CN109444311B - Separation and detection method of related components of ginseng and astragalus strengthening injection - Google Patents
Separation and detection method of related components of ginseng and astragalus strengthening injection Download PDFInfo
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Abstract
The invention provides a method for quickly separating and evaluating related components of a ginseng and astragalus strengthening injection, which comprises the steps of separating the ginseng and astragalus strengthening injection by a polyamide column, detecting by LC-MS, and extracting an extracted ion image (EIC) from a total ion current chromatogram (TIC) by using accurate molecular weights of 441.1678, 441.2007, 471.2083, 491.1195, 507.1508, 633.2553, 829.4591, 871.4697, 913.4650 and 991.5119 so as to determine the distribution condition of index components of codonopsis pilosula and astragalus in a preparation.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry and natural product chemistry, and particularly relates to a separation and detection method of related components of a ginseng and astragalus body resistance strengthening injection.
Background
Radix Codonopsis (Radix Codonopsis) has mild drug property, wide application, and multiple pharmacological activities, and has effects of promoting digestion, regulating cardiovascular system, regulating immune system function, and enhancing body resistance, with little toxicity. Radix astragali (Radix astragali) is a main qi-invigorating drug, and can significantly increase phagocytic function of macrophages, and can enhance phagocytic function of macrophages when used together with Radix Codonopsis.
The Shenqi Fuzheng injection is a unique Chinese medicinal variety in the pharmaceutical factory, is a pure Chinese medicinal preparation prepared by extracting radix codonopsitis and radix astragali, and is mainly used for tonifying qi and strengthening the body resistance. The radix codonopsitis and the astragalus root have the same properties, taste and channel tropism and basically same efficacy, and the two medicines are added to play the synergistic effect of monarch, minister, assistant and guide and mutual guide, thereby obviously enhancing the clinical curative effect.
However, in recent years, the traditional Chinese medicine injection has been controversial, and the clinical application range of the traditional Chinese medicine injection is limited. The reason for this is probably that the traditional Chinese medicine contains complex components and is difficult to perform quantitative and qualitative analysis. Aiming at the problem, research and development personnel of the ginseng-astragalus body resistance strengthening injection put forward the concept of digital traditional Chinese medicine, namely, the traditional Chinese medicine is guided by syndrome differentiation and treatment, and the theories and methods of subjects such as modern computer technology, phytochemistry, analytical chemistry, pharmacology and the like are adopted to digitally control the whole processes of planting, extraction, preparation, detection and the like of the traditional Chinese medicine, so that the quality of the traditional Chinese medicine is stable and controllable, and the clinical safety and effectiveness are ensured.
At present, various quality control and detection methods are established for the Shenqi body resistance strengthening injection; however, most of the methods only detect the index components of one medicinal material of the codonopsis pilosula or the astragalus, and lack a rapid, effective and comprehensive separation and evaluation method for the components of the codonopsis pilosula and the astragalus and the components of flavonoids and saponins in the astragalus.
Disclosure of Invention
Aiming at the problems in the prior art, the invention establishes a method for quickly separating and detecting the related components of the Shenqi Fuzheng injection. The method can simultaneously separate components of the ginseng and astragalus root strengthening injection from the codonopsis pilosula and the astragalus root and flavonoid and saponin components in the astragalus root, is convenient and quick to operate, and can provide a foundation for better controlling the quality of medicines.
In order to achieve the technical effects, the invention adopts the following technical scheme:
a method for quickly separating and evaluating related components of a Shenqi body resistance strengthening injection comprises the following steps: the Shenqi injection for strengthening body resistance is subjected to polyamide chromatography, and then component separation and evaluation are performed by LC-MS.
A method for quickly separating and evaluating related components of a ginseng and astragalus strengthening injection comprises the following steps:
(1) weighing polyamide, and filling 95% (v/v) ethanol into a column by a wet method to obtain a polyamide chromatographic column;
(2) precisely measuring the Shenqi body resistance strengthening injection, and directly loading the Shenqi body resistance strengthening injection to the polyamide chromatographic column obtained in the step (1);
(3) sequentially eluting with 3-5 times of column volume of water, 20% ethanol and 60% ethanol as eluents;
(4) receiving effluent liquid of each eluent, evaporating to dryness in a water bath, and dissolving residues respectively to obtain a test sample;
(5) and (4) performing component separation and evaluation on the test sample obtained in the step (4) by using LC-MS.
