Method for detecting contents of various components in puffball sample and application of method

Abstract

The invention discloses a method for detecting the content of various components in a puffball sample and application of the method, and the method comprises the following steps: taking puffball sample powder, adding an extraction solvent for extraction, and preparing to obtain a test solution; taking alpha-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid and ergosterol reference substances, and adding a solvent to prepare a mixed reference substance solution; respectively injecting the mixed reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, and comparing the maps of the reference substance solution and the test solution; and making a regression equation by using the sample size Ln value and the peak area Ln value of each component, and calculating the content of the components in the puffball sample. According to the quality detection method for the puffball sample, the contents of the six components can be simultaneously determined, the operation is simple, the accuracy is high, the recovery rate is reliable, the detection efficiency is high, and a basis is provided for standardizing the market of puffball medicinal materials and preparations and reasonably developing the puffball medicinal materials and preparations.

Description

Technical Field

[0001] This invention relates to the quality testing of puffballs, specifically to a method for detecting the content of multiple components in a puffball sample and its application. Background Technology

[0002] Puffballs are the dried fruiting bodies of the fungi *Calvatia pekinensis*, *Calvatia macrophylla*, or *Calvatia purpurea*, belonging to the family Lycopodiaceae. First recorded in the *Mingyi Bielu* (Records of Famous Physicians), they are used to clear the lungs and soothe the throat, and stop bleeding. Internally, they are used for sore throat, hoarseness, and cough due to wind-heat stagnation in the lungs; externally, they are used to treat epistaxis and traumatic bleeding. Modern research shows that puffballs contain amino acids, proteins, steroids, terpenes, and other components, possessing various pharmacological effects such as anti-inflammatory, hemostatic, anti-tumor, and antioxidant properties.

[0003] Regarding the quality control of puffball, the current edition of the Chinese Pharmacopoeia only includes items on properties, identification, inspection, and extracts, and does not yet include items on indicator components and content determination. Currently, there are many studies on the content determination of puffball, such as Yang Xiulu et al. determining the content of ergosterol and ergosterone in puffball with small stalks, Li Aixin et al. determining the content of mannitol in puffball with star-shaped hard skin, Zhou Xian et al. determining the content of ergosterol in puffball with star-shaped hard skin, and Meng Jingjing et al. determining the content of white linear cyclic lipopeptides in puffball with peeled skin. However, there are no reports on the determination of fatty acid components in puffball, especially no reports on methods for simultaneously determining multiple fatty acid components in puffball. Summary of the Invention

[0004] Purpose of the Invention: The purpose of this invention is to provide a method for detecting the content of multiple components in puffball samples and its application. Addressing the current limitations in the availability of fatty acid component indicators for quality evaluation of puffball samples (e.g., crude drugs, processed slices, and preparations), particularly the scarcity of multi-indicator fatty acid component content determination methods, this invention proposes to simultaneously apply six components—α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol—to the quality detection of puffball samples. A content determination method using high-performance liquid chromatography (HPLC) is established, providing a new analytical tool for controlling the intrinsic quality of puffball samples.

[0005] Technical solution: The method for detecting the content of multiple components in a puffball sample according to the present invention includes the following steps:

[0006] (1) Preparation of test solution: Take puffball sample powder, add extraction solvent to extract, and prepare test solution;

[0007] (2) Preparation of reference solution: Take α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid and ergosterol reference standards, add solvent to prepare a mixed reference solution;

[0008] (3) Chromatographic detection: The mixed reference solution and the test solution were injected into an ultra-high performance liquid chromatograph for detection, and the chromatograms of the reference solution and the test solution were compared.

[0009] (4) Content calculation: The content of the components in the puffball sample is calculated by using the injection amount Ln value and the peak area Ln value of the components to form a regression equation; the components are α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid and ergosterol.

