CN113406225B - Multi-component analysis method for standing circle - Google Patents
Multi-component analysis method for standing circle Download PDFInfo
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Abstract
The invention relates to a multi-component analysis method for a panoramic circle, which adopts UHPLC-QQQ MS and has the liquid phase conditions as follows: a chromatographic column: a C18 column; mobile phase: a: acetonitrile; b:0.1% aqueous formic acid, gradient elution, mobile phase gradient as follows: 0.00-0.5min: 8 percent of A and 92 percent of B; 0.05-8.00min: 50 percent of A and 50 percent of B; 8.00-10.00min: 70 percent of A and 30 percent of B; 10.00-10.50min: 8 percent of A and 92 percent of B; 10.5-13.00min, A; the mass spectrum conditions are as follows: an ion source: an ESI source; desolventizing line temperature: 250 ℃; temperature of the heating block: 400 ℃; flow rate of atomizing gas: 3 L.min ‑1 (ii) a Flow rate of drying gas: 15 L.min ‑1 (ii) a Positive ion mode detection voltage: +4.5kV; the negative ion mode detection voltage is-3.5 kV. The invention has the advantages that the method for simultaneously measuring the contents of 18 components in the Bingjing circus is provided, the method is stable and reliable, and a foundation is laid for the research of the drug effect components and the pharmacological action of the Bingjing circus, the research and the development of new drugs and the like.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, in particular to component analysis of a traditional Chinese medicine compound, and specifically relates to a multi-component analysis method of a standing circle.
Background
Zhu Jing Yuan, also called Zhu Jing Wan, is composed of dodder, prepared rehmannia root and plantain seed, and was recorded in the thirty-third volume of Tai Ping Sheng Hui Fang, which is recorded in the following manner: semen plantaginis preparata rehmanniae radix (three or two each) semen Cuscutae (soaked in wine, ground into powder, five or two each), and refined with Mel to make into pill, as large as Firmiana simplex seed. Thirty pills are taken every time, the pills are taken with warm wine, and the pills are taken twice a day before being eaten in the air and at night. It is indicated for deficiency of liver and kidney, dim and dark eyes, or nebula, blurred vision, and lacrimation in the wind. Can tonify eyes after long-term administration. Semen Cuscutae in the formula has effects of nourishing yin, invigorating kidney, replenishing vital essence, and improving eyesight; radix rehmanniae Preparata has qi and blood invigorating effect; che Qian Zi can clear heat, induce diuresis and improve vision. Eyes are the external condition of liver, and they can see liver blood, while kidney essence is injected upwards to improve vision. The prescription can be taken to ensure that the liver and the kidney are filled and the nebula is eliminated, so that the external beautiful scenery can reside in the eyes, and the name is 'stay in the round scene'.
The "Taiping Shenghui Fang" describes the action of the Hui Jing Yuan for treating eye diseases, and the "Taiping Hui Min He Ji Ju Fang" describes the action more precisely: it is indicated for deficiency of liver and kidney, dim and dark eyes, or nebula, blurred vision, and lacrimation in the wind. The eye nourishing ability after long-term taking is also shown in modern researches, the oval of residence can relieve retinopathy of an atrophic Age-related macular degeneration (AMD) model mouse, improve the oxidation resistance of the retina and reduce the oxidative damage of the retina of the mouse, the function is mainly realized by starting an Nrf2 signal channel for resisting oxidative stress, and meanwhile, the oval of residence can also up-regulate the expression of p62 protein and activate autophagy, so that the self-protection ability of retinal epithelial cells is realized, and double protection is provided for the retina.
In view of the better curative effect of the Bingjing circa, the multi-component analysis of the Bingjing circa is helpful for further defining the drug effect substance basis of the Bingjing circa, providing screening indexes for the extraction and purification of prescriptions, providing basis for the establishment of quality standards, and more importantly, providing an early research basis for the development of new drugs, but no deep report on the multi-component analysis method of the Bingjing circa is found at present.
Disclosure of Invention
The invention aims to provide a multi-component analysis method for a panoramic circle, aiming at the defects in the prior art.
Another objective of the present invention is to provide a quality control method for the scene circle.
