阅读说明：本技术 一种黄芪桂枝五物汤全成分的分析方法 (Analysis method of total components of astragalus mongholicus-cassia twig five-substance decoction ) 是由 姚琳 程红 陈永君 周颖虹 张敏宜 郭淇 姚雨彤 周娜 于 2019-12-19 设计创作，主要内容包括：本发明提供了一种黄芪桂枝五物汤全成分的分析方法,属于药物分析技术领域。本发明采用超快速高效液相色谱三重四级杆飞行时间串联质谱联用(HPLC-Q-TOF-MS/MS)技术对黄芪桂枝五物汤样品进行检测,同时采用药典标识性的5种化学成分——黄芪甲苷、毛蕊异黄酮、葡萄糖苷、桂皮醛、芍药苷、6-姜辣素对黄芪桂枝五物汤检测结果进行确认,明确识别了31种成分,实现了对黄芪桂枝五物汤复方成分的全面分析,提高了其质量控制的可靠性。(The invention provides an analysis method of the whole components of an astragalus-cassia twig five-substance decoction, belonging to the technical field of drug analysis. The invention adopts the ultra-fast high performance liquid chromatography triple quadrupole flight time tandem mass spectrometry (HPLC-Q-TOF-MS/MS) technology to detect the radix astragali and cassia twig five-substance decoction sample, and simultaneously adopts 5 chemical components identified by pharmacopeia-astragaloside, calycosin, glucoside, cinnamaldehyde, paeoniflorin and 6-gingerol to confirm the detection result of the radix astragali and cassia twig five-substance decoction, thereby clearly identifying 31 components, realizing the comprehensive analysis of the compound components of the radix astragali and cassia twig five-substance decoction and improving the reliability of the quality control.)

1. An analysis method of the whole components of the astragalus and cassia twig five-substance decoction is characterized in that the analysis method adopts an ultrafast high performance liquid chromatography triple quadrupole time of flight tandem mass spectrometry to detect an astragalus and cassia twig five-substance decoction sample;

wherein, the conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column, the length of the column is 100-250mm, the inner diameter of the column is 3.0-4.6mm, and the particle size is 1.5-5.0 μm; the flow rate of the mobile phase is 0.2-1 mL/min; the sample amount is 5-20 mu L; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.1% (v/v) formic acid, the mobile phase B is an acetonitrile solution containing 0.1% (v/v) formic acid, and the following gradient elution procedure is adopted: 1) reducing the volume percentage of the mobile phase A from 95% to 5% in 0-30 min; 2) 30-35 min, wherein the volume percentage of the mobile phase A is 5%; 3) the volume percentage of the mobile phase A is increased from 5% to 95% within 35-37 min; 4) the volume percentage of the mobile phase A is 95 percent for 37-45 min;

the triple quadrupole time-of-flight tandem mass spectrometry conditions are as follows: adopting an electrospray positive and negative ion mode, wherein the full scanning mass-to-charge ratio range is 50-1200, the pressure of atomizing gas is 35-75psi, the pressure of auxiliary gas is 37-75psi, the pressure of air curtain gas is 25-45psi, the atomizing temperature is 350-;

the astragalus and cassia twig five-ingredient decoction sample is prepared from astragalus, cassia twig, white paeony root, ginger and Chinese date.

2. The analytical method according to claim 1, wherein the chromatographic column has a column length of 150mm, a column inner diameter of 3.0mm, and a particle diameter of 2.8 μm; the flow rate of the mobile phase is 0.3 mL/min; the sample size was 10. mu.L.

3. The analytical method of claim 1, wherein the column temperature of the chromatographic column is 40 ℃.

4. The analytical method of claim 1, wherein the atomization gas pressure is 55psi, the assist gas pressure is 55psi, the curtain gas pressure is 35psi, the atomization temperature is 550 ℃, the collision energy is 40V, and the declustering voltage is 60V.

5. The analytical method of claim 1, wherein the electrospray ionization mode is employed at a spray voltage of 5500V; when the electrospray negative ion mode is adopted, the spray voltage is-4500V.

