CN110554103B - Radix astragali and radix puerariae particle component detection method and fingerprint spectrum construction method thereof - Google Patents

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a radix astragali and radix puerariae particle component detection method and a fingerprint spectrum construction method thereof. The detection method comprises the following steps: taking the astragalus-kudzuvine root granules to be tested and the standard substance for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: by C₁₈A chromatographic column, wherein a water phase containing a buffer solution is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, and gradient elution is carried out; according to HPLC detection results, the ingredients and contents of the astragalus and kudzuvine root granules are obtained. The spectrum measured by the method can comprehensively reflect the chemical components in the Qige particles, the separation of each spectrum peak is good, the base line is stable, the peak type is good, the repeatability is good, the method has good linear relation, precision, stability and recovery rate, the content of the components in the Qige particles can be accurately detected, and the quality of a sample can be objectively evaluated by the obtained contrast fingerprint spectrum.

Description

Radix astragali and radix puerariae particle component detection method and fingerprint spectrum construction method thereof

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a radix astragali and radix puerariae particle component detection method and a fingerprint spectrum construction method thereof.

Background

The astragalus and kudzu vine root granules are prepared from eight medicines of astragalus, kudzu vine root, white paeony root, szechuan lovage rhizome, cassia twig, turmeric, clematis root and chrysanthemum, have the effects of dispelling wind and clearing heat, warming channels and activating blood, and freeing Bi and freeing channels, and are used for treating neck and neck pain, shoulder and back pain, neck and neck movement difficulty, limb numbness, chilly and cold limbs and limb spasm caused by nerve root type cervical spondylosis and cold obstruction of channels.

With the rapid development of Chinese herbal medicine preparations in China, the quality management problem of the Chinese herbal medicine preparations is also widely concerned by people, and the quality of the Chinese herbal medicine preparations can directly influence the medical quality and the life safety of patients. In order to better control the quality of the medicine and ensure the clinical curative effect, a method for comprehensively evaluating the quality of the preparation needs to be established.

The Qige granule is a compound preparation and has complex chemical components. The analysis of chemical components of traditional Chinese medicines is one of the key problems of the clarification of medicinal effect substances of traditional Chinese medicines and the quality control, and the comprehensive analysis of the chemical components of astragalus-kudzuvine root granules is not reported at present, so that the method for controlling the quality of the astragalus-kudzuvine root granules has important practical significance.

Disclosure of Invention

The invention provides a radix astragali and radix puerariae particle component detection method and a fingerprint spectrum construction method thereof. The spectrum measured by the detection method can comprehensively reflect chemical components in the Qige particles, the separation of each spectrum peak is good, the base line is stable, the peak type is good, the repeatability is good, the method has good linear relation, precision, stability and recovery rate, the component content of the particles can be accurately detected, and the quality of a sample can be objectively evaluated by the obtained contrast fingerprint spectrum.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting active ingredients of astragalus and kudzuvine root granules, which is characterized by comprising the following steps: grinding radix astragali and radix Puerariae granules, and preparing 20mg/ml sample with methanol; taking a radix astragali and radix puerariae particle test sample and a reference substance for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: by C₁₈The chromatographic column uses a water phase containing buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and gradient washing is carried outThe removing procedure is as follows: 0-35 min, 5% -12% of B; 35-50 min, 12% -30% of B; 50-55 min, 30-75% of B; 55-60 min, 75% B; the flow rate is 0.8-1.5 mL/min; the column temperature is 25-40 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively; and obtaining the component information of the Qige granules or the component and content information thereof according to the HPLC detection result.

The invention provides a method for detecting active ingredients of astragalus and kudzuvine root granules, which is characterized by comprising the following steps: grinding radix astragali and radix Puerariae granules, and preparing 20mg/ml sample with methanol; taking a radix astragali and radix puerariae particle test sample and a reference substance for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: by C₁₈The chromatographic column takes an aqueous phase containing a buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and the gradient elution procedure comprises the following steps: 0-12 min, 5% -10% B; 12-20 min, 10% -14% of B; 20-30 min, 14% -20% of B; 30-40 min, 20-30% B; 40-46 min, 30-48% B; 46-47 min, 48% -70% B; 47-55 min, 70% -90% B; 55-70 min, 90% B; the column temperature is 25-40 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively; and obtaining the component information of the Qige granules or the component and content information thereof according to the HPLC detection result.