In order to achieve the first purpose, the invention adopts the technical scheme that:
a multi-component analysis method of a standing circle is used for simultaneously determining the content of 18C components of diglucoside D, isoacteoside, quercetin, baicalein, leonurin, kaempferol, hyperoside, plantain, geniposide, verbascoside, rutin, apigenin-7-O-glucoside, isoquercitrin, isorhamnetin, rhoifolin, apigenin, chlorogenic acid and isochlorogenic acid in the standing circle; using UHPLC-QQQ MS, wherein:
the liquid phase conditions were:
a chromatographic column: a C18 column; mobile phase: a: acetonitrile; b:0.1% aqueous formic acid, eluting with a gradient of the following mobile phase: 0.00-0.5min: 8 percent of A and 92 percent of B; 0.05-8.00min: 50 percent of A and 50 percent of B; 8.00-10.00min: 70 percent of A and 30 percent of B; 10.00-10.50min: 8 percent of A and 92 percent of B; 10.5-13.00min, A;
the mass spectrum conditions are as follows:
an ion source: an ESI source; scanning mode: apigenin-7-O-glucoside, apigenin, isoquercitrin, rhoifolin, and isorhamnetinAdopting a positive ion mode, and adopting a negative ion mode for other components; desolventizing line temperature: 250 ℃; temperature of the heating block: 400 ℃; flow rate of atomizing gas: 3 L.min -1 (ii) a Flow rate of drying gas: 15 L.min -1 (ii) a Positive ion mode detection voltage: +4.5kV; the negative ion mode detection voltage is-3.5 kV.
As a preferred example of the present invention, the liquid phase conditions further include: the flow rate was 0.4mL/min.
As another preferred example of the present invention, the liquid phase conditions further include: the injection volume was 2. Mu.L.
As another preferable example of the invention, the sample is a Zhengjing round square which is a medicine prepared from semen plantaginis, cooked dry rehmannia and semen cuscutae according to the weight ratio of 1 (1-2) to 1.5-2.5.
More preferably, the zhuangjing round formula is medicinal powder, extract, honeyed pill, concentrated pill, dripping pill, granule, tablet, capsule, mixture or decoction.
As another preferred example of the present invention, the sample is prepared as a sample solution for measuring the content of each component, and the preparation method of the sample solution comprises: adding methanol into the sample, ultrasonic extracting, filtering, collecting filtrate, evaporating to dryness, and fixing volume.
More preferably, the sample is medicinal powder of round-square living scenery, and the preparation method of the sample solution comprises the following steps: taking medicinal powder of ZHUANGJINGMIANFANG, adding 75% methanol, performing ultrasonic extraction at 50Hz frequency for 40min, filtering, collecting filtrate, evaporating to dry and fixing volume to obtain the final product; wherein the proportion of the medicinal powder of the Liaojing Yuan Fang to the added 75% methanol is 1g (80-120) mL.
As another preferred example of the present invention, the reference solution is: 20 mu g/mL of isorhamnetin, 25 mu g/mL of apigenin, 50 mu g/mL of each of quercetin and apigenin-7-O-glucoside, 100 mu g/mL of each of the other components, and methanol as a solvent.
In order to achieve the second object, the invention adopts the technical scheme that:
a quality control method of a scenic spot circle comprises a quantitative analysis step of 18 active ingredients in the scenic spot circle, wherein the quantitative analysis step of the 18 active ingredients in the scenic spot circle is as described in any one of the above steps.
The invention has the advantages that:
the method takes a classic famous prescription resident sight circle recorded in Taiping Shenghui Fang as a research object, adopts UHPLC-QQQ MS, selects proper liquid phase and mass spectrum conditions based on the abundant research experience of an inventor, is assisted with optimal chromatogram and mass spectrum conditions, measures the content of 20 compounds contained in the traditional Chinese medicine in a reasonable detection channel, and verifies the accuracy of the measuring method through the systematic methodology research of linear regression investigation, instrument stability investigation, precision investigation, accuracy investigation and sample-adding recovery experiment. The linear inspection results show that: selected 20 compounds were well linear, R 2 Are all larger than 0.99; determining the standing circle and a single medicine test sample thereof to obtain the content of 18 compounds in the standing circle; the compounds of the semen cuscutae, the prepared rehmannia root and the semen plantaginis can be detected respectively by the semen cuscutae test sample, and the content test is stable after multiple sample injections. Meanwhile, the research results of methodologies such as accuracy, stability, precision and the like reveal that the RSD is between 2% and 6%, and the sample adding recovery results show that the sample adding recovery rate is between 99% and 102% and the RSD is between 2% and 5%. The method is stable and reliable. The invention is helpful for further defining the drug effect substance basis of the Bingjing, provides a screening index for the extraction and purification of the prescription, provides a basis for establishing a quality standard, and more importantly provides an early research basis for the development of new drugs.
