A method for detecting contents of multiple index chemical components in phellinus baumii

Abstract

The application provides a detection method of multi-index chemical component content in Phellinus baumii, and relates to the technical field of analysis and detection.The application establishes a method for simultaneously detecting the content of multi-index chemical components (up to 14 kinds) in Phellinus baumii medicinal materials based on HPLC-MS / MS, adopts 70-100% low-grade alcohol as an extraction solvent, detects through HPLC-MS / MS, the response of the chromatographic peak is stronger, and after adding 0.08-0.12 vol% formic acid in the mobile phase, the peak tailing phenomenon can be well improved, and the content of multi-index chemical components in the Phellinus baumii medicinal materials is simultaneously determined.The detection method provided by the application is simple, efficient, high in sensitivity and strong in specificity, has good precision, repeatability and stability, can reflect the content difference of the measured chemical components in different batches of Phellinus baumii medicinal materials, and can provide a reference for the quality evaluation of the Phellinus baumii medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for detecting the content of multiple chemical components in Phellinus linteus. Background Technology

[0002] Sanghuang is the dried fruiting body of the fungus *Inonotus hispidus* (Bull.) P. Karst., belonging to the family Phyllostachyaceae. It possesses the effects of promoting blood circulation and stopping bleeding, strengthening the spleen and stopping diarrhea, tonifying liver yang, and aiding sleep and calming the mind. It is mainly used to treat hematuria, amenorrhea, spleen deficiency diarrhea, liver and kidney yin deficiency, insomnia, and excessive dreaming. Sanghuang has a complex chemical composition, mainly including polysaccharides, phenols, flavonoids, and terpenes, exhibiting significant antioxidant, anti-aging, anti-tumor, anti-inflammatory, and sleep-improving effects.

[0003] Due to the influence of geographical conditions, climate, and environment, the chemical composition of Sanghuang (Phellinus linteus) produced in different regions varies greatly, and the content of the same component also differs significantly, inevitably leading to inconsistencies in quality. Related technologies disclose the use of high-performance liquid chromatography (HPLC) to analyze the changes in the content of three compounds—protocatechuic acid, protocatechuic aldehyde, and ergosterol—in Sanghuang under different processing methods, in order to screen for more suitable processing methods. Although the aforementioned technologies involve HPLC detection of protocatechuic acid, protocatechuic aldehyde, and ergosterol in Sanghuang, they detect only a limited number of components and cannot provide comprehensive quality control for Sanghuang. Therefore, establishing an efficient and accurate method for determining the content of multiple components is of great significance for the quality evaluation of Sanghuang. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting the content of multiple chemical components in Phellinus linteus. The detection method provided by this invention can simultaneously achieve accurate and highly sensitive detection of the content of multiple chemical components in Phellinus linteus, thereby enabling quality monitoring of Phellinus linteus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for detecting the content of multiple chemical components in Phellinus linteus, comprising the following steps:

[0007] The powdered *Sanghuang* to be tested is extracted using an extractant to obtain a sample solution; the extractant includes a lower alcohol or an aqueous solution of a lower alcohol; the volume fraction of the lower alcohol in the extractant is 70-100%;

[0008] The sample solution to be tested was analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to obtain the detection results of the content of multiple chemical components in Sanghuang.

[0009] The multi-index chemical components include at least four of the following: hesperidin, tangerine, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milk tree alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin.

[0010] The high-performance liquid chromatography-tandem mass spectrometry detection includes high-performance liquid chromatography separation and mass spectrometry detection;

[0011] The high-performance liquid chromatography (HPLC) separation conditions include: mobile phase A is 0.08–0.12 vol% formic acid aqueous solution, mobile phase B is acetonitrile, and the elution method is gradient elution. The gradient elution program is as follows: 0–2 min, the volume fraction of mobile phase B increases from 15% to 40%; 2–9 min, the volume fraction of mobile phase B increases from 40% to 75%; 9–10 min, the volume fraction of mobile phase B increases from 75% to 100%.

[0012] Preferably, the lower alcohol includes methanol and / or ethanol.

[0013] Preferably, the solid-liquid ratio of the tested Phellinus linteus powder to the extractant is 0.19–0.21 g: 10 mL.

[0014] Preferably, the extraction includes ultrasonic extraction.

