Method for measuring content of multiple lipid components in ganoderma lucidum spore oil by quantitative analysis of multi-components by single marker

Abstract

The present invention relates to the technical field of quality standards and measurement of health-care food. Disclosed is a method for measuring content of multiple lipid components in Ganoderma lucidum spore oil by quantitative analysis of multi-components by single marker. According to the present invention, high-performance liquid chromatography is used, glycerol trioleate is used as an internal standard, correction factors for glycerol trioleate and other lipid components are established, and the correction factors are used to calculate the concentrations of multiple components to be measured in lipids in Ganoderma lucidum spore oil. According to the present invention, in the high-performance liquid chromatography, acetonitrile-acetone-isooctane is used as a mobile phase, and HPLC-UV combination has strong specificity.

Description

A method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil using a one-test, multiple-evaluation method Technical Field

[0001] The invention belongs to the technical field of quality standards and detection of health foods, and particularly relates to a method for detecting the contents of multiple lipid components in ganoderma lucidum spore oil. Background Art

[0002] Ganoderma lucidum spore oil is derived from the spores of the Polyporaceae fungus Ganoderma lucidum (Leyss. ex Fr.) Karst., obtained through cell wall destruction and supercritical extraction. Studies have shown that Ganoderma lucidum spore oil has beneficial effects in inhibiting tumors, enhancing immune function, improving memory, and lowering blood lipids. As modern people prioritize health and pursue a higher quality of life, health supplements containing Ganoderma lucidum spore oil are entering the market.

[0003] Ganoderma lucidum spore oil contains a variety of chemically active ingredients, including glycerides, sterols, and triterpenes. Among them, lipid components are the main components of Ganoderma lucidum spore oil. Currently, the content of lipid components in Ganoderma lucidum spore oil is mostly detected using high-performance liquid chromatography-eluting light evaporative detector (HPLC-ELSD). It uses the logarithm of concentration and the logarithm of peak area to fit the linear calculation process, which is relatively complicated and tedious with poor reproducibility. There are many types of lipid components in Ganoderma lucidum spore oil, but there are few studies on related content detection. There are known reports that HPLC-ELSD was used to measure 8 types of triglycerides in Ganoderma lucidum spore oil, which means that when detecting the content of lipid components in Ganoderma lucidum spore oil, the experimental operation steps are trivial and the experimental results are calculated with a large amount of effort. In addition, triglycerides are composed of different fatty acids combined with a glycerol backbone. Due to the different carbon chain lengths and degrees of saturation of fatty acids, the types of fatty acids are very large. Or there are also different types of sterols and triterpenoid components that are also bound to the backbone, and the combination has different geometric configurations on the triglycerides, enriching the types of lipid components. The physical and chemical properties of lipid components with different structures are different, which increases the difficulty of separation. At present, the content of lipid components in oils and fats is often detected using isopropanol-acetonitrile in different proportions as the mobile phase, using an ELSD detector, and using the linear fitting method of the logarithm of concentration and the logarithm of peak area for quantitative calculation.

[0004] The QAMS (Qualified Assessment Method) utilizes an internal reference substance and establishes correction factors for other components relative to the reference substance, enabling simultaneous determination of multiple components. This simplifies testing procedures and reduces experimental time and costs. In 2015, the Chinese Pharmacopoeia reported that the QAMS could be used for content determination in traditional Chinese medicines (TCMs), leading to increased research on these materials and Chinese patent medicines. While the QAMS has been reported for determining triterpenes in Ganoderma lucidum-related TCMs, other components, such as lipids, have yet to be determined using the QAMS. The five compounds with the highest content in the disclosed lipid components of Ganoderma lucidum spore oil are triolein (OOO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), cis-1-palmitoyl-2-oleoyl-3-linoleoyl glyceride (POL), cis-1,2-dioleoyl-3-linoleoyl glyceride (OOL), and 1,2-dilinoleoyl-3-oleoyl glyceride (LLO). However, since there are few domestic manufacturers producing lipid component-related reference substances, they are often ordered from abroad, resulting in a long delivery time and high prices for the reference substances.

[0005] Summary of the Invention

[0006] In view of the fact that lipid component reference substances are relatively rare and expensive, and the external standard method is cumbersome in calculation, the present invention provides for the first time a one-test-multiple-evaluation method for detecting the lipid component content in Ganoderma lucidum spore oil, which can accurately detect several lipid components that can be located.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The invention discloses a method for detecting the content of multiple lipid components in Ganoderma lucidum spore oil by a one-test-multiple-evaluation method, which is characterized by comprising adopting high performance liquid chromatography, using triolein as an internal reference, and combining HPLC-UV to establish correction factors for triolein and other lipid components, and using the correction factors to calculate the concentrations of multiple components to be tested in Ganoderma lucidum spore oil, wherein the chromatographic conditions of the high performance liquid chromatography include adopting acetonitrile-acetone-isooctane as the mobile phase.

[0009] As a preferred embodiment of the method of the present invention, the other lipid components include one or more of 1,2-dilinoleyl-3-oleyl glyceride, cis-1,2-dioleyl-3-linoleyl glyceride, cis-1-palmitoyl-2-oleoyl-3-linoleyl glyceride, and 1,2-dioleoyl-3-palmitoyl-rac-glycerol.

