Determination of 16 chemical components in Epimedium extract

The invention discloses a method for measuring the content of 16 chemical components in an epimedium extract, which adopts an HPLC-MS/MS method to simultaneously measure the content of 16 chemical components in the epimedium extract; the method comprises the following steps: (1) establishing a standard curve of 16 chemical compositions; (2) obtaining a multi-reaction ion chromatogram of each 16 chemical components in the epimedium extract; (3) determining the content of 16 chemical components in the epimedium extract; the method has the characteristics of high sensitivity, strong reliability, high efficiency and high speed, and can more effectively control the quality of the epimedium extract by measuring the content of 16 chemical components in the epimedium extract, thereby ensuring the clinical curative effect of the epimedium extract.

Technical Field

The invention relates to the field of traditional Chinese medicine quality analysis, in particular to a method for measuring the content of 16 chemical components in an epimedium extract.

Background

Epimedium Epimedii Herba is derived from Epimedium L.plant of Epimedium of Berberidaceae (Berberidaceae), is a Chinese traditional tonifying traditional Chinese medicine, and has a long history of medication. Dried leaves of Epimedium brevicornum Maxim, Epimedium sagittatum Sieb. et Zucc. et.C, Epimedium pubescens Maxim, and Epimedium koreanum nakai of Korean Epimedium are collected from the 'Chinese pharmacopoeia' 2015 edition. It is pungent, sweet and warm in nature, enters liver and kidney meridians, and has the actions of tonifying kidney yang, strengthening tendons and bones, and dispelling wind-damp. Herba Epimedii contains various chemical components such as flavonoids, lignanoids, phenol glycosides, alkaloids, and polysaccharides, and its main effective component is flavonoids. Modern pharmacological research shows that epimedium has pharmacological effects of enhancing immunity, improving cardiovascular system function, resisting inflammation, resisting tumor, promoting bone cell proliferation, etc. and has wide application foreground. But the content of total flavonoids in the epimedium medicinal materials is lower; with the increasing development of traditional Chinese medicine extraction technology, epimedium extract is gradually becoming an important raw material of numerous traditional Chinese medicine preparations as a processed product of epimedium medicinal materials. At present, the quality control of the epimedium extract mostly adopts methods such as high performance liquid chromatography, ultraviolet spectrophotometry and the like to measure the content of a single or a small amount of components in the epimedium extract; on one hand, the detection method has low sensitivity and specificity, and on the other hand, the epimedium extract contains a plurality of chemical components, and only one or a small amount of components are controlled, so that the quality of the epimedium extract cannot be effectively controlled, and therefore, a brand-new method for measuring the content of the chemical components in the epimedium extract is needed, so that the quality of the epimedium extract can be more reliably and comprehensively controlled.

Disclosure of Invention

The invention aims to provide a method for measuring the content of 16 chemical components in an epimedium extract so as to realize more credible and more comprehensive quality control of the epimedium extract. The specific technical scheme is as follows:

a method for measuring the content of 16 chemical components in herba Epimedii extract adopts HPLC-MS/MS method, and simultaneously measures the content of 16 chemical components in herba Epimedii extract; the 16 chemical compositions comprise: baohuoside II, baohuoside I, epimedin A, epimedin B, epimedin C, quercitrin, arrow holly A, arrow holly B, hyperin, chlorogenic acid, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, icariside I, icariin and astragalin, wherein the method comprises the following steps:

(1) establishing a standard curve of 16 chemical compositions

Precisely weighing reference substances including baohuoside II, baohuoside I, caohuoside A, caohuoside B, caohuoside C, quercetin, agastachside A, agastachside B, hyperoside, chlorogenic acid, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, icariside I, icariin and astragalin respectively, dissolving with 70-100% methanol, and making into each reference substance stock solution with known concentration; respectively and precisely sucking 16 reference substance stock solutions, mixing the reference substance stock solutions, diluting the reference substance stock solutions by using methanol with the volume fraction of 70-100%, and preparing reference substance mixed stock solutions respectively containing 16 chemical components with known concentrations;

diluting the reference substance mixed stock solution into a series of reference substance mixed solutions with 16 chemical components with different known concentrations by using 70-100% of methanol by volume fraction;

under the same preset chromatographic condition and mass spectrum condition, mixing V₁Injecting the volume of the reference substance mixed solution with each concentration into a high performance liquid chromatography-mass spectrometer to obtain a multi-reaction ion chromatogram of each reference substance under different concentrations;

taking the chromatographic peak area Y of each reference substance as a vertical coordinate and the concentration X of each reference substance as a horizontal coordinate, and obtaining a standard curve of each chemical component through linear regression operation;