[0015] Preferably, the ultrasonic extraction power is 280-320W, the frequency is 30-50kHz, and the time is 0.5-1.5h.

[0016] Preferably, the chromatographic column used in the high-performance liquid chromatography separation is a C18 column. 18 The chromatographic column was used at a temperature of 28–32°C.

[0017] Preferably, the injection volume for high performance liquid chromatography separation is 1.8–2.2 μL.

[0018] Preferably, the flow rate of the mobile phase for high-performance liquid chromatography separation is 0.28–0.32 mL / min.

[0019] Preferably, the mass spectrometry detection conditions include: the ion source is an electrospray ion source, the detection mode is multiple reaction ion monitoring, the scanning mode is positive and negative ion scanning mode, the gas temperature is 320℃, the gas flow rate is 10L / min, the nebulizer pressure is 40psig, the fragmentation voltage is 66~219V, and the collision energy is 8~40V.

[0020] Preferably, the quantitative ion pairs of the following ingredients are 403.1 / 373.3, 395.0 / 365.0, 166.1 / 120.1, 474.1 / 60.1, 153.0 / 109.0, 137.0 / 108.0, 645.1 / 301.1, 245.0 / 159.1, 179.0 / 135.0, 219 / 135.1, 177.0 / 134.1, 456.1 / 323.1, 717.1 / 321.0, and 579.2 / 271.0, respectively.

[0021] This invention establishes a method for the simultaneous determination of multiple chemical components (up to 14) in *Sanghuang* medicinal materials based on HPLC-MS / MS. Using 70–100% lower alcohols as the extraction solvent, the chromatographic peak response is stronger after HPLC-MS / MS detection. Furthermore, the addition of 0.08–0.12 vol% formic acid to the mobile phase effectively improves peak tailing, enabling the simultaneous determination of multiple chemical components in *Sanghuang* medicinal materials. The detection method provided by this invention is simple, efficient, highly sensitive, and specific, exhibiting good precision, repeatability, and stability. It can reflect the differences in the content of chemical components measured in different batches of *Sanghuang* medicinal materials, providing a reference for the quality evaluation of *Sanghuang* medicinal materials. Attached Figure Description

[0022] Figure 1 The MRM diagrams for the standard reference (A) and the test sample (B) are shown below. In the diagram, 1 is phenylalanine, 2 is protocatechuic acid, 3 is protocatechuic aldehyde, 4 is amygdalin, 5 is caffeic acid, 6 is scopolamine, 7 is scutellarin, 8 is milkweed alkaloid, 9 is rutin, 10 is hesperidin, 11 is salvianolic acid B, 12 is linalool A, 13 is citrus red, and 14 is betulinic acid. Detailed Implementation

[0023] This invention provides a method for detecting the content of multiple chemical components in Phellinus linteus, comprising the following steps:

[0024] The powdered *Sanghuang* to be tested is extracted using an extractant to obtain a sample solution; the extractant includes a lower alcohol or an aqueous solution of a lower alcohol; the volume fraction of the lower alcohol in the extractant is 70-100%;

[0025] The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry to obtain the detection results of the content of multiple chemical components in Sanghuang.

[0026] The multi-index chemical components include at least four of the following: hesperidin, tangerine, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milk tree alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin.

[0027] The high-performance liquid chromatography-tandem mass spectrometry detection includes high-performance liquid chromatography separation and mass spectrometry detection;

[0028] The high-performance liquid chromatography (HPLC) separation conditions include: mobile phase A is 0.08–0.12 vol% formic acid aqueous solution, mobile phase B is acetonitrile, and the elution method is gradient elution. The gradient elution program is as follows: 0–2 min, the volume fraction of mobile phase B increases from 15% to 40%; 2–9 min, the volume fraction of mobile phase B increases from 40% to 75%; 9–10 min, the volume fraction of mobile phase B increases from 75% to 100%.

[0029] This invention utilizes an extractant to extract the powdered Phellinus linteus to be tested, thereby obtaining a sample solution; the extractant comprises lower alcohols or aqueous solutions of lower alcohols; the volume fraction of the lower alcohols in the extractant is 70-100%.

[0030] In this invention, the tested Phellinus linteus powder is preferably obtained by crushing Phellinus linteus and then sieving it, and the sieving is preferably through a No. 3 sieve.