[0010] 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 in a volume ratio of 53-57.7:32-29.1:15-13.2; the stationary phase is a C18 chromatographic column, and the column temperature is 35-40°C.

[0011] As a preferred embodiment of the method of the present invention, the mobile phase is acetonitrile-acetone-isooctane in 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 the column temperature is 38°C.

[0012] As a preferred embodiment of the method of the present invention, the method comprises the following steps:

[0013] A. Preparation of test solution: Accurately weigh a sample of Ganoderma lucidum spore oil, dissolve it in a solvent, and dilute it to obtain a test solution;

[0014] B. Preparation of reference substance stock solution:

[0015] B1) accurately weighing the standard substances of 1,2-dilinoleic acid-3-oleic acid glyceride, cis-1,2-dioleic acid-3-linoleic acid glyceride, and cis-1-palmitic acid-2-oleic acid-3-linoleic acid glyceride, respectively, dissolving them with isopropanol, and diluting them to obtain 1,2-dilinoleic acid-3-oleic acid glyceride reference substance stock solution, cis-1,2-dioleic acid-3-linoleic acid glyceride reference substance stock solution, and cis-1-palmitic acid-2-oleic acid-3-linoleic acid glyceride reference substance stock solution, all at a concentration of about 3 mg / mL, for subsequent use;

[0016] B2) Accurately weighing the triolein and 1,2-dioleoyl-3-palmitoyl-rac-glycerol standards, dissolving them in acetonitrile-isopropanol at a volume ratio of 53:47, and diluting them to obtain a triolein reference stock solution and a 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference stock solution, each at a concentration of approximately 2 mg / mL, for later use;

[0017] C. Preparation of mixed standard solution: Accurately take the 1,2-dilinoleyl-3-oleylglycerol reference stock solution, the cis-1,2-dioleyl-3-linoleylglycerol reference stock solution, the cis-1-palmitoyl-2-oleoyl-3-linoleylglycerol reference stock solution, the triolein, and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol standard described in step B, mix them to obtain a mixed standard solution, and reserve it for testing and calculation of the correction factor;

[0018] D. Preparation of standard curve solution: Accurately pipette the triolein reference solution described in step B and dissolve it into triolein standard curve solutions of different concentrations;

[0019] E. Detection: Accurately pipette the triolein standard curve solutions of different concentrations described in step D and perform high performance liquid chromatography detection to prepare an internal reference standard curve; accurately pipette the test solution described in step A and perform high performance liquid chromatography detection to obtain a sample solution spectrum;

[0020] F. Calculation: The characteristic peaks of triolein and the other lipid components are present in the sample solution spectrum. The concentration of a component to be tested is calculated using the internal standard method using the correction factor and the internal reference standard curve described in step E. The calculation method is as follows:

[0021] F1) taking the test solution and performing high performance liquid chromatography to obtain a test solution spectrum,

[0022] F2) obtaining the concentration of triolein in the test solution according to the sample solution profile and the internal reference standard curve described in step E;

[0023] F3) triolein in the test solution is used as an internal reference, numbered 4x, and the number of the component to be measured in the test solution is set to ax. According to the test solution spectrum in step F1) and the triolein concentration in F2), the correction factor is used to calculate the concentration of the component to be measured. The formula is as follows:

[0024] f is the correction factor of a component to be measured,

[0025] A 4x is the peak area of ​​the internal reference substance in the test solution,

[0026] W 4x is the concentration of the internal reference substance in the test solution,

[0027] A ax is the peak area of ​​a component to be measured in the test solution,

[0028] W ax is the concentration of a component to be measured in the test solution.

[0029] Furthermore, the correction factor is obtained as follows:

[0030] a) injecting the mixed standard solution into a high performance liquid chromatograph to obtain a mixed standard solution spectrum;

[0031] b) According to the mixed standard solution atlas, triolein in the mixed standard solution is used as an internal reference and is numbered 4, and the component to be measured in the mixed standard solution is numbered a. The formula for the relative correction factor of triolein and the component to be measured is as follows:

[0032] f4a is the relative correction factor between triolein and a component to be measured,

[0033] A4 is the peak area of ​​the internal reference substance,

[0034] W4 is the concentration of the internal reference substance,

[0035] A a is the peak area of ​​a reference substance of a component to be tested,

[0036] W a is the concentration of the reference substance of a component to be tested;

[0037] c) injecting 10 μL, 20 μL, 25 μL, and 30 μL of the mixed standard solution respectively, calculating the relative correction factors of the triolein and the certain component to be measured, and taking the average value to obtain the correction factor of the certain component to be measured.

[0038] 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 it with acetonitrile-isopropanol in a volume ratio of 53:47 and diluting it to about 4.5 mg / mL to obtain the test solution.