Wherein, the chromatographic conditions are as follows:
stationary phase: bonded silica gel C18;
mobile phase: gradient elution is carried out by taking 0.1 percent (v/v) formic acid aqueous solution as a mobile phase A and 0.1 percent formic acid (v/v) acetonitrile solution as a mobile phase B; the elution procedure is shown in table 1:
TABLE 1 elution gradient
| Time (min) | Mobile phase A (%, v/v) | Mobile phase B (%, v/v) |
| 0～0.5 | 95 | 5 |
| 0.5～10 | 95→75 | 5→25 |
| 10～15 | 75→45 | 25→55 |
| 15～18 | 45→0 | 55→100 |
| 18～20 | 0 | 100 |
| 20～20.1 | 0→95 | 100→5 |
| 20.1～25 | 95 | 5 |
Column temperature: 40 ℃;
flow rate: 0.35 ml/min;
sample introduction amount: 5 μ l.
The mass spectrum conditions are as follows:
an ion source: ESI source, negative ion mode detection;
atomizing gas pressure: 35 psi;
temperature of the drying gas: 350 ℃;
flow rate of drying gas: 10L/min;
vcap capillary voltage: 3500V;
Fragmentor：135V。
the evaluation method comprises the following steps: extracted ion images (EICs) were extracted from total ion current chromatograms (TICs) with exact molecular weights 441.1678, 441.2007, 471.2083, 491.1195, 507.1508, 633.2553, 829.4591, 871.4697, 913.4650, and 991.5119, and integrated separately.
Preferably, the molecular weight of the polyamide is 14000-17000, and the specific surface area is 5-10 m2/g。
In the step (2), the sample loading amount of the ginseng and astragalus strengthening injection is the conventional amount in the field.
Preferably, in the step (1), after the polyamide is packed into the column by the wet method, the polyamide column is activated by 1-3 times of column volume of 5% (w/v) sodium hydroxide and 2-5 times of column volume of 10% (v/v) glacial acetic acid in sequence, and then washed to be neutral by water for later use.
Preferably, in the step (3), the elution is performed sequentially by using water, 20% ethanol and 60% ethanol as eluents, which are 4 times of the column volume respectively.
The elution in the step (3) can not be exchanged, and because the ginseng and astragalus strengthening injection solvent is water and contains salt components such as sodium chloride and the like, the elution by water is favorable for removing impurities; in view of the polarity difference of flavonoid and saponin components in radix codonopsitis and radix astragali, the radix codonopsitis and radix astragali are firstly eluted by water, and the concentration of ethanol is gradually increased, so that the separation of related components is facilitated.
Preferably, in the step (4), the effluent residues of the water eluent and the 20% ethanol eluent are dissolved by water and diluted to the sampling amount of the ginseng stilbene strengthening injection in the step (2), and the effluent of the 60% ethanol eluent is dissolved by 60% ethanol and diluted to the sampling amount of the ginseng stilbene strengthening injection in the step (2).
Preferably, in the step (5), the chromatographic column is an Agilent Zorbax Eclipse Plus C18 column, 2.1X 100mm, 1.8 μm.
The inventor takes astragalus extract (containing alcohol) and codonopsis pilosula extract (containing alcohol) as references, respectively screens the accurate molecular weight from the total ion chromatogram, and extracts the Total Ion Chromatogram (TIC) of each test sample of the ginseng and astragalus strengthening injection prepared by the method of the invention to obtain the extracted ion map. Wherein 491.1195 and 507.1508 are used for extracting astragalus flavonoid components, 829.4591, 871.4697, 913.4650 and 991.5119 are used for extracting astragalus saponin components, and 441.1678, 441.2007, 471.2083 and 633.2553 are used for extracting codonopsis pilosula components. The method of the invention determines the distribution condition of index components of radix codonopsitis and radix astragali in the preparation by positioning and identifying the representative components; and the separation situation of astragalosides (represented by astragaloside IV) and flavonoid (represented by calycosin glucoside) components can be visually shown.