6. The assay of claim 1, wherein the assay simultaneously qualitatively detects 31 components in the astragalus cinnamomi five-substance decoction sample, wherein the 31 components are astragaloside IV, astragaloside II, calycosin glucoside, formononetin, calycosin, formononetin, kaempferol, quercetin, astragaloside VII, isorhamnetin, quinine, cinnamaldehyde, cinnamic acid, coumarin, protocatechuic acid, protocatechuic aldehyde, cinnamyl alcohol, 2-methoxycinnamic acid, paeoniflorin, benzoylpaeoniflorin, paeonol, hydroxypaeoniflorin, gallic acid, paeoniflorin sulfite, galloyl paeoniflorin, 6-gingerol, zingerone, β -sesquiterpenene, oleanolic acid, scopoletin, and rutin.

7. The method of claim 6, wherein the same ultrafast high performance liquid chromatography triple quadrupole time of flight tandem mass spectrometry is used for detection and analysis of astragaloside IV, calycosin glucoside, cinnamaldehyde, paeoniflorin and 6-gingerol to verify the accuracy and reliability of the analysis result of the astragalus cinnamomi five-substance decoction sample.

8. The analysis method as claimed in claim 1, wherein the concentration of crude drug in the sample of the astragalus and cassia twig five-material decoction is 0.4-0.6 g/mL.

9. The assay method according to claim 1, wherein the mass ratio of the astragalus, the cassia twig, the white peony root, the ginger and the Chinese date is astragalus: cassia twig: white peony root: ginger: the Chinese date is 1:1:1:2: 1.

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an analysis method of the whole components of an astragalus-cassia twig five-substance decoction.

Background

The astragalus and cassia twig five-ingredient decoction is recorded for the first time in golden Kui Yao L ü e, which is prepared from 5 traditional Chinese medicines of astragalus, cassia twig, white paeony root, ginger and Chinese date, has the effects of tonifying qi, warming channels and regulating ying and freeing Bi, is a famous prescription for treating blood arthralgia consumptive disease, and is commonly used for treating diseases such as diabetic neuropathy, stroke sequela, thromboangiitis obliterans, cervical spondylosis and the like in modern clinic. The existing research on the components of the astragalus-cassia twig five-component decoction comprises the steps of carrying out content determination on main components of astragaloside IV, calycosin glycoside, paeoniflorin, albiflorin and cinnamic acid by using an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering method), an LC-MS (liquid chromatography-mass spectrometry combined method) and an UFLC (ultra-fast liquid chromatography) method so as to achieve the purpose of comprehensively evaluating the quality of the astragalus-cassia twig five-component decoction; and using HPLC methods^[4]A fingerprint of the traditional Chinese medicine of the astragalus-cassia twig five-ingredient decoction is established, and a basis is tried to be provided for the quality control of the astragalus-cassia twig five-ingredient decoction.

However, the research on the components of the astragalus-cassia twig five-ingredient decoction at present has certain limitations and disadvantages: (1) the content detection method mainly focuses on the content determination research of single medicinal materials and representative components in the formula; (2) the existing method for detecting the components of the astragalus and cassia twig five-component decoction is low in sensitivity, and the components with low content cannot be detected, for example, for researching the fingerprint of the astragalus and cassia twig five-component decoction traditional Chinese medicine, an HPLC method is adopted to only screen out 13 common peaks (Wei nationality, Cao Peng, Hodging, and the like; the astragalus and cassia twig five-component decoction HPLC fingerprint research [ J ]. Chinese traditional medicine journal, 2013(12): 2796-. However, the composition of the drug effect substances of the Chinese herbal compound is complex, the influence of the drug effect substances is not the cumulative effect of single components, but the interaction among the components is included, and the content of the single component cannot comprehensively reflect the quality of the compound; meanwhile, the existing method for detecting the components of the astragalus-cassia twig five-component decoction cannot detect low-content components and cannot reflect the overall situation of the compound prescription. Therefore, there is a need for comprehensive quality control using multi-technology methods in conjunction with multi-index component measurements.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides an analysis method for the whole components of the astragalus-cassia twig five-substance decoction.