(2) obtaining multiple reaction ion chromatogram of each 16 chemical components in epimedium extract

Dissolving herba Epimedii extract with mass M in 70-100% methanol to volume V₂Filtering the supernatant to obtain a sample solution;

taking the volume V under the same preset chromatographic and mass spectrometric conditions as in step (1) above₁Injecting the test solution into a high performance liquid chromatography-mass spectrometer to obtain a multi-reaction ion chromatogram of each chemical component in the epimedium extract;

(3) determining the content of 16 chemical components in herba Epimedii extract

Determining the content of each chemical component in the epimedium extract according to the peak area of the multi-reaction ion chromatogram of each chemical component in the epimedium extract and the standard curve of each chemical component.

In a specific embodiment of the present invention, the determining the content of each chemical component in the epimedium extract comprises:

determining the concentration C corresponding to the peak area of each chemical component in the herba Epimedii extract from the standard curve of each chemical component_{Concentration}Respectively calculating the contents of 16 chemical components in the sample according to the following formula;

in one embodiment of the present invention, the standard curve of each chemical component is obtained by linear regression operation, specifically:

and performing linear regression operation by adopting a weighted least square method to obtain a standard curve, wherein the weight coefficient is 1/X.

In one embodiment of the present invention, the predetermined chromatographic conditions comprise:

a chromatographic column: a stationary phase of octadecylsilane chemically bonded silica is adopted; mobile phase: the phase A is 0.05-0.5% formic acid-water, and the phase B is acetonitrile; gradient elution; column temperature: 25-35 ℃; flow rate: 0.3-1.0 mL/min; sample introduction amount: 2-10 μ L.

In one embodiment of the present invention, the predetermined chromatographic conditions comprise:

a chromatographic column: a stationary phase of octadecylsilane chemically bonded silica is adopted; mobile phase: phase A is 0.1% formic acid-water, phase B is acetonitrile; gradient elution; column temperature: 30 ℃; flow rate: 0.3 mL/min; sample introduction amount: 5 mu L of the solution;

the elution procedure was: 0-4min, 20% -50% B; 4-5min, 50% -60% B; 5-15min, 60% -70% B.

In one embodiment of the present invention, the preset mass spectrum conditions include:

an ionization mode: carrying out electrospray ionization; a multiple reactive ion monitoring mode, negative ion scanning; drying gas temperature: 330 ℃ to 370 ℃; flow rate of drying gas: 7-15L/min; spray gas pressure: 17-22 psi; drying gas and atomizing gas: nitrogen gas.

In one embodiment of the present invention, the preset mass spectrum conditions include:

an ionization mode: carrying out electrospray ionization; a multiple reactive ion monitoring mode, negative ion scanning; drying gas temperature: 350 ℃; flow rate of drying gas: 9L/min; spray gas pressure: 20 psi; drying gas and atomizing gas: nitrogen gas.

In one embodiment of the present invention, the mass spectrometry parameters are as follows:

in one embodiment of the present invention, the epimedium extract is obtained by the following method:

extracting folium Epimedii with 50-90% ethanol (preferably 70% ethanol) under reflux for 0.5-2 hr at a weight ratio of (14-20): 1; reflux extraction times are 1-4 times;

mixing extractive solutions, and drying under reduced pressure to obtain herba Epimedii extract.

In one embodiment of the invention, the methanol with a volume fraction of 70 to 100% is in particular methanol.

The invention adopts HPLC-MS/MS method to measure the content of 16 chemical components in the epimedium extract at the same time, has the characteristics of high sensitivity, strong reliability, high efficiency and high speed, and can more effectively control the quality of the epimedium extract by measuring the content of 16 chemical components in the epimedium extract, thereby ensuring the clinical curative effect of the epimedium extract.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1, panel A, is a multiple reactive ion chromatogram of a 16 chemical component control at a given concentration;

FIG. 1B is a multi-reactive ion chromatogram of each chemical component in herba Epimedii extract;

in fig. 1, each numeral symbol represents: 1. magnoflorine; 2. chlorogenic acid; 3. chlorogenic acid; 4. cryptochlorogenic acid; 5. astragalin; 6. quercetin; 7. hyperin; 8. (iii) baohuoside II; 9. b, baohuoside I; 10. icariside I; 11. fix towards houding B; 12. b, Arbutin B; 13. joading C; 14. fix towards Huo Ding A; 15. 1, Arbutin A; 16. icariin.