The method can separate the ingredients of the codonopsis pilosula and the astragalus membranaceus, and the flavonoid and saponin ingredients in the astragalus membranaceus in the finished product of the preparation and classify the flavonoid and saponin ingredients in the chromatogram. Simple operation, quick reaction and stable repeatability, and can be used as a method for quickly separating and detecting related components of the ginseng and astragalus strengthening injection.
Drawings
The invention will be further explained with reference to the drawings. In addition to fig. 13, each of the following figures is composed of two spectra, wherein the upper is a mass total ion flow chromatogram (TIC) of a test sample, and the lower is an extracted ion image (EIC) extracted with a precise molecular weight (441.1678, 441.2007, 471.2083, 491.1195, 507.1508, 633.2553, 829.4591, 871.4697, 913.4650, and 991.5119).
FIG. 1 shows the Codonopsis pilosula extract (alcoholic) profile.
FIG. 2 shows the Astragalus extract (alcohol containing) profile.
FIG. 3 shows a calycosin glucoside control profile.
FIG. 4 shows a lobetyolin control profile.
Figure 5 shows an astragaloside control map.
Fig. 6 shows a map of the ginseng and astragalus strengthening injection.
Fig. 7 shows a spectrum of the D101 macroporous resin adsorbed water eluent.
Figure 8 shows a graph of D101 macroporous resin adsorbing 20% ethanol eluate.
Figure 9 shows a profile of polyamide adsorbed water eluent.
Figure 10 shows a plot of polyamide adsorbing 20% ethanol eluate.
Fig. 11 shows a spectrum of polyamide adsorption water eluent of the ginseng and astragalus strengthening injection.
Fig. 12 shows a spectrum of adsorption of 20% ethanol eluate by polyamide of the ginseng and astragalus strengthening injection.
Figure 13 shows a graph of polyamide adsorption of 30% ethanol eluate from a ginseng and astragalus strengthening injection; the upper graph is a total ion flow chromatogram (TIC) of a mass spectrum of a test sample, the middle graph is extracted ion-extracted graphs (EICs) extracted from 441.1678, 441.2007, 471.2083 and 633.2553, and the lower graph is extracted ion-extracted graphs (EICs) extracted from 491.1195, 507.1508, 829.4591, 871.4697, 913.4650 and 991.5119.
Fig. 14 shows a spectrum of polyamide adsorption of 40% ethanol eluate of the ginseng and astragalus strengthening injection.
Fig. 15 shows a spectrum of polyamide adsorption of 60% ethanol eluate of the ginseng and astragalus strengthening injection.
Fig. 16 shows a spectrum of polyamide adsorption of 80% ethanol eluate of the ginseng and astragalus strengthening injection.
Fig. 17 shows a spectrum of polyamide adsorption of 95% ethanol eluate of the ginseng and astragalus strengthening injection.
Fig. 18 shows a spectrum of polyamide adsorption of 20% ethanol 40ml eluate of the Shenqi Fuzheng injection.
Fig. 19 shows a spectrum of polyamide adsorption 20% ethanol 60ml eluate of Shenqi Fuzheng injection.
FIG. 20 shows the absorption spectrum of 20% ethanol 80ml eluate of polyamide of SHENQIFUZHENG injection.
FIG. 21 shows the absorption spectrum of 20% ethanol 100ml eluate in polyamide injection.
Figure 22 shows a map of polyamide adsorbed water eluate of ginseng and astragalus strengthening injection lot No. 180211.
Fig. 23 shows a graph of adsorption of 20% ethanol eluate to polyamide of ginseng and astragalus strengthening injection lot No. 180211.