[0010] Preferably, in step (1), take the puffball sample powder, weigh it accurately, place it in a container, add the extraction solvent accurately, seal it tightly, weigh it, extract it, let it cool, weigh it again, use the extraction solvent to make up the lost weight, shake it well, filter it, take three-fifths of the volume of the filtrate, evaporate it to dryness, add the extraction solvent to the residue to redissolve it, transfer it to a quantitative container, add the extraction solvent to make up the volume, shake it well, filter it, take the filtrate, and the test solution is obtained.

[0011] Preferably, the extraction solvent in step (1) includes at least one of methanol, ethanol, ethyl acetate, and petroleum ether. More preferably, the extraction solvent is methanol.

[0012] Preferably, the mass-to-volume ratio of puffball sample powder to extraction solvent in step (1) is 1 g: (7.5-25 mL); if the sample concentration is too low or too high, it may exceed the linear relationship range.

[0013] Preferably, the extraction method in step (1) includes ultrasonic treatment, heating and reflux, or shaking. Preferably, the extraction method in step (1) is heating and reflux extraction.

[0014] Preferably, the extraction time in step (1) is 30 to 120 minutes.

[0015] More preferably, the solvent in step (2) is methanol, ethanol, ethyl acetate, or petroleum ether. Preferably, to simplify the practical steps, the solvent used to prepare the reference solution can be the same as the extraction solvent used to prepare the test solution, and the extraction solvent is preferably methanol.

[0016] Preferably, the conditions of the ultra-high performance liquid chromatograph in step (3) are as follows: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and 0.05-0.15% formic acid solution as mobile phase B, with gradient elution: 0-15 minutes, 92% A; 15-30 minutes, 92%-100% A; 30-35 minutes, 100% A; preferably 0.1% formic acid solution.

[0017] Preferably, the flow rate is 0.65 mL / min to 0.8 mL / min, the column temperature is 25℃ to 35℃, and an electro-fogging detector is used.

[0018] Further preferred options include a flow rate of 0.8 mL / min and a column temperature of 30-35 °C.

[0019] Further preferred options include TC C18(2) (Agilent, 4.5×250 mm, 5 μm), ZORBAX SB-C18 (Agilent, 4.6 x 250 mm, 5 μm), or Eclipse XDB-C18 (Agilent, 4.6 x 250 mm, 5 μm).

[0020] Preferably, in step (4), the regression equation for the injection amount Ln value of α-linolenic acid and the peak area Ln value is Y = 1.039X + 2.655, R ≥ 0.999; the regression equation for the injection amount Ln value of linoleic acid and the peak area Ln value is Y = 0.701X + 2.040, R ≥ 0.999; and the regression equation for the injection amount Ln value of palmitic acid and the peak area Ln value is Y = 0.874X + 1.863, R ≥ The regression equations for the injection volume Ln value and peak area Ln value of oleic acid are Y = 0.778X + 2.015, R ≥ 0.999; the regression equations for the injection volume Ln value and peak area Ln value of stearic acid are Y = 1.042X + 2.443, R ≥ 0.999; and the regression equations for the injection volume Ln value and peak area Ln value of ergosterol are Y = 0.888X + 2.076, R ≥ 0.999.

[0021] More preferably, in step (4), the linear range of α-linolenic acid is 0.049–0.410 μg / mL; the linear range of linoleic acid is 3.723–31.025 μg / mL; the linear range of palmitic acid is 0.883–7.359 μg / mL; the linear range of oleic acid is 1.543–12.859 μg / mL; the linear range of stearic acid is 0.119–0.992 μg / mL; and the linear range of ergosterol is 0.579–4.826 μg / mL.

[0022] The application of the method described in this invention in the quality control or quality testing of puffball samples.

[0023] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention applies fatty acid components to the quality control of puffball. By rationally controlling the conditions of liquid chromatography, a method for simultaneously determining the content of five fatty acid components (α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, etc.) and six components (ergosterol) in puffball samples has been established, providing a new analytical means for the quality detection of puffball samples; 2. The method of the present invention is simple to operate, highly accurate, has reliable recovery rate, and high detection efficiency, which can provide a basis for standardizing the market and rational development of puffball medicinal materials and preparations. Attached Figure Description

[0024] Figure 1 The liquid chromatograms are of reference standards for α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol, where the chromatographic peaks numbered 1 to 6 are, in order, α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol.