Drawings
FIG. 1: taiping Shenghui Fang and Jujing Yuan Yao.
FIG. 2: total Ion Current (TIC) profile for 21 compounds.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
Example 1
Section 1 Basic information of scene circle
1 Hu Jing Yuan literature examination and Fang Jie
1.1 standing circle document examination certificate
The zhuojing yuan, also known as zhuojing pill, is composed of three medicines of dodder, prepared rehmannia root and plantain seed, and is recorded in the thirty-third volume of taiping shenghuifang, which is recorded as: semen plantaginis preparata rehmanniae radix (three or two each) semen Cuscutae (soaked in wine, ground into powder, five or two), and refined with honey into pills, such as Firmiana simplex seed. Thirty pills are taken every time, the pills are taken with warm wine, and the pills are taken twice a day before being eaten in the air and at night. It is indicated for deficiency of liver and kidney, dim and dark eyes, or nebula, blurred vision, and lacrimation in the wind. Can tonify eyes after long-term administration.
1.2 round and square solution of standing scenery
In the formula, the dodder is pungent, sweet and neutral in nature; it enters kidney, liver and spleen meridians. Nourishing yin and tonifying kidney, replenishing vital essence and improving eyesight, the product is pungent in flavor and dry in flavor and sweet in flavor and tonifying deficiency, is a product for balancing yin and yang, has the functions of tonifying kidney yang, replenishing kidney essence to secure essence and reduce urination, and is good for nourishing liver and kidney, replenishing vital essence and nourishing blood to improve eyesight, so the product can be used for treating deficiency of liver and kidney and dim eyesight, and the expression in Shen nong Ben Cao Jing is that: dodder seed, semen cuscutae, is taken for a long time to improve eyesight. In the recipe, cooked rehmannia root, radix rehmanniae is sweet in taste and slightly warm in nature; the liver and kidney meridians entered, the functions of enriching blood and nourishing yin, replenishing essence and benefiting marrow; the plantain seed is sweet in flavor and slightly cold in nature; it enters liver, kidney, lung and small intestine meridians; it is good at clearing liver heat to improve vision, and inducing diuresis to treat stranguria without flowing essence and qi (compendium of materia Medica). The section of the materia medica detailed section: two orifices respectively cover the yin of the male and the female, one orifice leads to essence, the other orifice leads to water, the two orifices do not open, the water orifice opens to discharge damp heat, the ministerial fire is Chang Ning, the seminal orifice is normally closed, and the long-term seminal emission is sufficient. Plantain seed from famous medical records can nourish lung, strengthen yin and nourish essence. Eyes are the external condition of liver, and they can see liver blood, while kidney essence is injected upwards to improve vision. The whole formula is combined, so the medicine has the effects of tonifying liver and kidney, clearing heat and improving eyesight, and can tonify but not greasy, and purge but not disperse. The prescription can be taken to ensure that the liver and the kidney are filled and the nebula is eliminated, so that the external beautiful scenery can reside in the eyes, and the name is 'stay in the round scene'.
2 stationary circularity component determination
And finally determining 21 compounds as target quantitative substances of the to-be-detected oval landscape by consulting the literature and combining the current research situations of the semen cuscutae, the prepared rehmannia root and the semen plantaginis.