[0031] In this invention, the volume fraction of lower alcohols in the extractant is preferably 70-90%, more preferably 70-80%, and even more preferably 70-75%; the lower alcohols include methanol and / or ethanol, more preferably methanol.

[0032] In this invention, the solid-liquid ratio of the tested Phellinus linteus powder (dry weight) to the extractant is preferably 0.19-0.21 g:10 mL, more preferably 0.2 g:10 mL.

[0033] In this invention, the extraction preferably includes ultrasonic extraction; the power of the ultrasonic extraction is preferably 280-320W, more preferably 290-310W, and even more preferably 300W; the frequency of the ultrasonic extraction is preferably 30-50kHz, more preferably 35-45kHz, and even more preferably 40kHz; the time of the ultrasonic extraction is preferably 0.5-1.5h, and even more preferably 1h.

[0034] After the extraction is completed, the present invention preferably further includes: filtering the obtained extract through a 0.22 μm microporous membrane to obtain a test solution.

[0035] After obtaining the sample solution to be tested, the present invention performs high performance liquid chromatography-tandem mass spectrometry on the sample solution to obtain the detection results of the content of multiple chemical components in Sanghuang.

[0036] In this invention, the multi-index chemical components include at least four of the following: noriheptacorlin, hesperidin, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milk tree alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin. Specifically, it is preferred to include four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen of the above substances.

[0037] In this invention, the high performance liquid chromatography-tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection.

[0038] In this invention, the high-performance liquid chromatography separation conditions include: the chromatographic column is preferably C12. 18 Chromatographic column, preferably CORTECS A C18 column; the column temperature is preferably 28–32℃, more preferably 29–31℃, and even more preferably 30℃; mobile phase A is 0.08–0.12 vol% formic acid aqueous solution, more preferably 0.09–0.11 vol% formic acid aqueous solution, and even more preferably 0.1 vol% formic acid aqueous solution; mobile phase B is acetonitrile; the mobile phase flow rate is preferably 0.28–0.32 mL / min, more preferably 0.29–0.31 mL / min, and even more preferably 0.3 mL / min; the elution method is gradient elution, and the gradient elution program is as follows: 0–2 min, the volume fraction of mobile phase B increases from 15% to 40%; 2–9 min, the volume fraction of mobile phase B increases from 40% to 75%; 9–10 min, the volume fraction of mobile phase B increases from 75% to 100%; the injection volume is preferably 1.8–2.2 μL, more preferably 1.9–2.1 μL, and even more preferably 2 μL.

[0039] In this invention, the preferred mass spectrometry detection conditions include: an electrospray ionization (ESI) source, a multiple reaction monitoring (MRM) detection mode, a positive and negative ion scanning mode, a gas temperature of 320°C, a gas flow rate of 10 L / min, a nebulizer pressure of 40 psig, a fragmentation voltage of 66–219 V, a collision energy of 8–40 V, and a collision gas preferably high-purity nitrogen; the nebulizer gas is preferably high-purity nitrogen; the quantitative analysis ion pairs, mass spectrometry parameters, and ion modes are shown in Table 1.

[0040] Table 1. Mass Spectrometry Parameters of 14 Chemical Components

[0041]

[0042]

[0043] In this invention, the preferred method for obtaining the content of multiple chemical components is the standard curve method. Specifically, the mixed reference linear solution is detected under the conditions of ultra-high performance liquid chromatography-tandem mass spectrometry to obtain the chromatographic peak area. A standard curve is plotted with the concentration of the mixed reference linear solution as the independent variable and the chromatographic peak area as the dependent variable to obtain the standard curve regression equation. The chromatographic peak area of ​​the sample solution to be tested is then substituted into the standard curve regression equation to obtain the detection results of the content of multiple chemical components.

[0044] In this invention, the method for preparing the mixed reference linear solution preferably includes the following steps: preparing a standard reference stock solution, diluting the reference stock solution to obtain a mixed reference solution; and gradually diluting the mixed reference solution to obtain a mixed reference linear solution.

[0045] In this invention, the preferred method for preparing the reference stock solution includes the following steps: dissolving the reference standards of noriheptacorlin, hesperidin, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milkweed alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin in a 70 vol% methanol aqueous solution to prepare a reference stock solution of 14 single reference standards, each with a concentration of 1 mg / mL.