[0025] Figure 2 Liquid chromatograms of puffball extract with different extraction solvents;

[0026] Figure 3 Liquid chromatograms of puffball medicinal materials extracted using different methods;

[0027] Figure 4 Liquid chromatograms of puffball extract at different extraction volumes;

[0028] Figure 5 Liquid chromatograms of puffball herbs extracted at different times;

[0029] Figure 6 Standard curves for six component reference standards;

[0030] Figure 7 Liquid chromatogram of the specificity test results for the determination of puffball medicinal content;

[0031] Figure 8 Liquid chromatograms of puffball herbs at different flow rates;

[0032] Figure 9 The liquid chromatograms of puffball medicinal materials were determined using different column temperatures.

[0033] Figure 10 Liquid chromatography columns used to determine the properties of puffball medicinal materials using different chromatographic columns;

[0034] Figure 11 The liquid chromatograms of puffball medicinal materials were determined using formic acid solutions of different concentrations. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings. Unless otherwise stated, the instruments, reagents, materials, etc. used in the present invention can all be obtained through conventional commercial means.

[0036] Example 1: Construction of a method for simultaneous determination of six components in puffball

[0037] 1. Instruments, reagents and samples

[0038] Thermo Vanquish ultra-high performance liquid chromatograph; Thermo Vanquish electro-cavitation detector (CAD); Chromeleam 7.2SR4 Chameleon workstation; Agilent TC-C18(2) column (column length: 250mm, inner diameter: 4.6mm, particle size: 5μm); KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); METTLER TOLEDO XP6 part per million balance (Mettler-Toledo (Shanghai) Instrument Co., Ltd.); ME204E / 02 electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); pure water system (Sartorius); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.). Acetonitrile (chromatographic grade, Thermo Fisher Scientific); water was ultrapure water; formic acid (chromatographic grade, Aladdin Scientific); all other reagents were of analytical grade.

[0039] Alpha-linolenic acid (China National Institutes for Food and Drug Control, purity 99.8%, batch number 111631-201605), linoleic acid (China National Institutes for Food and Drug Control, purity 99.6%, batch number 111622-202105), palmitic acid (Chengdu Pusi Biotechnology Co., Ltd., purity 98%, batch number PS020930), oleic acid (China National Institutes for Food and Drug Control, purity 99.3%, batch number 111621-202409), stearic acid (Shanghai Shidande Standard Technical Service Co., Ltd., purity 98%, batch number 14706), ergosterol (China National Institutes for Food and Drug Control, purity 96.2%, batch number 111845-201604) are for content determination and do not require pretreatment.

[0040] The samples of puffball medicinal materials were collected by Jiangyin Tianjiang Pharmaceutical Co., Ltd. from Heilongjiang, Jilin and other regions, totaling 12 batches (numbered D1 to D12 respectively). They were identified as the dried fruiting bodies of the fungus Calvatia gigantea (Batsch ex Pers.) Lloyd of the family Calvatiaceae.

[0041] 2. Preparation of reference solution

[0042] Accurately weigh appropriate amounts of α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol reference standards, and dissolve them in methanol to prepare mixed solutions with concentrations of 16.4171 μg / mL, 1241.016 μg / mL, 294.434 μg / mL, 514.374 μg / mL, 39.69 μg / mL, and 193.0253 μg / mL, respectively. The liquid chromatography results are shown in the figure. Figure 1 .

[0043] 3. Preparation of the test solution

[0044] Take about 2g of puffball powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of methanol, stopper tightly, weigh it, heat under reflux for 90 minutes, cool it, weigh it again, make up the lost weight with the appropriate solvent, shake well, filter, take 15mL of the filtrate, evaporate to dryness, dissolve the residue in methanol and make up to 5mL, shake well, filter, and take the filtrate to obtain the product.