Fig. 24 shows a graph of polyamide 180211 polyamide adsorbed 60% ethanol eluate.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified. Wherein, some reagents and instruments are purchased as follows:
ginseng and astragalus injection for strengthening body resistance: lizhu group Limin pharmaceutical factory;
radix Codonopsis alkynoside: chinese food & drug assay research institute, lot number: 111732-201607;
calycosin glucoside: chinese food & drug assay research institute, lot number: 111920-201606;
astragaloside IV: chinese food & drug assay research institute, lot number: 110781-;
LC-Ms：Agilent 1290—Q-TOF6520；
polyamide: cangzhou BaoEn sorbent materials science and technology, Inc. Specification: polyamide resin for chromatography; molecular weight: 14000 to 17000; specific surface area: 5 to 10m2(ii)/g; pH value: 6-7;
ethanol, sodium hydroxide, glacial acetic acid, acetonitrile, formic acid: are all commercially available, chromatographic grade;
purifying water: and (4) self-making.
Example 1Total Ion Chromatogram (TIC) and extracted ion map (EIC) of radix Codonopsis extract (containing alcohol)
Heating and extracting about 10g of radix Codonopsis with 100ml, 80ml and 70ml of water for 1h, 1h and 0.5h respectively. Mixing the extractive solutions for 3 times, concentrating, precipitating with 75% and 85% ethanol for 2 times.
About 5g of polyamide was weighed, degassed with 95% ethanol and then packed into a column by a wet method (column height about 10cm, inner diameter 1 cm). After activation with 40ml of 5% sodium hydroxide and 80ml of 10% glacial acetic acid in this order, the mixture was washed with water for injection to neutrality. Precisely weighing 25ml of the above prepared radix Codonopsis extractive solution, loading onto activated polyamide chromatographic column, and sequentially eluting with 80ml of water, 80ml of 20% ethanol and 80ml of 60% ethanol. 60% ethanol eluate was received, evaporated to dryness in a water bath and the residue was dissolved in 60% ethanol and diluted to 25 ml. Component analysis by LC-MS:
chromatographic conditions are as follows:
a chromatographic column: agilent Zorbax Eclipse Plus C18 column (2.1X 100mm, 1.8 μm);
column temperature: 40 ℃;
mobile phase: taking 0.1% formic acid aqueous solution as a mobile phase A; 0.1% formic acid acetonitrile solution is used as mobile phase B, and the elution gradient is shown in Table 1;
the flow rate is 0.35 ml/min;
the sample amount is 5 mul;
mass spectrum conditions: the ion source is an ESI source, and the detection is carried out in a negative ion mode. Atomizing gas pressure: 35psi, dry air temperature: 350 ℃, flow rate of drying gas: 10l/min, Vcap capillary voltage: 3500V, fragment: 135V.
The determination method comprises the following steps: precisely sucking 5 μ l of the test solution, injecting into a liquid chromatograph-mass spectrometer, measuring, and recording the chromatogram, as shown in figure 1.
Using the total ion flow diagram (see upper diagram of fig. 1) of the codonopsis pilosula extract (containing alcohol) sample obtained in the example to the total ion diagram (see upper diagram of fig. 2) of the astragalus extract (containing alcohol) obtained in the example 2, screening specific peaks of codonopsis pilosula, and extracting the specific molecular weights of the peaks respectively as 441.1678, 441.2007, 471.2083 and 633.2553 to obtain an extracted ion diagram (EIC) of codonopsis pilosula extract (see lower diagram of fig. 1).
Example 2Mass spectrum total ion current chromatogram (TIC) and Extracted Ion Current (EIC) of radix astragali extract
Heating and extracting about 10g of radix astragali with 100ml, 80ml and 70ml of water for 1h, 1h and 0.5h respectively. Mixing the extractive solutions for 3 times, concentrating, precipitating with 75% and 85% ethanol for 2 times.
About 5g of polyamide was weighed, degassed with 95% ethanol and then packed into a column by a wet method (column height about 10cm, inner diameter 1 cm). After activation with 40ml of 5% sodium hydroxide and 80ml of 10% glacial acetic acid in this order, the mixture was washed with water for injection to neutrality. Precisely weighing 25ml of the radix astragali extractive solution, loading onto activated polyamide chromatographic column, and sequentially eluting with 80ml of water, 80ml of 20% ethanol and 80ml of 60% ethanol. 60% ethanol eluate was received, evaporated to dryness in a water bath and the residue was dissolved in 60% ethanol and diluted to 25 ml. Component analysis using LC-MS:
chromatographic conditions, mass spectrometry conditions and assay: the same as example 1;
the map is shown in FIG. 2.