In order to achieve the purpose, the invention adopts the technical scheme that: the invention provides an analysis method of the whole components of the astragalus and cassia twig five-component decoction, which adopts an HPLC-Q-TOF-MS/MS method (ultra-fast high performance liquid chromatography triple quadrupole flight time tandem mass spectrometry) to detect an astragalus and cassia twig five-component decoction sample; wherein, the conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column, the length of the column is 100-250mm, the inner diameter of the column is 3.0-4.6mm, and the particle size is 1.5-5.0 μm; the flow rate of the mobile phase is 0.2-1 mL/min; the sample amount is 5-20 mu L; the mobile phase consisted of mobile phase a, which was an aqueous solution containing 0.1% (v/v) formic acid, and mobile phase B, which was an acetonitrile solution containing 0.1% (v/v) formic acid, using the gradient elution procedure as shown in table 1:

table 1 mobile phase gradient elution procedure

The triple quadrupole time-of-flight tandem mass spectrometry conditions are as follows: adopting an electrospray positive and negative ion mode, wherein the full scanning mass-to-charge ratio range is 50-1200, the pressure of atomizing gas is 35-75psi, the pressure of auxiliary gas is 37-75psi, the pressure of air curtain gas is 25-45psi, the atomizing temperature is 350-;

the HPLC-Q-TOF-MS/MS method can realize qualitative analysis of 31 components such as astragaloside IV, astragaloside II, calycosin glucoside, formononetin, calycosin, formononetin, kaempferol, quercetin, astragaloside VII, isorhamnetin, genistin, cinnamaldehyde, cinnamic acid, coumarin, protocatechuic acid, protocatechualdehyde, cinnamyl alcohol, 2-methoxycinnamic acid, paeoniflorin, benzoylpaeoniflorin, paeonol, paeoniflorin, hydroxypaeoniflorin, gallic acid, paeoniflorin sulfite, galloyl paeoniflorin, 6-gingerol, zingerone, β -sesquiterpenene, oleanolic acid, scopoletin, rutin and the like in the astragalus-cassia twig five-substance decoction.

As a preferred embodiment of the analytical method of the present invention, the column of the chromatographic column has a column length of 150mm, a column inner diameter of 3.0mm, and a particle diameter of 2.8. mu.m; the flow rate of the mobile phase is 0.3 mL/min; the sample size was 10. mu.L.

As a preferred embodiment of the analytical method according to the invention, the column temperature of the chromatographic column is 40 ℃.

As a preferred embodiment of the analysis method of the present invention, the atomization gas pressure is 55psi, the auxiliary gas pressure is 55psi, the gas curtain gas pressure is 35psi, the atomization temperature is 550 ℃, the collision energy is 40V, and the declustering voltage is 60V.

As a preferred embodiment of the analytical method of the present invention, when an electrospray positive ion mode is adopted, the spray voltage is 5500V; when the electrospray negative ion mode is adopted, the spray voltage is-4500V.

As a preferred embodiment of the analysis method, the analysis method also adopts the same ultrafast high performance liquid chromatography triple quadrupole time-of-flight tandem mass spectrometry to carry out detection and analysis on the standard substances of astragaloside IV, calycosin glucoside, cinnamaldehyde, paeoniflorin and 6-gingerol so as to verify the accuracy and reliability of the analysis result of the astragalus cassia twig five-substance decoction sample.

As a preferred embodiment of the analysis method, the concentration of the crude drug in the sample of the astragalus and cassia twig five-material decoction is 0.4-0.6 g/mL.

As a more preferable embodiment of the analysis method of the invention, the injection crude drug concentration of the astragalus and cassia twig five-material decoction sample is 0.4 g/mL.

As a preferred embodiment of the analysis method of the present invention, the mass ratio of the astragalus root, the cassia twig, the white peony root, the ginger and the Chinese date is astragalus: cassia twig: white peony root: ginger: the Chinese date is 1:1:1:2: 1.

Compared with the prior art, the invention has the following advantages and beneficial effects: the invention adopts HPLC-Q-TOF-MS/MS method to carry out full component detection on the radix astragali and cassia twig five-component decoction, can identify 31 effective components, not only can accurately, comprehensively and reliably analyze the quality of the radix astragali and cassia twig five-component decoction preparation, but also is efficient and rapid; in addition, the accuracy of the astragalus-cassia twig five-substance decoction can be verified by comparing the analysis result of the astragalus-cassia twig five-substance decoction with the analysis results of5 pharmacopoeia component standards of astragaloside, calycosin glucoside, cinnamaldehyde, paeoniflorin and 6-gingerol.