[0045] 4. Determination of analytical methods

[0046] 4.1 Chromatographic conditions

[0047] A TC-C18(2) (Agilent, 4.6 mm × 250 mm, 5 μm) column was used. Methanol was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed: 0–15 min, 92% A; 15–30 min, 92%–100% A; 30–35 min, 100% A. The column temperature was 35℃, and the flow rate was 0.8 mL / min. An electro-fogging detector was used. 1 μL and 25 μL of the reference solution and 25 μL of the test solution were accurately pipetted into the liquid chromatograph and analyzed. The result was calculated using the logarithmic equation of the two-point external method.

[0048] The results showed that the detection baseline was stable, the target components were well separated, and the detection could be completed within 35 minutes.

[0049] 4.2 Investigation on the preparation of the test solution

[0050] (1) Investigation of extraction solvent

[0051] Methods: Approximately 2g of powdered puffball (D4) was accurately weighed into four groups, with two replicates per group. Each group was placed in a stoppered conical flask, and 25mL of methanol, ethanol, ethyl acetate, and petroleum ether were accurately added to each flask. The flasks were sealed, weighed, and heated under reflux for 90 minutes. After cooling, the flasks were weighed again. The lost weight was replenished with the appropriate solvent, and the mixture was shaken well and filtered. 15mL of the filtrate was collected, evaporated to dryness, and the residue was dissolved in methanol and brought to a final volume of 5mL. The mixture was shaken well, filtered, and the filtrate was collected. 25μL of each test solution was accurately injected into the liquid chromatograph under the chromatographic conditions described in section “4.1”. The contents of α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol were calculated. The results are shown in Table 1. Figure 2 .

[0052] Table 1 Comparison of different extraction solvents

[0053]

[0054] Conclusion: The total content of the six components was relatively low when petroleum ether was used as the extraction solvent, while the total content was higher when extracted with methanol, ethanol, or ethyl acetate; all three can be used as extraction solvents. Considering that the total content of the six components was highest when methanol was used, methanol is the preferred extraction solvent.

[0055] (2) Examination of extraction methods

[0056] Methods: Approximately 2g of powdered puffball (D4) was taken, divided into three groups (two replicates per group). The samples were accurately weighed and placed in stoppered conical flasks. 25mL of methanol was accurately added, the flasks were sealed, and the samples were weighed. The samples were then subjected to ultrasonic treatment (250W, 40kHz), reflux heating, and shaking extraction for 90 minutes. After cooling, the samples were weighed again. The lost weight was replenished with the appropriate solvent, the mixture was shaken well, and filtered. 15mL of the filtrate was collected, evaporated to dryness, and the residue was dissolved in methanol and brought to a final volume of 5mL. The mixture was shaken well, filtered, and the filtrate was collected. 25μL of each test solution was accurately injected into the liquid chromatograph under the chromatographic conditions described in section “4.1”. The contents of α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol were calculated. The results are shown in Table 2. Figure 3 .

[0057] Table 2 Comparison of Extraction Methods

[0058]

[0059] Conclusion: The total content of the six components was relatively high when heating and reflux were used, therefore heating and reflux extraction was chosen.

[0060] (3) Investigation of the volume of the extraction solvent

[0061] Methods: Approximately 2g of powdered puffball (D4) was taken, divided into three groups (two replicates per group). The powders were accurately weighed and placed in stoppered conical flasks. 15mL, 25mL, and 50mL of methanol were accurately added to each flask, respectively. The flasks were sealed, weighed, and heated under reflux for 90 minutes. After cooling, the flasks were weighed again. The lost weight was replenished with the appropriate solvent, and the mixture was shaken well and filtered. 10mL, 15mL, and 25mL of the filtrate were collected, respectively, and evaporated to dryness. The residue was dissolved in methanol and diluted to a final volume of 5mL. The mixture was shaken well, filtered, and the filtrate was collected. 25μL of each test solution was accurately injected into the liquid chromatograph under the chromatographic conditions described in section “4.1”. The contents of α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol were calculated. The results are shown in Table 3. Figure 4 .