[0046] In this invention, the preferred method for preparing the mixed reference solution includes the following steps: accurately measuring each reference stock solution and placing it in the same volumetric flask, then diluting it with 70 vol% methanol aqueous solution to obtain the mixed reference solution; the mixed reference solution contains 12.5 μg / mL of salvianolic acid B, 9 μg / mL of milkweed alkaloid, 6 μg / mL of betulinic acid, 4 μg / mL of sennae and linalool A, 3 μg / mL of protocatechuic aldehyde, 2 μg / mL of amygdalin and naringin, 1 μg / mL of protocatechuic acid, caffeic acid, phenylalanine and hesperidin, and 0.08 μg / mL of norihesperidin and citrus red pigment.

[0047] In this invention, the preferred method for preparing the linear solution of the mixed reference standard includes the following steps: diluting the mixed reference standard solution by 2 times with a 70 vol% methanol aqueous solution to obtain a linear solution 1; further diluting the linear solution 1 of the mixed reference standard by 2 times with a 70 vol% methanol aqueous solution to obtain a linear solution 2 of the mixed reference standard; further diluting the linear solution 2 of the mixed reference standard by 2.5 times with a 70 vol% methanol aqueous solution to obtain a linear solution 3 of the mixed reference standard; further diluting the linear solution 3 of the mixed reference standard by 2 times with a 70 vol% methanol aqueous solution to obtain a linear solution 4 of the mixed reference standard; and further diluting the linear solution of the mixed reference standard by 2 times with a 70 vol% methanol aqueous solution to obtain a linear solution 4 of the mixed reference standard. Liquid 4 was further diluted 2.5 times with 70 vol% methanol aqueous solution to obtain mixed reference linear solution 5; mixed reference linear solution 5 was further diluted 2 times with 70 vol% methanol aqueous solution to obtain mixed reference linear solution 6; mixed reference linear solution 6 was further diluted 2.5 times with 70 vol% methanol aqueous solution to obtain mixed reference linear solution 7; mixed reference linear solution 7 was further diluted 2 times with 70 vol% methanol aqueous solution to obtain mixed reference linear solution 8; mixed reference linear solution 8 was further diluted 2 times with 70 vol% methanol aqueous solution to obtain mixed reference linear solution 9.

[0048] To further illustrate the present invention, the following detailed description of the detection method for the content of multiple chemical components in Phellinus linteus provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0049] In embodiments of the present invention, the specific instruments are as follows: Agilent 1200 high performance liquid chromatograph (Agilent Technologies, USA); Agilent 6430 triple quadrupole tandem mass spectrometer (Agilent Technologies, USA); Agilent MassHunter analysis software (Agilent Technologies, USA); Milli-Q IQ 7005 ultrapure water preparation system (Millipore); 5424R high-speed centrifuge (Eppendorf, Germany); AS 60 / 220.R2 0.0001 g balance (Radiwag, Poland); G3KT 18273 vortex mixer (Thermo Fisher Scientific).

[0050] The standard reference standards are as follows: rutin (batch number: DST211101-098), betulinic acid (batch number: DST2200509-026), salvianolic acid B (batch number: DSTDD000903), protocatechuic acid (batch number: DSTDY008101), amygdalin (batch number: DST200710-004), scutellarin (batch number: DSTDZ013801), linalool A (batch number: DST230411-616), caffeic acid (batch number: DST1) The following substances were purchased from Chengdu Desite Biotechnology Co., Ltd. and Sichuan Weikeqi Biotechnology Co., Ltd., and their purity was greater than 98%.

[0051] The reagents used were as follows: methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific, USA; formic acid was purchased from ROE, USA; and ultrapure water was prepared using a Milli-Q ultrapure water preparation system.

[0052] Sanghuang: The dried fruiting body of the fungus Inonotus hispidus (Bull.) P.Karst. of the family Inonotusceae. See Table 2 for specific source information of Sanghuang.

[0053] Table 2. Specific Source Information of Sanghuang Medicinal Material

[0054]

[0055]

[0056] The HPLC chromatographic conditions are as follows:

[0057] The chromatographic column was a CORTECS C18 (2.1 × 50 mm, 2.7 μm; Waters); mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile; gradient elution was used, with the elution gradient program as follows: 0–2 min, the volume fraction of mobile phase B increased from 25% to 40%; 2–9 min, the volume fraction of mobile phase B increased from 40% to 75%; 9–10 min, the volume fraction of mobile phase A increased from 75% to 100%; the mobile phase flow rate was 0.3 mL / min; the column temperature was 30 °C; and the injection volume was 2.0 μL.

