CN113588801A - Method for evaluating quality of Tibetan medicine rhododendron anthopogonoides by one-test-multiple evaluation and application - Google Patents

Method for evaluating quality of Tibetan medicine rhododendron anthopogonoides by one-test-multiple evaluation and application Download PDF

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CN113588801A
CN113588801A CN202011549272.9A CN202011549272A CN113588801A CN 113588801 A CN113588801 A CN 113588801A CN 202011549272 A CN202011549272 A CN 202011549272A CN 113588801 A CN113588801 A CN 113588801A
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component
solution
detected
test
rhododendron
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李顺祥
李娟�
吴尚洁
彭瑶
雷思
孙智娜
何秀琴
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Qinghai Provincial Prison Administration Central Hospital (qinghai Red Cross Hospital)
Second Xiangya Hospital of Central South University
Hunan University of Chinese Medicine
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Qinghai Provincial Prison Administration Central Hospital (qinghai Red Cross Hospital)
Second Xiangya Hospital of Central South University
Hunan University of Chinese Medicine
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G01N30/8634—Peak quality criteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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Abstract

The invention relates to the technical field of traditional Chinese medicine detection, and discloses a method for evaluating the quality of Tibetan medicine rhododendron anthopogonoides by one test and multiple tests and application thereof, wherein the method comprises the following steps: preparing a reference substance solution and a test substance solution; carrying out chromatographic analysis; determining the position of a chromatographic peak of a target compound; the content of the target compound is calculated. According to the method, hyperoside is selected as an internal standard substance, the content of main active components in a sample to be detected is calculated by adopting relative correction factors of rutin, quercitrin, quercetin and kaempferol, and a high performance liquid chromatography technology and a one-detection-multiple-evaluation method are combined, so that the experiment cost and the detection period can be reduced, the method has the characteristics of simplicity, convenience, high efficiency, high accuracy and good repeatability, and the improvement and reasonable and efficient utilization of the quality standard of rhododendron anthopogonoide can be effectively promoted.

Description

Method for evaluating quality of Tibetan medicine rhododendron anthopogonoides by one-test-multiple evaluation and application
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for evaluating the quality of Tibetan medicine rhododendron anthopogonoides by one test and an application thereof.
Background
The Rhododendron anthopogonoides is dry tender branches, leaves and flowers of Rhododendron anthopogonoides (Rhododendron anthopogonoides Maxim.) or Rhododendron moulmainense (Rhododendron cephalum Franch.) of Ericaceae, also called folium Eriobotryae, Passiflora incana, ramulus et folium Eriobotryae, wild fructus Eriobotryae or Rhododendron Jujulianum, and is one of the sources of Tibetan medicines "dary".
Tender branches and leaves and flowers of rhododendron anthopogonoides are commonly used as medicines in Tibetan medicines, have the effects of clearing heat, reducing swelling and tonifying kidney, are used for treating tracheitis, emphysema, edema, dyspepsia, gastroptosis, weakness, water and soil inadequacy and the like, and are also used for treating sore pestilence when being externally used. Modern pharmaceutical research shows that rhododendron anthopogonoides has pharmacological effects of relieving cough and asthma, eliminating phlegm, inhibiting bacteria, resisting inflammation and the like.
A large amount of literature data and preliminary research of subject groups show that rhododendron anthopogonoide contains various flavonoid compounds, wherein 5 components such as hyperoside, rutin, quercitrin, quercetin, kaempferol and the like are main drug effect substances of the rhododendron anthopogonoide for playing anti-inflammatory, cough relieving, asthma relieving and bacteriostasis roles, and the content of the hyperoside in the rhododendron anthopogonoide medicinal material can reach 5.8mg/g and is higher than the content of other flavonoid components.
Rhododendron anthopogonoides is recorded in the "Ministry of health medicine Standard Tibetan medicine booklet" (WS)3BC-0078-95) and Qinghai province Tibetan medicine processing standard (2010 edition), the former only measures the volatile oil in rhododendron anthopogonoide, and the latter only measures the content of a single component quercetin in rhododendron anthopogonoide decoction pieces.
Because the main material composition of the rhododendron anthopogonoide playing the effects is not a single component, and quercetin is used as an index component, the quercetin exists in the rhododendron anthopogonoide and also exists in bupleurum, mulberry leaves, sophora fruit, inula flowers, hawthorn and other medicinal materials, the content of the quercetin is only measured, the specificity is lacked, and the quality of the rhododendron anthopogonoide medicinal material cannot be effectively controlled.
At present, the content determination method of rhododendron anthopogonoides is reported in the existing literature, an HPLC method is adopted, determination and data processing are carried out through an external standard method, the types of reference substances needed in the experiment are many, the consumption is large, the reference substances are difficult to obtain, and the experiment process is complicated.
Disclosure of Invention
The invention aims to solve the problems of lack of specificity, complicated experiment and high cost in the existing rhododendron anthopogonoides quality control technology.
In order to achieve the above object, the present invention provides, in a first aspect, a method for evaluating the quality of a Tibetan medicine rhododendron anthopogonoide by multiple tests, the method comprising:
(1) preparing a reference solution and a test solution:
preparation of control solutions: mixing a hyperin reference substance with a solvent I and fixing the volume to obtain a reference substance solution;
preparation of a test solution: mixing rhododendron anthopogonoides with a solvent II, and fixing the volume to obtain the test solution;
(2) chromatographic conditions are as follows: respectively carrying out high performance liquid chromatography detection analysis on the reference solution and the test solution to obtain a high performance liquid chromatogram; the conditions of the high performance liquid chromatography detection analysis comprise: the chromatographic column is C18A chromatographic column; the mobile phase isGradient elution of acetonitrile with 0.1-0.3 vol% phosphoric acid; the detection wavelength is 190-400 nm; the column temperature is 25-35 ℃; the sample amount is 1-20 mu L;
(3) determining the position of the chromatographic peak of the target compound: determining the chromatographic peak position of hyperoside in the sample solution with the chromatographic peak of hyperoside reference substance in the reference substance solution as reference, and determining the retention time t of at least one component to be tested selected from rutin, hyperoside, quercetin and kaempferol in the sample solution according to formula (I)iDivided by the retention time t of the internal standard substance hyperin in the test solutionsCalculating to obtain the relative retention time t of the component to be tested in the test solutionfAnd the theoretical relative retention time is ti/sSaid t isfAnd said ti/sSatisfies the following conditions: | tf-ti/s|/ti/sDetermining the corresponding chromatographic peak position of the component to be detected in the high performance liquid chromatogram of the test solution, wherein the chromatographic peak position is less than or equal to 5%;
tf＝ti/tsformula (I);
wherein, in the formula (I), tfActual relative retention time; t is tiThe retention time of the component to be detected in the test solution is min; t is tsThe retention time of the internal standard substance hyperin in the test solution is min;
when the component to be detected is hyperin, the ti/sIs 1.000;
when the component to be detected is rutin, the t isi/s0.9638;
when the component to be detected is quercitrin, the ti/sIs 1.357;
when the component to be tested is quercetin, ti/sIs 1.852;
when the component to be detected is kaempferol, t isi/s2.085;
(4) calculating the content of the target compound:
calculating the content C of the component to be detected in the test solution according to the formula (II)iThe components to be detected are hyperin, rutin and quercetinQuercetin or kaempferol;
Ci＝fs/i×Ai×Cs/Asformula (II);
wherein f iss/iAs a relative correction factor, AsThe peak area of hyperin in the high performance liquid chromatogram of the reference solution is shown; csThe unit is the mass concentration of the hyperin in the reference solution, and the unit is mu g/mL; a. theiThe peak area of the component to be detected in the high performance liquid chromatogram of the test solution is shown; ciThe unit is the mass concentration of the component to be detected, and the unit is mu g/mL;
when the component to be detected is hyperin, the fs/iIs 1.000;
when the component to be tested is rutin, the fs/iIs 1.243;
when the component to be detected is quercitrin, the fs/i0.9905;
when the component to be tested is quercetin, fs/i0.5350;
when the component to be detected is kaempferol, fs/iIs 0.7813.
