be used to calculate the content of a certain analyte: Content of analyte = Concentration of analyte × Volume of test solution / Mass of test sample. Where: the unit of content is mg / g, the unit of concentration is mg / mL, the volume of the test solution in Example 1 is 10 mL, and the mass of the test sample is the actual measured value in Example 1.

[0108] Example 5: Separation between components in the mixed standard solution

[0109] Prepare the mixed standard solution according to the steps in Example 2 (III), and perform detection under the high-performance liquid chromatography conditions of Example 3. Inject 20 μL, record the chromatogram, and see [see details]. Figure 5 The separation degree between the peaks of each component was calculated according to the elution order. The results are shown in Table 5, which shows that the separation degree of the mixed standard solution of the present invention is very good under the above conditions.

[0110] Table 5 Separation rate of mixed standard solution

[0111]

[0112]

[0113] Example 6 Sample Resolution

[0114] Take the same batch of samples and prepare test solutions according to the method in Example 1. Use acetonitrile-acetone-isooctane (53:32:15) and acetonitrile-acetone-isooctane (57.7:29.1:13.2) as mobile phases, respectively. The other conditions are the same as those in Example 3 for high performance liquid chromatography. Inject 20 μL, record the chromatogram, and calculate the resolution between the component peaks and adjacent peaks, as shown in Table 6.

[0115] The resolution between each component peak and its adjacent peak was greater than 1, indicating that using a mobile phase system with a certain ratio to separate the lipid components in Ganoderma lucidum spore oil can effectively separate LLO, OOL, POL, OOO, and OOP lipid components with good separation effect. Among the two mobile phase ratios, acetonitrile-acetone-isooctane (57.7:29.1:13.2) showed better separation effect.

[0116] Table 6 Sample Resolution

[0117]

[0118] Example 7: Confirmation of the mobile phase

[0119] Take the same batch of samples and prepare test solutions according to the method in Example 1. Use acetonitrile-isopropanol (55:45) and acetonitrile-acetone-isooctane (57.7:29.1:13.2) as mobile phases, respectively. Perform detection under the high-performance liquid chromatography (HPLC) conditions of Example 3, collect chromatograms, and use the same integration method. The results are as follows: The chromatogram of lipid components separated by the acetonitrile-isopropanol (55:45) system shows 13 chromatographic peaks. Figure 1 The chromatogram of lipid components separated by the acetonitrile-acetone-isooctane (57.7:29.1:13.2) system showed that 25 chromatographic peaks were effectively separated. (See attached image.) Figure 2 .

[0120] This indicates that using an acetonitrile-acetone-isooctane (57.7:29.1:13.2) mobile phase system to separate lipid components in oils can effectively separate several adjacent lipid components, resulting in better separation performance.

[0121] Example 8 Detector Confirmation

[0122] Take the same batch of samples and prepare the test solution according to the method in Example 1.

[0123] The UV detector and the ELSD detector are used in series.

[0124] UV detector: Take the test solution and perform detection according to the high performance liquid chromatography conditions of Example 3.

[0125] ELSD detector: The test solution was taken for detection. The chromatographic conditions were the same as those in Example 3: instrument, column, flow rate, column temperature, injection volume, etc. The ELSD settings were: drift tube temperature 70℃, gas flow 2.0L / min, gain 1, split mode.

[0126] Record the chromatogram. It is evident that, under the same mobile phase, different detectors produce varying separation results. In terms of resolution, while the ELSD detector can separate the peak between OOL and POL, the separation between the peaks between OOO and OOP is poor; the same applies to the separation between LLO and its adjacent peaks. In terms of the number of separated peaks, the ELSD detector separates 17 peaks (see...). Figure 3 The UV detector separated 25 peaks (see...). Figure 4 This indicates that, under the same mobile phase, the UV detector outperforms the ELSD detector in this experiment.

[0127] Example 9 Method Comparison

[0128] A method for detecting triglycerides in Job's tears using a single-analysis, multiple-evaluation method has been studied. The isocratic mobile phases used include methanol-acetonitrile (95:5 or 90:10) and acetonitrile-dichloromethane (65:35), while the gradient mobile phase is isopropanol-n-hexane. An evaporative light detector is used. The following comparison between the method of this invention and the existing "method for detecting triglycerides in Job's tears using a single-analysis, multiple-evaluation method" is shown. Unless otherwise specified, the samples and their processing methods not described in this invention are identical to those described in this invention.