[0058] The mass spectrometry conditions were as follows: the ion source was an electrospray ionization source (ESI); the detection mode was multiple reaction monitoring (MRM); the scanning mode was positive and negative ion scanning mode; the gas temperature (Gas Temp) was 320℃; the gas flow rate (Gas Flow) was 10 L / min; the nebulizer pressure (Nebulizer) was 40 psig; the quantitative analysis ion pairs, mass spectrometry parameters, and ion modes are shown in Table 1.

[0059] Example 1

[0060] Methodological Examination—Standard Curve Regression Equation

[0061] Preparation of reference stock solutions: The reference standards of noriheptacortin, hesperidin, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milk alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin were dissolved in 70 vol% methanol aqueous solution to prepare reference stock solutions of 14 single reference standards with a concentration of 1 mg / mL.

[0062] Preparation of the mixed reference solution: Accurately measure each reference stock solution and place it in the same volumetric flask. Dilute with 70 vol% methanol aqueous solution to obtain the mixed reference solution. The concentrations of the mixed reference solution are as follows: tanshinone B is 12.5 μg / mL, milkweed alkaloid is 9 μg / mL, betulinic acid is 6 μg / mL, scutellarin and linalool A are both 4 μg / mL, protocatechuic aldehyde is 3 μg / mL, amygdalin and rutin are both 2 μg / mL, protocatechuic acid, caffeic acid, phenylalanine and hesperidin are both 1 μg / mL, and norihesperidin and citrus red are both 0.08 μg / mL.

[0063] Preparation of linear solutions of mixed reference standards: The mixed reference standard solution was diluted 2-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 1. Mixed reference standard linear solution 1 was further diluted 2-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 2. Mixed reference standard linear solution 2 was further diluted 2.5-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 3. Mixed reference standard linear solution 3 was further diluted 2-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 4. Mixed reference standard linear solution 4 was further diluted 2.5-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 5. Mixed reference standard linear solution 5 was further diluted 2-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 6. Mixed reference standard linear solution 6 was further diluted 2.5-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 7. Mixed reference standard linear solution 7 was further diluted 2-fold with 70 vol% methanol aqueous solution to obtain mixed reference standard linear solution 8. The mixed reference linear solution 8 was further diluted 2 times with 70 vol% methanol aqueous solution to obtain the mixed reference linear solution 9.

[0064] Preparation of the test solution: Accurately weigh 200.0 mg of coarse powder of Sanghuang (passed through a No. 3 sieve), place it in a 10 mL volumetric flask, add 70 vol% methanol aqueous solution to make up to the mark, sonicate at 300 W and 40 kHz for 1 h, cool, make up the weight loss, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0065] Take the above solutions and analyze them by HPLC-MS / MS. Weighted least squares regression was performed, with the x-axis (X) representing the analyte concentration and the y-axis (Y) representing the analyte peak area, and the weighting coefficient being 1 / X. 2 Linear regression equations were used to determine the concentrations of hesperidin, chrysanthemum, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milkweed alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin. The concentrations of the reference standards calculated with a signal-to-noise ratio (S / N) of 10 were used as the limits of quantitation (LLOQ). The results are shown in Table 3.

[0066] Table 3. Standard curve regression equations and limits of quantification (LLOQ) for 14 chemical components.

[0067]

[0068] Figure 1The image shows the MRM chromatograms of the standard reference (A) and the test sample (B), where 1 represents phenylalanine, 2 represents protocatechuic acid, 3 represents protocatechuic aldehyde, 4 represents amygdalin, 5 represents caffeic acid, 6 represents scopolamine, 7 represents scutellarin, 8 represents milkweed alkaloid, 9 represents rutin, 10 represents hesperidin, 11 represents salvianolic acid B, 12 represents linalool A, 13 represents hesperidin, and 14 represents betulinic acid. Figure 1 It can be seen that the peaks of each compound have good shapes and do not interfere with each other.

[0069] Example 2

[0070] Methodological investigation—precision testing

[0071] Precision (within day): The test solution was prepared according to Example 1. The sample was analyzed 6 times by consecutive injections of HPLC-MS / MS, and the RSD values ​​of the peak areas of each compound were calculated. The results are shown in Table 4. In Tables 4-6, 1-6 represent the 1st to 6th injection analyses, indicating good intra-day precision of the instrument.