Detailed Description

The invention adopts HPLC-MS/MS method (high performance liquid chromatography-tandem mass spectrometry) to simultaneously determine the content of 16 chemical components in the epimedium extract.

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

First, it should be noted that the term "precisely defined" as used in the present invention: precision weighing means that the weight should be weighed to exactly one thousandth of the weight taken. "precision absorption": it is to be understood that the accuracy of the measured volume should meet the accuracy requirements of the measuring instrument in the national standard.

1. Instruments, reagents and materials

1.1 Instrument:

agilent model 6430 triple quadrupole mass spectrometer (Agilent corporation, usa); agilent model 1200 high performance liquid chromatograph (Agilent corporation, usa); AgilentMassHunter analysis software (Agilent, USA); a one hundred thousand balance of the type AX 205 (mettler toledo, switzerland); Milli-Q ultrapure water preparation apparatus (Millipore Corp.); model 3K15 high speed centrifuge (Sigma, usa); XW-80A vortex mixer (Shanghai province of analytical instruments).

1.2 reagent:

the reference products of baohuoside II, baohuoside I, epimedin A, epimedin B, epimedin C, quercitrin, sagoroside A, sagoroside B, hyperoside, chlorogenic acid, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, icariside I, icariin and astragalin are all purchased from Tianjin-Yi Zhongkang pharmaceutical technology Limited. Methanol (chromatographically pure), acetonitrile (chromatographically pure) were obtained from Fisher corporation, USA, and formic acid (chromatographically pure) was obtained from ROE corporation, USA.

1.3 materials

Herba Epimedii extract prepared by the following method:

weighing 1kg of folium Epimedii (produced in Gansu province), adding 16kg of 70% ethanol (prepared by mixing ethanol and water at a volume ratio of 7: 3), heating and reflux-extracting for two times (each for 1.5 hr), mixing extractive solutions, and drying under reduced pressure to obtain 156.1g of herba Epimedii extract. It should be noted that the epimedium extract prepared by other existing methods can be used for implementing the technical scheme of the invention.

2. Chromatographic and mass spectral conditions

2.1 chromatographic conditions

2.2 Mass Spectrometry conditions

An ionization mode: electrospray ionization (ESI); detection mode: performing negative ion scanning in a multiple reactive ion monitoring mode (MRM); drying gas temperature: 350 ℃; flow rate of drying gas: 9L/min; spray gas pressure: 20 psi; dry gas and atomizing gas in the experiment: nitrogen gas; the quantitative analysis of ion pairs, mass spectral parameters and ion modes are shown in table 1.

TABLE 1 Mass Spectrometry parameters for 16 chemical constituents in Epimedium herb extract

2.3 preparation of the solution

2.3.1 preparation of control solutions

Respectively and precisely weighing reference substances including baohuoside II, baohuoside I, epimedin A, epimedin B, epimedin C, quercitrin, sagittoside A, sagittoside B, hyperoside, chlorogenic acid, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, icariside I, icariin and astragalin, adding methanol into 1mg of each to fix the volume to 1mL, preparing reference substance stock solutions of 1mg/mL, and storing the reference substance stock solutions in a refrigerator at 4 ℃ for later use.

2.3.2 preparation of test solutions

Precisely weighing herba Epimedii extract 5mg (mass M is 5mg) prepared under item "1.3", dissolving in 25mL volumetric flask with methanol, and fixing to the scale mark (volume V)₂25mL), filtering the supernatant with 0.45 μm organic microporous membrane, and storing the stock solution in a refrigerator at 4 deg.C.

