CN111505180A

CN111505180A - Method for simultaneously determining contents of multiple flavonoid aglycones in radix puerariae

Info

Publication number: CN111505180A
Application number: CN202010388778.XA
Authority: CN
Inventors: 侯建波; 谢文; 史颖珠; 李�杰; 陆顺; 毛壬熠; 汪鹏
Original assignee: Hangzhou Customs Technical Center
Current assignee: Hangzhou Customs Technical Center
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-07
Anticipated expiration: 2040-05-09
Also published as: CN111505180B

Abstract

The invention relates to a method for simultaneously measuring the content of a plurality of flavonoid aglycones in kudzuvine root, in particular to a method for simultaneously measuring the content of 11 flavonoid aglycones in kudzuvine root by hydrolyzing flavonoid glycoside, carrying out solid-phase extraction and purification and liquid chromatography-tandem mass spectrometry. A method for simultaneously measuring the contents of multiple flavonoid aglycones in radix Puerariae comprises the steps of extracting, purifying and measuring, wherein the extraction adopts a hydrochloric acid ethanol solution hydrolysis extraction method, and T-butyl-1, 4-phenol is added into a sample as an antioxidant during extraction. According to the method, T-butyl-1, 4-phenol is added into a sample as an antioxidant during extraction, and researches show that the loss rate of each aglycone in the hydrolysis process is lower than 20%. Furthermore, in order to solve the technical problem of comprehensively and accurately measuring the flavonoid compounds in the radix puerariae, the application provides a method for simultaneously measuring the contents of 11 flavonoid aglycones in the radix puerariae by hydrochloric acid hydrolysis, solid-phase extraction purification, liquid chromatography and tandem mass spectrometry, and the method has the characteristics of simplicity, convenience, rapidness, small resource consumption and low detection cost.

Description

Method for simultaneously determining contents of multiple flavonoid aglycones in radix puerariae

Technical Field

The invention relates to a method for simultaneously measuring the content of a plurality of flavonoid aglycones in kudzuvine root, in particular to a method for simultaneously measuring the content of 11 flavonoid aglycones in kudzuvine root by hydrolyzing flavonoid glycoside, carrying out solid-phase extraction and purification and liquid chromatography-tandem mass spectrometry.

Background

Kudzu roots, represented by kudzu (dried root of kudzu vine (kudzu vine) belonging to the family Leguminosae), and kudzu (kudzu vine) belonging to the family Leguminosae), have the effects of relieving muscles and fever, promoting the production of body fluid to quench thirst, promoting eruption, invigorating yang to arrest diarrhea, clearing and activating the channels and collaterals, relieving alcoholism and the like, and are therefore commonly used for treating exogenous fever headache, neck and back pain, thirst, diabetes, measles imperviousness, dysentery with heat, diarrhea, dizziness headache, stroke hemiplegia, chest stuffiness and cardialgia, and alcoholic injury, and the kudzu roots are officially approved as medicinal and edible plants by the national ministry of health in 2000.

The flavonoid compounds represented by puerarin (puerarin), daidzin (daidzin), daidzein (daidzein), genistein (genistein) and genistin (genistein) are important effective components of radix puerariae, wherein the highest content is puerarin, and the Chinese pharmacopoeia takes the content of puerarin as an important index for measuring the quality of radix puerariae. Based on the Chinese pharmacopoeia, the content determination method of flavonoid compounds in the root of kudzu vine reported in the literature at present mainly comprises the steps of measuring the content of total flavone and the content of puerarin by a spectrophotometry, measuring and analyzing the content of compounds such as puerarin, daidzein and daidzein in the root of kudzu vine by a liquid chromatography, and carrying out structural analysis and screening detection on the flavonoid compounds by a liquid chromatography-tandem mass spectrometer or a liquid chromatography-high resolution mass spectrometer.