[0072] Precision (daytime): The test solution was prepared according to Example 1. HPLC-MS / MS analysis was repeated twice, with injections performed continuously over 3 days. The RSD values ​​of the peak areas of each compound were calculated, and the results are shown in Table 5, indicating good daytime precision of the instrument.

[0073] Table 4. Intra-day precision results for 14 chemical components (n=6)

[0074]

[0075] Table 5. Daytime precision results for 14 chemical components (n=6)

[0076]

[0077]

[0078] Example 3

[0079] Methodological investigation—repeatability testing

[0080] Six test solutions were prepared in parallel according to Example 1, and analyzed by HPLC-MS / MS. The RSD values ​​of each compound concentration were calculated. The results are shown in Table 6, indicating that the method has good repeatability.

[0081] Table 6. Repeatability results for 14 chemical components (n = 6, ng / mL)

[0082]

[0083]

[0084] Example 4

[0085] Methodological investigation—stability testing

[0086] The test solution prepared according to Example 1 was analyzed by HPLC-MS / MS at 0h, 2h, 4h, 8h, 12h and 24h respectively. The RSD value of the peak area of ​​each compound was calculated. The results are shown in Table 7, indicating that each compound has good stability.

[0087] Table 7. Stability results of 14 chemical components (n=6)

[0088]

[0089] Example 5

[0090] Methodological investigation—spiking recovery test

[0091] Preparation of spiked test solution: Accurately weigh 100.0 mg of coarse powder of Sanghuang medicinal material (passed through a No. 3 sieve), place it in a 10 mL volumetric flask, add different amounts (see Table 8) of the mixed reference solution prepared in Example 1, add 70 vol% methanol aqueous solution to make up to the mark, sonicate at 300 W and 40 kHz for 1 h, cool, make up the weight loss, shake well, and filter through a 0.22 μm microporous membrane to obtain the spiked test solution.

[0092] Each spiked test solution was analyzed by HPLC-MS / MS six times, and the recovery rate of each compound was calculated. The results are shown in Table 8.

[0093] Table 8. Recovery results of 14 chemical components (n=6)

[0094] compound Measured value / ng Actual value / ng Add value / ng Average recovery rate / % RSD / % Phenylalanine 3999.10 2024.01 2000.00 98.76 1.27 Protocatechuic acid 2971.51 1508.00 1500.00 97.57 2.22 Protocatechuic aldehyde 5313.84 2590.54 2600.00 104.74 1.34 amygdalin 319.37 160.38 150.00 105.99 5.08 caffeic acid 3095.61 1538.21 1500.00 103.82 1.73 Osmidone 21644.82 10946.66 10900.00 98.15 3.39 Milk Tree Alkaloids 18092.90 9150.51 9150.00 97.73 0.83 Rutin 236.21 123.95 120.00 93.56 2.78 hesperidin 5046.95 2584.11 2600.00 94.72 1.59 Tanshinone B 1275.21 637.68 600.00 106.27 2.53 Phellinus A 11350.72 5942.10 5950.00 90.90 2.48 hesperidin 11.08 6.12 6.00 81.01 6.10 Sichuan tangerine peel extract 25.50 13.20 13.00 94.56 3.60 Betulinic acid 4947.71 2463.70 2400.00 103.50 2.70

[0095] Example 6

[0096] Content determination

[0097] Accurately weigh each batch (S1~S10) of Sanghuang medicinal material crude powder, prepare test solution according to Example 1, and analyze by HPLC-MS / MS. Record the concentration of 14 compounds and calculate the content. The results are shown in Table 9.