TABLE 1 reference information sheet
Section 2 Information of test sample
1 test article
The Suofitabine round medicinal powder (batch number: 20201228-1,20201228-2, 2104091, 2104092, 2104093, 2104094, 2104095, 2104096, 2104097) is brown to brown, and the specific preparation method is as follows: weighing the medicinal materials according to the prescription amount (250 g of semen cuscutae, 150g of prepared rehmannia root and 150g of semen plantaginis), crushing, and sieving with a No. 4 sieve (65 meshes) to obtain the traditional Chinese medicine. The preparation method of the Zhuangjing Yuanqiuan reference medicinal material (batch number: 20210410-1) comprises the following steps: weighing the medicinal materials according to the prescription amount (250 g of standard semen cuscutae, 150g of standard radix rehmanniae preparata and 150g of standard semen plantaginis), crushing, and sieving with a No. 4 sieve (65 meshes) to obtain the traditional Chinese medicine. Prepared rehmannia root extract (batch number: SD 20201228), dodder extract (batch number: TS 20201228) and plantain seed extract (batch number: CQ 20201228), prepared rehmannia root drug (batch number: 121196-202007), dodder drug (batch number: 121232-201403) and plantain seed drug (batch number: 121628-201001).
Third section UHPLC-QQQ MS condition determination
1 laboratory instruments, consumables and reagents
1.1 Experimental instruments
Triple Quad 4500 quadrupole mass spectrometer (SCIEX, usa); model CBX-20A high performance liquid chromatography (Waters, usa); DHX type ii ultrasonic vibration apparatus (kunshan, china); one in ten thousand electronic balances (mettler, switzerland); DHG type blast drying ovens (shanghai, china).
1.2 consumables and reagents
1000. Mu.L, 500. Mu.L and 100. Mu.L pipette (Ai Bende, germany); 10mL, 50mL, and 100mL volumetric flasks; 1mL and 100 muL disposable tips; filter paper, funnel, evaporating dish, etc.; formic acid, acetonitrile, methanol and the like are all in mass spectrum level.
2 preparation of control solution
Weighing appropriate amount of the above 21 reference substances, respectively, adding methanol to obtain standard mother liquor containing 1.0mg per 1mL (wherein isorhamnetin 0.2mg/mL, apigenin 0.25mg/mL, quercetin, and apigenin-7-O-glucoside 0.5 mg/mL).
Precisely measuring 100 μ L of the above mother solution, adding 900 μ L of methanol, and making into reference solution with appropriate concentration.
3 determination of the Mass Spectrometry method
An ion source: an ESI source; desolventizing line temperature: 250 ℃; temperature of the heating block: 400 ℃; flow rate of atomizing gas: 3 L.min -1 (ii) a Flow rate of drying gas: 15 L.min -1 (ii) a Positive ion mode detection voltage: +4.5kV; negative ion mode detection voltage: -3.5kV.
And determining the contents of 21 target chemical components by adopting a positive ion mode and a negative ion mode. Through experimental investigation, the apigenin-7-O-glucoside, the apigenin, the isoquercitrin, the rhoifolin and the isorhamnetin have better stability in a positive ion mode, and have higher response value and better peak shape. Other chemical components are more stable in the negative ion mode, and the peak shape and the response value are better.
TABLE 2 optimized Mass Spectrometry Condition parameters
4 determination of the liquid phase method
4.1 mobile phase gradient determination
According to the requirements of system adaptability experiments and the guaranteed separation degree, the mobile phase gradient is inspected (table 3), the differences of different mobile phase conditions are inspected by taking the separation degrees of geniposide, verbascoside, isoacteoside and other components as indexes, and the optimal mobile phase gradient is optimized.
TABLE 3 mobile phase gradient setup table
Note: the sample injection volume is 2 mu L; mobile phase A: acetonitrile; mobile phase B:0.1% aqueous formic acid; column temperature: at 40 ℃.
The optimal mobile phase gradient is optimized by a method of repeating 3 batches of sample injection and calculating the average value of index components, and experimental results show that the separation degree of isoacteoside and verbascoside is poor under the mobile phase method 1, and the separation degree of isoacteoside and verbascoside is better under the mobile phase method 2. Therefore, mobile phase method 2 is selected in consideration of the combination.
4.2 column selection
The column was investigated on the premise of optimal mobile phase gradient, using an acquired UHPLC BEH C18 silica gel bonded phase column (150 x 1.7 μm,
) And an ACQUITY UHPLC BEH C18 silica gel bonded phase chromatography column (150 x 3.5 μm,
) Two kinds of chromatographic columns, and the separation effect of different chromatographic columns is inspected by taking the separation degree of geniposide, verbascoside, isoactein and other components as indexes.