Since the flavonoid compounds are more in variety, aglycones can be ubiquitous with glycosides in different forms from saccharides in plants, and many standard substances are difficult to obtain, so that other analysis means are difficult to obtain more comprehensive information in the existing liquid chromatography-high resolution mass spectrometry analysis. Therefore, the method has important significance for comprehensively reacting the total amount of flavonoids represented by each aglycone by hydrolyzing representative flavonoid glycosides (puerarin-6' -O-xyloside, liquiritin, daidzin, scutellarin, daidzin, rutin, tectoridin, genistin, kaempferol-3-O-rutinoside, baicalin and formononetin) in the kudzuvine root and detecting the puerarin, the liquiritin, the daidzein, the scutellarin, the daidzein, the quercetin, the tectorigenin, the genistein, the kaempferol, the baicalein and the formononetin in the aglycone form and accurately measuring the content of the flavonoids.

TABLE 1 basic information on the various classes of compounds

The Chinese patent application (CN107389814A) discloses a method for rapidly determining the content of catechin, rutin, myricetin, quercetin, kaempferol and isorhamnetin in sea buckthorn by using a reversed-phase high performance liquid chromatography diode array detection, the method comprises the steps of hydrolyzing flavonoid glycoside by acid hydrolysis, releasing independent free aglycone, and accurately quantifying the free aglycone by RP-HP L C-DAD to obtain accurate quality control data of the flavonoid extract.

Disclosure of Invention

In order to solve the technical problem of low recovery rate of flavonoid aglycone extracted by a hydrochloric acid hydrolysis method, the application aims to provide a method for simultaneously measuring the content of a plurality of flavonoid aglycones in kudzuvine root, T-butyl-1, 4-phenol is added into a sample during extraction as an antioxidant, and researches show that the loss rate of each flavonoid aglycone in the hydrolysis process is lower than 20%. Furthermore, in order to solve the technical problem of comprehensively and accurately measuring the flavonoid compounds in the radix puerariae, the application provides a method for simultaneously measuring the contents of 11 flavonoid aglycones in the radix puerariae by hydrochloric acid hydrolysis, solid-phase extraction purification, liquid chromatography and tandem mass spectrometry, and the method has the characteristics of simplicity, convenience, rapidness, small resource consumption and low detection cost.

In order to achieve the above object, the present application adopts the following technical solutions:

a method for simultaneously measuring the contents of multiple flavonoid aglycones in radix Puerariae comprises the steps of extracting, purifying and measuring, wherein the extraction adopts a hydrochloric acid ethanol solution hydrolysis extraction method, and T-butyl-1, 4-phenol is added into a sample as an antioxidant during extraction.

Furthermore, the addition amount of the T-butyl-1, 4-phenol is 3-10 times of the mass of the sample.

Further, the extraction hydrolysis conditions are 10% hydrochloric acid, 50VO L% ethanol solution, and T-butyl-1, 4-phenol and nitrogen protection, and hydrolysis is carried out for 120 min.

As a specific embodiment, the extraction process comprises the steps of weighing 0.1g of sample, accurately obtaining 0.01g of sample, placing the sample in a 150m L round bottom flask, adding 0.5g of T-butyl-1, 4-phenol, 36m L50 VO L% ethanol solution and 4.0m L concentrated hydrochloric acid, heating and refluxing the mixture in a water bath for 120min under the protection of nitrogen, cooling the mixture under the protection of nitrogen, and adding water to the mixture until the volume is 50m L.

Further, the purification adopts an H L B solid phase extraction column.

Taking 1m L of the extraction supernatant, adding 9m L of water, uniformly mixing, transferring to an H L0B solid phase extraction column, sequentially activating by using 5m L1 of methanol and 5m L25 VO L3% of methanol solution, adding 5m L420 VO L% of methanol solution for leaching, draining, adding 10m L of methanol for elution, controlling the flow rate to be 1m L/min-2 m L/min, collecting all eluent, fixing the volume to 10m L, uniformly shaking, filtering by using a 0.22 mu m organic filter membrane, taking 0.1m L of fixed volume solution, adding 40VO L% of methanol solution for dilution and fixing the volume to 10m L.

Further, the flavonoid aglycone is one or more of puerarin, liquiritigenin, daidzein, scutellarein, daidzein, quercetin, tectorigenin, genistein, kaempferol, baicalein, and formononetin.