[0098] Table 9. Content of 14 chemical components in different batches of Sanghuang medicinal materials (μg / g, n=3)

[0099]

[0100]

[0101] Example 7

[0102] Investigation of extractant and mobile phase

[0103] Preparation of the test solution: Accurately weigh 200.0 mg of coarse powder of Sanghuang medicinal material (passed through a No. 3 sieve), place it in a 10 mL volumetric flask, add extraction solvent (30 vol% methanol aqueous solution, 50 vol% methanol aqueous solution, 70 vol% methanol aqueous solution and methanol respectively) and dilute to the mark, sonicate at 300 W and 40 kHz for 1 h, cool, make up the weight loss, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0104] The test sample was analyzed by HPLC-MS / MS. Mobile phases A through B were water-methanol, 0.1 vol% formic acid aqueous solution-methanol, water-acetonitrile, and 0.1 vol% formic acid aqueous solution-acetonitrile, respectively. The results showed that when 70–100 vol% acetonitrile-water was used as the extraction solvent, the chromatographic peak response was stronger, and the addition of 0.1% formic acid to the mobile phase improved the peak tailing phenomenon. Therefore, an HPLC-MS / MS method was established using 70–100% methanol as the extraction solvent and 0.1% formic acid aqueous solution-acetonitrile as the mobile phase to achieve simultaneous determination of multiple chemical components in Sanghuang medicinal material.

[0105] In summary, this invention establishes a method for the simultaneous determination of multiple chemical components (up to 14) in *Sanghuang* medicinal materials based on HPLC-MS / MS, and applies it to the content determination of different batches of *Sanghuang* medicinal materials. The established detection method is simple, efficient, highly sensitive, and specific, with good precision, repeatability, and stability. It can reflect the differences in the content of chemical components measured in different batches of *Sanghuang* medicinal materials, and can provide a reference for the quality evaluation of *Sanghuang* medicinal materials.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the content of multiple chemical components in Phellinus linteus, comprising the following steps: The sample solution was obtained by ultrasonic extraction of the powdered Phellinus linteus to be tested using an extractant; the extractant included a lower alcohol or an aqueous solution of a lower alcohol, wherein the volume fraction of the lower alcohol in the extractant was 70-100%, and the lower alcohol was methanol; The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry to obtain the detection results of the content of multiple chemical components in Sanghuang. The multi-index chemical components include hesperidin, tangerine, phenylalanine, betulinic acid, protocatechuic acid, protocatechuic aldehyde, hesperidin, milk tree alkaloid, caffeic acid, linalool A, scutellarin, amygdalin, salvianolic acid B, and rutin. The high-performance liquid chromatography-tandem mass spectrometry detection includes high-performance liquid chromatography separation and mass spectrometry detection; The high-performance liquid chromatography separation conditions include: a CORTECS C10 column. 18 The chromatographic column was 2.1 × 50 mm, 2.7 μm. Mobile phase A was 0.08–0.12 vol% formic acid aqueous solution, and mobile phase B was acetonitrile. The elution method was gradient elution, and the gradient elution program was as follows: 0–2 min, the volume fraction of mobile phase B increased from 15% to 40%; 2–9 min, the volume fraction of mobile phase B increased from 40% to 75%; 9–10 min, the volume fraction of mobile phase B increased from 75% to 100%.

2. The detection method according to claim 1, characterized in that, The solid-liquid ratio of the tested Phellinus linteus powder to the extractant was 0.19~0.21g:10mL.

3. The detection method according to claim 1, characterized in that, The ultrasonic extraction power is 280~320W, the frequency is 30~50kHz, and the time is 0.5~1.5h.

4. The detection method according to claim 1, characterized in that, The column temperature for the high-performance liquid chromatography separation is 28~32℃.

5. The detection method according to claim 1, characterized in that, The injection volume for high-performance liquid chromatography separation is 1.8~2.2 μL.

6. The detection method according to claim 1, 4, or 5, characterized in that, The flow rate of the mobile phase for high-performance liquid chromatography separation is 0.28~0.32 mL / min.

7. The detection method according to claim 1, characterized in that, The mass spectrometry detection conditions include: the ion source is an electrospray ion source, the detection mode is multiple reaction ion monitoring, the scanning mode is positive and negative ion scanning mode, the gas temperature is 320℃, the gas flow rate is 10L / min, the nebulizer pressure is 40psig, the fragmentation voltage is 66~219V, and the collision energy is 8~40V.

8. The detection method according to claim 1 or 7, characterized in that, The quantitative ion pairs of the following compounds are 403.1 / 373.3, 395.0 / 365.0, 166.1 / 120.1, 474.1 / 60.1, 153.0 / 109.0, 137.0 / 108.0, 645.1 / 301.1, 245.0 / 159.1, 179.0 / 135.0, 219.0 / 135.1, 177.0 / 134.1, 456.1 / 323.1, 717.1 / 321.0, and 579.2 / 271.0, respectively.

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