2.4 methodological considerations

2.4.1 Standard Curve, detection Limit, quantification Limit

Accurately measuring a proper amount of reference stock solution under the item of '2.3.1', and preparing a reference mixed stock solution containing magnoflorine, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, hyperoside, epimedin A, epimedin C, hodroside B, hodroside I and icariin 5 mug/mL and containing astragalin, quercitrin, hodroside II, icariside I, epimedin B and hodroside A500 ng/mL by using methanol. Diluting the solution with methanol for 2.5, 2, 5, 2, 2.5 times to obtain 7 reference substance mixed solutions, sampling 5 μ L of each reference substance mixed solution under the chromatographic condition of "2.1" and the mass spectrum condition of "2.2" for analysis, and obtaining multiple reaction ion chromatograms of each reference substance at different concentrations, wherein the multiple reaction ion chromatogram after 3 rd dilution is shown as A in FIG. 1. And taking the chromatographic peak area Y of each reference substance as a vertical coordinate and the concentration X of the reference substance as a horizontal coordinate, and obtaining the standard curve of each chemical component reference substance by adopting weighted least square normal weighted regression with the weight coefficient of 1/X. The concentrations of each control were taken as the lowest limit of detection (LLOD) and the lowest limit of quantitation (LLOQ) at S/N (signal-to-noise ratio) of 3 and S/N of 10, respectively, and the results are shown in table 2.

Table 216 regression equation of standard curve of chemical composition, quantitative limit and detection limit

2.4.2 precision test

Taking the reference substance stock solution prepared under the item 2.3.1, diluting by 10 times, continuously injecting samples for 6 times according to the chromatographic condition under the item 2.1 and the mass spectrum condition of the item 2.2, measuring the concentration of each reference substance, calculating the RSD value, and showing that the result is shown in Table 3, thereby indicating that the method has good precision.

Table 316 ingredient precision experimental data table (n ═ 6)

2.4.3 repeatability test

Accurately weighing 6 parts of epimedium extract, 5mg of each part, preparing a sample solution under the item of '2.3.2', respectively injecting 5 mu L of sample under the chromatographic condition under the item of '2.1' and the mass spectrum condition of '2.2', recording the concentration of 16 components, calculating the RSD value, and showing the result in table 4, which indicates that the method has better repeatability.

Table 416 type composition repeatability test data table (n ═ 6)

2.4.4 stability test

Precisely weighing 5mg of epimedium extract, preparing a sample solution under the item '2.3.2', respectively carrying out sample injection for 5 mu L under the chromatographic condition and the mass spectrum condition '2.2' under the items '2.1' and the conditions of 0, 2, 6, 12 and 24h, recording the concentration of 16 chemical components, investigating the standing stability at room temperature, calculating the RSD value, and showing that the result is shown in Table 5 and shows that the 16 chemical components in the sample are stable under the condition of standing for 12h at room temperature.

TABLE 516 chemical compositions stability at room temperature for 24h

2.4.5 sample recovery test

Precisely weighing 6 parts of epimedium extract, each part being 2.5mg, adding a certain amount of mixed reference solution, fixing the volume to 25mL by using methanol, preparing in parallel under the item '2.3.2', injecting 5 mu L of sample according to the chromatographic condition and the mass spectrum condition '2.2' under the item '2.1', and calculating the sample injection recovery rate, wherein the result is shown in Table 6.

TABLE 616 recovery of sample addition for ingredients Experimental data Table (n ═ 6)

2.5 sample determination

Preparing 6 parts of test solution in parallel from herba Epimedii extract prepared under item "1.3" according to method under item "2.3.2", and determining according to the chromatographic conditions and mass spectrum conditions under item "2.1" to obtain multiple reactive ion chromatogram of each chemical component in herba Epimedii extract, as shown in figure 1B; determining the concentration C corresponding to the peak area of each chemical component in the herba Epimedii extract from the standard curve of each chemical component in Table 2_{Concentration}Respectively calculating the contents of 16 chemical components in the sample according to the following formula; the results are shown in Table 7.

TABLE 7 Epimedium extract contains 16 chemical components (μ g/g)

From the results, the invention adopts an HPLC-MS/MS method to simultaneously determine the content of 16 chemical components in the epimedium extract, has the characteristics of high sensitivity, strong reliability, high efficiency and high speed, and can more effectively control the quality of the epimedium extract by determining the content of 16 chemical components in the epimedium extract, thereby ensuring the clinical curative effect of the epimedium extract.