The total ion flow diagram (figure 2 upper diagram) of the astragalus extract (containing alcohol) sample obtained in the embodiment is compared with the total ion diagram (figure 1 upper diagram) of the codonopsis pilosula extract (containing alcohol) obtained in the embodiment 1 to screen the specific peaks of the astragalus, extract the accurate molecular weight of the peaks, extract the flavonoid components of the astragalus by 491.1195 and 507.1508, extract the saponins components of the astragalus by the accurate molecular weight of 829.4591, 871.4697, 913.4650 and 991.5119, and obtain the extracted ion diagram (EIC) of the astragalus extract (figure 2 lower diagram).
Example 3TIC and EIC maps of representative ingredients of astragalus and codonopsis pilosula
Precisely weighing about 5mg of radix Codonopsis alkynyloside, 12.5mg of calycosin glucoside and 10mg of astragaloside IV, respectively dissolving with water, 30% methanol and methanol to obtain solutions of 1mg/ml, 5 μ g/ml and 0.01 mg/ml. Component analysis using LC-MS:
chromatographic conditions, mass spectrometry conditions and assay: the same as example 1;
the maps are shown in fig. 3, fig. 4 and fig. 5, respectively.
Example 4Selection of related evaluation components of ginseng and astragalus body resistance strengthening injection
About 5g of polyamide was weighed, degassed with 95% ethanol and then packed into a column by a wet method (column height about 10cm, inner diameter 1 cm). After activation with 40ml of 5% sodium hydroxide and 80ml of 10% glacial acetic acid in this order, the mixture was washed with water for injection to neutrality. Precisely weighing 25ml of Shenqi Fuzheng injection (batch No. 180211), loading onto activated polyamide chromatographic column, and sequentially eluting with 80ml of water, 80ml of 20% ethanol and 80ml of 60% ethanol. And (3) receiving the effluent of each eluent, evaporating to dryness in a water bath, dissolving the effluent residues of the water eluent and the 20% ethanol eluent in water and diluting to 25ml, and dissolving the effluent of the 60% ethanol eluent in 60% ethanol and diluting to 25 ml. The total ion current spectrum was obtained by performing the composition analysis by LC-MS according to the conditions and methods described in example 1 (see upper panel of FIG. 6).
Comparing the total ion current chromatogram with radix Codonopsis extract (containing alcohol) shown in figure 1, radix astragali extract (containing alcohol) shown in figure 2, calycosin glucoside shown in figure 3, radix Codonopsis alkynoside shown in figure 4, and mass spectrum total ion current chromatogram (TIC) and extracted ion map (EIC) of astragaloside shown in figure 5, and locating with radix Codonopsis alkynoside as radix Codonopsis representative component, calycosin glucoside as flavonoid representative component, and astragaloside as saponin representative component; and further, according to the comparison result, 2 flavonoid components are extracted from the total ion current chromatogram (TIC) of fig. 6 with accurate molecular weights of 491.1195 and 507.1508, 9 saponin components are extracted from the total ion current chromatogram (TIC) with accurate molecular weights of 991.5119, 829.4591, 871.4697 and 913.4650, and 4 codonopsis pilosula components are extracted from the total ion current chromatogram (TIC) with accurate molecular weights of 633.2553, 471.2083, 441.1678 and 441.2007, so as to obtain an extracted ion map (EIC) of the ginseng and astragalus strengthening injection, as shown in the lower graph of fig. 6.
Example 5Comparative investigation of different adsorbents
The adsorption conditions of polyamide and D101 macroporous resin on related components of the Shenqi injection for strengthening the body resistance are compared through experiments.