The results of the two columns showed that the obtained (obtained) UPLC BEH C18 silica gel bonded phase column (150 x 3.5 μm,
) The separation effect of the material due to the excessive particle size is better than that of an ACQUITY UPLC BEH C18 silica gel bonded phase chromatographic column (150 x 1.7 μm,
) Poor (fig. 2). In view of separation efficiency and resolution, an acquired UPLC BEH C18 silica gel bonded phase chromatography column (150 x 1.7 μm,
) As a chromatographic column for the next separation.
Section 4 Single factor investigation and methodology investigation of stationary scenery circle component extraction method
1 laboratory instruments, consumables and reagents
1.1 Experimental instruments
Triple Quad 4500 quadrupole liquid mass spectrometer (SCIEX, usa); CBX-20A high performance liquid chromatograph (SCIEX, USA); DHX ii ultrasonic vibration apparatus (kunshan, china); one in ten thousand electronic balances (mettler, switzerland); DHG type blast drying ovens (shanghai, china).
1.2 consumables and reagents
1000. Mu.L, 500. Mu.L and 100. Mu.L pipette (Ai Bende, germany); 10mL, 50mL, and 100mL volumetric flasks; 1mL and 100 muL disposable tips; filter paper, funnel, evaporating dish, etc., absolute ethyl alcohol, methanol; the above are analytical grades, such as acetonitrile, formic acid, methanol and the like; the above are all mass spectrum levels.
1.3 preparation of control solutions
Precisely measuring the above 21 reference substances, respectively, and adding methanol to obtain standard mother liquor containing 1.0mg per 1mL (wherein isorhamnetin 0.2mg/mL, apigenin 0.25mg/mL, quercetin, and apigenin-7-O-glucoside 0.5 mg/mL). Taking 100 μ L of the above standard mother liquor, respectively, adding 900 μ L of methanol to make into control solution with appropriate concentration.
2 method of experiment
2.1 liquid phase and Mass Spectrometry methods
2.1.1 Mass Spectrometry method
An ion source: an ESI source; desolventizing line temperature: 250 ℃; temperature of the heating block: 400 ℃; flow rate of atomizing gas: 3 L.min -1 (ii) a Flow rate of drying gas: 15 L.min -1 (ii) a Positive ion mode detection voltage: +4.5kV; negative ion mode detection voltage: -3.5kV.
2.1.2 liquid phase Process
A chromatographic column: ACQUITY UPLC TM BEH C18 silica gel bonded phase chromatography column (150 x 1.7 μm,
) (ii) a Sample introduction volume: 2 mu L of the solution; flow rate: 0.4mL/min; mobile phase: a: acetonitrile; b:0.1% aqueous formic acid; elution was performed with a gradient of mobile phase as follows: 0.00-0.5min: 8 percent of A and 92 percent of B; 0.05-8.00min: 50 percent of A and 50 percent of B; 8.00-10.00min：A:70％，B:30％；10.00-10.50min：A:8％，B:92％；10.5-13.00min，A:8％，B:92％。
2.2 Single-factor investigation experiment for optimizing preparation conditions of test sample solution
2.2.1 extraction time study
According to an effective component extraction method in a prescription similar to Suo Jing Yuan in the 'Chinese pharmacopoeia' 2020 edition, the influence of ultrasonic time on the dissolution efficiency of the effective component is fully considered, and ultrasonic time of 30min, 40min, 45min and 75min is specially set for single-factor investigation. The sample treatment method of the test sample comprises the following steps:
taking 1g of a standing circle sample, precisely weighing, and placing 4 parts of the sample in a 250mL conical flask; adding 100mL of methanol, placing into an ultrasonic oscillation extractor, and performing ultrasonic treatment at 50Hz for 30min, 40min, 45min and 75min respectively; and cooling, filtering, evaporating the filtrate to dryness, adding 5mL of methanol for dissolving, and metering to 10mL, wherein the volume is marked as sample 1, sample 2, sample 3 and sample 4 respectively for later use.
2.2.2 examination of extraction solvent
Taking 1g of a standing circle sample, precisely weighing and placing 2 parts of the standing circle sample in a 250mL conical flask; respectively adding 100mL of methanol and 75% of methanol, placing the mixture into an ultrasonic oscillation extractor, and extracting at the frequency of 50Hz according to the optimal extraction time; and cooling, filtering, evaporating the filtrate to dryness, adding 5mL of methanol to dissolve, and metering to 10mL, wherein the volume is marked as a sample 1 and a sample 2 respectively for later use.