In a preferred embodiment of the present invention, in the above detection method, the flow rate of HPLC detection is 1.0mL/min, and the column temperature is 30 ℃; the methanol is 50% methanol.

In a preferred embodiment of the present invention, in the above detection method, the buffer solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof, and polybasic weak acids and salts thereof; preferably one or more of trifluoroacetic acid, formic acid, ammonium formate, acetic acid, sodium acetate, ammonium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, sodium citrate, glycine, hydrochloric acid, phthalic acid; more preferably 0.2% formic acid.

In a preferred embodiment of the present invention, in the above-mentioned detection method, said C₁₈The chromatographic column is Kramosil C₁₈Chromatographic column, Agilent Zorbax SB-C₁₈Chromatographic column, Phenomenex Gemini C₁₈Chromatography column, Thermo syncronis C₁₈Chromatographic column, Agilent 5TC-C₁₈Chromatographic column, Kromasil 100-5C₁₈Column, Waters symmetry C₁₈Chromatographic column or Phenomenex Luna C₁₈A chromatographic column.

In a preferred embodiment of the present invention, in the above detection method, the reference substance is one or more of puerarin, daidzin, formononetin, astragaloside IV, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, 3 '-hydroxypuerarin, 3' -methoxypugenin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin, albiflorin, benzoylpaeoniflorin, apigenin-7-O-beta-D-glucopyranoside, farnesin, senkyunolide A, isoferulic acid, luteolin, apigenin or angelicin A. And establishing a standard curve of one or more reference substances by using the detection conditions, and calculating the content of the effective components according to the detection result of the test sample.

In a preferred embodiment of the invention, after the Qige granule fingerprint is established, the fingerprint of a Qige granule sample to be detected is determined according to the same method, and then the similarity is calculated and compared with the fingerprint of the Qige granule by traditional Chinese medicine chromatography fingerprint similarity evaluation system software provided by the national pharmacopoeia committee, wherein the similarity is not lower than 0.90.

The invention also provides a method for constructing the fingerprint of the Qige particles, which is characterized in that,

preparation of a test solution: grinding radix astragali and radix Puerariae granules, and preparing 20mg/ml sample with methanol;

preparation of control solutions: precisely weighing puerarin, daidzin, formononetin, astragaloside IV, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, 3 '-hydroxypuerarin, 3' -methoxy puerarin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin, albiflorin benzoylpaeoniflorin, apium graveolensThe preparation method comprises the steps of adding 50% methanol into brassinodin-7-O-beta-D-glucopyranoside, farnesin, senkyunolide A, isoferulic acid, luteolin, apigenin and levistilide A to prepare the mixture with the concentration of each component being 20-25 mug.mL^-1Single or mixed standard solutions of (a).

The chromatographic conditions for HPLC detection were: by C₁₈The chromatographic column takes an aqueous phase containing a buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and the gradient elution procedure is as follows: 0-35 min, 5% -12% of B; 35-50 min, 12% -30% of B; 50-55 min, 30-75% of B; 55-60 min, 75% B; the flow rate is 0.8-1.5 mL/min; the column temperature is 25-40 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively;

and (4) obtaining the fingerprint of the Qige granules according to the HPLC detection result.

Preferably, the gradient elution procedure described above may also be replaced by: 0-12 min, 5% -10% B; 12-20 min, 10% -14% of B; 20-30 min, 14% -20% of B; 30-40 min, 20-30% B; 40-46 min, 30-48% B; 46-47 min, 48% -70% B; 47-55 min, 70% -90% B; 55-70 min, 90% B.

In a preferred embodiment of the invention, the flow rate for HPLC detection is 1.0mL/min, the column temperature is 30 ℃; the methanol is 50% methanol; the mobile phase was 0.2% aqueous formic acid.