[0129] 1. The method using methanol-acetonitrile (95:5) as the mobile phase and an evaporative light detector showed no significant difference in separation performance compared to the isopropanol-acetonitrile method, only separating 7 triglyceride peaks. Using an isopropanol-n-hexane gradient as the mobile phase with gradient elution and evaporative light detection optimized the original method, separating 10 triglyceride peaks. However, in the above methods, no other peaks were detected between the OOL and POL peaks, or between the OOO and OOP peaks. The method of this invention, using acetonitrile-acetone-isooctane (57.7:29.1:13.2) as the mobile phase and an ultraviolet detector, not only detected the triglyceride peaks measured in other systems, but also detected an unknown peak between the OOL and POL peaks, and between the OOO and OOP peaks.

[0130] 2. Similarly, in the drug standard for Kanglaite injection, the fingerprint chromatogram of the emulsion was detected using an evaporative light detector with octadecylsilane-bonded silica gel as the filler, acetonitrile-dichloromethane (65:35) as the mobile phase, a flow rate of 0.5 mL / min, and a column temperature of 35℃. Under the same conditions, Ganoderma lucidum spore oil was processed, and the detection results are as follows: Although an unknown peak was detected between OOL and POL, no unknown peak was detected between OOO and OOP. The chromatogram is shown above using acetonitrile-dichloromethane (65:35) as the mobile phase. Figure 6 Thirteen chromatographic peaks were separated; the elution method described in this invention, using acetonitrile-acetone-isooctane (57.7:29.1:13.2) as the mobile phase, effectively separated 25 chromatographic peaks, as shown in the chromatogram. Figure 2 or Figure 4 .

[0131] The methods used to detect triglycerides in Ganoderma lucidum spore oil, such as methanol-acetonitrile (95:5), acetonitrile-dichloromethane (65:35), and isopropanol-n-hexane, showed that some peaks were encapsulated by adjacent peaks, resulting in a reduction in the total number of peaks detected. Compared to the above three methods, the method of this invention is superior in terms of peak separation.

[0132] Example 10 Precision Test

[0133] Take the same batch of samples and prepare the test solution according to the steps in Example 1 to make 6 reproducible sample solutions.

[0134] The reference stock solutions of each component were prepared according to the method in Example 2(a), and diluted separately with acetonitrile-isopropanol (53:47) to obtain the standard curve solutions of each component. Their concentrations are as follows (unit: mg / mL):

[0135] LLO standard curves 1–5: 0.06630, 0.1326, 0.2652, 0.5304, 1.0608

[0136] OOL standard curves 1-5: 0.05845, 0.1169, 0.2337, 0.4673, 0.9346

[0137] POL standard curves 1–5: 0.07770, 0.1554, 0.3108, 0.6216, 1.2432

[0138] OOO standard curves 1-5: 0.4341, 0.8682, 1.3023, 1.7364, 2.1705

[0139] OOP standard curves 1–5: 0.3209, 0.6417, 0.9626, 1.2834, 1.6043

[0140] The standard curve solution and the test solution were taken separately and analyzed under the high performance liquid chromatography conditions of Example 3. The chromatograms were recorded, and the RSDs were calculated using the external standard method. The RSDs for LLO content were 1.85%, OOL content was 1.44%, POL content was 1.92%, OOO content was 1.04%, and OOP content was 1.37%. The results are shown in Table 7.

[0141] Table 7 Precision Calculation

[0142]

[0143] Example 11 Accuracy Test

[0144] Recovery rates of LLO, OOL, POL, OOO, and OOP components in the test solution were calculated at three levels: 80%, 100%, and 120%. Approximately 18 mg, 22 mg, and 26 mg of Ganoderma lucidum spore oil samples were weighed and placed in separate 10 mL volumetric flasks for each level (80%, 100%, and 120%). Five reference standards (LLO, OOL, POL, OOO, and OOP) were added to each volumetric flask for each level, with the mass of each reference standard added being equivalent to the background mass of the sample taken at that level. Each volumetric flask was diluted with acetonitrile-isopropanol (53:47) and brought to volume.