In a second aspect, the invention provides the use of the method of the first aspect in the first-test multi-evaluation quality evaluation of Tibetan medicine rhododendron anthopogonoide.
Compared with the existing rhododendron anthopogonoide quality control technology, the method for evaluating the quality of the Tibetan medicine rhododendron anthopogonoide by one test and multiple tests provided by the invention has at least the following advantages:
(1) according to the detection method provided by the invention, hyperoside is selected as an internal standard substance, and the content of the main active components in the sample to be detected is calculated by adopting relative correction factors of rutin, quercitrin, quercetin and kaempferol, so that the experiment cost and the detection period can be reduced, and the quantitative detection method of the active components in rhododendron anthopogonoide is simplified;
(2) the detection method provided by the invention combines a high performance liquid chromatography technology with a one-test-multiple evaluation method, has the characteristics of simplicity, high efficiency, high accuracy and good reproducibility, and can effectively promote the improvement and reasonable and efficient utilization of the quality standard of rhododendron anthopogonoides medicinal materials.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
FIG. 1 is a high performance liquid chromatogram of a 5-component control mixed solution described in test example 1;
FIG. 2 is a high performance liquid chromatogram of a rhododendron anthopogonoide sample S1 from example 1.
Description of the reference numerals
1 rutin 2 Hyperoside
3 Quercetin 4 Quercetin
5 Kaempferol
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
As described above, the first aspect of the present invention provides a method for evaluating the quality of a Tibetan medicine rhododendron anthopogonoide by one-test-multiple evaluation, comprising:
(1) preparing a reference solution and a test solution:
preparation of control solutions: mixing a hyperin reference substance with a solvent I and fixing the volume to obtain a reference substance solution;
preparation of a test solution: mixing rhododendron anthopogonoides with a solvent II, and fixing the volume to obtain the test solution;
(2) chromatographic conditions are as follows: respectively carrying out high performance liquid chromatography detection analysis on the reference solution and the test solution to obtain a high performance liquid chromatogram; the conditions of the high performance liquid chromatography detection analysis comprise: the chromatographic column is C18A chromatographic column; the mobile phase is acetonitrile and 0.1-0.3 volume percent phosphoric acid gradient elution; the detection wavelength is 190-400 nm; the column temperature is 25-35 ℃;the sample amount is 1-20 mu L;
(3) determining the position of the chromatographic peak of the target compound: determining the chromatographic peak position of hyperoside in the sample solution with the chromatographic peak of hyperoside reference substance in the reference substance solution as reference, and determining the retention time t of at least one component to be tested selected from rutin, hyperoside, quercetin and kaempferol in the sample solution according to formula (I)iDivided by the retention time t of the internal standard substance hyperin in the test solutionsCalculating to obtain the relative retention time t of the component to be tested in the test solutionfAnd the theoretical relative retention time is ti/sSaid t isfAnd said ti/sSatisfies the following conditions: | tf-ti/s|/ti/sDetermining the corresponding chromatographic peak position of the component to be detected in the high performance liquid chromatogram of the test solution, wherein the chromatographic peak position is less than or equal to 5%;
tf＝ti/tsformula (I);
wherein, in the formula (I), tfActual relative retention time; t is tiThe retention time of the component to be detected in the test solution is min; t is tsThe retention time of the internal standard substance hyperin in the test solution is min;
when the component to be detected is hyperin, the ti/sIs 1.000;
when the component to be detected is rutin, the t isi/s0.9638;
when the component to be detected is quercitrin, the ti/sIs 1.357;
when the component to be tested is quercetin, ti/sIs 1.852;
when the component to be detected is kaempferol, t isi/s2.085;
(4) calculating the content of the target compound:
calculating the content C of the component to be detected in the test solution according to the formula (II)iThe component to be detected is hyperoside, rutin, quercetin, quercitrin or kaempferol;
Ci＝fs/i×Ai×Cs/Asformula (II);
wherein f iss/iAs a relative correction factor, AsThe peak area of hyperin in the high performance liquid chromatogram of the reference solution is shown; csThe unit is the mass concentration of the hyperin in the reference solution, and the unit is mu g/mL; a. theiThe peak area of the component to be detected in the high performance liquid chromatogram of the test solution is shown; ciThe unit is the mass concentration of the component to be detected, and the unit is mu g/mL;
when the component to be detected is hyperin, the fs/iIs 1.000;
when the component to be tested is rutin, the fs/iIs 1.243;
when the component to be detected is quercitrin, the fs/i0.9905;
when the component to be tested is quercetin, fs/i0.5350;
when the component to be detected is kaempferol, fs/iIs 0.7813.
In the invention, the quality evaluation method of the Tibetan medicine rhododendron anthopogonoide by multiple tests is a method for detecting the content of active ingredients in the rhododendron anthopogonoide.
In order to obtain the reference solution or the sample solution having a desired concentration, the mixing and volume-fixing operations may be performed 1 or 2 or more times, and the present invention is not limited to the number of times, and the scope of the present invention encompasses 1 or 2 or more times.
In the present invention, the aforementioned method for determining the position of the chromatographic peak of the target compound is also the following method:
taking the chromatographic peak of the hyperin reference substance in the reference substance solution as a reference, and defining the chromatographic peak as an S peak; calculating the theoretical retention time t of at least one component to be measured selected from rutin, hyperoside, quercetin and kaempferol in the test solution according to the formula (III)i1(ii) a According to the theoretical retention time ti1Determining the corresponding chromatogram of the component to be detected in the high performance liquid chromatogram of the test solutionPeak position to obtain the actual retention time t of the component to be measuredf1And said ti1And said tf1Satisfies the following conditions: | tf1-ti1|/ti1≤5％；
ti1＝ti1/s1×ts1Formula (III);
wherein, in the formula (III), ti1/s1Is the relative retention time; t is ti1The theoretical retention time of the component to be measured in the test solution is min; t is ts1The retention time of the hyperin reference substance in the reference substance solution is min;
when the component to be detected is hyperin, the ti1/s1Is 1.000;
when the component to be detected is rutin, the t isi1/s10.9638;
when the component to be detected is quercitrin, the ti1/s1Is 1.357;
when the component to be tested is quercetin, ti1/s1Is 1.852;
when the component to be detected is kaempferol, t isi1/s1Is 2.085.