Drawings

Fig. 1 is a total ion flow diagram of astragalus cinnamomi five-ingredient decoction samples with different crude drug concentrations obtained by different processing methods under an anion mode, wherein the crude drug concentration of the astragalus cinnamomi five-ingredient decoction concentrated solution sample in fig. 1A is 0.562g/mL, and the crude drug concentration of the astragalus cinnamomi five-ingredient decoction freeze-dried powder aqueous solution sample in fig. 1B is 0.4 g/mL;

fig. 2 is a total ion flow diagram of an astragalus and cassia twig five-ingredient decoction freeze-dried powder water solution sample, wherein fig. 2A is a negative ion mode, and fig. 2B is a positive ion mode.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

1. Instruments, reagents and materials

1.1 instruments

A novel sealed grinder (500g sealed oscillating grinder), a second sieve (24 meshes), a fourth sieve (65 meshes), a volatile oil extraction device (comprising a KDM type temperature-adjusting electric heating jacket, a 5000mL round-bottom flask, a volatile oil extractor, a condenser tube), a rotary evaporator (IKA, model RV10, including a vacuum pump and a low-temperature cooling liquid circulating pump), a low-temperature vacuum drier (LABCONCO), an ultra-fast high-performance liquid chromatograph (LC-20AD-XR binary pump, SIL-20AD-XR automatic sample injector, CTO-20A column incubator, Shimadzu corporation); four-stage rod-time-of-flight mass spectrometer (Triple TOF5600+, AB SCIEX, USA), ultra-pure water device (Millipore, USA, model Simplicity).

1.2 reagents

Comparison products: astragaloside IV (purity is more than or equal to 98.0 wt.%), calycosin glucoside (purity is more than or equal to 98.0 wt.%), cinnamaldehyde (purity is more than or equal to 98.0 wt.%), paeoniflorin (purity is more than or equal to 98.0 wt.%), and 6-gingerol (purity is more than or equal to 98.0 wt.%) were all purchased from Dormant Biotech, Inc.

Methanol (analytical, Guangdong Guanghua chemical Co., Ltd.), acetonitrile (chromatographic, Fisher Scientific Co., Ltd.), formic acid (Sigma Co., batch No.: 0001408600).

1.3 materials

(1) Source and treatment of medicinal materials: the traditional Chinese medicinal materials of the astragalus, the cassia twig, the white paeony root, the ginger and the Chinese date are from Kangmei pharmaceutical industry, Inc. Pulverizing radix astragali, ramulus Cinnamomi, and radix Paeoniae alba in a pulverizer, and sieving with a second sieve and a fourth sieve to obtain radix astragali coarse powder, ramulus Cinnamomi coarse powder, and radix Paeoniae alba coarse powder. Cutting rhizoma Zingiberis recens into small pieces of about 2cm x 2cm, and cutting fructus Jujubae into small pieces of about 2cm x 2cm (core of fructus Jujubae).

(2) Obtaining a compound water decoction: weighing 45g of coarse powder of the astragalus, the cassia twig and the white paeony root, 90g of ginger blocks and 45g of Chinese date dices (added with the weight of date pits) respectively, placing the mixture into a 5000mL round-bottom flask, and performing compound decoction by adopting a volatile oil extraction device: heating by an electric heating sleeve, adding water, decocting twice, adding 10 times (2700 mL) of water for the first time, namely the mass of the water is 10 times of the total mass of the 5 substances, namely the astragalus, the cassia twig, the white paeony root, the ginger and the Chinese date, decocting for 1h, filtering to obtain a first filtrate, collecting aromatic water once every half hour during the first filtrate, and collecting twice in total; adding 6 times of water (1620mL pure water) 6 times of the total mass of5 materials such as radix astragali, ramulus Cinnamomi, radix Paeoniae alba, rhizoma Zingiberis recens and fructus Jujubae for the second time, decocting for 45min, filtering to obtain a second filtrate, and collecting the first aromatic water. Mixing the two filtrates to obtain decoction of radix astragali and ramulus Cinnamomi, and storing at 4 deg.C. Three aromatic hydrates were combined and stored in 100mL Erlenmeyer flasks hermetically to give total aromatic water, which was stored at 4 ℃ until use.