[0145] The detection was performed and recorded under the high-performance liquid chromatography conditions of Example 3. The standard curve was the same as that in Example 10.

[0146] The calculated accuracy data are as follows: the recovery rate of POL is 94.16%, with an RSD of 1.6%; the recovery rate of OOO is 101.44%, with an RSD of 1.5%; the recovery rate of OOP is 99.87%, with an RSD of 1.1%; the recovery rate of LLO is 97.17%, with an RSD of 1.6%; and the recovery rate of OOL is 100.09%, with an RSD of 2.3%. This indicates that the accuracy of LLO, OOL, POL, OOO, and OOP in the sample is good, and the method is reliable. The results are shown in Table 8.

[0147] Table 8 Accuracy Calculation

[0148]

[0149] Example 12: Confirmation of the Relative Correction Factor

[0150] The mixed standard solution from Example 4 was prepared with concentrations of LLO (0.1212 mg / mL), OOL (0.1246 mg / mL), POL (0.1243 mg / mL), OOO (0.4341 mg / mL), and OOP (0.3209 mg / mL). Detection was performed under the high-performance liquid chromatography (HPLC) conditions described in Example 3, with injections of 10 μL, 20 μL, 25 μL, and 30 μL, respectively. Chromatographic values ​​were recorded, and the calculations were performed as follows:

[0151] Following the calculation of relative correction factors in Example 4, the correction factors of LLO, OOL, POL, and OOP relative to OOO were calculated for each injection volume. The results show that different injection volumes within the range of 10–30 μL have no significant effect on the correction factors. The relative correction factor for LLO is 0.1783 with an RSD of 0.78%; for OOL, it is 0.3012 with an RSD of 0.94%; for POL, it is 0.2996 with an RSD of 0.61%; and for OOP, it is 1.4017 with an RSD of 1.25%. See Table 9 for details.

[0152] Table 9 Calculation of Correction Factors

[0153]

[0154] Example 13: Comparison of External Standard Method and One-Measure-Multiple-Evaluation Method

[0155] The replicate solutions 1-6 from the precision test in Example 10 were used in the calculation method described in Example 4, with OOO as the internal reference. The contents of the test components LLO, OOL, POL, and OOP were calculated and compared with the data obtained by the external standard method. The results are as follows: the RSD of LLO content is 3.21%; the RSD of OOL content is 3.86%; the RSD of POL content is 2.58%; and the RSD of OOP content is 1.60%. This indicates that the RSD of each component obtained by multiple measurements and calculations is less than 4%, and there is no significant difference between the two content calculation methods. Therefore, the method of determining the lipid content in Ganoderma lucidum spore oil using the one-test-multiple-evaluation method is reliable. See Table 10 for details.

[0156] Table 10 Comparison of External Standard Method and One Measurement Multiple Evaluation Method

[0157]

[0158]

[0159] Different batches of Ganoderma lucidum spore oil were collected, and the lipid content was obtained using both the single-analysis-multiple-evaluation method (calculation) and the external standard method (determination). The single-analysis-multiple-evaluation method used OOO as an internal reference and was performed according to the method in Example 4. The specific contents obtained are shown in Table 11. The results are as follows: In each batch, the RSD of the contents obtained by the external standard method and the single-analysis-multiple-evaluation method for different components was less than 5%, indicating a small difference. This demonstrates that the single-analysis-multiple-evaluation method can be widely used for measuring the lipid content in Ganoderma lucidum spore oil.

[0160] Table 11 Contents of five lipid components in different batches (unit: mg / g)

[0161]

[0162]

[0163] Example 14: Comparison of the application of the one-test-multiple-evaluation method to the detection of other oils.

[0164] Ganoderma lucidum spore oil, olive oil, soybean oil, and medium-chain triglycerides were used to prepare a test solution according to the steps in Example 1. The solution was then analyzed under the high-performance liquid chromatography (HPLC) conditions of Example 3, with an injection volume of 20 μL. The chromatograms were recorded as follows. Figure 7 (1 is olive oil, 2 is soybean oil, 3 is medium-chain triglyceride oil, and 4 is Ganoderma lucidum spore oil).