Preferably, in the step (1), the mass concentration of the hyperin in the control solution is 15.40-308.0 μ g/mL.
More preferably, the mass concentration of the hyperin in the control solution is 100-.
Preferably, in the step (1), the rhododendron anthopogonoide is a powdered rhododendron anthopogonoide, and the powdered rhododendron anthopogonoide is obtained by sieving crushed rhododendron anthopogonoide, and the sieve is a second sieve, a fourth sieve or a fifth sieve. More preferably, the screen is a No. four screen.
Preferably, in step (1), the solvent I is methanol; the solvent II is methanol water solution and/or ethanol water solution.
Preferably, in the step (1), the mass ratio of the solvent II to the rhododendron anthopogonoides is 20-40: 1.
preferably, in the step (1), in the solvent II, the methanol aqueous solution is an aqueous solution containing 50 to 100 vol% of methanol, and the ethanol aqueous solution is an aqueous solution containing 50 to 100 vol% of ethanol.
More preferably, in the solvent II, the methanol aqueous solution is an aqueous solution containing 80 to 100 vol% of methanol, and the ethanol aqueous solution is an aqueous solution containing 80 to 100 vol% of ethanol.
Preferably, in step (1), the rhododendron anthopogonoides is mixed with the solvent II under ultrasonic conditions in the preparation of the test solution.
More preferably, the conditions of the ultrasound include: the time is 20-60min, preferably 30-40 min; the frequency is 30-50 KHz.
Further preferably, the conditions of the ultrasound include: the time is 30min and the frequency is 40 KHz.
In the present invention, in step (1), there is no particular limitation on the preparation method of the sample solution, and a preparation method known to those skilled in the art can be adopted, and the present invention is not described herein again, and a specific operation is listed hereinafter, and those skilled in the art should not be construed as a limitation to the present invention.
Preferably, in step (2), the conditions of the high performance liquid chromatography detection analysis include: the chromatographic column is selected from CAPCELL PAK C18、Venusil XBP C18And Phenomenex Luna C18At least one of;
the mobile phase is acetonitrile and 0.2 volume percent phosphoric acid gradient elution;
the detection wavelength is 254nm or 365 nm;
the column temperature is 30 ℃; the sample amount is 10-20 μ L.
In the invention, in the step (2), the reference solution and the test solution are preferably filtered by microporous filter membranes respectively, and then subjected to high performance liquid chromatography detection and analysis.
In the present invention, the microfiltration membrane is preferably a 0.22 μm microfiltration membrane.
Preferably, in step (2), the elution gradient of the mobile phase of the hplc assay is: 0-15min, 10-15 vol% of said acetonitrile; 15-50min, 15-20 vol% of said acetonitrile; 50-60min, 20-30 vol% of said acetonitrile; 60-70min, 30-35 vol% of said acetonitrile; 70-80min, 35-60 vol% of said acetonitrile; 80-90min, 60-10 vol% of the acetonitrile.
In the present invention, the mobile phase consists of acetonitrile and 0.2 vol% phosphoric acid, and in the elution gradient, the remaining amount of the mobile phase is 0.2 vol% phosphoric acid except the amount of the acetonitrile.
The present invention is not particularly limited to the specific operation of the elution gradient of the mobile phase in the hplc analysis, and can be performed by the operation methods known to those skilled in the art, and the present invention is not described herein again.
As mentioned above, the second aspect of the present invention provides the use of the method of the first aspect in the first-test multi-evaluation quality evaluation of Tibetan medicine rhododendron anthopogonoide.
The present invention will be described in detail below by way of examples.
In the following examples, unless otherwise specified, the laboratory instruments and raw materials are commercially available.
Laboratory apparatus
Waters2695 high performance liquid chromatograph: waters corporation, USA;
agilent 1260Infinity hplc: agilent, USA;
CAPCELL PAK C18a chromatographic column: 4.6X 250mm, 5 μm, Japan Seisakusho Co;
Venusil XBP C18a chromatographic column: 4.6X 250mm, 5 μm, Aijer USA;
Phenomenex Luna C18a chromatographic column: 4.6X 250mm, 5 μm, Fenomei, USA;
model JS15-01 electronic balance: shanghai Puchun Meter, Inc.;
BSA124S-CW model 1/10 ten thousand electronic balance: sidoris scientific instruments, Inc.
The external standard method referred to in the following examples is a method of obtaining the content of the component to be detected by using a reference substance of the component to be detected as a reference substance and comparing response signals (i.e., peak area size in a chromatogram) of the reference substance and the component to be detected in a sample, and the specific operation of the method is not particularly limited in the present invention, and can be performed by using an operation method known to those skilled in the art, and the method is not described herein again.
Raw materials
Hyperin: lot number 111521-;
rutin: the batch number is 100080 and 201811 for content determination, China institute for food and drug assay;
and (3) quercetin: the batch number is 100080 and 201811 for content determination, China institute for food and drug assay;
kaempferol: the batch No. 110861-202013 for content determination, China institute for testing food and drug;
and (3) quercetin: lot number P19D10F106420 for content determination, shanghai yuan leaf biotechnology limited;
acetonitrile: chromatographically pure, TEDIA corporation, usa;
purified water: yibao Corp;
the other reagents are analytically pure, national drug group chemical reagent limited;
the rhododendron anthopogonoides medicinal materials: entrusted to Tibetan medicinal material decoction piece processing Limited company in Qinghai snow region, 12 batches of Rhododendron anthopogonoide medicinal materials are collected from different areas of Qinghai province, the information of the medicinal materials is shown in Table 1, and dried tender leaves and flowers of Rhododendron anthopogonoide (Rhododendron anthopogonoides Maxim.) belonging to Ericaceae are identified by Trauda college of traditional Chinese medicine resource, university of Hunan China.
Each sample in table 1 was pulverized and sieved through a No. four sieve to obtain a powder of each sample.