Further, the determination adopts a liquid chromatography-tandem mass spectrometry method to carry out quantitative determination on the flavonoid aglycone, and comprises the following steps:

① sample introduction of standard working solution and sample solution under the set condition of liquid chromatogram tandem mass spectrum, taking percentage concentration X as abscissa, taking peak area ratio Y as ordinate, taking peak area as ordinate for compound quantified by external standard method, drawing 6-point standard working curve, quantifying sample by using standard working curve, response value of each compound in sample solution should be in linear range detected by instrument;

② under the chromatographic condition, judging whether there is corresponding object in the sample, if the retention time of the mass spectrum peak appearing in the sample solution is consistent with the mixed matrix standard working solution, the allowable deviation is less than + -2.5%, the relative abundance of the compound corresponding to the chromatographic peak in the set mass spectrum qualitative ion is consistent with the relative abundance of the mixed matrix standard working solution with the equivalent concentration, the deviation of the relative abundance is not more than the set regulation, then the compound can be determined to be contained.

As a specific embodiment, the specific parameters of the chromatographic column are as follows:

as a specific embodiment, the mass spectrum specific parameters are as follows:

other mass spectral parameters are shown in the following table:

is a quantitative ion pair.

By adopting the technical scheme, flavonoid glycoside in kudzuvine root is hydrolyzed by hydrochloric acid ethanol solution, flavonoid aglycone is extracted, H L B solid phase extraction column purification, liquid chromatography-tandem mass spectrometry detection and external standard method quantification are carried out, the content of representative flavonoid aglycone in kudzuvine root is measured, the method has the limit of quantification (calculated by S/N is more than 10) of 0.10 percent (namely 1.0 mg/g: puerarin, daidzein and baicalein) and 0.05 percent (namely 0.5 mg/g: liquiritigenin, scutellarein, daidzein, quercetin, tectorigenin, genistein, kaempferol and formononetin), a 0-2.5 percent linear working curve is prepared, the linear correlation coefficient is more than 0.996, each flavonoid glycoside is added into kudzuvine root powder (equivalent to 0.25 percent, 0.5 percent and 1.0 percent of the content of the corresponding flavonoid aglycone after hydrolysis, the total average recovery rate is 75.1-93.8 percent, the total flavonoid aglycone is relatively good, the method is applicable to the measurement of flavonoid aglycone, the method is applicable to the conventional method, the method has the detection, the method has the advantages of providing a good detection, the method is applicable to the measurement of flavonoid aglycone, the method has the advantages of the method has the detection of the method has the.

Drawings

FIGS. 1-3 show the total ion flow diagram (solution concentration 10ng/m L) of the target compound, wherein FIG. 1 shows puerarin, liquiritigenin, daidzein, scutellarein, daidzein, quercetin, tectorigenin, genistein, kaempferol, baicalein and formononetin, FIG. 2 shows puerarin-6' -O-xyloside, liquiritin, scutellarin, rutin, tectoridin, genistin, kaempferol-3-O-rutinoside and baicalin, and FIG. 3 shows daidzin, daidzin and formononetin.

FIGS. 4-7 are selective ion flow diagrams (solution concentration 10ng/m L) for the target compound.

FIG. 8 is a comparison graph of purification of different solid phase extraction columns, 1 puerarin, 2 glycyrrhizin, 3 daidzein, 4 scutellarein, 5 daidzein, 6 quercetin, 7 tectorigenin, 8 genistein, 9 kaempferol, 10 scutellarein, 11 formononetin, (A. biocommah L BSPEColumn; B.Waters H L BSPEColumn; C.Waters C18 SPEColumn; D.CNWC18SPEColumn).

Detailed Description

1 experimental part

1.1 instruments, reagents and materials

API model 4000 triple quadrupole tandem mass spectrometer (electric spray ion source ESI, AB, usa), model 1100 liquid chromatograph (Agilent, usa), Milli-QSynergy185 ultra pure water device (Millipore, usa), IKAMS3Basic type vortexer (IKA, germany), 24-well solid phase extraction device (Supelco, usa), heraeus multifuge x1R type bench top centrifuge (Thermo, usa), G & GJJ500 type electronic balance (bijie, china), mettlere 260(mettler toledo, switzerland).