Taking a proper amount of the pretreated macroporous resin D101 (pretreatment of the macroporous resin, namely adding sufficient water to swell the macroporous resin until the volume of the macroporous resin is not increased any more, then pouring the macroporous resin into a chromatographic column, so that the resin amount in the column does not exceed 1/2 of the length of the column, removing resin particles suspended on the liquid level of an aqueous solution, washing the column with 95% ethanol until the effluent is not white and turbid after being mixed with 2 times of water, and finally washing with water to remove ethanol completely for later use), and performing wet packing on the column to obtain a D101 macroporous resin column (the height of the column is about 15cm, and the inner diameter of the column is 1cm) for later use. 25ml of Shenqi Fuzheng injection (batch No. 180211) was applied to the D101 macroporous resin column. The elution was carried out with 80ml of water and 80ml of 20% ethanol in this order. Respectively receiving effluent liquid of each eluent, evaporating to dryness in a water bath, dissolving residues in water, diluting to 25ml, and performing component analysis by using LC-MS: the chromatographic conditions, mass spectrometric conditions and assay were the same as in example 1. The maps are shown in FIG. 7 and FIG. 8, respectively.
The column was packed and activated by the wet method as described in example 1 to obtain a polyamide column for use. 25ml of the Shenqi Fuzheng injection (batch No. 180211) was applied to an activated polyamide column. The elution was carried out with 80ml of water and 80ml of 20% ethanol in this order. The effluent of each eluent is respectively received, evaporated to dryness in a water bath, and the residue is dissolved in water and diluted to 25 ml. Component analysis using LC-MS: the chromatographic conditions, mass spectrometric conditions and assay were the same as in example 1. The maps are shown in FIGS. 9 and 10.
The results show that after loading the D101 macroporous resin, when water washing and 20% ethanol elution are carried out, the monitoring component is eluted, and separation cannot be achieved. The adsorption effect of the polyamide resin on related components is obviously better than that of D101 macroporous resin, and the polyamide resin is determined to be possibly used for separating the components of the ginseng and astragalus strengthening injection.
Example 6Examination of ethanol elution concentration
The column was packed and activated by the wet method as described in example 1 to obtain a polyamide column for use. 25ml of the Shenqi Fuzheng injection (batch No. 180211) was applied to an activated polyamide column. Eluting with 2 times of column water, 20%, 30%, 40%, 60%, and 80% ethanol, respectively, collecting eluates, evaporating in water bath, dissolving the residue in water, and filtering with 0.22 μm filter membrane. Component analysis using LC-MS: the chromatographic conditions, mass spectrometric conditions and assay were the same as in example 1. The maps are shown in FIGS. 11 to 17.
The results show that: the best elution solvents for separation are water, 20% ethanol, 60% ethanol, respectively. The water eluent mainly contains the components with strong water solubility of the codonopsis pilosula; the 20% ethanol eluate mainly contains flavonoids represented by calycosin glucoside and pterocarpan glycoside in radix astragali, and the 60% ethanol eluate mainly contains saponins represented by astragaloside IV in radix astragali. When the extract is eluted by 30% ethanol, the calycosin glucoside, the pterocarpan glycoside and the astragaloside are simultaneously eluted; the 40% ethanol eluate was similar to the 30% ethanol eluate. After 60% ethanol elution, 80% and 95% ethanol eluates have no detectable components. Therefore, the optimal elution solvent is determined to be water, 20% ethanol and 60% ethanol in sequence.
Example 7Examination of the amount of eluting solvent
And (3) respectively inspecting the complete elution condition and the separation effect of the elution solvent with the column volumes of 2 times, 3 times, 4 times and 5 times by utilizing the determined optimal elution concentration. Namely: eluting with 40ml, 60ml, 80ml and 100ml 20% ethanol as eluent respectively. The component analysis of each test sample was performed by LC-MS: the chromatographic conditions, mass spectrometric conditions and assay were the same as in example 1. The maps are shown in FIGS. 18 to 21. The peak areas of the representative components of flavonoids and saponins in the codonopsis pilosula and the astragalus are selected as evaluation indexes, and the integration results are shown in table 2 by comparing the evaluation indexes with the stock solution of the ginseng and astragalus body resistance strengthening injection.