TABLE 1
Sample numbering Producing area Time of purchase
S1 Menyuan Hui Autonomous County 2018-09
S2 Menyuan Hui Autonomous County 2018-10
S3 Hualong Hui Autonomous County 2019-08
S4 Hualong Hui Autonomous County 2019-09
S5 Datong county of Xining City 2020-09
S6 Datong county of Xining City 2020-10
S7 Mutually-aided counties of Shandong City 2020-08
S8 Mutually-aided counties of Shandong City 2020-09
S9 Yushu Tibetan Autonomous Prefecture 2020-07
S10 Yushu Tibetan Autonomous Prefecture 2020-09
S11 Qilian county of North China sea 2020-07
S12 Qilian county of North China sea 2020-09
Test example 1: measurement of precision of method
(1) Preparing a hyperin reference substance solution and a 5-component reference substance mixed solution:
preparation of hyperin reference solution: weighing 7.7mg of hyperin, placing the hyperin in a 10mL volumetric flask, mixing with methanol and fixing the volume; transferring 4mL of the solution, placing the solution in a 10mL volumetric flask, mixing the solution with methanol and fixing the volume to obtain the reference substance solution with the mass concentration of the hyperin being 308 mu g/mL;
preparation of 5-component reference substance mixed solution: respectively weighing rutin 4.6mg, hyperin 7.7mg, quercetin 5.0mg, quercetin 4.9mg and kaempferol 1.0mg, placing in a 10mL volumetric flask, mixing with methanol and fixing volume; transferring 4mL of the solution, placing the solution in a 10mL volumetric flask, mixing the solution with methanol and fixing the volume to obtain 5-component reference substance mixed solution with the mass concentrations of the rutin, the hyperoside, the quercetin, the quercitrin and the kaempferol being 184, 308, 200, 196 and 40 mu g/mL respectively;
(2) chromatographic conditions are as follows: performing high performance liquid chromatography detection analysis (analysis conditions are described below) on the hyperin reference solution and the 5-component reference mixed solution respectively to obtain a high performance liquid chromatogram, wherein the high performance liquid chromatogram of the 5-component reference mixed solution is shown in fig. 1;
the conditions of the high performance liquid chromatography detection analysis comprise: the high performance liquid chromatography system is a Waters2695 liquid chromatography system, and the chromatographic column is Venusil XBP C18(ii) a The mobile phase is acetonitrile and 0.2 volume percent phosphoric acid gradient elution; the detection wavelength is 254 nm; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 10 mu L;
the elution gradient of the mobile phase is: 0-15min, 10-15 vol% of said acetonitrile; 15-50min, 15-20 vol% of said acetonitrile; 50-60min, 20-30 vol% of said acetonitrile; 60-70min, 30-35 vol% of said acetonitrile; 70-80min, 35-60 vol% of said acetonitrile; 80-90min, 60-10 vol% of the acetonitrile.
(3) Determination of the position of the chromatographic peak of each active compound: determining the chromatographic peak position of the hyperin in the 5-component reference mixed solution by taking the chromatographic peak of the hyperin in the hyperin reference solution as a reference, and then determining the chromatographic peak positions of the rest 4 components in the chromatogram of the 5-component reference mixed solution according to the retention time of each absorption peak in the chromatogram and by combining the formula (I); the corresponding t in the chromatogram of the 5-component reference substance mixed solution can be obtained through calculationfAnd t of the inventioni/sSatisfies the following conditions: | tf-ti/s|/ti/s≤5％；
(4) The content of each active compound was calculated:
calculating the content C of each active component in the 5-component reference substance mixed solution according to the formula (II)iThe components are hyperin, rutin, quercetin, quercitrin and kaempferol respectively;
Ci＝fs/i×Ai×Cs/Asformula (II);
obtaining peak area A from the high performance liquid chromatogram of the hyperin reference solutionsAnd the hyperin reference solution prepared as aboveMass concentration of Cs308 μ g/mL; respectively obtaining peak areas A of the components from the high performance liquid chromatogram of the 5-component reference substance mixed solutioniAnd the mass concentration of each active component in the 5-component reference substance mixed solution is obtained by calculation as follows:
Chyperoside A＝306μg/mL；
CRutin＝188μg/mL；
CQuercetin＝195μg/mL；
CQuercetin＝193μg/mL；
CKaempferol＝38μg/mL；
Comparison of C calculated by the method of the inventioniThe value and the actual mass concentration value of the 5-component reference substance mixed solution prepared in the previous step are known, and the RSD is less than 2.0%, namely, the method has good precision.
In the following examples, the methodology investigation was carried out using the following methods:
preparation of 5-component reference substance mixed solution: respectively weighing rutin 4.6mg, hyperin 7.7mg, quercetin 5.0mg, quercetin 4.9mg and kaempferol 1.0mg, placing in a 10mL volumetric flask, mixing with methanol and fixing volume; transferring 4mL of the solution, placing the solution in a 10mL volumetric flask, mixing the solution with methanol and fixing the volume to obtain 5-component reference substance mixed solution with the mass concentrations of the rutin, the hyperoside, the quercetin, the quercitrin and the kaempferol being 184, 308, 200, 196 and 40 mu g/mL respectively;
preparation of a test solution: about 0.5g of the powder of crushed rhododendron anthopogonoide sample S1 (sieved through a sieve four) was precisely weighed, placed in a stopper-cone-shaped bottle, and mixed with 15mL of an aqueous solution containing 80 vol% of methanol, weighed to mass, and then subjected to ultrasonic treatment under conditions including: the time is 30min, and the frequency is 40 KHz; weighing the sample solution again, supplementing the loss mass with an aqueous solution containing 80 vol% of methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the sample solution;
chromatographic conditions are as follows: the high performance liquid chromatography system is a Waters2695 liquid chromatography system,the chromatographic column is Venusil XBP C18(ii) a The mobile phase is acetonitrile and 0.2 volume percent phosphoric acid gradient elution; the detection wavelength is 254 nm; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 10 mu L;
the elution gradient of the mobile phase is: 0-15min, 10-15 vol% of said acetonitrile; 15-50min, 15-20 vol% of said acetonitrile; 50-60min, 20-30 vol% of said acetonitrile; 60-70min, 30-35 vol% of said acetonitrile; 70-80min, 35-60 vol% of said acetonitrile; 80-90min, 60-10 vol% of said acetonitrile;
(a) investigation of linear relationships
And (3) precisely absorbing the 5-component reference substance mixed solutions respectively, diluting the 5-component reference substance mixed solutions by adding methanol to prepare 5-component reference substance mixed solutions with series concentrations, carrying out sample injection measurement according to the chromatographic conditions, and taking the peak area (Y) as a vertical coordinate and the concentration (X) of each solution to be measured as a horizontal coordinate to obtain a regression equation, a linear range and a regression coefficient of each solution to be measured, wherein the result shows that the linear relation of each component in a certain concentration range is good, and the result is shown in Table 2.
TABLE 2
Composition (I) Regression equation R Concentration range of μ g/mL-1
Rutin Y＝24221X+8796.9 0.9999 9.200～184.0
Hyperoside A Y＝30295X－358149 0.9996 15.40～308.0
Quercetin Y＝30517X－2352.3 1.0000 9.800～196.0
Quercetin Y＝56483X－4467.9 0.9999 10.00～200.0
Kaempferol Y＝38689X－1749 0.9999 2.000～40.00
(b) Precision test
Precisely absorbing the 5-component reference substance mixed solution, continuously sampling for 6 times according to the chromatographic conditions, wherein the sampling amount is 10 mu L each time, recording peak areas of rutin, hyperoside, quercetin and kaempferol, and calculating RSD of the peak areas. As a result, RSD of each fraction was 0.58%, 0.63%, 0.52%, 0.57% and 0.53%, respectively, indicating good precision of the apparatus.