(3) And (3) rotationally evaporating the concentrated solution to obtain: and (3) carrying out rotary evaporation concentration on the decoction of the five-substance decoction of astragalus and cassia twig by using a rotary evaporator, cooling the circulating water at the temperature of below 0 ℃, carrying out rotary evaporation at the rotating speed of 100rpm and the rotary evaporation at the temperature of 60 ℃, concentrating the decoction to 250mL of 200-.

(4) Obtaining freeze-dried powder: subpackaging the radix astragali and ramulus Cinnamomi five-substance decoction concentrated solution in a freeze-drying tray, placing in a refrigerator at-80 deg.C for freezing for more than 4h, taking out, placing in a low-temperature vacuum drier, and drying for 24h to obtain radix astragali and ramulus Cinnamomi five-substance decoction freeze-dried powder.

2. Chromatographic and mass spectral conditions

2.1 chromatographic conditions

A chromatographic column: a C18 column (150X 3.0mm,2.8 μm, Phenomenex Co.); the flow rate is 0.3 mL/min; the column temperature was 40 ℃; the sample volume is 10 mu L; the mobile phase consisted of mobile phase A, which was an aqueous solution containing 0.1% (v/v) formic acid, and mobile phase B, which was an acetonitrile solution containing 0.1% (v/v) formic acid, and was subjected to gradient elution according to the elution procedure shown in Table 1.

2.2 Mass Spectrometry conditions

An ion source: ESI (electrospray); analysis software: analyst software (PeakView, Version 2.1, abciex). Mass spectrum conditions: the detection is carried out by adopting a positive ion mode and a negative ion mode respectively, the full scanning mass-to-charge ratio m/z range is 50-1200, the atomization air pressure is 55psi, the auxiliary air pressure is 55psi, the air curtain air pressure is 33psi, the atomization temperature is 550 ℃, the spray voltage is 5500V or-4500V, the collision energy is 40V, and the cluster removing voltage is 60V.

3. Determination of detection target compound:

according to records and literature reports about the determination of the ingredients of astragalus, cassia twig, white paeony root, ginger and Chinese date in pharmacopoeia 2015 edition of the people's republic of China, China's knowledge network and PubMed foreign language website, 47 possible target detection compounds in the astragalus-cassia twig five-material decoction are preliminarily determined, and the details are shown in table 2.

TABLE 2 radix astragali and ramulus Cinnamomi decoction target detection compound Table

4. Preparation of the solution

4.1 preparation of Mixed control solutions

Preparation of mixed reference: adding 1mL methanol into astragaloside IV (20mg), calycosin glucoside (20mg), paeoniflorin (20mg), cinnamaldehyde (20 μ L), and 6-gingerol (20mg) respectively to obtain single control stock solution of 20mg/mL (cinnamaldehyde is 20 μ L/mL), and storing in refrigerator at 4 deg.C; mixing the single stock solutions, diluting, mixing, centrifuging, collecting supernatant, and filtering with 0.45 μm organic microporous membrane to obtain mixed reference substance, wherein the concentrations of astragaloside IV, calycosin glucoside, paeoniflorin and 6-gingerol are all 0.02mg/mL, and the concentration of cinnamaldehyde is 0.02 μ L/mL.

4.2 preparation of test articles

Dissolving 0.4g of the radix astragali and ramulus Cinnamomi five-substance decoction lyophilized powder in pure water to obtain a sample water solution with crude drug concentration of 0.4g/mL, mixing, centrifuging, collecting supernatant, and filtering with 0.45 μm organic microporous membrane to obtain the sample of the desired sample.