In a preferred embodiment of the present invention, the flow buffer solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof, and polybasic weak acids and salts thereof; preferably one or more of trifluoroacetic acid, formic acid, ammonium formate, acetic acid, sodium acetate, ammonium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, sodium citrate, glycine, hydrochloric acid, phthalic acid; more preferably 0.2% formic acid.

In a preferred embodiment of the present invention, said C₁₈The chromatographic column is Kramosil C₁₈Chromatographic column, Agilent Zorbax SB-C₁₈Chromatographic column, Phenomenex Gemini C₁₈Chromatography column, Thermo syncronis C₁₈Chromatographic column, Agilent 5TC-C₁₈Chromatographic column, Kromasil 100-5C₁₈Chromatographic column、Waters symmetry C₁₈Chromatographic column or Phenomenex Luna C₁₈A chromatographic column.

The spectrum measured by the method can comprehensively reflect the chemical components in the Qige particles, the separation of each spectrum peak is good, the base line is stable, the peak type is good, the repeatability is good, the method has good linear relation, precision, stability and recovery rate, the content of the components in the Qige particles can be accurately detected, and the obtained fingerprint spectrum can objectively evaluate the quality of a sample. At most 82 compounds can be detected, 78 compounds including flavone and its glycosides, saponins, organic acids, lactone and its dimers can be identified and speculated, wherein 29 compounds are verified by a reference substance. The result lays a foundation for clarifying the drug effect substance basis of the Qige granules and provides reference for quality control.

Drawings

Fig. 1 is a total ion flow diagram of positive and negative ion modes of a astragalus pueraria lobata particle sample under 1.1 detection conditions (a is the negative ion mode, and B is the positive ion mode).

FIG. 2 is a HPLC chart of different gradient elution.

FIG. 3 is a HPLC chart of different column temperatures.

FIG. 4 is a HPLC plot of different flow rates.

Fig. 5 is a total ion flow diagram of positive and negative ion modes of a radix puerariae particle sample under 2.1 detection conditions (a is the negative ion mode, and B is the positive ion mode).

Detailed Description

The invention discloses a radix astragali and radix puerariae particle component detection method and a fingerprint spectrum construction method thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

If not specifically stated, the medicines, reagents and instruments used in the method for detecting the ingredients of the Qige particles and the method for constructing the fingerprint spectrum thereof provided by the invention can be purchased from the market:

instrument and reagent

Agilent 1290 hplc, DAD detector, Agilent corp;

agilent 6538Q-TOF mass spectrometer, Agilent Inc. USA;

mettler Toledo XP6 electroanalytical balance, Mettler corporation, switzerland;

sartorius BSA224S electronic analytical balance, Sartorius, germany;

HH digital display constant temperature water bath, changzhou guoyu instruments manufacturing ltd;

TG16MW desk-top high speed centrifuge, hokken instruments & equipments limited, hannan;

Milli-Q Academic Water purification machines, Milli-Q Academic, Milli, USA;

the reference ferulic acid, isoferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, puerarin, daidzin, luteolin, formononetin, apigenin, penoniflorin, astragaloside IV, ligustilide, and levistilide A are provided by Chinese food and drug testing institute; the reference substances of neochlorogenic acid, 1, 5-dicaffeoylquinic acid, 3 '-hydroxy puerarin, 3' -methoxy puerarin, puerarin apioside and senkyunolide A are provided by Dopperdie biotechnology, Inc.; the daidzein and genistin reference substances are provided by identification of Chinese medicinal biological products; the albiflorin reference substance is provided by VIKEQI Biotech limited of Sichuan province; paeontolactone, benzoylpaeoniflorin, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cryptochlorogenic acid, apigenin-7-O-beta-D-glucopyranoside, and acacetin reference substance are provided by WUDEMANSI Biotechnology GmbH.

The methanol and the formic acid are both chromatographically pure; the water is ultrapure water; the other reagents are analytically pure.