(c) Stability test
Sucking the sample solution, detecting according to the chromatographic conditions for 0, 2, 4, 8, 12 and 24h, recording peak area, and calculating RSD. The results show that the RSD of rutin, hyperin, quercitrin, quercetin and kaempferol is 1.8%, 2.1%, 2.0% and 1.4%, respectively, and the quality of the test solution is stable within 24 h.
(d) Repeatability test
6 parts of test solution is prepared in parallel according to the method, sample injection is carried out according to the chromatographic conditions, the mass fraction of 5 components to be measured is measured, and RSD is calculated. The results show that the average mass fractions of rutin, hyperoside, quercetin and kaempferol are 2.083, 5.998, 0.406, 0.739 and 0.096mg/g, and the RSD is 1.5%, 2.3%, 1.3%, 2.0% and 1.3%, respectively, which indicates that the method has good repeatability.
(e) Sample application recovery test
Precisely weighing 6 parts of rhododendron anthopogonoides (S1) with known content, each part is about 0.25g, precisely adding appropriate amount of the 5-component reference substance mixed solution, respectively, preparing a sample solution according to the method, performing sample injection analysis according to the chromatographic conditions, measuring the sample recovery rate of the 5 components, and calculating RSD. The sample recovery rates of rutin, hyperoside, quercetin and kaempferol are 103.3%, 98.69%, 102.8%, 100.5% and 102.2%, and the RSD are 1.6%, 2.0%, 0.85%, 1.7% and 1.1%, respectively, which indicates that the recovery rate of the method is good, and the results are shown in Table 3.
TABLE 3
Figure BDA0002857364130000141
Figure BDA0002857364130000151
In the following examples, the relative correction factor (f)s/i) The calculation of (a) is carried out by the following method:
using a multipoint correction method, based on a relative correction factor (f)s/i) Calculation formula fs/i＝fs/fi＝As×Ci/(Ai×Cs)(AsPeak area of the internal standard substance in the 5-component reference substance mixed solution; csThe mass concentration of an internal standard substance in the 5-component reference substance mixed solution is in the unit of mu g/mL; a. theiIs the peak area of the other components to be measured except the internal standard substance in the 5-component reference substance mixed solution；CiIs the mass concentration of the other components to be measured except the internal standard substance in the 5-component reference substance mixed solution, the unit is mu g/mL), f of each component is obtained by calculating a plurality of concentration pointss/iTaking the average value as f for quantifications/i。
Precisely sucking the 5-component reference substance mixed solution, continuously injecting 1, 2, 4, 6, 8, 10 and 20 μ L according to the chromatographic conditions, recording peak areas of the components, taking hyperoside (S) as an internal standard substance, and calculating correction factors (f) of the rest 4 componentss/i) Wherein f iss/AA correction factor for rutin, fs/BIs a correction factor for quercetin, fs/CCorrection factor for Quercetin, fs/DAs a correction factor for kaempferol, the results showed f for the remaining 4 componentss/i1.2426, 0.9905, 0.5350, 0.7813, RSD 0.16%, 0.71%, 0.34%, 0.25%, respectively, all less than 2.0%, the results are shown in Table 4.
TABLE 4
Sample volume (μ L) fs/A fs/B fs/C fs/D
20 1.2421 0.9851 0.5359 0.7788
10 1.2422 0.9892 0.5373 0.7825
8 1.2432 0.9850 0.5363 0.7813
6 1.2440 0.9888 0.5354 0.7784
4 1.2413 0.9885 0.5346 0.7823
2 1.2396 0.9909 0.5333 0.7827
1 1.2458 1.0057 0.5320 0.7832
Mean value of 1.2426 0.9905 0.5350 0.7813
RSD(％) 0.16 0.71 0.34 0.25
In the following examples, the relative correction factor (f)s/i) The durability test of (1) was carried out by the following method:
(a) different HPLC and column pairs fs/iInfluence of (2)
2 different liquid chromatography systems, namely a Waters2695 liquid chromatography system, an active 1260 liquid chromatography system, and 3 different brands of chromatography columns, namely CAPCELL PAK C, were used, respectively18(4.6 mm. times.250 mm, 5 μm) column, Venusil XBP C18(4.6 mm. times.250 mm, 5 μm) column and Phenomenex Luna C18(4.6 mm. times.250 mm, 5 μm) column, and different HPLC chromatographs and column pairs f were examineds/iThe influence of (c).
Measuring the content of rutin, quercetin and kaempferol in different instruments and chromatographic columnss/iThe results show that different HPLC and different chromatographic columns are used for f of active ingredients in rhododendron anthopogonoidess/iThe effect was not significantly different (RSD < 3.0%), and the results are shown in Table 5.
TABLE 5
Figure BDA0002857364130000161
(b) Column temperature pair fs/iInfluence of (2)
Using other conditions than the column temperature in the chromatographic conditions described aboveDetermination of rutin, Quercetin and Kaempferol at column temperature (20, 25, 30 and 35 deg.C)s/iRSD of 0.57%, 0.53%, 0.56% and 0.51%, respectively, and the results show that different column temperatures are vs. fs/iThe effects of (A) were not significantly different (RSD < 2.0%), and the results are shown in Table 6.
TABLE 6
Column temperature deg.C fs/A fs/B fs/C fs/D
20 1.2396 0.9836 0.5416 0.7834
25 1.2434 0.9845 0.5419 0.7793
30 1.2278 0.9737 0.5358 0.7758
35 1.2417 0.9840 0.5420 0.7844
Mean value of 1.2381 0.9814 0.5403 0.7807
RSD(％) 0.57 0.53 0.56 0.51
In the following embodiments, the positioning of the chromatographic peak of the component to be measured in the one-test multi-evaluation method is performed by the following method:
precisely sucking the 5-component reference substance mixed solution, and inspecting the Waters2695 liquid chromatography system, the Aglient 1260 liquid chromatography system and the CAPCELL PAK C according to the chromatographic conditions18(4.6 mm. times.250 mm, 5 μm) column, Venusil XBP C18(4.6 mm. times.250 mm, 5 μm) column, Phenomenex Luna C18(4.6 mm. times.250 mm, 5 μm) column pair ts/iThe influence of (c). Taking hyperoside (S) as an internal standard, calculating retention time (t) of the other 4 componentss/i) Wherein t iss/AThe retention time of rutin, ts/BIs the retention time of quercetin, ts/CIs the retention time of quercetin, ts/DIs the retention time of kaempferol.
As a result, it was found that ts/iThe fluctuation is small, and no significant difference (RSD is less than 5.0 percent) exists, which indicates that different instruments and chromatographic columns can be usedTo use ts/iThe components to be tested were located and the results are shown in Table 7.
TABLE 7
Figure BDA0002857364130000171
In the following examples, the comparison between the one-test-multiple-evaluation method and the external standard method was performed by the following method:
preparing test solution according to the method, determining according to the chromatographic conditions, and establishing internal standard substance of hyperoside and rutin, quercitrin, quercetin and kaempferol by one-test-multiple-evaluation methods/iAnd calculating the content of each component in 12 batches of rhododendron anthopogonoide samples, and simultaneously measuring the content of hyperoside, rutin, quercetin and kaempferol by adopting an external standard method, wherein the result is shown in a table 8.