[0039] Furthermore, the preparation of the mixed standard solution in step C of the method of the present invention comprises the following steps: accurately taking the 1,2-dilinoleic acid-3-oleic acid glyceride reference substance stock solution, the cis-1,2-dioleic acid-3-linoleic acid glyceride reference substance stock solution, the cis-1-palmitic acid-2-oleic acid-3-linoleic acid glyceride reference substance stock solution, the triolein reference substance stock solution, and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference substance stock solution in step B, and mixing them in a volume ratio of 53:4 7% acetonitrile-isopropanol to prepare the mixed standard solution with a 1,2-dilinoleo-3-oleoyl glyceryl concentration of about 0.12 mg / mL, a cis-1,2-dioleo-3-linoleoyl glyceryl concentration of about 0.12 mg / mL, a cis-1-palmitoyl-2-oleoyl-3-linoleoyl glyceryl concentration of about 0.12 mg / mL, a triolein concentration of about 0.4 mg / mL, and a 1,2-dioleoyl-3-palmitoyl-rac-glycerol concentration of about 0.4 mg / mL.

[0040] Furthermore, the preparation of the standard curve solution in step D of the method of the present invention comprises the following steps: accurately pipetting the triolein reference substance stock solution in step B into a 5 mL volumetric flask, diluting it with acetonitrile-isopropanol in a volume ratio of 53:47 to prepare the triolein standard curve solution with a concentration of approximately 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.2 mg / mL, respectively.

[0041] 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 equipped with a UV detector, the UV detection wavelength is selected at 205-206 nm, the injection volume is 20 μL, the mobile phase equilibrium flow rate is 1.0 mL / min, and the mobile phase is maintained for up to 60 minutes.

[0042] Furthermore, in addition to being applied to Ganoderma lucidum spore oil, the method of the present invention is also applicable to the separation, purification, detection of lipid component content, identification of component structure or qualitative identification of other oils (such as olive oil, soybean oil, medium-chain oil, etc.).

[0043] While researching the detection of lipid components in Ganoderma lucidum spore oil, the inventors discovered that the determination of lipid components was more stable under ultraviolet light. After extensive experimental exploration, they discovered that using a mobile phase system with a certain ratio to separate lipid components in Ganoderma lucidum spore oil resulted in better separation of structurally similar components, and the separation effect was improved compared to a system using isopropyl alcohol and acetonitrile as the mobile phase. When using a single-measurement, multiple-evaluation method to detect the content of lipid components in Ganoderma lucidum spore oil, the inventors selected triolein (OOO) as an internal reference and successfully established correction factors for LLO, OOL, POL, and OOP using it for calculation.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1) For the first time, the mobile phase was modified with acetonitrile-acetone-isooctane in a volume ratio of (53-57.7):(32-29.1):(15-13.2), which resulted in better separation and enabled the effective separation and quantitative detection of overlapping components, facilitating the study of the structure of the prototype component in the product.

[0046] 2) In view of the current situation that the HPLC-ELSD logarithmic fitting linear method is cumbersome and has poor reproducibility, the HPLC-UV coupling method has strong specificity, small detection error, accurate and reliable results, good reproducibility of results, and a simpler calculation process;

[0047] 3) The present invention adopts a one-test-multiple-evaluation method for determining the content of lipid components in Ganoderma lucidum spore oil for the first time. As lipid component reference substances are relatively expensive and the application of the one-test-multiple-evaluation method is lacking, the present invention fills the gap in the field and uses triolein (OOO) as an internal reference substance, thereby reducing the use of other expensive reference substances. This method can save costs while ensuring controllable product quality and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 is a high performance liquid chromatogram of the lipid components of the Ganoderma lucidum spore oil sample using acetonitrile-isopropanol (55:45) as the mobile phase;

[0049] FIG2 is a high performance liquid chromatogram of the lipid components of the Ganoderma lucidum spore oil sample using acetonitrile-acetone-isooctane (57.7:29.1:13.2) as the mobile phase;

[0050] FIG3 is a high performance liquid chromatogram of the lipid components of the Ganoderma lucidum spore oil sample separated by an acetonitrile-acetone-isooctane (57.7:29.1:13.2)-ELSD system;

[0051] FIG4 is a high performance liquid chromatogram of the lipid components of the Ganoderma lucidum spore oil sample separated by acetonitrile-acetone-isooctane (57.7:29.1:13.2)-UV system;

[0052] FIG5 is a high performance liquid chromatogram of the mixed standard solution in Example 5;

[0053] FIG6 is a high performance liquid chromatogram of the lipid components of the Ganoderma lucidum spore oil sample separated by an acetonitrile-dichloromethane (65:35)-ELSD system;

[0054] Figure 7 is a high performance liquid chromatogram 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 DESCRIPTION

[0055] To enable those skilled in the art to better understand and implement the present invention, the following embodiments are not intended to limit the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are within the scope of the present invention.

[0056] Tables 1 to 4 show the information of the relevant test instruments, standards, reagents and samples used in the examples:

[0057] Table 1 Information of reagents used in the present invention

[0058] Table 2 Instrument information used in the present invention

[0059] Table 3 Information of reference substances (standard substances) used in the present invention

[0060] Table 4 Sample information used in the present invention

[0061] Example 1 Preparation of test solution

[0062] Take 45 mg of Ganoderma lucidum spore oil sample, accurately weigh its mass, dissolve it in a 10 mL volumetric flask with acetonitrile-isopropanol (53:47) and dilute it to the scale, shake well, and use it as the test solution.