[0062] Table 3 Comparison of extraction volumes

[0063]

[0064] Conclusion: The total content of the six components obtained when the extraction volume was 25 mL was relatively high, indicating that the extraction was sufficient when the extraction solvent was 25 mL. Therefore, the solvent addition amount was selected as 25 mL.

[0065] (4) Examination of extraction time

[0066] Methods: Approximately 2g of powdered puffball (D4) was taken, divided into 4 groups (two replicates per group). The powder was accurately weighed and placed in stoppered conical flasks. 25mL of methanol was accurately added to each flask, which was then sealed and weighed. The flasks were heated under reflux for 30, 60, 90, and 120 minutes respectively, cooled, and weighed again. The lost weight was replenished with the appropriate solvent, shaken well, and filtered. 15mL of the filtrate was collected, evaporated to dryness, and the residue was dissolved in methanol and diluted to 5mL. The filtrate was shaken well, filtered, and collected as the final product. 25μL of each test solution was accurately injected into the liquid chromatograph under the chromatographic conditions described in section "4.1". The contents of α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol were calculated. The results are shown in Table 4. Figure 5 .

[0067] Table 4 Comparison of extraction times

[0068]

[0069] Conclusion: The total content of the six components obtained from different heating reflux times did not differ significantly, indicating that extraction times of 30–120 minutes are acceptable. Considering that the total content of the six components is relatively higher at 90 minutes, 90 minutes is the preferred extraction time.

[0070] Based on the above investigation, the method for preparing the test solution is determined as follows:

[0071] Take approximately 2g of puffball powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of methanol, seal tightly, weigh, heat under reflux for 90 minutes, cool, weigh again, replenish the lost weight with the appropriate solvent, shake well, filter, take 15mL of the filtrate, evaporate to dryness, dissolve the residue in methanol and make up to 5mL, shake well, filter, and take the filtrate to obtain the final product.

[0072] (5) Testing of different batches

[0073] Twelve batches of samples from different locations were collected and prepared according to the method for preparing the test solution specified in section "3". The samples were then analyzed under the chromatographic conditions specified in section "4.1". The specific results are shown in Table 5.

[0074] Table 5. Content and total content of six components in different batches of puffball samples.

[0075]

[0076] The results showed that the contents of α-linolenic acid in different batches of puffball samples (numbered D1-D12) ranged from 0.017 to 0.053 mg / g, linoleic acid from 0.675 to 3.422 mg / g, palmitic acid from 0.417 to 0.954 mg / g, oleic acid from 0.523 to 0.920 mg / g, stearic acid from 0.049 to 0.098 mg / g, and ergosterol from 0.176 to 0.691 mg / g. The total content of the six components ranged from 2.165 to 5.960 mg / g, indicating that the contents of the six components varied among different batches. Among them, the content of linoleic acid fluctuated the most, with the maximum value being about 5 times the minimum value, indicating that linoleic acid best reflects the quality differences of puffballs.

[0077] Example 2: Methodological validation of the content determination method

[0078] 1. Linear relationship

[0079] Accurately pipette 3, 5, 8, 10, 15, 20, and 25 μL of α-linolenic acid (16.4171 μg / mL) reference solution, 3, 5, 8, 10, 15, 20, and 25 μL of linoleic acid (1241.016 μg / mL) reference solution, 3, 5, 8, 10, 15, 20, and 25 μL of palmitic acid (294.343 μg / mL) reference solution, and 3, 5, 8, 10, and 15 μL of oleic acid (514.374 μg / mL) reference solution, respectively. 20, 25 μL of stearic acid (concentration 39.69 μg / mL) reference solution and 3, 5, 8, 10, 15, 20, 25 μL of ergosterol (concentration 193.0253 μg / mL) reference solution were injected into the high-performance liquid chromatograph (HPLC) and analyzed under the chromatographic conditions described in Example 1, "4.1". A standard curve was plotted with peak area (Ln) as the ordinate and injection volume (Ln) as the abscissa. The regression equation was obtained, and the results are shown in Table 6. The chromatogram is shown in [Figure 1]. Figure 6 .