Methanol (chromatographically pure, TEDIA corporation), formic acid (Mass Spectroscopy grade, Scharlau corporation), acetonitrile (chromatographically pure, Scharlau corporation), ethanol (analytically pure, Anhuite food products Ltd.), hydrochloric acid (Uygol, Yonghua chemical technology (Jiangsu) Co., Ltd.), T-butyl-1, 4-phenol (tert-butyl hydroquinone, analytically pure, Aladdin corporation), butylhydroxyanisole (butylhydroxyanisole, BHA for short, analytically pure, Macklin corporation), 2, 6-Di-tert-butyl-p-cresol (2,6-Di-tert-butyl-4-methylphenol, WC: BHT for short, analytically pure, Macklin corporation), L- (+) -ascorbic acid ((+) -L Ascorbicacid, analytically pure, Guangdong-Guanghua-GmbH, Ltd.), CND (CND, CND for short, Inc. (+) -C L Ascorbicacid, analytically pure, Guangdong-Guanghua-Industy-Industies, Inc.), and₁₈solid phase extraction column (6m L, 500mg, Angel Corp.), C₁₈Solid phase extraction column (6m L, 500mg, Waters)Company), H L B solid phase extraction column (6m L, 200mg, Waters corporation), H L B solid phase extraction column (6m L, 200mg, Biocomma corporation).

The standard substances include daidzein (purity 98.2%, Chromadex corporation), daidzein (purity 99.0%, Chromadex corporation), quercetin (purity 96.4%, Chromadex corporation), formononetin (purity 98.5%, Chromadex corporation), genistein (purity 99.0%, China institute for pharmaceutical and biological products), liquiritigenin (purity 96.9%, Chromadex corporation), baicalein (purity 99.2%, ANPE L corporation), scutellarein (purity 96.2%, Chromadex corporation), puerarin (purity 87.0%, Chromadex corporation), kaempferol (purity 95.7%, China institute for food and drug analysis), iritin (purity 98.1%, Phyto L ab corporation), daidzin (purity 92.9%, Chromadex corporation), daidzin (purity 93.3%, China institute for food and drug analysis), rutin (95.0%, TRC (TRC L ab corporation), baicalin (purity 399.9%, purity 99%, Desmadin 99.6%, Desorpin corporation), and purity 99.6%, purity of Chromadin (purity), and purity of Desomadex 368%, Desorpin-96.6%, purity), and purity of pure, purity of Chromadin (purity), purity of Chromadex 368%, Desorpin-96.6%, purity), and purity of pure, purity of pure methanol, purity of pure, Desomadex 368, Desmophilgarcino-96, and diluted by using standard substance.

1.2 Experimental part

1.2.1 sample pretreatment

Weighing 0.1g of sample (accurate to 0.01g) and placing the sample in a 150m L round bottom flask, adding 0.5g of T-butyl-1, 4-phenol, 36m L50% ethanol solution and 4.0m L0 concentrated hydrochloric acid, heating and refluxing in a water bath for 120min under the protection of nitrogen, cooling under the protection of nitrogen, adding water to fix the volume to 50m L1, taking supernatant 1m L2, adding 9m L3 water, mixing uniformly, transferring to an H L4B solid phase extraction column (activated by 5m L5 methanol and 5m L5% methanol solutions in sequence), adding 5m L20% methanol solution to elute, draining, adding 10m L methanol to elute, controlling the flow rate to 1m L/min-2 m L/min, collecting all eluents, feeding to 10m L, shaking uniformly, passing through a 0.22 μm organic filter membrane, taking 0.1m L solution, adding 40% methanol solution to fix the volume to L/min, diluting to a volume to L m, and measuring by a mass spectrometer.

1.2.2 chromatographic parameters

A chromatographic column of Mightysill RP-18 with the diameter of 3 mu m and the diameter of 4.6 × 150mm, a mobile phase of methanol (A) -0.15 percent formic acid solution (B, B is water when the daidzin, daidzin and formononetin are measured), a mobile phase gradient elution procedure of 40 percent (A) linear increase to 75 percent when 0-10.0 min, 75 percent (A) linear increase to 90 percent when 10.0-15.0 min, 90 percent (A) constant when 15.0-22.0 min, 22.0-23.0 min linear decrease to 40 percent (A), 23.0-30.0 min40 percent (A) carry out system equilibrium, the flow rate is 0.4m L/min, the sample introduction amount is 20 mu L, and the column temperature is 25 ℃.