[0063] Example 2 Preparation of standard solution

[0064] (1) Preparation of reference substance stock solution

[0065] LLO reference stock solution: Accurately take LLO reference, place it in a 25 mL volumetric flask, dissolve it with isopropanol, and dilute to the scale, the concentration is 3.0308 mg / mL.

[0066] OOL reference substance stock solution: accurately take OOL reference substance, place it in a 25mL volumetric flask, dissolve it with isopropanol, and dilute to the scale, the concentration is 3.1152mg / mL.

[0067] POL reference substance stock solution: accurately take POL reference substance, place it in a 25 mL volumetric flask, dissolve it with isopropanol, and dilute to the scale, the concentration is 3.1080 mg / mL.

[0068] OOO reference substance stock solution: accurately take OOO reference substance, place it in a 25mL volumetric flask, dissolve it in acetonitrile-isopropanol (53:47), and dilute to the scale, the concentration is 2.1705mg / mL.

[0069] OOP reference substance stock solution: accurately take the OOP reference substance, place it in a 25 mL volumetric flask, dissolve it in acetonitrile-isopropanol (53:47), and dilute to the scale to a concentration of 1.6043 mg / mL.

[0070] (2) Preparation of OOO standard curve solution

[0071] Take the OOO reference substance stock solution in this Example (1) as the OOO standard curve solution 5, accurately pipette 4 mL, 3 mL, 2 mL, and 1 mL of the OOO reference substance stock solution into 5 mL volumetric flasks, respectively, dilute to the scale with acetonitrile-isopropanol (53:47), shake well, and obtain OOO standard curve solutions 4 to 1.

[0072] (III) Preparation of mixed standard solution

[0073] Accurately pipette 0.2 mL of the LLO reference stock solution, 0.2 mL of the OOL reference stock solution, 0.2 mL of the POL reference stock solution, 1.0 mL of the OOO reference stock solution, and 1.0 mL of the OOP reference stock solution in this 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.

[0074] Example 3 Chromatographic determination and chromatographic conditions

[0075] Take acetonitrile-isopropanol (53:47) as a blank solution, take 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) according to actual needs, and inject them into the chromatograph for measurement according to the following high-performance liquid chromatography conditions. The high-performance liquid chromatography conditions are as follows:

[0076] Chromatographic column: YMC-Triart-C18 250*4.6mm DS-3μm 12nm

[0077] Instrument: Thermo Fisher U3000 with UV detector

[0078] Mobile phase: acetonitrile-acetone-isooctane (57.7:29.1:13.2)

[0079] Flow rate: constant flow rate 1.0mL / min

[0080] Elution: Keep the mobile phase for up to 60 minutes.

[0081] Column oven: 38°C

[0082] Injection volume: 20 μL (the injection volumes of the mixed standard solution are 10 μL, 20 μL, 25 μL, and 30 μL respectively)

[0083] Detection wavelength: 205-206 nm

[0084] Example 4 Calculation of the content of the component to be measured in the "one test, multiple evaluations" method

[0085] Using triolein (OOO) as the internal reference, 5-1 of the OOO standard curve solution prepared in Example 2 (ii) was taken and injected into the chromatograph according to the HPLC determination and chromatographic conditions of Example 3. The spectrum was recorded to establish an OOO standard curve with concentrations of 0.4341 mg / mL, 0.8682 mg / mL, 1.3023 mg / mL, 1.7364 mg / mL, and 2.1705 mg / mL, respectively.

[0086] A mixed standard solution was prepared according to the steps in Example 2 (iii). The concentration of LLO in the mixed standard solution was 0.1212 mg / mL, the concentration of OOL was 0.1246 mg / mL, the concentration of POL was 0.1243 mg / mL, the concentration of OOO was 0.4341 mg / mL, and the concentration of OOP was 0.3209 mg / mL. The remaining conditions were the same as the HPLC determination and chromatographic conditions of Example 3, and a chromatogram of the mixed standard solution was collected. In the mixed standard results, OOO was selected as the internal reference and numbered 4. The other components were numbered as follows: LLO is 1, OOL is 2, POL is 3, and OOP is 5. The relative correction factor was calculated using the following formula:

[0087] f4a is the relative correction factor between triolein and a component to be measured,

[0088] A4 is the peak area of ​​the internal reference substance;

[0089] W4 is the concentration of the internal reference substance;

[0090] A a is the peak area of ​​a reference substance of a component to be tested;

[0091] W a is the concentration of the reference substance of a component to be tested.

[0092] Inject 10 μL, 20 μL, 25 μL, and 30 μL of the mixed standard solution respectively. After calculating the relative correction factors of different injection volumes of each component, take the average value, which is the correction factor of the component.

[0093] The calculated LLO correction factor is 0.1783, the OOL correction factor is 0.3012, the POL correction factor is 0.2996, and the OOP correction factor is 1.4017 (Table 9).