The source of the medicine is as follows: the Qige granules used in the invention are produced by Jiangsu Kangyuan pharmaceutical industry GmbH.

Example 1

1. Method and results

1.1 detection conditions

(1) The chromatographic conditions are Kramosil C18 chromatographic column (4.6X 150mm, 3.5 μm), the mobile phase is 0.2% formic acid (A) -acetonitrile (B), and the gradient elution is carried out (0-35 min, 5-12% B, 35-50 min, 12-30% B, 50-55 min, 30-75% B, 55-60 min, 75% B). Flow rate 1.0mL/min^-1The column temperature is 30 ℃, the sample injection amount is 10 mu L, and the detection wavelengths are 250nm and 325nm respectively through dual-wavelength detection.

(2) Mass spectrum condition electrospray ionization (ESI), positive and negative ion mode, and scanning range m/z of 100-3000; the temperature of the drying gas is 350 ℃; volume flow of drying gas 10L min^-1(ii) a Atomization gas pressure 40 psig; capillary voltage 4000V; a fragment voltage of 135V; the taper hole voltage is 65V; the collision energy in MS/MS analysis is set at 10-55 eV according to the requirements of different compounds.

1.2 preparation of control solutions

Precisely weighing puerarin, daidzin, formononetin, astragaloside IV, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid C, 3 '-hydroxy puerarin, 3' -methoxy puerarin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin, albiflorin and isochlorogenic acid B, and adding 50% methanol to obtain extract containing 20-25 μ g/mL^-1The single standard solution of (1).

1.3 preparation of test solutions

Taking 5 bags of the product, grinding, precisely weighing about 1g, placing in a flat-bottomed flask, precisely adding 50mL of 50% methanol, weighing, heating and refluxing for 30min, cooling, weighing again, supplementing lost weight with 50% methanol, shaking, and 12000 r.min^-1Centrifuging for 5min, filtering the supernatant with 0.22 μm filter membrane, and collecting the filtrate.

1.4 identification and resolution of Compounds

Detecting the sample solution and the reference solution by UPLC-Q-TQF-MS under the condition of '1.1', and analyzing the chemical components in the Qige granules to obtain a total ion flow diagram (TIC) of positive and negative ion modes, which is specifically shown in the attached figure 1(A is a positive ion mode and B is a negative ion mode).

Identifying 44 compounds by attributing each mass spectrum peak in a prescription through each single medicine, combining a reference substance, an excimer ion peak and retention time provided by a primary mass spectrum, fragment ion information provided by a secondary mass spectrum and related documents, and identifying the target compound, wherein 2, 3, 5, 6, 7, 9, 10, 12, 14, 16-21, 23, 28, 32-36 and 38 are flavone and glycoside compounds; 1, 4, 11, 15, 24, 26, 27 and 30 are organic acid compounds; 39-44 is ligustilide compound; 8, 13 and 37 are saponin compounds, and specific results are shown in table 1.

TABLE 1 RRLC/Q-TOF/MS/MS analysis of compounds in Qige granules

Note the identified components are compared by control.

After the Qige particle atlas is established according to the conditions, the atlas of a Qige particle sample to be detected is measured according to the same method, and then the atlas of the Qige particle sample is compared with the atlas of the Qige particle sample by calculation of traditional Chinese medicine chromatogram fingerprint atlas similarity evaluation system software provided by the State pharmacopoeia committee, wherein the similarity is not lower than 0.90.

Precisely absorbing the same reference substance solution and sample solution, continuously injecting according to the detection condition of the invention, calculating relative retention time and relative peak area, RSD is less than 0.5%, fingerprint similarity is more than 0.9, indicating that the method has good precision, detecting according to the condition of the invention at 0h, 2h, 6h, 10h, 14h, 18h and 24h respectively, and indicating that the stability of the reference substance solution and the sample solution is good within 24 hours at room temperature, preparing different batches of solutions by the same method, and indicating that the method has good repeatability and good recovery rate. According to different reference substances, the condition established by the method is detected to obtain a corresponding curve, and a corresponding content detection method is established.