2.3 methodological investigation
2.3.1 Linear relationship examination
Injecting the reference substance solutions with different concentrations into UHPLC-QQQ-MS/MS for analysis, and analyzing the data of each component by adopting a linear regression analysis method to obtain a linear equation.
2.3.2 preparation of control solutions
Precisely measuring the above 21 reference substances, and adding methanol (chromatogram) to obtain standard mother liquor containing 1.0mg per 1mL (wherein isorhamnetin 0.2mg/mL, apigenin 0.25mg/mL, quercetin, and apigenin-7-O-glucoside 0.5 mg/mL).
100 μ L of the above standard mother liquor is taken respectively, 900 μ L of methanol is added to prepare a reference solution with appropriate concentration.
2.3.3 preparation of test samples
Taking 1g of a standing circle sample, precisely weighing 6 parts, placing the sample in a 250mL conical flask, adding 100mL of 75% methanol, and placing the sample in an ultrasonic extractor for ultrasonic extraction for 40min at the frequency of 50 Hz; filtering with funnel, collecting filtrate, placing in evaporating dish, and evaporating in water bath; then adding 5mL of 75% methanol for redissolution, transferring the redissolved solution into a 10mL volumetric flask, and fixing the volume to the scale for later use.
4 results of the experiment
4.1 Single factor investigation experiment
4.1.1 extraction time study
The samples under different ultrasonic extraction times are measured by adopting a Triple Quad 4500 quadrupole liquid-phase mass spectrometer, and the content of hyperin, geniposide, verbascoside and digitoxin D in a standing circle is taken as a survey index, so that the experimental result shows that the content of four substances in the sample 1 is lower than that of other three samples, which indicates that the components cannot be completely dissolved out within 30min of extraction time; and the contents of four substances in other three samples have no great difference, and 40min is selected as the optimal extraction time according to the comprehensive experiment result. (Table 4)
Table 4 extraction time study results (n =2,ng/mg)
4.1.2 examination of extraction solvent
Samples under different extraction solvents are measured by adopting a Triple Quad 4500 quadrupole liquid-phase mass spectrometer, and the content of hyperin, geniposide, verbascoside and digitoxin D in the Huoshan circle are used as investigation indexes. Finally, the four substances in sample 1 extracted with 75% methanol were found to be higher in content. Thus 75% methanol was selected as the extraction solvent.
TABLE 5 examination of extraction solvent
4.2 methodological observations
4.2.1 results of Linear examination
The results show that the linear correlation coefficient R of geniposide and rhoifolin is removed 2 Greater than 0.999, linear correlation coefficient R of other substances 2 Are all larger than 0.990; linear range of the digitoxin D is 2.5-1000ng/mL, quercetin, baicalein, kaempferol, homoplantain, catechin, plantaginin, rutin, apigenin-7-O-glucoside, isorhamnetin, rhoifolin, apigenin, and isochlorogenic acid C is 2.5-2500ng/mL, isoacteoside, leonuroside, hypericin, isoquercitrin, verbascoside is 10-10000ng/mL, and geniposide and chlorogenic acid are 20-20000ng/mL. The above results show that 20 compounds have good detection results in respective channels, and the method is reliable.
TABLE 6 results of standard curves
4.2.2 precision investigation
The reference substance solution (concentration: 1000 ng/mL) of 18 substances detected in the panoramic circle is injected into the mass spectrometer for 9 times at different time points, the integral average value is counted, and the experimental result shows that the RSD value of the 18 compounds is between 2 and 6 percent, which indicates that the floating change after multiple injection is small and the precision of the instrument is reliable.
Table 7 results of instrumental precision measurement (n = 9)
4.2.3 repeatability Studies
According to the preparation method of the test sample solution, 6 parts of the test sample solution is prepared in parallel by taking the same sample, and the determination result shows that the RSD of the content of the 18 compounds is 2-6 percent, which shows that the sample has good uniformity and the preparation method of the test sample is stable and reliable.