[0094] Take the test solution prepared in Example 1, and perform the HPLC determination and chromatographic conditions according to Example 3 for the remaining conditions, and record the chromatogram. The actual concentration of OOO in the test solution is obtained by the OOO standard curve; the concentrations of the remaining components to be tested are calculated by the relative correction factor, as follows:

[0095] f is the correction factor of a component to be measured,

[0096] A 4x is the peak area of ​​the internal reference substance in the test solution,

[0097] W 4x is the concentration of the internal reference substance in the test solution,

[0098] A ax is the peak area of ​​a component to be measured in the test solution,

[0099] W ax is the concentration of a component to be measured in the test solution.

[0100] The concentration and content conversion formula can be used to calculate the content of a component to be tested: Content of a component to be tested = Concentration of a component to be tested × Volume of test solution / Mass of the test sample. Wherein: Content is in mg / g, concentration is in mg / mL, the volume of the test solution in Example 1 is 10 mL, and the mass of the test sample is the actual value weighed according to Example 1.

[0101] Example 5 Separation between components in mixed standard solution

[0102] A mixed standard solution was prepared according to the steps in Example 2 (iii). The remaining HPLC conditions were the same as those in Example 3. A 20 μL injection was performed, and the chromatogram was recorded (see Figure 5). The separation between the component peaks was calculated according to the order of peak appearance. The results are shown in Table 5, demonstrating that the mixed standard solution of the present invention exhibited excellent separation under the above conditions.

[0103] Table 5 Separation of mixed standard solution

[0104] Example 6 Sample Separation

[0105] Take the same batch of samples and prepare the test solution 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 the mobile phase, respectively. The other conditions are detected according to the high performance liquid chromatography conditions of Example 3. Inject 20uL, record the spectrum, and calculate the separation between the component peak and the adjacent peak, as shown in Table 6.

[0106] The resolution between each component peak and adjacent peaks was greater than 1, indicating that using a mobile phase system with a certain ratio can effectively separate the lipid components LLO, OOL, POL, OOO, and OOP in Ganoderma lucidum spore oil, with good separation results. Among the two mobile phase ratios, acetonitrile-acetone-isooctane (57.7:29.1:13.2) provided the best separation results.

[0107] Table 6 Sample separation

[0108] Example 7 Confirmation of mobile phase

[0109] The same batch of samples was taken and the test solution was prepared according to the method in Example 1. Acetonitrile-isopropanol (55:45) and acetonitrile-acetone-isooctane (57.7:29.1:13.2) were used as the mobile phase, respectively. The other conditions were detected according to the high performance liquid chromatography conditions of Example 3, and the chromatograms were collected. The same integration processing method was used, and the results were as follows: the chromatogram of the acetonitrile-isopropanol (55:45) system for separating lipid components separated 13 chromatographic peaks, as shown in Figure 1; the chromatogram of the acetonitrile-acetone-isooctane (57.7:29.1:13.2) system for separating lipid components effectively separated 25 chromatographic peaks, as shown in Figure 2.

[0110] It is shown that the use of an acetonitrile-acetone-isooctane (57.7:29.1:13.2) mobile phase system to separate lipid components in oils and fats can effectively separate several adjacent lipid components, and the separation effect is better.

[0111] Example 8 Detector Verification

[0112] Take the same batch of samples and prepare the test solution according to the method of Example 1.

[0113] A UV detector was used in series with an ELSD detector.

[0114] UV detector: Take the test solution and detect it according to the HPLC conditions of Example 3.

[0115] ELSD detector: The test solution was taken for detection. The chromatographic conditions: instrument, chromatographic column, flow rate, column temperature, injection volume, etc. were the same as the HPLC conditions in Example 3; ELSD settings were: drift tube temperature 70°C, air flow 2.0 L / min, gain 1, split mode.

[0116] Record the chromatograms. It can be seen that different detectors have different separation effects under the same mobile phase. In terms of resolution, while the ELSD detector can separate the peaks between OOL and POL, the peaks between OOO and OOP are poorly separated, and the same situation occurs between LLO and its adjacent peak. In terms of the number of separated peaks, the ELSD detector separated 17 peaks (see Figure 3), while the UV detector separated 25 peaks (see Figure 4). This indicates that in this experiment, under the same mobile phase, the UV detector performed better than the ELSD detector.

[0117] Example 9 Method Comparison

[0118] A single-detection, multiple-evaluation method for detecting triglycerides in coix seed has been studied. The isocratic mobile phases used include methanol-acetonitrile (95:5 or 90:10) and acetonitrile-dichloromethane (65:35), the gradient mobile phases include isopropanol-n-hexane, and the detector is an evaporative light detector. The following compares the present method with the prior art method for detecting triglycerides in coix seed using a single-detection, multiple-evaluation method. Unless otherwise noted, the following samples and their processing methods used in the non-present method are the same as those used in the present method:

[0119] 1. The separation effect of the method using methanol-acetonitrile (95:5) as the mobile phase and an evaporative light detector was not significantly different from that of isopropanol-acetonitrile, and only 7 triglyceride peaks were separated. Using an isopropanol-n-hexane gradient as the mobile phase and gradient elution, and using an evaporative light detector for detection, the original method was optimized and 10 triglyceride peaks were separated. However, in the above method, no other peaks were detected between the OOL and POL peaks, or between the OOO and OOP peaks. The method of the present invention uses acetonitrile-acetone-isooctane (57.7:29.1:13.2) as the mobile phase and a UV detector for detection. It can not only detect the triglyceride peaks detected in other systems, but also detect an unknown peak between the OOL and POL peaks, and between the OOO and OOP peaks.