TABLE 2 peak area integration results
| Retention time | Ginseng and astragalus injection for strengthening body resistance | 40ml | 60ml | 80ml | 100ml |
| 7.968 | 641367 | 77603 | 474168 | 715269 | 766876 |
| 12.958 | 1218992 | 1201410 | 1198605 | 1217765 | 1254676 |
As a result, when the amount of the elution solvent is 80ml, the peak area of each index peak is similar to that of the stock solution of the ginseng and astragalus strengthening injection, and the amount of the elution solvent is 100ml, the peak area of each index peak is not obviously increased. Therefore, the optimum amount of the eluting solvent was determined to be 80 ml. I.e. 4 column volumes were used for optimal elution solvent.
Example 8Experimental reproducibility investigation:
the column was packed and activated by the wet method as described in example 1 to obtain a polyamide column for use. The Shenqi Fuzheng injection (batch No. 180211) was applied to an activated polyamide column. Eluting with 80ml of water, 80ml of 20% ethanol and 80ml of 60% ethanol in sequence. Respectively receiving the effluent of each eluent, evaporating to dryness in water bath, dissolving the effluent residues of the water eluent and the 20% ethanol eluent in water, and diluting to 25ml, and dissolving the effluent of the 60% ethanol eluent in 60% ethanol solvent, and diluting to 25 ml. Component analysis using LC-MS:
chromatographic conditions, mass spectrometry conditions and assay: the same as in example 1.
The total ion current profile (TIC) of each elution fraction is shown in the upper panels of FIGS. 22 to 24. From the total ion current chromatograms (TIC) in fig. 22 to 24, 2 flavonoid component extraction ion graphs (EIC) were extracted with the accurate molecular weights (491.1195, 507.1508), 9 saponin component extraction ion graphs (EIC) were extracted with the accurate molecular weights (991.5119, 829.4591, 871.4697, 913.4650) in combination, and 4 codonopsis pilosula component extraction ion graphs (EIC) were extracted with the accurate molecular weights (633.2553, 471.2083, 441.1678, 441.2007), as shown in the lower graphs in fig. 22 to 24, the results of integration were respectively shown in table 3.
Table 3 peak area results for 180211 batches of shenqi injection for strengthening body resistance:
and (4) conclusion: by the method, the ginseng and astragalus body resistance strengthening injection can well reserve related components in the codonopsis pilosula and the astragalus, so that the ginseng and astragalus body resistance strengthening injection can fully exert the medicinal effects of the codonopsis pilosula and the astragalus.
The method provided by the invention can separate the components from the codonopsis pilosula and the astragalus root in the ginseng and astragalus root strengthening injection by using the polyamide. Meanwhile, flavonoid and saponin components in the astragalus can be separated. The method is simple to operate, and can be used as a method for quickly separating related components of the ginseng and astragalus strengthening injection.
Claims (5)
1. A method for quickly separating and evaluating related components of a Shenqi body resistance strengthening injection comprises the following steps: the ginseng and astragalus body resistance strengthening injection is subjected to polyamide chromatography, and then component separation and evaluation are performed by LC-MS; the method specifically comprises the following steps:
(1) weighing polyamide, and filling the polyamide and 95% v/v ethanol into a column by a wet method to obtain a polyamide chromatographic column;
(2) precisely measuring the ginseng and astragalus body resistance strengthening injection, and directly loading the sample to the polyamide chromatographic column obtained in the step (1);
(3) sequentially eluting with 4 times of column volume of water, 20% ethanol and 60% ethanol as eluents;
(4) receiving effluent liquid of each eluent, evaporating to dryness in a water bath, and dissolving residues respectively to obtain a test sample; wherein, the effluent liquid residues of the water eluent and the 20 percent ethanol eluent are dissolved and diluted by water until the sampling amount of the radix astragali body resistance strengthening injection in the step (2) is reached, and the effluent liquid of the 60 percent ethanol eluent is dissolved and diluted by 60 percent ethanol until the sampling amount of the radix astragali body resistance strengthening injection in the step (2) is reached;