[0080] Table 6. Linear regression equations for each component

[0081]

[0082] The results show that the injection amount Ln value of the above components has a good linear relationship with the peak area Ln value.

[0083] 2. Precision test

[0084] Method of investigation: Take samples from the same batch (number D4), prepare test solutions according to the test solution preparation method determined in section "3" of Example 1, accurately pipette 3 μL of test solution, inject into ultra-high performance liquid chromatograph, inject under the chromatographic conditions in section "4.1" of Example 1, inject continuously 6 times, record the peak areas of the six components, calculate the relative standard deviation, and the results are shown in Table 7.

[0085] Table 7 Instrument Precision Test (Peak Area)

[0086]

[0087] Results: The peak area RSD of all six components was less than 2%, and the instrument precision test was good.

[0088] 3. Repeatability test

[0089] Methods of investigation: Take about 2g of the same batch of puffball sample (number: D4), accurately weigh it, make 6 parallel samples, prepare the sample according to the preparation method of the test solution under section "3" of Example 1, and analyze it according to the chromatographic conditions under section "4.1" of Example 1. Measure the peak area of ​​the six components, calculate the content and RSD value. The results are shown in Table 8.

[0090] Table 8 Repeatability Tests of Puffball Content in Samples

[0091]

[0092]

[0093] Results: The RSD of each component was less than 3%, and the repeatability test was good.

[0094] 4. Stability test

[0095] Methods of investigation: One sample of puffball (number D4) was taken, and the test solution was prepared according to the preparation method of the test solution determined in Section 1 "3". 25 μL of the sample was injected every 4 hours, and the peak area was measured and the RSD value was calculated. The results are shown in Table 9.

[0096] Table 9. Results of stability test (peak area)

[0097]

[0098] Results: The RSD of the peak area of ​​each component was less than 3% within 24 hours, indicating that the test solution had good stability within 24 hours.

[0099] 5. Accuracy Test

[0100] Methods of Investigation: Six replicates of a known concentration (α-linolenic acid 0.0047%, linoleic acid 0.3213%, palmitic acid 0.0989%, oleic acid 0.0908%, stearic acid 0.0093%, ergosterol 0.0426%) were accurately weighed and mixed. α-linolenic acid (0.003831 mg / mL), linoleic acid (0.217128 mg / mL), and palmitic acid (0.063543 mg / mL) were added to each replicate, respectively. 15 mL of reference solutions of oleic acid (0.074078 mg / mL), stearic acid (0.006164 mg / mL), and ergosterol (0.028745 mg / mL) were prepared according to the preparation method of the test solution determined in Section 1 "3" of Example 1. The samples were then injected under the chromatographic conditions in Section 1 "4.1" of Example 1, with 25 μL injected respectively. The recovery rate and RSD were calculated using the following formulas. The results are shown in Table 10.

[0101]

[0102] Table 10 Accuracy Experiment

[0103]

[0104]

[0105]

[0106] Results: The average recoveries of α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, and ergosterol were 95.74%, 105.89%, 94.86%, 102.56%, 98.77%, and 98.34%, respectively, and the accuracy test was good.

[0107] 6. Specificity test

[0108] Method of investigation: The test solution prepared from blank solvent and puffball sample (number D4) (prepared according to the method for preparing the test solution determined under section "3" of Example 1) was injected under the chromatographic conditions under section "4.1" of Example 1. The results are shown in [Figure 1]. Figure 7 .