[0120] 2. Similarly, in the Kanglaite injection drug standard, the emulsion fingerprint was tested 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°C. Ganoderma lucidum spore oil was also treated under the above conditions, and the test results were as follows: although an unknown peak was detected between OOL and POL, no unknown peak was detected between OOO and OOP. As above, using acetonitrile-dichloromethane (65:35) as the mobile phase, the chromatogram is shown in Figure 6, with 13 chromatographic peaks separated. The present invention's elution method using acetonitrile-acetone-isooctane (57.7:29.1:13.2) as the mobile phase effectively separated 25 chromatographic peaks, as shown in Figures 2 or 4.

[0121] The results show that when using methanol-acetonitrile (95:5), acetonitrile-dichloromethane (65:35), and isopropanol-n-hexane as mobile phases to detect triglycerides in Ganoderma lucidum spore oil, some peaks are encapsulated by adjacent peaks, resulting in a decrease in the total number of peaks during detection. Compared with the above three methods, the method of the present invention is superior in the separation effect between peaks.

[0122] Example 10 Precision Test

[0123] Take the same batch of samples and prepare the test solution according to the steps in Example 1 to make 6 repeated sample solutions.

[0124] Prepare the reference stock solutions of each component according to the method of Example 2 (I) and dilute them with acetonitrile-isopropanol (53:47) to obtain the standard curve solutions of each component. The concentrations are as follows (unit: mg / mL):

[0125] LLO standard curves 1 to 5: 0.06630, 0.1326, 0.2652, 0.5304, 1.0608

[0126] OOL standard curves 1 to 5: 0.05845, 0.1169, 0.2337, 0.4673, 0.9346

[0127] POL standard curves 1 to 5: 0.07770, 0.1554, 0.3108, 0.6216, 1.2432

[0128] OOO standard curve 1-5: 0.4341, 0.8682, 1.3023, 1.7364, 2.1705

[0129] OOP standard curves 1 to 5: 0.3209, 0.6417, 0.9626, 1.2834, 1.6043

[0130] The standard curve solution and the test solution were separately tested using the HPLC conditions of Example 3. The chromatograms were recorded and calculated using the external standard method. The RSDs for LLO content were 1.85%, OOL content 1.44%, POL content 1.92%, OOO content 1.04%, and OOP content 1.37%. The results are shown in Table 7.

[0131] Table 7 Precision calculation

[0132] Example 11 Accuracy Test

[0133] The 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 10 mL volumetric flasks at the three levels of 80%, 100%, and 120%, respectively. Five reference standards (standards) (LLO, OOL, POL, OOO, and OOP) were added to each level, i.e., each volumetric flask. The mass of each reference standard added was equivalent to the background value of the sample taken at that level. Each volumetric flask was diluted with acetonitrile-isopropanol (53:47) and brought to volume.

[0134] Detection and recording were performed according to the HPLC conditions of Example 3. The standard curve was the same as that in Example 10.

[0135] The calculated accuracy data were: recovery of POL was 94.16% with an RSD of 1.6%; recovery of OOO was 101.44% with an RSD of 1.5%; recovery of OOP was 99.87% with an RSD of 1.1%; recovery of LLO was 97.17% with an RSD of 1.6%; and recovery of OOL was 100.09% with an RSD of 2.3%. This indicates that the method has good accuracy for LLO, OOL, POL, OOO, and OOP in the sample, and is reliable. The results are shown in Table 8.

[0136] Table 8 Accuracy calculation

[0137] Example 12 Confirmation of relative correction factor

[0138] Take the mixed standard solution from Example 4, with LLO concentrations of 0.1212 mg / mL, OOL concentrations of 0.1246 mg / mL, POL concentrations of 0.1243 mg / mL, OOO concentrations of 0.4341 mg / mL, and OOP concentrations of 0.3209 mg / mL. Perform the HPLC test according to the conditions of Example 3, injecting 10 uL, 20 uL, 25 uL, and 30 uL of the solution, respectively. Record the chromatograms and calculate the concentrations as follows:

[0139] Following the calculation of relative correction factors in Example 4, the correction factors for LLO, OOL, POL, and OOP relative to OOO were calculated for each injection volume. The results show that injection of different injection volumes within the range of 10 to 30 μL had no significant effect on the correction factors. The relative correction factor for LLO was 0.1783, with an RSD of 0.78%; the relative correction factor for OOL was 0.3012, with an RSD of 0.94%; the relative correction factor for POL was 0.2996, with an RSD of 0.61%; and the relative correction factor for OOP was 1.4017, with an RSD of 1.25%. See Table 9 for details.