1.2.3 Mass Spectrometry parameters

An ion source: electrospray ion source (ESI); scanning mode: scanning negative ions; the monitoring mode is as follows: multiple Reaction Monitoring (MRM); electrospray voltage (IS): -4500V; atomization gas pressure (GS 1): 289kpa (42 psi); air curtain pressure (GS 2): 310kPa (45 psi); auxiliary airflow rate (CUR): 172kpa (25 psi); ion source Temperature (TEM): 540 ℃; other mass spectral parameters are shown in table 2.

TABLE 2 basic information of the respective compounds and conditions for mass spectrometry

Is a quantitative ion pair.

2 results and discussion

2.1 selection of hydrolysis and extraction conditions

Generally, two modes of extraction and reflux extraction are adopted for extracting effective components in Chinese herbal medicines, and by referring to the method of Chinese pharmacopoeia, cross experiments are carried out on flavonoid glycoside under the conditions of 30 percent of ethanol content, 50 percent and 70 percent of ethanol content, 5 percent of hydrochloric acid concentration, 10 percent and 12.5 percent of hydrochloric acid concentration, heating and refluxing are carried out in a water bath for 30min, 60min, 90min, 120min and 180min under the protection of nitrogen, and compared with the protection conditions of four different antioxidants of T-butyl-1, 4-phenol, BHA, BHT and ascorbic acid, the results show that the recovery rates of quercetin, kaempferol and astragalin in an antioxidant are lower than 30 percent under the condition of no antioxidant, the recovery rates of quercetin and kaempferol under the condition of existence of ascorbic acid are not obviously improved, the recovery rates of quercetin and kaempferol after the addition of T-butyl-1, 4-phenol can reach more than 70 percent (shown in Table 3), the concentration of the aglycon in the ethanol solution has no obvious influence on the results, the dissolution rate of flavonoid glycoside and the isoflavone glycoside is found in the test, the hydrolysis rate is lower than 120 percent under the condition of hydrolysis of the hydrolysis of 10 percent under the condition of the hydrolysis of the ethanol solution under the condition of 3510 percent under the condition of hydrolysis of 10 percent under the hydrolysis of the hydrolysis of.

Table 3 shows the recovery of different antioxidants

Compound (I)	Without adding antioxidant	Adding T-butyl-1, 4-phenol	Adding L- (+) ascorbic acid
				Puerarin and its preparation method	73.1％	75.2％	69.8％
Liquiritigenin	69.5％	74.8％	78.3％
				Soyabean extract	94.7％	95.1％	102％
Scutellarein	68.8％	76.4％	66.4％
				Daidzein	105％	106％	109％
Quercetin	22.9％	78.3％	45.2％
				Tectorigenin	85.4％	108％	110％
Genistein	70.8％	107％	104％
				Kaempferol	24.9％	88.8％	51.2％
Baicalein	27.0％	71.9％	63.6％
				Formononetin	102％	104％	98.6％

2.2 selection of purification conditions

When the liquid chromatography-mass spectrometry/mass spectrometry method is used for quantitative measurement, the liquid-liquid extraction purification, solid-phase extraction purification, QuEChERS and other purification methods are usually adopted to reduce the interference of the background, reduce the influence of matrix effect and improve the accuracy of the detection result. This test investigated C₁₈Adding 100ng/m L standard solution of flavonoid aglycone into 50% ethanol solution (containing 10% hydrochloric acid and 0.1g of T-butyl-1, 4-phenol), adding 9m L water into 1m L, uniformly mixing, transferring into each solid phase extraction column, and respectively eluting with 10%, 20%, 30%, 40%, 50% methanol solution and methanol, and the result shows that C is a compound of the formula₁₈The solid phase extraction column is used for purification, puerarin can be eluted when the methanol content is 20-30%, daidzein and liquiritigenin can be eluted when the methanol content is 50%, puerarin can be eluted when the methanol content is 50% and other compounds can not be eluted when the H L B solid phase extraction column is used for purification, the recovery rates of H L B of Waters and Biocomma are not obviously different, the stability of Waters H L B is slightly good, the condition of comprehensive extraction of hydrolysate is adopted, and the experiment adopts the Waters H L B solid phase extraction column for carrying out methodology investigation.