1.5 results of the experiment

The experiment adopts RRLC/Q-TOF-MS/MS technology to research the chemical components of the Qige particles, identifies and speculates 44 chemical components in the Qige particles, wherein 3 flavones and 21 glycosides from radix puerariae, radix astragali and chrysanthemum, 8 organic acids from chrysanthemum and ligusticum wallichii, 6 lactones and dimers thereof from ligusticum wallichii, and 3 saponins from radix astragali and radix paeoniae alba, and simultaneously adopts a reference substance to determine 21 components; the method adopts RRLC/Q-TOF-MS/MS technology, combines single medicinal materials to attribute each mass spectrum peak, comprehensively and systematically analyzes chemical components of the Qige particles through an excimer ion peak obtained by a primary mass spectrum, researches and summarizes the cracking rule of flavonoids, flavonoid glycosides, organic acids of chrysanthemum and ligusticum wallichii, ligusticum wallichii phthalide and dimers and saponins thereof in the mass spectrum, analyzes the chemical components in the Qige particles, and provides a theoretical basis for the deep research of the quality control and pharmacodynamic material basis of the Qige particles. The method has good linear relation, precision, stability and recovery rate, can accurately detect the component content of the astragalus-kudzuvine root granules, and can objectively evaluate the quality of a sample by comparing with a fingerprint spectrum.

Example 2

Chromatographic condition screening

(1) Selection of mobile phase:

by taking acetonitrile-0.2% formic acid as a mobile phase and investigating different elution procedures (the elution procedures are shown in tables 1-3), the results show that: the two chromatographic peaks 4 and 5 in the chromatogram of the elution program 1 are not separated ideally, and the peak shape of the chromatographic peak 20 in the chromatogram of the elution program 3 is poor and contains impurity peaks, so the elution program 2 is preferably selected as a mobile phase, and the result is shown in fig. 2.

TABLE 1

TABLE 2

TABLE 3

(2) Selection of column temperature:

when the fixed flow rate was 1ml/min, the chromatographic behavior was examined at a column temperature of 35 ℃ and 30 ℃ and at a column temperature of 25 ℃ and the results are shown in FIG. 3. The results show that under the condition of 35 ℃, the separation of No. 5 and No. 6 peaks, No. 13 and No. 14 peaks in the chromatogram is not ideal, and under the condition of 25 ℃, the separation of No. 4 and No. 5 peaks, No. 6 and No. 7 peaks is not ideal, and the peak shape of the chromatographic peak within 28-34 min is poor, so the column temperature is preferably selected to be 30 DEG C

(3) Selection of flow rate:

when the fixed column temperature was 30 ℃, the chromatographic behavior was examined under the conditions of flow rates of 0.9ml/min, 1ml/min and 1.1ml/min, and the results are shown in FIG. 4. As a result, it was found that at a flow rate of 1.1ml/min, the separation of peaks 13 and 14 and the peak shape of peaks 25 and 26 in the chromatogram were not satisfactory, and that at a flow rate of 0.9ml/min, the separation of peaks 4 and 5 and the peak shape of peaks 25 and 26 in the chromatogram were not satisfactory, and therefore, a flow rate of 1ml/min was preferred.

(4) Precision: the same sample solution is continuously injected for 6 times, the relative retention time and the relative peak area result are shown in the table, and the RSD of both is less than 1 percent, which indicates that the precision of the instrument is good.

(5) Repeatability: 6 parts of test solution to be tested are prepared and analyzed, the relative retention time and the relative peak area result are shown in the table, and the RSD of both is less than 1 percent, which indicates that the method has good repeatability.

(6) Stability: the same test solution is injected in 0, 3, 6, 9, 12, 15, 18, 21 and 24 hours respectively, the relative retention time and the relative peak area result are shown in the table, and the RSD of both is less than 2%, which indicates that the test solution has good stability within 24 hours at room temperature.