1. The method for measuring the content of 16 chemical components in the epimedium extract is characterized in that an HPLC-MS/MS method is adopted to simultaneously measure the content of 16 chemical components in the epimedium extract; the 16 chemical compositions comprise: baohuoside II, baohuoside I, epimedin A, epimedin B, epimedin C, quercitrin, arrow holly A, arrow holly B, hyperin, chlorogenic acid, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, icariside I, icariin and astragalin, wherein the method comprises the following steps:

(1) establishing a standard curve of 16 chemical compositions

Precisely weighing reference substances including baohuoside II, baohuoside I, caohuoside A, caohuoside B, caohuoside C, quercetin, agastachside A, agastachside B, hyperoside, chlorogenic acid, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, icariside I, icariin and astragalin respectively, dissolving with 70-100% methanol, and making into each reference substance stock solution with known concentration; respectively and precisely sucking 16 reference substance stock solutions, mixing the reference substance stock solutions, diluting the reference substance stock solutions by using methanol with the volume fraction of 70-100%, and preparing reference substance mixed stock solutions respectively containing 16 chemical components with known concentrations;

diluting the reference substance mixed stock solution into a series of reference substance mixed solutions with 16 chemical components with different known concentrations by using 70-100% of methanol by volume fraction;

under the same preset chromatographic condition and mass spectrum condition, mixing V₁Injecting the volume of the reference substance mixed solution with each concentration into a high performance liquid chromatography-mass spectrometer to obtain a multi-reaction ion chromatogram of each reference substance under different concentrations;

taking the chromatographic peak area Y of each reference substance as a vertical coordinate and the concentration X of each reference substance as a horizontal coordinate, and obtaining a standard curve of each chemical component through linear regression operation;

(2) obtaining multiple reaction ion chromatogram of each 16 chemical components in epimedium extract

Dissolving herba Epimedii extract with mass M in 70-100% methanol to volume V₂Filtering the supernatant to obtain a sample solution;

taking the volume V under the same preset chromatographic and mass spectrometric conditions as in step (1) above₁Injecting the test solution into a high performance liquid chromatography-mass spectrometer to obtain a multi-reaction ion chromatogram of each chemical component in the epimedium extract;

(3) determining the content of 16 chemical components in herba Epimedii extract

Determining the content of each chemical component in the epimedium extract according to the peak area of the multi-reaction ion chromatogram of each chemical component in the epimedium extract and the standard curve of each chemical component;

the preset chromatographic conditions include:

a chromatographic column: a stationary phase of octadecylsilane chemically bonded silica is adopted; mobile phase: phase A is 0.1% formic acid-water, phase B is acetonitrile; gradient elution; column temperature: 30 ℃; flow rate: 0.3 mL/min; sample introduction amount: 5 mu L of the solution;

the elution procedure was: 0-4min, 20% -50% B; 4-5min, 50% -60% B; 5-15min, 60% -70% B;

the mass spectrum parameters were as follows:

2. the method of claim 1, wherein the determining the content of each chemical component in the epimedium extract comprises:

determining the concentration C corresponding to the peak area of each chemical component in the herba Epimedii extract from the standard curve of each chemical component_{Concentration}Respectively calculating the contents of 16 chemical components in the sample according to the following formula;

3. the method of claim 1, wherein the standard curve for each chemical component is obtained by linear regression, specifically:

and performing linear regression operation by adopting a weighted least square method to obtain a standard curve, wherein the weight coefficient is 1/X.

4. The method of claim 1, wherein the preset mass spectrometry conditions comprise:

an ionization mode: carrying out electrospray ionization; a multiple reactive ion monitoring mode, negative ion scanning; drying gas temperature: 330 ℃ to 370 ℃; flow rate of drying gas: 7-15L/min; spray gas pressure: 17-22 psi; drying gas and atomizing gas: nitrogen gas.

5. The method of claim 4, wherein the preset mass spectrometry conditions comprise:

an ionization mode: carrying out electrospray ionization; a multiple reactive ion monitoring mode, negative ion scanning; drying gas temperature: 350 ℃; flow rate of drying gas: 9L/min; spray gas pressure: 20 psi; drying gas and atomizing gas: nitrogen gas.

6. The method of claim 1, wherein the epimedium extract is obtained by the following method:

extracting folium Epimedii with 50-90% ethanol under reflux for 0.5-2 hr, wherein the weight ratio of ethanol to folium Epimedii is (14-20): 1; reflux extraction times are 1-4 times;

mixing extractive solutions, and drying under reduced pressure to obtain herba Epimedii extract.

7. The method of claim 6, wherein the icariin leaves are heated under reflux in ethanol with a volume fraction of 70%.

8. The process according to claim 1, wherein the methanol, in particular methanol, is present in a volume fraction of 70 to 100%.