2.3 examination of chromatographic conditions

The results of the experiments comparing two types of columns, namely Mightysil RP-18, 3 mu m, 4.6 × 150mm and Inertsil ODS-3, 3 mu m, 4.6 × 150mm, with methanol or acetonitrile as an organic phase and 0.15% formic acid solution or water as an aqueous phase show that under the same chromatographic conditions, the Mightysil RP-18 has a slightly better separation effect, wherein the signals of baicalin and scutellarin are improved by 3 times and 5 times when the Mightysil RP-18 is used as the separation column, and the Mightysil RP-18 is finally used as the separation column to analyze the target compound.

Under the same chromatographic column and mobile phase gradient elution program, the signal response and peak shape of most compounds are better than those of an acetonitrile organic phase when methanol is an organic phase (wherein the signal response of rutin and kaempferol-3-O-rutinoside is improved by 5 times when methanol is a mobile phase), and the experiment adopts methanol as an organic phase to separate from a target compound in consideration of effective connection with pretreatment purification. Under the condition, scutellarein (285.1/117.1) and kaempferol (285.1/117.1) can realize effective separation.

Comparing the separation of the methanolic organic phase, water and 0.15% formic acid solution as aqueous phase under a mightysil rp-18 column, it was found that most of the compounds showed better peak shape and response in the 0.15% formic acid solution than in water (wherein baicalin and scutellarin showed tongue extending peaks when water was mobile phase), whereas daidzin, daidzin and formononetin showed different phenomena, the signal response of daidzin was increased by nearly 10 times when water was mobile phase, and the signal response of daidzin and formononetin was increased by nearly 20 times when water was mobile phase. Experiments show that water is adopted as a mobile phase for daidzin, daidzin and formononetin, and a 0.15% formic acid solution is adopted as a mobile phase for other daidzin and aglycon.

Therefore, in the experiment, Mightysill RP-18 is used as a separation column, methanol is used as an organic phase, water is used as a mobile phase for analysis of daidzin, daidzin and formononetin, and a 0.15% formic acid solution is used as a mobile phase for analysis of other daidzin and aglycone.

2.4 examination of Mass Spectrometry conditions

The experiment respectively dilutes standard solutions of flavonoid glycoside and aglycone, adopts a flow injection mode to perform mother ion full scanning in an anion mode, determines a molecular ion peak, and respectively uses the molecular ion of a compound to be detected as the mother ion to perform full scanning on the daughter ion. According to the requirement of EU EC/657 instruction, 4 identification points (parent ion 1 point, characteristic daughter ion 1.5 points/piece) are required to be satisfied when the compound quantitative confirmation is carried out, and two characteristic daughter ions are selected, wherein ion pairs with high signal-to-noise ratio, good peak shape and small interference are taken as quantitative ion pairs. Various mass spectral parameters were optimized in a multiple reaction monitoring negative ion mode, and the optimal mass spectral conditions obtained are shown in table 1.

2.5 Linear relationship and quantitative limits of the method

Determining the spectral peak area of each aglycone in the analysis result under the determined experimental condition, drawing a working curve of the mixed standard solution of each compound by taking the peak area Y of the standard sample as the ordinate and the mass concentration X (%) of the substance to be detected as the abscissa, and determining the correlation coefficient r of the linear equation of each compound²Greater than 0.997. The method has the quantitative limit (calculated by S/N more than 10) of 0.10 percent (namely 1.0 mg/g: puerarin, daidzein and baicalein) and 0.05 percent (namely 0.5 mg/g: liquiritigenin, scutellarein, daidzein, quercetin, tectorigenin, genistein, kaempferol and formononetin), and meets the judgment requirements of Chinese pharmacopoeia on the root of kudzu vine and the root of kudzu vine.

2.6 method recovery and precision experiment

The hydrolysis and purification analysis of kudzu root powder under the determined experimental conditions, it was determined that one kudzu root powder contained 0.5% puerarin and 0.1% daidzein, each flavonoid glycoside (corresponding to 0.25%, 0.5% and 1.0% of the corresponding flavonoid aglycone content after hydrolysis) was added to the kudzu root powder, 3 concentration level addition recovery experiments were performed, and 6 replicates were taken for each concentration level. The results are shown in table 4, and the overall recovery of the process, quantified by external standard method, is 67.8% -102%, relative standard deviation: 3.5 to 12.0 percent.