2. Method and results

2.1 detection conditions

(1) The chromatographic condition is a Kramosil C18 chromatographic column (4.6 multiplied by 150mm, 3.5 mu m), the mobile phase is 0.2% formic acid (A) -acetonitrile (B), and the gradient elution is carried out (0-12 min, 5-10% B, 12-20 min, 10-14% B, 20-30 min, 14-20% B, 30-40 min, 20-30% B, 40-46 min, 30-48% B, 46-47 min, 48-70% B, 47-55 min, 70-90% B, 55-70 min and 90% B). Flow rate 1.0mL/min^-1The column temperature is 30 ℃, the sample injection amount is 10 mu L, and the detection wavelengths are 250nm and 325nm respectively through dual-wavelength detection.

(2) Mass spectrum condition electrospray ionization (ESI), positive and negative ion mode, and scanning range m/z of 100-3000; the temperature of the drying gas is 350 ℃; volume flow of drying gas 10L min^-1(ii) a Atomization gas pressure 40 psig; capillary voltage 4000V; a fragment voltage of 135V; the taper hole voltage is 65V; collision energy in MS/MS analysisThe setting is 10-55 eV according to the requirements of different compounds.

2.2 preparation of control solutions

Precisely weighing puerarin, daidzin, formononetin, astragaloside, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, 3 '-hydroxypuerarin, 3' -methoxy puerarin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin, albiflorin, benzoylpaeoniflorin, apigenin-7-O-beta-D-glucopyranoside, farnesin, senkyunolide A, isoferulic acid, luteolin, apigenin and angelicin A, adding 50% methanol to obtain a mixture containing 20-25 μ g/mL of each component^-1Single or mixed standard solutions of (a).

2.3 preparation of test solutions

Taking 5 bags of the product, grinding, precisely weighing about 1g, placing in a conical flask, precisely adding 50mL of 50% methanol, weighing, ultrasonically treating for 15min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, and 12000 r.min^-1Centrifuging for 5min, filtering the supernatant with 0.22 μm filter membrane, and collecting the filtrate.

2.4 identification and resolution of Compounds

Detecting the sample solution and the reference solution by UPLC-Q-TQF-MS under the condition of '2.1', and analyzing the chemical components in the Qige granules to obtain a total ion flow diagram (TIC) of positive and negative ion modes, which is shown in the attached figure 5(A is a negative ion mode and B is a positive ion mode).

Identifying target compounds by the aid of excimer ion peaks provided by a primary mass spectrum, comparison of a reference substance, fragment ion information provided by a secondary mass spectrum and related documents, and identifying or deducing 78 compounds from 82 compounds in total, wherein the identification comprises the following steps: 46 flavonoids and glycosides, 11 organic acids, 11 lactones and dimers, and 10 saponins, and the specific results are shown in Table 2.

TABLE 2 RRLC/Q-TOF-MS/MS analysis of compounds in Qige granules

Note that the identified components were compared by control, Glu for glucose, Ap for apiose, Xyl for xylose, CQA for monocaffeoylquinic acid, DCQA for dicaffeoylquinic acid, CA for caffeic acid, and QA for quinic acid.

After the Qige particle atlas is established according to the conditions, the atlas of a Qige particle sample to be detected is measured according to the same method, and then the atlas of the Qige particle sample is compared with the atlas of the Qige particle sample by calculation of traditional Chinese medicine chromatogram fingerprint atlas similarity evaluation system software provided by the State pharmacopoeia committee, wherein the similarity is not lower than 0.90.

Precisely absorbing the same reference substance solution and sample solution, continuously injecting according to the detection condition of the invention, calculating relative retention time and relative peak area, RSD is less than 0.5%, fingerprint similarity is more than 0.9, indicating that the method has good precision, detecting according to the condition of the invention at 0h, 2h, 6h, 10h, 14h, 18h and 24h respectively, and indicating that the stability of the reference substance solution and the sample solution is good within 24 hours at room temperature, preparing different batches of solutions by the same method, and indicating that the method has good repeatability and good recovery rate. According to different reference substances, the condition established by the method is detected to obtain a corresponding curve, and a corresponding content detection method is established.