[0109] Conclusion: The results show that the blank solvent did not interfere with the determination of the six components, indicating that this method has high specificity.

[0110] 7. Durability test

[0111] 7.1 Investigation of different flow velocities

[0112] Methods: One sample of puffball (number D4) was prepared according to the method for preparing the test solution as determined in Example 1, Section 3. A TC C18(2) (Agilent, 4.5×250mm, 5μm) column was used as the chromatographic column. Methanol was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed: 0–15 min, 92% A; 15–30 min, 92%–100% A; 30–35 min, 100% A. The column temperature was 35℃. An electro-fogging detector was used, and the theoretical plate number calculated based on linoleic acid should not be less than 5000. 25 μL of the test solution was accurately pipetted and injected into the high-performance liquid chromatograph to investigate the effect of different flow rates (0.6 mL / min, 0.65 mL / min, 0.7 mL / min, 0.75 mL / min, 0.8 mL / min, and 1.0 mL / min) on the separation of chromatographic peaks. The results are shown in […]. Figure 8 .

[0113] Results: At flow rates of 0.6 mL / min and 1.0 mL / min, peak 6 ergosterol easily overlapped with other chromatographic peaks; at flow rates of 0.65 mL / min to 0.8 mL / min, all components could be well separated.

[0114] 7.2 Column Temperature Investigation

[0115] Methods of Investigation: One sample of puffball (number D4) was prepared according to the preparation method of the test solution determined in Section 1 "3" of Example 1. A TC C18(2) (Agilent, 4.5×250mm, 5μm) column was used as the chromatographic column. Methanol was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed: 0-15 min, 92% A; 15-30 min, 92%-100% A; 30-35 min, 100% A. The flow rate was 0.8 mL / min. An electro-fogging detector was used, and the theoretical plate number calculated based on linoleic acid should not be less than 5000. 25 μL of the test solution was accurately pipetted and injected into the high performance liquid chromatograph. The results were investigated at different temperatures, including 25℃, 30℃, 33℃, 35℃, and 40℃. Figure 9 .

[0116] Results: At a column temperature of 40℃, peak 6 ergosterol overlapped with other chromatographic peaks, resulting in poor separation. When the column temperature was between 25℃ and 35℃, all components could be separated well.

[0117] 7.3 Investigation of different chromatographic columns

[0118] Methods of Investigation: One sample of puffball (number D4) was taken and a test solution was prepared according to the preparation method of the test solution determined in Section 1 "3" of Example 1. The separation effects of different chromatographic columns such as TC C18(2) (Agilent, 4.5×250 mm, 5 μm), ZORBAX SB-C18 (Agilent, 4.6x250 mm, 5 μm), and Eclipse XDB-C18 (Agilent, 4.6x250 mm, 5 μm) were investigated. Methanol was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed for 0–15 min, 92% A; 15–30 min, 92%–100% A; 30–35 min, 100% A. The flow rate was 0.8 mL / min. An electro-fogging detector was used, and the theoretical plate number calculated based on linoleic acid should not be less than 5000. Accurately pipette 25 μL of the test solution and inject it into the high-performance liquid chromatograph. The results are shown in the figure. Figure 10 .

[0119] Results: When using the above three chromatographic columns for content determination, all components could be well separated.

[0120] 7.4 Investigation of formic acid solutions of different concentrations

[0121] Methods of Examination: One sample of puffball (number D4) was prepared according to the preparation method of the test solution determined in Section 1 "3" of Example 1. A TC C18(2) (Agilent, 4.5×250mm, 5μm) column was used as the chromatographic column. Methanol was used as mobile phase A, and 0.05% formic acid solution, 0.1% formic acid solution, and 0.15% formic acid solution were used as mobile phase B, respectively. Gradient elution was performed for 0-15 minutes, 92% A; 15-30 minutes, 92%-100% A; 30-35 minutes, 100% A. The flow rate was 0.8 mL / min. An electro-fogging detector was used, and the theoretical plate number calculated based on linoleic acid should not be less than 5000. 25 μL of the test solution was accurately pipetted and injected into the high performance liquid chromatograph to examine the separation effect of 0.05% formic acid solution, 0.1% formic acid solution, and 0.15% formic acid solution. The results are shown in the figure. Figure 11 .