4.3 selection of the concentration of the test sample

(1) Because the compound extract of the astragalus-cassia twig five-substance decoction contains various insoluble components and can interfere with determination, the sample concentration of a sample to be detected is selected by taking the maximum dissolution of the water-soluble components as one of standards, and the maximum solubility is determined by observing the color shade of the sample and the clarity of the sample after centrifugation. The specific method comprises the following steps: preparing the same batch of astragalus and cassia twig five-ingredient decoction freeze-dried powder samples with different masses into water solutions with different crude drug concentrations (1g crude drug is 0.188g freeze-dried powder, namely the ratio of the total mass of the crude drugs to the total mass of the freeze-dried powder is 0.188), comparing the clarity of the water solutions with different crude drug concentrations, and when the crude drug concentration of the astragalus and cassia twig five-ingredient decoction freeze-dried powder water solution exceeds 0.4g/mL, the clarity is lower; when the crude drug concentration of the astragalus and cassia twig five-ingredient decoction freeze-dried powder aqueous solution is below 0.4g/mL, the solution is clear and is suitable for HPLC-Q-TOF-MS/MS sample loading, and in consideration of detection of components with lower content, the sample loading concentration of the astragalus and cassia twig five-ingredient decoction freeze-dried powder aqueous solution is selected to be the maximum concentration under the clear condition, namely the crude drug concentration is 0.4g/mL, and the details are shown in Table 3.

TABLE 3 comparison table of crude drug concentration and color clarity

(2) Respectively detecting the radix astragali and ramulus Cinnamomi penta-drug decoction concentrated solution (prepared by the steps (1) - (3) described in the section of material 1.3 without the step (4), namely freeze-dried powder) and the radix astragali and ramulus Cinnamomi penta-drug decoction freeze-dried powder aqueous solution with the crude drug concentration of 0.4g/mL by using an HPLC-Q-TOF-MS/MS method (the detection conditions are the same as the section of 2, chromatographic conditions and mass spectrum conditions), wherein the obtained mass spectrum is shown in a figure 1A and a figure 1B, and the comparison shows that the sample with the crude drug concentration of 0.4g/mL has normal peak appearance and the peak appearance number is more than that of the sample with the crude drug concentration of 0.562g/mL, so that the crude drug concentration of 0.4g/mL is determined as the final sample appearance concentration of the radix astragali and ramulus Cinnamomi penta-drug sample, and the results are determined to be 7, and the methodological investigation shows that the appearance concentration of the radix astragali and ramulus Cinnamomi penta-drug sample in the section is 0.4g/mL (crude drug concentration) .

5. Sample assay

Preparing a sample of the test sample according to the part of '4 and solution preparation', and determining according to the conditions of the part of '2 and chromatographic conditions and mass spectrum conditions' to obtain a total ion flow diagram of chemical components in positive and negative ion modes of the astragalus and cassia twig five-substance decoction freeze-dried powder, as shown in figure 2.

6. Determination of results

Detecting by HPLC-Q-TOF-MS/MS method, and recording and comparing T of molecular formulas and chromatographic peaks of target compounds of the mixed reference substance and the radix astragali and ramulus Cinnamomi decoction sample_R(retention time) and m/z information (wherein the measured value refers to the measured value of the molecular weight of the fragment of the parent ion, and the theoretical value refers to the theoretical value of the molecular weight of the fragment of the parent ion), and the relevant map and data are stored as electronic versions. Table 4 shows 31 common chemical components T of the astragalus and cassia twig five-ingredient decoction detected by HPLC-Q-TOF-MS/MS technology_RAnd m/z information, wherein 11 chemical components belong to astragalus, 7 chemical components are derived from cassia twig, 7 chemical components belong to white paeony root, 3 chemical components belong to ginger, and 3 chemical components belong to Chinese date.

TABLE 4 Mass spectrum data of chemical components in Huangqi Guizhi Wu-Tang

7. Methodology investigation

7.1 principle

1) The accuracy evaluation indexes of the HPLC-Q-TOF-MS/MS method are as follows: selecting T of five standard substances (astragaloside IV, calycosin glucoside, paeoniflorin, cinnamaldehyde, and 6-gingerol)_RFound at mass (molecular weight of parent ion fragment of sample) and MS²Difference values of three main detection parameters of mass spectrum (main secondary ion fragments) and corresponding three parameters in the astragalus and cassia twig five-ingredient decoction freeze-dried powder aqueous solution, namely △ T_R(T_RDifference of (d), △ Mass (difference of found at Mass), and △ MS²(MS²Difference value) as a reference index for determining whether the HPLC-Q-TOF-MS/MS detection method and component results are accurate and reliable, the invention considers △ T_R(units min), △ Mass and △ MS²The value is within the range of + -0.1Belonging to the normal error range.