TABLE 8 results of repeated investigation
4.2.4 stability Studies
The same sample solution in the repeatability test is taken and subjected to sample injection analysis for 0,2,4,8, 12 and 24 hours respectively according to the chromatographic conditions, the RSD of the content of each component is calculated to be 1.3% -5.0%, and the result shows that the sample solution is stable within 24 hours at room temperature.
Table 9 stability test results
4.2.5 sample application recovery experiment
According to the content of 18 substances in the Happy ending, precisely weighing the standard substance with the same quality, adding the standard substance into a test solution, performing operation by adopting the same preparation method of the test solution to obtain a sample recovery test sample, detecting by adopting the optimal chromatographic condition, calculating that the average sample recovery rate of 18 components is between 99 and 102 percent, and the RSD is between 2 and 5 percent, and indicating that the recovery rate of the method is good.
TABLE 10 sample recovery test results
Section 5 Content determination of main chemical components in standing circle
The 9 batches of the zao jing yuan and the zao jing yuan prepared by the reference medicinal materials are measured according to the established measuring method. The experimental result shows that only 18 substances can be detected from 21 selected compounds, wherein the contents of the homoplantaside, the catechin and the aucubin are extremely low and are not detected. Among the 18 compounds detected, hyperin, verbascoside and geniposide were higher, while rutin was the least, only 0.15ng/mg. The above test results fully indicate that the three medicinal materials in the Zhengjing yuan contain less rutin, hyperin is the main effective component of the dodder, and the dodder is the monarch drug of the Zhengjing yuan, and the high content of the dodder also indicates the rationality of the compatibility of the flavor of the Zhengjing yuan drugs to a certain extent.
Meanwhile, the content determination results of the active ingredients of the dodder, the prepared rehmannia root and the plantain seed are analyzed, and the hyperin in the dodder is 0.124 percent, the digitoxin D in the prepared rehmannia root is 0.061 percent, the geniposide in the plantain seed is 0.712 percent, and the verbascoside is 0.538 percent, and the content of the three is higher than the limit specified in the 2020 version of Chinese pharmacopoeia.
TABLE 11 survey results of the circle of residence
TABLE 12 determination of the contents of the three effective components
The content determination limit is determined according to the medicinal material items of semen plantaginis, semen Cuscutae and radix rehmanniae Preparata in 2020 edition of Chinese pharmacopoeia, and the obtained data are analyzed. As a result, the geniposide content in the plantago seed is 7116.90 mug/g, the verbascoside content is 5378.03 mug/g, which respectively account for 0.71169% and 0.537803%, and are far higher than the limits of 0.5% and 0.4% specified in pharmacopoeia; the content of hyperin in semen Cuscutae is 1240.30 μ g/g, and the ratio is 0.12403%, which meets the regulation of not less than 0.1%; meanwhile, the content of the digitonin D in the prepared rehmannia root reaches 609.53 mu g/g, and accounts for 0.060953 percent of the mass of the medicinal material, and the content is far higher than the limit of 0.02 percent.
Section 6 Discussion and summary
The project adopts UHPLC-QQQ MSMS to carry out accurate quantitative analysis on the special components in the standing circle and the single medicine thereof, and combines the methodology investigation of the system to finally determine the contents of 18 components in the standing circle. In view of the above experimental results and in combination with the compatibility principle of the scene circle, the following discussion and summary are now made:
1. the three medicinal materials of the dodder, the prepared rehmannia root and the plantain seed all contain various compounds, wherein the content of the components is determined by the pharmacopoeia, and the hyperin in the dodder, the verbascoside in the prepared rehmannia root and the geniposide in the plantain seed are all higher than the limit specified by the pharmacopoeia, which shows that the medicinal materials used in the research have better quality.
2. The methodology investigation result shows that the preparation method of the standing circle sample is scientific, the test method is reliable, and the measurement result is credible. The linear investigation result reveals a linear correlation coefficient R of removing geniposide and rhoifolin 2 Greater than 0.999, linear correlation coefficient R of other substances 2 Are all larger than 0.990; linear range of the digitoxin D is 2.5-1000ng/mL, quercetin, baicalein, kaempferol, homoplantain, catechin, plantaginin, rutin, apigenin-7-O-glucoside, isorhamnetin, rhoifolin, apigenin, and isochlorogenic acid C is 2.5-2500ng/mL, isoacteoside, leonuroside, hypericin, isoquercitrin, verbascoside is 10-10000ng/mL, and geniposide and chlorogenic acid are 20-20000ng/mL. Peach leaves with different concentrationsThe corallin reference substance does not show good linearity after being detected for many times, so the corallin reference substance is not listed, and is not taken as an effective component of the scene-staying pill to be subjected to the next experiment.