[0140] Table 9 Calculation of correction factors

[0141] Example 13 Comparison of External Standard Method and One-Test-Multiple-Evaluation Method

[0142] The replicate solutions 1 to 6 in the precision test of Example 10 were calculated using the single-measurement, multiple-evaluation method (calculation) in Example 4, with OOO as the internal reference, to calculate the contents of the components LLO, OOL, POL, and OOP. The results were then compared with the data obtained by the external standard method (determination). The results were as follows: the RSD of the LLO content was 3.21%; the RSD of the OOL content was 3.86%; the RSD of the POL content was 2.58%; and the RSD of the OOP content was 1.60%. This indicates that the RSDs of the contents of each component obtained by multiple measurements and calculations were all less than 4%, and there was no significant difference between the two content calculation methods. This indicates that the single-measurement, multiple-evaluation method is reliable for determining the lipid content in Ganoderma lucidum spore oil. The details are shown in Table 10.

[0143] Table 10 Comparison between external standard method and one test multiple evaluation method

[0144] Take different batches of spore oil, and use the one-measurement-multiple-evaluation method (calculation) and the external standard method (determination) to obtain the lipid component content respectively. The one-measurement-multiple-evaluation method uses OOO as the internal reference and is calculated according to the method of Example 4. The obtained content is specifically shown in Table 11, and the results are as follows: in each batch, the RSDs of the contents obtained by the determination of different components using the external standard method and the calculation using the one-measurement-multiple-evaluation method are all less than 5%, and the difference is small, indicating that the one-measurement-multiple-evaluation method can be widely used in the content measurement of lipid components in Ganoderma lucidum spore oil.

[0145] Table 11 Lipid content of 5 kinds of lipid components in different batches (unit: mg / g)

[0146] Example 14: Comparison of the application of the one-test-multiple-evaluation method to the detection of other oils and fats

[0147] Take Ganoderma lucidum spore oil, olive oil, soybean oil, and medium-chain oil, and prepare test solutions according to the steps in Example 1. Detection is carried out according to the HPLC conditions of Example 3, 20 uL is injected, and the recorded spectrum is shown in Figure 7 (1 is olive oil, 2 is soybean oil, 3 is medium-chain oil, and 4 is Ganoderma lucidum spore oil).

[0148] The test results show significant differences between soybean oil, medium-chain oil, and Ganoderma lucidum spore oil, primarily in the 5-15 minute and 25-40 minute intervals. Differences between olive oil and Ganoderma lucidum spore oil are located in the 5-15 minute and 18.5-25 minute intervals. Figure 7 demonstrates that, in addition to Ganoderma lucidum spore oil, this method effectively separates lipid components in olive oil, soybean oil, and other oils, providing a theoretical basis for the content determination or qualitative identification of other oils.

[0149] The above disclosure is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for detecting the contents of multiple lipid components in Ganoderma lucidum spore oil by the method of multi-component determination with a single marker, characterized in that, It includes using high performance liquid chromatography combined with ultraviolet detection, taking triolein as an internal reference substance, establishing correction factors for triolein and other lipid components, and calculating the concentrations of various components to be measured in Ganoderma lucidum spore oil by using the correction factors. The chromatographic conditions of the high performance liquid chromatography include using acetonitrile-acetone-isooctane as the mobile phase.

2. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the one-test-multiple-evaluation method according to claim 1, wherein The other lipid components include one or more of 1,2-dilinoleoyl-3-oleoyl-glycerol, cis-1,2-dioleoyl-3-linoleoyl-glycerol, cis-1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol, and 1,2-dioleoyl-3-palmitoyl-rac-glycerol.

3. The method for detecting the contents of various lipid components in ganoderma spore oil by the one-test multi-evaluation method according to claim 1, characterized in that, 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 chromatographic column, and the column temperature is 35-40 °C.

4. The method for detecting the contents of various lipid components in ganoderma spore oil by the one-test multi-evaluation method according to claim 3, characterized in that, The chromatographic conditions of the high performance liquid chromatography 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 chromatographic column with a specification of 250*4.6mm DS-3μm 12nm, and the column temperature is 38 °C.

5. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the one-test multi-evaluation method according to any one of claims 1 to 4, characterized in that, It includes the following steps: A. Preparation of the test solution: Weigh precisely a sample of Ganoderma lucidum spore oil, dissolve and dilute it in a solvent to obtain the test solution. B. Preparation of the stock solution of reference substances: B1) Weigh precisely the reference substances of 1,2-dilinoleoyl-3-oleoyl-glycerol, cis-1,2-dioleoyl-3-linoleoyl-glycerol, and cis-1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol, dissolve them respectively in isopropanol, and dilute to obtain the stock solutions of 1,2-dilinoleoyl-3-oleoyl-glycerol reference substance, cis-1,2-dioleoyl-3-linoleoyl-glycerol reference substance, and cis-1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol reference substance for standby. B2) Weigh precisely the reference substances of triolein and 1,2-dioleoyl-3-palmitoyl-rac-glycerol, dissolve them respectively in acetonitrile-isopropanol with a volume ratio of 53:47, and dilute to obtain the stock solutions of triolein reference substance and 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference substance for standby. C. Preparation of the mixed standard solution: Precisely take the stock solutions of 1,2-dilinoleoyl-3-oleoyl-glycerol reference substance, cis-1,2-dioleoyl-3-linoleoyl-glycerol reference substance, cis-1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol reference substance, the reference substances of triolein and 1,2-dioleoyl-3-palmitoyl-rac-glycerol in step B, mix and prepare to obtain the mixed standard solution, which is reserved for detection and calculation of the correction factor. D. Preparation of the standard curve solution: Precisely absorb the stock solution of triolein reference substance in step B, dissolve and prepare standard curve solutions of triolein with different concentrations. E. Detection: Accurately pipette the triglyceride standard curve solutions with different concentrations described in step D for high-performance liquid chromatography (HPLC) detection respectively to prepare an internal standard curve; accurately pipette the test solution described in step A for HPLC detection to obtain the test solution chromatogram. F. Calculation: Characteristic peaks of triglyceride and other lipid components appear in the test solution chromatogram. Using the internal standard method and through the correction factor and the internal standard curve in step E, calculate the concentration of a certain component to be measured.

6. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the one-test-multiple-evaluation method according to claim 5, characterized in that, The calculation method for the concentration of a certain component to be measured in step F is as follows: F1) Take the test solution and perform HPLC detection to obtain the test solution chromatogram. F2) According to the test solution chromatogram, obtain the triglyceride concentration in the test solution through the internal standard curve in step E. In the test sample solution, triolein is used as an internal reference and is numbered 4x. The number of a certain component to be measured in the test sample solution is set as ax. According to the test sample solution chromatogram described in step F1) and the triolein concentration described in F2), the calculation is performed through the correction factor. The formula for the concentration of a certain component to be measured is as follows: f is the correction factor of a certain component to be measured. A 4x is the peak area of the internal reference substance in the test solution, W 4x is the concentration of the internal reference substance in the test solution, A ax is the peak area of a certain component to be measured in the test solution, W ax is the concentration of a certain component to be measured in the test sample solution; The correction factor in step F is obtained through the following method: a) Inject the mixed standard solution into the HPLC instrument to obtain the mixed standard solution chromatogram. b) According to the chromatogram of the mixed standard solution, triolein in the mixed standard solution is used as an internal reference substance with the number 4, and the number of a certain component to be measured in the mixed standard solution is set as a. The formula for the relative correction factor between triolein and the certain component to be measured is as follows: f 4a is the relative correction factor of triolein to a certain component to be measured, A4 is the peak area of the internal standard reference substance in the mixed standard solution. W4 is the concentration of the internal standard reference substance in the mixed standard solution. A a is the peak area of the reference substance of a certain component to be measured in the mixed standard solution, W a is the concentration of the reference substance of a certain component to be measured in the mixed standard solution; c) Inject the mixed standard solution in volumes of 10 μL, 20 μL, 25 μL, and 30 μL respectively. After obtaining the relative correction factors of the triglyceride and the certain component to be measured, take the average value to obtain the correction factor of the certain component to be measured.

7. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the method of multi - component determination with one measurement as claimed in claim 5, characterized in that, The preparation of the test solution described 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 with a volume ratio of 53:47 to obtain the test solution; the concentrations of the 1,2-dilinoleoyl-3-oleoyl glycerol reference substance stock solution, cis-1,2-dioleoyl-3-linoleoyl glycerol reference substance stock solution, and cis-1-palmitoyl-2-oleoyl-3-linoleoyl glycerol reference substance stock solution in step B1) are all 3 mg / mL; the concentrations of the triglyceride reference substance stock solution and 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference substance stock solution in step B2) are all 2 mg / mL.

8. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the multi-component assay method according to claim 5, characterized in that, The preparation of the mixed standard solution described in step C includes the following steps: Precision pipette the 1,2-dilinoleoyl-3-oleoyl-glycerol reference substance stock solution, the cis-1,2-dioleoyl-3-linoleoyl-glycerol reference substance stock solution, the cis-1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol reference substance stock solution, the triolein reference substance stock solution, and the 1,2-dioleoyl-3-palmitoyl-rac-glycerol reference substance stock solution described in step B, and dilute with acetonitrile-isopropanol with a volume ratio of 53:47 to prepare a mixed standard solution with a concentration of 0.12 mg / mL for 1,2-dilinoleoyl-3-oleoyl-glycerol, a concentration of 0.12 mg / mL for cis-1,2-dioleoyl-3-linoleoyl-glycerol, a concentration of 0.12 mg / mL for cis-1-palmitoyl-2-oleoyl-3-linoleoyl-glycerol, a concentration of 0.4 mg / mL for triolein, and a concentration of 0.4 mg / mL for 1,2-dioleoyl-3-palmitoyl-rac-glycerol.

9. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the one-test multi-evaluation method according to claim 5, characterized in that, The preparation of the standard curve solution described in step D includes the following steps: Precision pipette the triolein reference substance stock solution described in step B, and dilute with acetonitrile-isopropanol with a volume ratio of 53:47 to prepare triolein 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.

10. The method for detecting the contents of various lipid components in Ganoderma lucidum spore oil by the multi-component determination method with one measurement as claimed in any one of claims 1 to 4, characterized in that, The chromatographic conditions of the high performance liquid chromatography method are as follows: The chromatograph used in the high performance liquid chromatography method includes the Thermo Fisher U3000 high performance liquid chromatograph, the injection volume is 20 μL, the constant flow rate of the mobile phase is 1.0 mL / min, and the mobile phase is maintained for 60 minutes; the wavelength of the ultraviolet detection is selected from 205 to 206 nm.

Images