2.5 results of the experiment

The experiment adopts RRLC/Q-TOF-MS/MS technology to research the chemical components of the Qige particles, totally detects 82 chemical components in the Qige particles, identifies and speculates 78 compounds, and comprises the following steps: 46 flavonoids and glycosides, 11 organic acids, 11 lactones and dimers thereof, and 10 saponins, wherein 29 components are determined by adopting a reference substance; the method provided by the invention utilizes an RRLC/Q-TOF-MS/MS technology, carries out comprehensive and systematic analysis on the chemical components of the Qige particles by virtue of an excimer ion peak obtained by a primary mass spectrum, and combines comparison of a reference substance, secondary fragment ion information, related documents and the like, researches and summarizes the cracking rule of flavonoids and glycosides thereof, organic acids, lactones and dimers and saponins thereof in the mass spectrum, analyzes the chemical components in the Qige particles, and provides a theoretical basis for the quality control of the Qige particles and the deep research of the drug effect substance basis. The method has good linear relation, precision, stability and recovery rate, can accurately detect the component content of the astragalus-kudzuvine root granules, and can objectively evaluate the quality of a sample by comparing with a fingerprint spectrum.

Claims (9)

1. A detection method of radix astragali and radix Puerariae granule comprises radix astragali, radix Puerariae, radix Paeoniae alba, rhizoma Ligustici Chuanxiong, ramulus Cinnamomi, Curcuma rhizome, radix Clematidis and flos Chrysanthemi; the method is characterized by comprising the following steps: taking a radix astragali and radix puerariae particle test sample and a reference substance for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: by C₁₈The chromatographic column takes 0.2% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and the gradient elution procedure is as follows: 0-35 min, 5% -12% of B; 35-50 min, 12% -30% of B; 50-55 min, 30-75% of B; 55-60 min, 75% B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively;

and obtaining the component information of the Qige granules or the component and content information thereof according to the HPLC detection result.

2. The detection method of claim 1, wherein C is₁₈The chromatographic column is Kramosil C₁₈Chromatographic column, Agilent Zorbax SB-C₁₈Chromatographic column, Phenomenex Gemini C₁₈Chromatography column, Thermo syncronis C₁₈Chromatographic column, Agilent 5TC-C₁₈Chromatographic column, Kromasil 100-5C₁₈Column, Waters symmetry C₁₈Chromatographic column or Phenomenex Luna C₁₈A chromatographic column.

3. A detection method of radix astragali and radix Puerariae granule comprises radix astragali, radix Puerariae, radix Paeoniae alba, rhizoma Ligustici Chuanxiong, ramulus Cinnamomi, Curcuma rhizome, radix Clematidis and flos Chrysanthemi; the method is characterized by comprising the following steps: taking a radix astragali and radix puerariae particle test sample and a reference substance for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: by C₁₈The chromatographic column takes 0.2% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and the gradient elution procedure is as follows: 0-12 min, 5% -10% B; 12-20 min, 10% -14% of B; 20-30 min, 14% -20% of B; 30-40 min, 20-30% of B, 40-46 min, 30-48% of B, 46-47 min, 48-70% of B, 47-55 min, 70-90% of B, 55-70 min and 90% of B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively;

and obtaining the component information of the Qige granules or the component and content information thereof according to the HPLC detection result.

4. The detection method of claim 3, wherein C is₁₈The chromatographic column is Kramosil C₁₈Chromatographic column, Agilent Zorbax SB-C₁₈Chromatographic column, Phenomenex Gemini C₁₈Chromatography column, Thermo syncronis C₁₈Chromatographic column, Agilent 5TC-C₁₈Chromatographic column, Kromasil 100-5C₁₈Column, Waters symmetry C₁₈Chromatographic column or Phenomenex Luna C₁₈A chromatographic column.

5. The assay of claim 3, wherein the control is selected from one or more of puerarin, daidzin, formononetin, astragaloside, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, 3 '-hydroxypuerarin, 3' -methoxypugenin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin, albiflorin, benzoylpaeoniflorin, apigenin-7-O- β -D-glucopyranoside, farnesin, senolide A, isoferulic acid, luteolin, apigenin, or angelicin A.