Comparing the RSD of the one-test-multiple-evaluation method with that of the external standard method shows that the calculation results of the two methods have no significant difference (RSD is less than 2.0 percent), which shows that the one-test-multiple-evaluation method (QAMS) can replace the External Standard Method (ESM) to carry out multi-component quality evaluation.
TABLE 8
Figure BDA0002857364130000181
Table 8 (continuation watch)
Figure BDA0002857364130000182
In the following comparative examples, relative correction factor (f)s/i) The calculation of (a) is carried out by the following method:
using a multipoint correction method, based on a relative correction factor (f)s/i) Calculation formula fs/i＝fs/fi＝As×Ci/(Ai×Cs)(AsPeak area of the internal standard substance in the 5-component reference substance mixed solution; csThe mass concentration of an internal standard substance in the 5-component reference substance mixed solution is in the unit of mu g/mL; a. theiIs the peak area of the other components to be measured except the internal standard substance in the 5-component reference substance mixed solution; ciIs the mass concentration of the other components to be measured except the internal standard substance in the 5-component reference substance mixed solution, the unit is mu g/mL), f of each component is obtained by calculating a plurality of concentration pointss/iTaking the average value as f for quantifications/i。
Precisely sucking the 5-component reference substance mixed solution, continuously injecting 1, 2, 4, 6, 8, 10 and 20 μ L according to the chromatographic conditions, recording peak area of each component, taking quercetin (S) as an internal standard substance, and calculating correction factors (f) of the rest 4 componentss/i) Wherein f iss/AA correction factor for rutin, fs/BIs a correction factor for hyperin, fs/CIs a correction factor for quercetin, fs/DAs a correction factor for kaempferol, the results showed f for the remaining 4 componentss/i2.323, 1.869, 1.852, 1.460, RSD 0.41%, 0.34%, 1.01%, 0.50%, respectively, the results are shown in Table 9.
TABLE 9
Sample volume (μ L) fS/A fS/B fS/C fS/D
20 2.3178 1.8661 1.8383 1.4533
10 2.3119 1.8612 1.8412 1.4564
8 2.3180 1.8646 1.8366 1.4568
6 2.3236 1.8679 1.8470 1.4540
4 2.3220 1.8706 1.8490 1.4634
2 2.3224 1.8750 1.8578 1.4675
1 2.3419 1.8798 1.8905 1.4722
Mean value of 2.3228 1.8693 1.8515 1.4605
RSD(％) 0.41 0.34 1.01 0.50
In the following comparative examples, relative correction factor (f)s/i) The durability test of (1) was carried out by the following method:
(a) different HPLC and column pairs fs/iInfluence of (2)
2 different liquid chromatography systems, namely a Waters2695 liquid chromatography system, an active 1260 liquid chromatography system, and 3 different brands of chromatography columns, namely CAPCELL PAK C, were used, respectively18(4.6 mm. times.250 mm, 5 μm) column, Venusil XBP C18(4.6 mm. times.250 mm, 5 μm) column and Phenomenex Luna C18(4.6 mm. times.250 mm, 5 μm) column, and different HPLC chromatographs and column pairs f were examineds/iThe influence of (c).
Measuring rutin, hyperoside, quercetin and kaempferol in different instruments and chromatographic columnss/iThe results show that different HPLC and different chromatographic columns are used for f of active ingredients in rhododendron anthopogonoidess/iThere was some effect (> 2.0% RSD) and the results are shown in Table 10.
Watch 10
Figure BDA0002857364130000201
(b) Column temperature pair fs/iInfluence of (2)
By usingThe f of rutin, hyperoside, quercetin and kaempferol was measured at different column temperatures (20, 25, 30 and 35 ℃) under the conditions other than the column temperature among the aforementioned chromatographic conditionss/iRSD of 0.10%, 0.57%, 0.05% and 0.32%, respectively, and the results show that different column temperatures are vs. fs/iThe effects of (A) were not significantly different (RSD < 2.0%), and the results are shown in Table 11.
TABLE 11
Column temperature deg.C fS/A fS/B fS/C fS/D
20 2.2888 1.8464 1.8162 1.4465
25 2.2945 1.8453 1.8167 1.4381
30 2.2916 1.8665 1.8174 1.4479
35 2.2909 1.8450 1.8154 1.4472
Mean value of 2.2915 1.8508 1.8164 1.4449
RSD(％) 0.10 0.57 0.05 0.32
In the following comparative examples, the positioning of the chromatographic peak of the component to be measured in the one-test-multiple-evaluation method was carried out by the following method:
precisely sucking the 5-component reference substance mixed solution, and inspecting the Waters2695 liquid chromatography system, the Aglient 1260 liquid chromatography system and the CAPCELL PAK C according to the chromatographic conditions18(4.6 mm. times.250 mm, 5 μm) column, Venusil XBP C18(4.6 mm. times.250 mm, 5 μm) column, Phenomenex Luna C18(4.6 mm. times.250 mm, 5 μm) column pair ts/iThe influence of (c). Using quercetin (S) as internal standard substance, calculating retention time (t) of the other 4 componentss/i) Wherein t iss/AThe retention time of rutin, ts/BThe retention time of hyperin, ts/CIs the retention time of quercetin, ts/DIs the retention time of kaempferol. The results are shown in Table 12.
TABLE 12
Figure BDA0002857364130000211
In the following comparative examples, the comparison between the one-test-multiple-evaluation method and the external standard method was performed by the following method:
preparing test solution according to the method, determining according to the chromatographic conditions, and establishing internal standard substances of quercetin and rutin, hyperoside, quercetin and kaempferol by one-test-multiple-evaluation methods/iAnd the content of each component in 12 batches of rhododendron anthopogonoides samples is calculated, and the content of hyperoside, rutin, quercetin and kaempferol is measured by adopting an external standard method, and the result is shown in a table 13. Comparing the RSD of the one-test-multiple-evaluation method with that of the external standard method, the calculated results of the two methods have certain difference (RSD is less than 2.0%).
However, the contents of rutin, hyperoside, quercetin and kaempferol measured by a one-test-multiple-evaluation method with quercetin as an internal standard substance are all higher than the result measured by an external standard method, and the RSD value of part of detection values is more than 2%.
Taking S1 as an example, the total content of rutin, hyperoside, quercetin and kaempferol in the S1 sample measured by a one-test-multiple-evaluation method taking quercetin as an internal standard substance is 9.3404 mg/g; compared with the total content of the rutin, the hyperoside, the quercitrin, the quercetin and the kaempferol in the S1 sample measured by an external standard method, which is 9.1699mg/g, the total content measured by the one-measurement-multiple-evaluation method is higher; the total content of rutin, hyperoside, quercetin and kaempferol in the S1 sample measured by a one-test-multiple-evaluation method with hyperoside as an internal standard substance is 9.1932mg/g, which is closer to the measured value of an external standard method. Therefore, the one-test-multiple-evaluation method taking the hyperoside as the internal standard substance is more suitable for measuring the content of the rhododendron anthopogonoide medicinal material.