3. After 75% methanol extraction is carried out on the Yuanjing round sample, the obtained sample is analyzed by UHPLC-QQQ MS, and the content of 18 compounds is obtained.
4. The content results of the 18 compounds of the Huizhiyuan are combined, the main components of the dodder, the prepared rehmannia root and the plantain seed also have obvious advantages in content, and meanwhile, the content of the components of the dodder is higher than that of the effective components of the other two medicines, so that the content is directly related to the compatibility proportion of the three medicinal materials in the prescription. Therefore, the difference of the contents of the components is related to the compatibility of the herbs in the Zhengjing Yuan.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (4)
1. A multi-component analysis method for a resident landscape circle is characterized in that the multi-component analysis method for the resident landscape circle is used for simultaneously measuring the content of 18 components including diglucoside D, isoacteoside, quercetin, baicalein, leonurin, kaempferol, hyperoside, plantaginin, geniposide, verbascoside, rutin, apin-7-O-glucoside, isoquercitrin, isorhamnetin, rhoifolin, apigenin, chlorogenic acid and isochlorogenic acid in the resident landscape circle; using UHPLC-QQQ MS, wherein:
the liquid phase conditions were:
a chromatographic column: c18 A column; mobile phase: a: acetonitrile; b:0.1% aqueous formic acid, eluting with a gradient of the following mobile phase: 0.00-0.5min: 8 percent of A and 92 percent of B; 0.05-8.00min: 50 percent of A and 50 percent of B; 8.00-10.00min: 70 percent of A and 30 percent of B; 10.00-10.50min: 8 percent of A and 92 percent of B; 10.5-13.00min, A;
c18 The column was an ACQUITY UPLC BEH C18 silica gel bonded phase chromatography column, 150 x 1.7 μm, 130A,
the mass spectrum conditions are as follows:
an ion source: an ESI source; scanning mode: apigenin-7-O-glucoside, apigenin, isoquercitrin, rhoifolin and isorhamnetin in positive ion mode, and other components in negative ion mode; desolventizing line temperature: 250. DEG C; temperature of the heating block: 400. DEG C; flow rate of atomizing gas: 3L. Min -1 (ii) a Flow rate of drying gas: 15 L.min -1 (ii) a Positive ion mode detection voltage: +4.5kV; the negative ion mode detection voltage is-3.5 kV, and the liquid phase condition further comprises: a flow rate of 0.4mL/min, the liquid phase conditions further comprising: the sample injection volume is 2 mu L, the sample is a round prescription for standing landscape, the round prescription for standing landscape is a medicine prepared by semen plantaginis, cooked dry rehmannia root and semen cuscutae according to the weight ratio of 1 (1-2) to 1.5-2.5,
the test sample is medicinal powder of a round and square potted landscape, and the preparation method of the test sample solution comprises the following steps: taking medicinal powder of Liangjing round square, adding 75% methanol, performing ultrasonic extraction at 50Hz frequency for 40min, filtering, collecting filtrate, evaporating to dry and fixing volume to obtain the final product; wherein, the proportion of the medicinal powder of the Huajing Yuan Fang and the added 75 percent methanol is 1g (80-120) mL.
2. The multi-component analysis method of the zhuanjing pill as claimed in claim 1, wherein the zhuanjing pill is selected from the group consisting of powder, extract, honeyed pill, concentrated pill, dripping pill, granule, tablet, capsule, mixture and decoction.
3. The multi-component analysis method of the panoramic circle of claim 1, wherein the reference solution is: 20 mu g/mL of isorhamnetin, 25 mu g/mL of apigenin, 50 mu g/mL of each of quercetin and apigenin-7-O-glucoside, 100 mu g/mL of each of the other components, and methanol as a solvent.
4. A quality control method of a scene circle, characterized in that the quality control method of the scene circle comprises a quantitative analysis step of 18 active ingredients in the scene circle, and the quantitative analysis step of 18 active ingredients in the scene circle is as claimed in any one of claims 1 to 3.
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