6. A radix astragali and radix Puerariae granule fingerprint chromatogram construction method comprises radix astragali, radix Puerariae, radix Paeoniae alba, rhizoma Ligustici Chuanxiong, ramulus Cinnamomi, Curcuma rhizome, radix Clematidis and flos Chrysanthemi; it is characterized in that the preparation method is characterized in that,

preparation of a test solution: grinding radix astragali and radix Puerariae granules, and preparing 20mg/ml sample with methanol;

preparation of control solutions: precisely weighing puerarin, daidzin, formononetin, astragaloside IV, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, 3 '-hydroxypuerarin, 3' -methoxy puerarin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin and albiflorin, adding 50% methanol to obtain extract with each component concentration of 20-25 μ g/mL^-1Single or mixed standard solutions of (a);

the chromatographic conditions for HPLC detection were: by C₁₈The chromatographic column takes 0.2% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and the gradient elution procedure is as follows: 0-35 min, 5% -12% of B; 35-50 min, 12% -30% of B; 50-55 min, 30-75% of B; 55-60 min, 75% B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively;

and (4) obtaining the fingerprint of the Qige granules according to the HPLC detection result.

7. The method of claim 6, wherein C is₁₈The chromatographic column is Kramosil C₁₈Chromatographic column, Agilent Zorbax SB-C₁₈Chromatographic column, Phenomenex Gemini C₁₈Chromatography column, Thermo syncronis C₁₈Chromatographic column, Agilent 5TC-C₁₈Chromatographic column, Kromasil 100-5C₁₈Column, Waters symmetry C₁₈Chromatographic column or Phenomenex Luna C₁₈A chromatographic column.

8. A radix astragali and radix Puerariae granule fingerprint chromatogram construction method comprises radix astragali, radix Puerariae, radix Paeoniae alba, rhizoma Ligustici Chuanxiong, ramulus Cinnamomi, Curcuma rhizome, radix Clematidis and flos Chrysanthemi; it is characterized in that the preparation method is characterized in that,

preparation of a test solution: grinding radix astragali and radix Puerariae granules, and preparing 20mg/ml sample with methanol;

preparation of control solutions: precisely weighing puerarin, daidzin, formononetin, astragaloside IV, paeoniflorin, ligustilide, ferulic acid, chlorogenic acid, 1, 3-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, 3 '-hydroxypuerarin, 3' -methoxy puerarin, neochlorogenic acid, cryptochlorogenic acid, 1, 5-dicaffeoylquinic acid, puerarin apioside, daidzein, genistin, albiflorin, benzoylpaeoniflorin, apigenin-7-O-beta-D-glucopyranoside, farnesin, senkyunolide A, isoferulic acid, luteolin, apigenin and levistilbenide A, adding 50% methanol to obtain extract with each component concentration of 20-25 μ g.mL^-1Single or mixed standard solutions of (a);

the chromatographic conditions for HPLC detection were: by C₁₈The chromatographic column takes 0.2% formic acid as a mobile phase A and acetonitrile as a mobile phase B, and the gradient elution procedure is as follows: 0-12 min, 5% -10% B; 12-20 min, 10% -14% of B; 20-30 min, 14% -20% of B; 30-40 min, 20-30% of B, 40-46 min, 30-48% of B, 46-47 min, 48-70% of B, 47-55 min, 70-90% of B, 55-70 min and 90% of B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; detecting with dual wavelengths, wherein the detection wavelengths are 250nm and 325nm respectively;

and (4) obtaining the fingerprint of the Qige granules according to the HPLC detection result.

9. The method of claim 8, wherein C is₁₈The chromatographic column is Kramosil C₁₈Chromatographic column, Agilent Zorbax SB-C₁₈Chromatographic column, Phenomenex Gemini C₁₈Chromatography column, Thermo syncronis C₁₈Chromatographic column, Agilent 5TC-C₁₈Chromatographic column, Kromasil 100-5C₁₈Column, Waters symmetry C₁₈Chromatographic column or Phenomenex Luna C₁₈A chromatographic column.

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