[0165] The test results show that soybean oil, medium-chain triglycerides, and Ganoderma lucidum spore oil have significant differences, mainly in the 5-15 min and 25-40 min ranges of the chromatogram information; the differences between olive oil and Ganoderma lucidum spore oil are in the 5-15 min and 18.5-25 min ranges of the chromatogram information. Figure 7 This indicates that, in addition to Ganoderma lucidum spore oil, this method is also effective in separating lipid components in oils such as olive oil and soybean oil, providing a theoretical reference for the content determination or qualitative identification of other oils.

[0166] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for determining the content of multiple lipid components in Ganoderma lucidum spore oil using a single-method-multiple-evaluation approach, characterized in that, The method includes using high performance liquid chromatography coupled with ultraviolet detection, using triglyceride as an internal reference, establishing a correction factor for triglyceride and other lipid components, and using the correction factor to calculate the concentration of various analytes in Ganoderma lucidum spore oil. The chromatographic conditions of the high performance liquid chromatography include using acetonitrile-acetone-isooctane as the mobile phase. The other lipid components include 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dilinoleic acid-3-oleic acid glyceride, cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride and 1,2-dioleoyl-3-palmitoyl-rac-glyceride; The chromatographic conditions of the high performance liquid chromatography method include: the mobile phase is acetonitrile-acetone-isooctane with a volume ratio of 57.7:29.1:13.2; the stationary phase is a YMC-Triart-C18 column with a specification of 250×4.6mm DS-3μm 12nm; The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using the one-test-multiple-evaluation method includes the preparation of the test solution: accurately weigh the Ganoderma lucidum spore oil sample, dissolve and dilute it in acetonitrile-isopropanol to obtain the test solution; The wavelength for ultraviolet detection is selected to be 205~206nm.

2. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-method, multi-evaluation approach as described in claim 1, characterized in that, Column temperature 38℃.

3. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-method, multi-evaluation approach as described in any one of claims 1 to 2, characterized in that, Includes the following steps: A. Preparation of the test solution: Accurately weigh the Ganoderma lucidum spore oil sample, dissolve and dilute it in acetonitrile-isopropanol to obtain the test solution; B. Preparation of reference stock solution: B1) Accurately weigh the standards of 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dioleic acid-3-linoleic acid glyceride, and cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride, dissolve them in isopropanol, and dilute them to obtain the reference stock solutions of 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dioleic acid-3-linoleic acid glyceride, and cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride, for later use; B2) Accurately weigh the standards of glycerol trioleate and 1,2-dioleoyl-3-palmitoyl-rac-glycerol, dissolve them in acetonitrile-isopropanol at a volume ratio of 53:47, and dilute them to obtain glycerol trioleate reference stock solution and 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference stock solution for later use; C. Preparation of mixed standard solution: Accurately take the 1,2-dilinoleic acid-3-oleic acid glyceride reference standard stock solution, the cis-1,2-dilinoleic acid-3-oleic acid glyceride reference standard stock solution, the cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride reference standard stock solution, the trioleic acid ester, and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol standard, mix them to prepare a mixed standard solution, which will be used for detection and calculation of the correction factor; D. Preparation of standard curve solutions: Accurately pipette the stock solution of triglyceride reference standard described in step B, and dissolve it to prepare triglyceride standard curve solutions of different concentrations; E. Detection: Accurately pipette the standard curve solutions of triglycerides of different concentrations described in step D and perform high performance liquid chromatography (HPLC) to detect them, and prepare an internal reference standard curve; accurately pipette the test solution described in step A and perform HPLC to detect it, and obtain the chromatogram of the test solution; F. Calculation: The test solution spectrum shows characteristic peaks of triglycerides and other lipid components. Using the internal standard method, the concentration of a certain analyte is calculated by comparing the correction factor with the internal reference standard curve in step E.

4. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-test, multi-evaluation method as described in claim 3, characterized in that, The concentration of a certain analyte in step F is calculated as follows: F1) Take the test solution and perform high-performance liquid chromatography (HPLC) to obtain the chromatogram of the test solution. F2) Based on the spectrum of the test sample solution, the concentration of triglyceride in the test sample solution is obtained through the standard curve of the internal reference in step E; F3) The triglyceride ester in the test solution is used as an internal reference, designated 4x. The analyte in the test solution is designated ax. Based on the chromatogram of the test solution in step F1) and the triglyceride ester concentration in F2), the concentration of the analyte is calculated using the correction factor. The formula for the concentration of the analyte is as follows: This is a correction factor for a certain analyte. A 4x The peak area of ​​the internal reference in the test solution. W 4x The concentration of the internal reference in the test solution. A ax The peak area of ​​a certain analyte in the test solution. W ax The concentration of a certain analyte in the test solution; The correction factor mentioned in step F is obtained as follows: a) Inject the mixed standard solution into a high-performance liquid chromatograph to obtain the chromatogram of the mixed standard solution; b) Based on the chromatogram of the mixed standard solution, triglyceride ester in the mixed standard solution is used as an internal reference and is numbered 4. The number of the analyte in the mixed standard solution is denoted as a. The formula for the relative correction factor between triglyceride ester and the analyte is as follows: This is the relative correction factor between triglyceride and a certain analyte. A4 represents the peak area of ​​the internal reference standard in the mixed standard solution. W4 represents the concentration of the internal reference standard in the mixed standard solution. A a This represents the peak area of ​​a reference standard for a specific analyte in the mixed standard solution. W a This represents the concentration of a reference standard for a specific analyte in the mixed standard solution. c) Inject 10µL, 20µL, 25µL and 30µL of the mixed standard solution respectively, calculate the relative correction factor between the triglyceride ester and the analyte, and take the average value to obtain the correction factor of the analyte.

5. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-method, multi-evaluation approach as described in claim 3, characterized in that... The preparation of the test solution in step A includes the following steps: Accurately weigh the Ganoderma lucidum spore oil sample, dissolve and dilute it to 4.5 mg / mL with acetonitrile-isopropanol at a volume ratio of 53:47 to obtain the test solution; the concentrations of the 1,2-dilinoleic acid-3-oleic acid glyceride reference stock solution, cis-1,2-dioleic acid-3-linoleic acid glyceride reference stock solution, and cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride reference stock solution in step B1) are all 3 mg / mL; the concentrations of the glyceryl trioleate reference stock solution and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference stock solution in step B2) are all 2 mg / mL.

6. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-test, multi-evaluation method as described in claim 3, characterized in that, The preparation of the mixed standard solution in step C includes the following steps: accurately pipetting the 1,2-dilinoleic acid-3-oleic acid glyceride reference stock solution, the cis-1,2-dioleic acid-3-linoleic acid glyceride reference stock solution, the cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride reference stock solution, the trioleic acid ester reference stock solution, and the 1,2-dioleoyl-3-palmitoyl-rac-glyceride reference stock solution from step B, at a volume ratio of 53:

4. Dilute with acetonitrile-isopropanol to prepare a mixed standard solution with the following concentrations: 1,2-dilinoleic acid-3-oleic acid glyceride at 0.12 mg / mL, cis-1,2-dioleic acid-3-linoleic acid glyceride at 0.12 mg / mL, cis-1-palmitoyl-2-oleic acid-3-linoleic acid glyceride at 0.12 mg / mL, trioleic acid trioleate at 0.4 mg / mL, and 1,2-dioleoyl-3-palmitoyl-rac-glycerol at 0.4 mg / mL.

7. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-method, multi-evaluation approach as described in claim 3, characterized in that... The preparation of the standard curve solution in step D includes the following steps: accurately pipetting the stock solution of the triglyceride reference standard in step B, diluting it with acetonitrile-isopropanol at a volume ratio of 53:47, and preparing the triglyceride standard curve solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.2 mg / mL, respectively.

8. The method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a single-method, multi-evaluation approach as described in any one of claims 1 to 2, characterized in that, The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatogram used in the high performance liquid chromatography method includes a Thermo Fisher U3000 high performance liquid chromatograph, the injection volume is 20µL, the mobile phase flow rate is 1.0mL / min, and the mobile phase is maintained for 60 minutes.

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