Watch 13
Figure BDA0002857364130000221
Watch 13 (continuation watch)
Figure BDA0002857364130000222
Example 1
The content of active ingredients in rhododendron anthopogonoides sample S1 was measured as follows. Specifically, the method comprises the following steps:
(1) preparing a reference solution and a test solution:
preparation of control solutions: weighing 7.7mg of hyperin, placing the hyperin in a 10mL volumetric flask, mixing with methanol and fixing the volume; transferring 4mL of the solution, placing the solution in a 10mL volumetric flask, mixing the solution with methanol and fixing the volume to obtain the reference substance solution with the mass concentration of the hyperin being 308 mu g/mL;
preparation of a test solution: about 0.5g of the powder of crushed rhododendron anthopogonoide sample S1 (sieved through a sieve four) was precisely weighed, placed in a stopper-cone-shaped bottle, and mixed with 15mL of an aqueous solution containing 80 vol% of methanol, weighed to mass, and then subjected to ultrasonic treatment under conditions including: the time is 30min, and the frequency is 40 KHz; weighing the sample solution again, supplementing the loss mass with an aqueous solution containing 80 vol% of methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the sample solution;
(2) chromatographic conditions are as follows: performing high performance liquid chromatography detection analysis on the reference solution and the test solution respectively to obtain a high performance liquid chromatogram, wherein the high performance liquid chromatogram of the rhododendron anthopogonoides sample S1 is shown in figure 2;
the conditions of the high performance liquid chromatography detection analysis comprise: the high performance liquid chromatography system is a Waters2695 liquid chromatography system, and the chromatographic column is Venusil XBP C18(ii) a The mobile phase is acetonitrile and 0.2 volume percent phosphoric acid gradient elution; the detection wavelength is 254 nm; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 10 mu L;
the elution gradient of the mobile phase is: 0-15min, 10-15 vol% of said acetonitrile; 15-50min, 15-20 vol% of said acetonitrile; 50-60min, 20-30 vol% of said acetonitrile; 60-70min, 30-35 vol% of said acetonitrile; 70-80min, 35-60 vol% of said acetonitrile; 80-90min, 60-10 vol% of said acetonitrile;
(3) it doesDetermining the chromatographic peak position of the target compound: determining the chromatographic peak position of the hyperoside in the sample solution by taking the chromatographic peak of the hyperoside reference substance in the reference substance solution as a reference, and then determining the chromatographic peak positions of the rest 4 components in the chromatogram of the sample solution according to the retention time of each absorption peak in the chromatogram and by combining the formula (I); calculating to obtain the corresponding t in the chromatogram of the test solutionfAnd t of the inventioni/sSatisfies the following conditions: | tf-ti/s|/ti/s≤5％；
(4) Calculating the content of the target compound: calculating the content C of the component to be detected in the test solution according to the formula (II)iThe components to be detected are hyperoside, rutin, quercetin, quercitrin and kaempferol respectively;
Ci＝fs/i×Ai×Cs/Asformula (II);
wherein f iss/iAs a relative correction factor, AsThe peak area of hyperin in the high performance liquid chromatogram of the reference solution is shown; csThe unit is the mass concentration of the hyperin in the reference solution, and the unit is mu g/mL; a. theiThe peak area of the component to be detected in the high performance liquid chromatogram of the test solution is shown; ciThe unit is the mass concentration of the component to be detected, and the unit is mu g/mL;
when the components to be detected are respectively hyperin, rutin, quercetin and kaempferol, the f iss/i1.000, 1.243, 0.9905, 0.5350, and 0.7813, respectively.
The same method is adopted to detect the content of active ingredients in rhododendron anthopogonoides samples S2-S12.
Meanwhile, the content of hyperoside, rutin, quercitrin, quercetin and kaempferol in the sample S1-S12 is measured by adopting the external standard method, and then the RSD of the two methods is compared, so that the calculated result of the content of each component has no obvious difference (the RSD is less than 2.0%).
Example 2
This example was carried out in a similar manner to example 1, except that the column was CAPCELL PAK C18。
Comparing the RSD of the method with that of an external standard method, the calculated result of the content of each component has no obvious difference (the RSD is less than 2.0 percent).
Example 3
This example was carried out in a similar manner to example 1, except that the column was Phenomenex Luna C18。
Comparing the RSD of the method with that of an external standard method, the calculated result of the content of each component has no obvious difference (the RSD is less than 2.0 percent).
Example 4
This example was carried out in a similar manner to example 1, except that the column temperature was 25 ℃.
Comparing the RSD of the method with that of an external standard method, the calculated result of the content of each component has no obvious difference (the RSD is less than 2.0 percent).
Example 5
This example was carried out in a similar manner to example 1, except that the amount of sample was 1. mu.L.
Comparing the RSD of the method with that of an external standard method, the calculated result of the content of each component has no obvious difference (the RSD is less than 2.0 percent).
Comparative example 1
This comparative example was conducted in a similar manner to example 1, except that the control solution was prepared using quercetin. Specifically, the method comprises the following steps:
(1) preparing a reference solution and a test solution:
preparation of control solutions: weighing 5.0mg of quercetin, placing the quercetin in a 10mL volumetric flask, mixing the quercetin with methanol and fixing the volume; transferring 4mL of the solution, placing the solution in a 10mL volumetric flask, mixing the solution with methanol and fixing the volume to obtain the reference substance solution with the mass concentration of the quercetin of 200 mug/mL;
the test solution was prepared in the same manner as in example 1;
(2) chromatographic conditions are as follows: same conditions as in example 1;
(3) determining the position of the chromatographic peak of the target compound: determining the chromatographic peak position of the quercetin in the test solution by taking the chromatographic peak of the quercetin reference in the reference solution as a reference, and then determining the chromatographic peak positions of the rest 4 components in the chromatogram of the test solution according to the retention time of each absorption peak in the chromatogram and by combining the formula (I);
relative retention time t used in this comparative examplei/sIs obtained by taking quercetin as internal standard substance, when the components to be tested are hyperoside, rutin, quercitrin, quercetin and kaempferol respectively, the t isi/s0.5405, 0.5210, 0.7334, 1.000 and 1.125, respectively, which are calculated to show ti/sThe fluctuation is large, and a certain difference exists (RSD is less than 5%);
(4) the content of each active compound was calculated:
calculating the content C of each component to be detected in the test solution according to the formula (II)iThe components are hyperin, rutin, quercetin, quercitrin and kaempferol respectively;
Ci＝fs/i×Ai×Cs/Asformula (II);
relative correction factor f used in this comparative examples/iIs obtained by taking quercetin as an internal standard substance, and when the components to be detected are hyperoside, rutin, quercitrin, quercetin and kaempferol respectively, the f iss/iThe RSD values of the partial detection values of the sample solutions were calculated to be 1.869, 2.323, 1.852, 1.000 and 1.460, respectively, and were higher than 4% compared to the case where hyperin was used as the internal standard.