Table 4 spiked recovery and relative standard deviation at 3 levels in puerariae radix (n ═ 6)

The recovery rate was (measured value-blank)/added concentration × 100%.

3 conclusion

The method comprises the steps of hydrolyzing flavonoid glycoside in kudzuvine root by using an ethanol solution of hydrochloric acid, extracting flavonoid aglycone, purifying an H L B solid-phase extraction column, detecting by using a liquid chromatography-tandem mass spectrometry method, and quantifying by using an external standard method, so that effective hydrolysis of representative flavonoid glycoside (puerarin-6' -O-xyloside, liquiritin, daidzin, scutellarin, daidzin, rutin, iritin, genistin, kaempferol-3-O-rutinoside, baicalin and formononetin) in kudzuvine root, and determination of the content of puerarin, liquiritin, daidzein, scutellarin, daidzein, quercetin, tectorigenin, genistein, kaempferol, baicalein and formononetin are realized.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously measuring the contents of multiple flavonoid aglycones in kudzuvine root comprises the steps of extraction, purification and measurement, wherein the extraction adopts a hydrochloric acid ethanol solution hydrolysis extraction method, and is characterized in that T-butyl-1, 4-phenol is added into a sample as an antioxidant during extraction.

2. The method as claimed in claim 1, wherein the amount of T-butyl-1, 4-phenol added is 3-10 times the mass of the sample.

3. The method for simultaneously measuring the content of the flavonoid aglycones in the kudzuvine root according to claim 2, wherein the extraction hydrolysis conditions comprise hydrolysis for 120min under the protection of 10 wt% hydrochloric acid, 50VO L% ethanol solution, T-butyl-1, 4-phenol and nitrogen.

4. The method of claim 3, wherein the simultaneous determination of the content of the flavonoid aglycones in radix Puerariae comprises weighing 0.1g of the sample to 0.01g, placing in a 150m L round bottom flask, adding 0.5g of T-butyl-1, 4-phenol, 36m L50 ethanol solution of 50VO L% and 4.0m L concentrated hydrochloric acid, heating in water bath under reflux for 120min under nitrogen protection, cooling under nitrogen protection, and adding water to 50m L.

5. The method of claim 1, wherein H L B solid phase extraction column is used for purification.

6. The method for simultaneously measuring the content of the multiple flavonoid aglycones in the kudzu roots according to claim 5, wherein the purification step comprises the steps of taking 1m L of the extracted supernatant, adding 9m L water, uniformly mixing, transferring to an H L0B solid phase extraction column, sequentially activating with 5m L1 methanol and 5m L25 VO L3% methanol solution, adding 5m L420 VO L% methanol solution for leaching, draining, adding 10m L methanol for elution, controlling the flow rate to be 1m L/min-2 m L/min, collecting all eluents, fixing the volume to 10m L, shaking uniformly, passing through a 0.22 mu m organic filter membrane, taking 0.1m L constant volume solution, adding 40VO L% methanol solution for dilution to be 10m L.

7. The method of claim 1, wherein the flavonoid aglycone is one or more of puerarin, liquiritigenin, daidzein, scutellarin, daidzein, quercetin, tectorigenin, genistein, kaempferol, baicalein, and formononetin.

8. The method for simultaneously measuring the contents of a plurality of flavonoid aglycones in the kudzuvine root according to any one of claims 1 to 7, wherein the measurement adopts a liquid chromatography-tandem mass spectrometry method to carry out quantitative measurement on the flavonoid aglycones, and comprises the following steps:

9. The method for simultaneously measuring the contents of a plurality of flavonoid aglycones in radix puerariae according to claim 8, wherein the specific parameters of a chromatographic column are as follows:

10. the method for simultaneously measuring the contents of the flavonoid aglycones in the kudzuvine root according to claim 8, wherein the specific parameters of mass spectrum are as follows:

other mass spectral parameters are shown in the following table:

is a quantitative ion pair.