(5) separating and evaluating components of the test sample obtained in the step (4) by using LC-MS;
the chromatographic conditions are as follows:
stationary phase: bonded silica gel C18;
mobile phase: gradient elution is carried out by taking 0.1% v/v formic acid aqueous solution as a mobile phase A and 0.1% formic acid v/v acetonitrile solution as a mobile phase B; the elution procedure is as follows:
0-0.5 min, 95% v/v mobile phase A and 5% v/v mobile phase B;
0.5-10 min, 95% v/v → 75% v/v mobile phase A, 5% v/v → 25% v/v mobile phase B;
10-15 min, 75% v/v → 45% v/v mobile phase A, 25% v/v → 55% v/v mobile phase B;
15-18 min, 45% v/v → 0%% v/v mobile phase A, 55% v/v → 100% v/v mobile phase B;
18-20 min, 0% v/v mobile phase A and 100% v/v mobile phase B;
20-20.1 min, 0% v/v → 95% v/v mobile phase A, 100% v/v → 5% v/v mobile phase;
20.1-25 min, 95% v/v mobile phase and 5% v/v mobile phase;
column temperature: 40 ℃;
flow rate: 0.35 ml/min;
sample introduction amount: 5 mu l of the solution;
the evaluation method comprises the following steps:
extracted ion images (EICs) were extracted from total ion current chromatograms (TICs) with exact molecular weights 441.1678, 441.2007, 471.2083, 491.1195, 507.1508, 633.2553, 829.4591, 871.4697, 913.4650, and 991.5119, and integrated separately.
2. The method for rapidly separating and evaluating related components of Shenqi Fuzheng injection as claimed in claim 1, wherein the chromatographic column is Agilent Zorbax Eclipse Plus C18 column, 2.1X 100mm, 1.8 μm.
3. The method for rapidly separating and evaluating related components of the ginseng and astragalus strengthening injection according to claim 1, wherein the mass spectrum conditions are as follows:
an ion source: ESI source, negative ion mode detection;
atomizing gas pressure: 35 psi;
temperature of the drying gas: 350 ℃;
flow rate of drying gas: 10L/min;
vcap capillary voltage: 3500V;
Fragmentor：135V。
4. the method for rapidly separating and evaluating related components of the ginseng and astragalus strengthening injection according to claim 1, wherein the molecular weight of the polyamide is 14000-17000, and the specific surface area is 5-10 m2/g。
5. The method for rapidly separating and evaluating the related components of the ginseng and astragalus strengthening injection according to claim 1, wherein in the step (1), after the polyamide is filled into the column by a wet method, the polyamide column is activated by 5% w/v sodium hydroxide in an amount of 1-3 times the column volume and 10% v/v glacial acetic acid in an amount of 2-5 times the column volume in sequence, and then washed to be neutral by water for later use.
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Citations (4)
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|---|---|---|---|---|
| CN1546152A (en) * | 2003-12-02 | 2004-11-17 | 张正生 | Freeze-dried 'Shenqifuzheng' powder for injection and its preparing process |
| CN101084972A (en) * | 2006-06-08 | 2007-12-12 | 天津天士力之骄药业有限公司 | Method for identifying codonopsis pilosula and preparation containing codonopsis pilosula |
| CN101352478A (en) * | 2007-07-26 | 2009-01-28 | 丽珠集团利民制药厂 | Quality control method of ginseng and astragalus injection for strengthening body |
| CN103808840A (en) * | 2012-11-02 | 2014-05-21 | 丽珠集团利民制药厂 | Shenqi Fuzheng injection fingerprint atlas establishing method |
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| CN1546152A (en) * | 2003-12-02 | 2004-11-17 | 张正生 | Freeze-dried 'Shenqifuzheng' powder for injection and its preparing process |
| CN101084972A (en) * | 2006-06-08 | 2007-12-12 | 天津天士力之骄药业有限公司 | Method for identifying codonopsis pilosula and preparation containing codonopsis pilosula |
| CN101352478A (en) * | 2007-07-26 | 2009-01-28 | 丽珠集团利民制药厂 | Quality control method of ginseng and astragalus injection for strengthening body |
| CN103808840A (en) * | 2012-11-02 | 2014-05-21 | 丽珠集团利民制药厂 | Shenqi Fuzheng injection fingerprint atlas establishing method |
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