[0122] Results: When the mobile phase B was 0.05% formic acid solution, 0.1% formic acid solution, and 0.15% formic acid solution, the analytes could be well separated. Among them, the 0.1% formic acid solution had the best separation of each chromatographic peak. Therefore, the mobile phase B can be preferably 0.1% formic acid solution.

Claims

1. A method for detecting the content of multiple components in a puffball sample, characterized in that, Includes the following steps: (1) Preparation of test solution: Take puffball sample powder, add extraction solvent to extract, and prepare test solution; (2) Preparation of reference solution: Take α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid and ergosterol reference standards, add solvent to prepare a mixed reference solution; (3) Chromatographic detection: The mixed reference solution and the test solution were injected into an ultra-high performance liquid chromatograph for detection, and the chromatograms of the reference solution and the test solution were compared. (4) Content calculation: The content of the components in the puffball sample is calculated by using the injection amount Ln value and the peak area Ln value of the components to form a regression equation; the components are α-linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid and ergosterol.

2. The method according to claim 1, characterized in that, In step (1), take the puffball sample powder, weigh it accurately, place it in a container, add the extraction solvent accurately, seal it tightly, weigh it, extract it, let it cool, weigh it again, use the extraction solvent to make up the lost weight, shake it well, filter it, take three-fifths of the volume of the filtrate, evaporate it to dryness, add the extraction solvent to the residue to redissolve it, transfer it to a quantitative container, add the extraction solvent to make up the volume, shake it well, filter it, take the filtrate, and the test solution is obtained.

3. The method according to claim 1, characterized in that, The extraction solvent mentioned in step (1) includes at least one of methanol, ethanol, ethyl acetate, and petroleum ether.

4. The method according to claim 1, characterized in that, The mass-to-volume ratio of the puffball sample powder to the extraction solvent in step (1) is 1 g: (7.5-25 mL).

5. The method according to claim 1, characterized in that, The extraction methods described in step (1) include ultrasonic treatment, heating and reflux, or shaking.

6. The method according to claim 1, characterized in that, The extraction time described in step (1) is 30 to 120 minutes.

7. The method according to claim 1, characterized in that, The conditions for the ultra-high performance liquid chromatograph in step (3) are as follows: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and 0.05-0.15% formic acid solution as mobile phase B, with gradient elution: 0-15 minutes, 92% A; 15-30 minutes, 92%-100% A; 30-35 minutes, 100% A.

8. The method according to claim 7, characterized in that, The flow rate was 0.65 mL / min to 0.8 mL / min, the column temperature was 25℃ to 35℃, and an electro-fogging detector was used.

9. The method according to claim 1, characterized in that, In step (4), the regression equation for the injection amount Ln value and peak area Ln value of α-linolenic acid is Y = 1.039X + 2.655, R ≥ 0.999; the regression equation for the injection amount Ln value and peak area Ln value of linoleic acid is Y = 0.701X + 2.040, R ≥ 0.999; and the regression equation for the injection amount Ln value and peak area Ln value of palmitic acid is Y = 0.874X + 1.863, R ≥ 0. The regression equations for the injection volume Ln value and peak area Ln value of oleic acid are Y = 0.778X + 2.015, R ≥ 0.999; the regression equations for the injection volume Ln value and peak area Ln value of stearic acid are Y = 1.042X + 2.443, R ≥ 0.999; the regression equations for the injection volume Ln value and peak area Ln value of ergosterol are Y = 0.888X + 2.076, R ≥ 0.

999.

10. The application of the method according to any one of claims 1-9 in the quality control or quality inspection of puffball samples.

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