Comparing the RSD of the method with that of an external standard method, the calculation results of the content of part of components have obvious difference, wherein the RSD of the hyperoside is 0.74-4.36%, the RSD of the rutin is 2.31-2.70%, the RSD of the quercitrin is 1.26-2.10%, and the RSD of the kaempferol is 0.78-1.98%. The accuracy of the method using quercetin as an internal standard substance is low.
The results show that the detection method provided by the invention has good precision and repeatability, has no obvious difference from the results obtained by an external standard method, and can replace the external standard method to carry out multi-component quality evaluation.
According to the method, hyperoside is selected as an internal standard substance, the content of main active components in a sample to be detected is calculated by adopting relative correction factors of rutin, quercitrin, quercetin and kaempferol, and a high performance liquid chromatography technology and a one-detection-multiple-evaluation method are combined, so that the experiment cost and the detection period can be reduced, the method has the characteristics of simplicity, convenience, high efficiency, high accuracy and good repeatability, and the improvement and reasonable and efficient utilization of the quality standard of rhododendron anthopogonoide can be effectively promoted.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (10)

1. A method for evaluating the quality of Tibetan medicine rhododendron anthopogonoides by one-test-multiple evaluation is characterized by comprising the following steps:
(1) preparing a reference solution and a test solution:
preparation of control solutions: mixing a hyperin reference substance with a solvent I and fixing the volume to obtain a reference substance solution;
preparation of a test solution: mixing rhododendron anthopogonoides with a solvent II, and fixing the volume to obtain the test solution;
(2) chromatographic conditions are as follows: respectively carrying out high performance liquid chromatography detection analysis on the reference solution and the test solution to obtain a high performance liquid chromatogram; the conditions of the high performance liquid chromatography detection analysis comprise: the chromatographic column is C18A chromatographic column; the mobile phase is acetonitrile and 0.1-0.3 volume percent phosphoric acid gradient elution; the detection wavelength is 190-400 nm; the column temperature is 25-35 ℃; the sample amount is 1-20 mu L;
(3) determining the position of the chromatographic peak of the target compound: determining the chromatographic peak position of hyperoside in the sample solution with reference to the chromatographic peak of hyperoside reference in the reference solution, wherein the sample solution is selected from rutin, hyperoside, and quercus albumRetention time t of at least one component to be tested selected from the group consisting of hesperidin, quercetin and kaempferoliDivided by the retention time t of the internal standard substance hyperin in the test solutionsCalculating to obtain the relative retention time t of the component to be tested in the test solutionfAnd the theoretical relative retention time is ti/sSaid t isfAnd said ti/sSatisfies the following conditions: | tf-ti/s|/ti/sDetermining the corresponding chromatographic peak position of the component to be detected in the high performance liquid chromatogram of the test solution, wherein the chromatographic peak position is less than or equal to 5%;
tf＝ti/tsformula (I);
wherein, in the formula (I), tfActual relative retention time; t is tiThe retention time of the component to be detected in the test solution is min; t is tsThe retention time of the internal standard substance hyperin in the test solution is min;
when the component to be detected is hyperin, the ti/sIs 1.000;
when the component to be detected is rutin, the t isi/s0.9638;
when the component to be detected is quercitrin, the ti/sIs 1.357;
when the component to be tested is quercetin, ti/sIs 1.852;
when the component to be detected is kaempferol, t isi/s2.085;
(4) calculating the content of the target compound:
calculating the content C of the component to be detected in the test solution according to the formula (II)iThe component to be detected is hyperoside, rutin, quercetin, quercitrin or kaempferol;
Ci＝fs/i×Ai×Cs/Asformula (II);
wherein f iss/iAs a relative correction factor, AsThe peak area of hyperin in the high performance liquid chromatogram of the reference solution is shown; csThe unit is the mass concentration of the hyperin in the reference solution, and the unit is mu g/mL; a. theiThe peak area of the component to be detected in the high performance liquid chromatogram of the test solution is shown; ciThe unit is the mass concentration of the component to be detected, and the unit is mu g/mL;
when the component to be detected is hyperin, the fs/iIs 1.000;
when the component to be tested is rutin, the fs/iIs 1.243;
when the component to be detected is quercitrin, the fs/i0.9905;
when the component to be tested is quercetin, fs/i0.5350;
when the component to be detected is kaempferol, fs/iIs 0.7813.
2. The method according to claim 1, wherein in step (1), the mass concentration of the hyperoside in the control solution is 15.40-308.0 μ g/mL;
preferably, the mass concentration of the hyperin in the control solution is 100-.
3. The method according to claim 1 or 2, wherein, in step (1), the rhododendron is a powdered rhododendron and the powdered rhododendron is obtained by sieving a pulverized rhododendron, and the sieve is a second sieve, a fourth sieve or a fifth sieve; preferably, the sieve is a No. four sieve.
4. The process according to any one of claims 1 to 3, wherein, in step (1), the solvent I is methanol; the solvent II is methanol water solution and/or ethanol water solution.
5. The method according to any one of claims 1 to 4, wherein in the step (1), the mass ratio of the solvent II to the rhododendron anthopogonoides is 20-40: 1.
6. the process according to claim 4 or 5, wherein, in the solvent II in the step (1), the methanol aqueous solution is an aqueous solution containing 50-100 vol% of methanol, and the ethanol aqueous solution is an aqueous solution containing 50-100 vol% of ethanol;
preferably, in the solvent II, the methanol aqueous solution is an aqueous solution containing 80 to 100 vol% of methanol, and the ethanol aqueous solution is an aqueous solution containing 80 to 100 vol% of ethanol.
7. The method according to any one of claims 1 to 6, wherein in step (1), in the preparation of the test solution, the rhododendron anthopogonoides are mixed with the solvent II under ultrasonication conditions;
preferably, the conditions of the ultrasound include: the time is 20-60min, preferably 30-40 min; the frequency is 30-50 KHz.
8. The method of any one of claims 1-7, wherein in step (2), the conditions of the high performance liquid chromatography detection assay comprise: the chromatographic column is selected from CAPCELL PAK C18、Venusil XBP C18And Phenomenex Luna C18At least one of;
the mobile phase is acetonitrile and 0.2 volume percent phosphoric acid gradient elution;
the detection wavelength is 254nm or 365 nm;
the column temperature is 30 ℃; the sample amount is 10-20 μ L.
9. The method according to any one of claims 1 to 8, wherein in step (2), the elution gradient of the mobile phase of the HPLC detection analysis is: 0-15min, 10-15 vol% of said acetonitrile; 15-50min, 15-20 vol% of said acetonitrile; 50-60min, 20-30 vol% of said acetonitrile; 60-70min, 30-35 vol% of said acetonitrile; 70-80min, 35-60 vol% of said acetonitrile; 80-90min, 60-10 vol% of the acetonitrile.
10. Use of the method of any one of claims 1 to 9 in the one-test-multiple-evaluation quality assessment of the Tibetan medicine rhododendron anthopogonoide.
CN202011549272.9A 2020-12-24 2020-12-24 Method for evaluating quality of Tibetan medicine rhododendron anthopogonoides by one-test-multiple evaluation and application Pending CN113588801A (en)

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