2) Selection of secondary ion fragments: in the invention, 1 or more secondary ions with the best signal and the highest stability under the optimized mass spectrum condition are selected as the study objects to evaluate the stability of the study objects.

7.2 test result accuracy verification experiment of HPLC-Q-TOF-MS/MS method

In order to test the accuracy of the results of the detection of the total components of the astragalus-cassia twig five-component decoction by the HPLC-Q-TOF-MS/MS method established in the above embodiment, 5 components (astragaloside, calycosin glucoside, cinnamaldehyde, paeoniflorin, 6-gingerol) identified by pharmacopeia are selected, a mixed reference substance and a sample to be tested are prepared according to the part of '4 and solution preparation', and the sample loading determination is carried out according to the conditions of the part of '2, chromatographic conditions and mass spectrum conditions'. Recording T of5 components corresponding to 5 mixed reference substances and the five-substance decoction of radix astragali and ramulus Cinnamomi_RMolecular weight of parent ion fragment, MS of main secondary fragment²Calculate △ T_RMin, △ Mass and △ MS²The statistical results are shown in Table 5, which shows △ T of the control and the Astragalus and Cassia twig five-ingredient decoction sample_R/min(T_{R, control}-T_{Decoction of five drugs R})、△Mass(Mass_Control-Mass_{Five-material decoction})、△MS²(MS² _Control-MS² _{Five-material decoction}) The difference values are within the error range of +/-0.1, so that the result of detecting the total components of the astragalus-cassia twig five-component decoction by the HPLC-Q-TOF-MS/MS method is accurate and reliable.

TABLE 5 comparison table of quality spectrum information of mixed comparison of Huangqi Guizhi Wu Tang and pharmacopoeia

7.3 repeatability experiment of HPLC-Q-TOF-MS/MS:

1) in order to detect the full ingredient knot of the astragalus and cassia twig five-ingredient decoction by an HPLC-Q-TOF-MS/MS methodAccording to the repeatability of fruits, 3 batches of raw materials are respectively prepared into freeze-dried powder according to the method of the part of 1.3 materials, then are respectively prepared into sample of the sample to be tested according to the method of the part of 4.2 sample preparation, and then are subjected to sample loading and measurement according to the detection conditions of the parts of 2, chromatographic conditions and mass spectrum conditions. Recording T of detection components of 3 batches of astragalus and cassia twig five-substance decoction samples_RMolecular weight of parent ion, MS of main secondary fragment²The data comparison result shows that the repeatability of the sample data of the three batches of the decocted astragalus and cassia twig five-ingredient decoction is high, the repeatability of the result of detecting the whole components of the astragalus and cassia twig five-ingredient decoction by the HPLC-Q-TOF-MS/MS method is good, and the specific comparison result is shown in Table 6.

TABLE 6 comparison analysis table of mass spectrum data of 3 batches of decoction samples of astragalus, cassia twig and wu-tang decoction

2) The invention further relates to T of three batches of samples_RThe repeatability standard deviation S is subjected to comparative analysis, and the specific analysis method comprises the following steps:

wherein, S-standard deviation (%); n-total number of samples or number of measurements; i-each measured value of a certain component in the material can be any one number from 1 to n. In the present invention, it is considered that the standard deviation (S) of reproducibility within the range of. + -. 0.1 falls within the normal error range. The statistical result shows that the repeatability standard deviation (S) of each detection compound of three batches of decoction samples of the astragalus, the cassia twig and the five-material is less than 0.1, which indicates that the data repeatability is high. The results of the analysis are shown in Table 7.

TABLE 7 three batches of Astragalus and Cassia twig five-herb decoction T_RStatistical table of standard deviation of/min repeatability

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.