CN109061002B - Method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles - Google Patents
Method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles Download PDFInfo
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- CN109061002B CN109061002B CN201811084546.4A CN201811084546A CN109061002B CN 109061002 B CN109061002 B CN 109061002B CN 201811084546 A CN201811084546 A CN 201811084546A CN 109061002 B CN109061002 B CN 109061002B
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- LQGUBLBATBMXHT-UHFFFAOYSA-N chrysophanol Chemical compound C1=CC=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O LQGUBLBATBMXHT-UHFFFAOYSA-N 0.000 title claims abstract description 154
- FFWOKTFYGVYKIR-UHFFFAOYSA-N physcion Chemical compound C1=C(C)C=C2C(=O)C3=CC(OC)=CC(O)=C3C(=O)C2=C1O FFWOKTFYGVYKIR-UHFFFAOYSA-N 0.000 title claims abstract description 154
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- 239000010282 Emodin Substances 0.000 title claims abstract description 81
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- NTGIIKCGBNGQAR-UHFFFAOYSA-N Rheoemodin Natural products C1=C(O)C=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1O NTGIIKCGBNGQAR-UHFFFAOYSA-N 0.000 title claims abstract description 81
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles, which comprises the following steps: preparing a plurality of groups of standard working solutions, testing the working solutions by an ultra-high performance liquid chromatograph, calculating the obtained standard curves to obtain linear regression equations of baicalin, emodin, physcion and chrysophanol, preparing a test solution by using Qiqing toxin-vanquishing particles, testing the test solution on a computer, respectively recording corresponding peak areas, substituting the peak areas into the obtained respective linear regression equations, and calculating the contents of the baicalin, the emodin, the physcion and the chrysophanol. The invention has the beneficial effects that: the detection limit and the quantification limit of the method are reduced by using the UPLC-PDA to detect under specific chromatographic conditions; the method is economical, rapid, sensitive and good in reproducibility, saves time and mobile phase compared with the common liquid chromatography time, reduces the use of laboratory reagents and the discharge of waste liquid, and accords with the principles of energy conservation and emission reduction.
Description
Technical Field
The invention relates to the field of veterinary drug supervision and detection, in particular to a method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles.
Background
Ultra Performance Liquid Chromatography (UPLC) covers brand new technologies such as small particle packing, very low system volume, rapid detection means and the like by means of the theory and principle of HPLC (high Performance Liquid Chromatography), and increases the flux, sensitivity and chromatographic peak capacity of analysis. Compared with the traditional HPLC, the speed, the sensitivity and the separation degree of the UPLC are respectively 9 times, 3 times and 1.7 times of the HPLC. At present, the UPLC is mostly applied to metabonomics analysis and other biochemical fields, and the application of UPLC in the analysis of natural products is gradually rising, because the deep research in these fields needs higher analysis precision.
The Qiqing Baidu granules are a common prescription, mainly comprise scutellaria baicalensis, polygonum cuspidatum, Chinese pulsatilla root, radix sophorae flavescentis, isatis root, rhizoma dryopteris crassirhizomae, folium isatidis, honeysuckle, fructus forsythiae, hypericin, polyinosinic polycytidylic acid, a stomach invigorating and appetite increasing agent and the like, and have the effects of clearing away heat and toxic materials, strengthening body resistance to eliminate pathogenic factors, promoting diuresis to remove jaundice, resisting bacteria and viruses and the like.
The sixth China animal pharmacopoeia Committee in 5 months in 2017 is established in Jing, and the compilation work of 'China animal pharmacopoeia' in 2020 edition is formally started. The conference indicates that the standard work of veterinary drugs needs to be practically done, the scientificity, the practicability, the advancement and the uniformity of the 2020 animal pharmacopoeia are further enhanced, the formulation of the 2020 animal pharmacopoeia is pushed to a new height, relevant problems existing in the 2015 animal pharmacopoeia are corrected, and some main component indexes in the veterinary drugs are further quantified. In the Chinese veterinary pharmacopoeia of 2015, although there are three thin-layer identification items, the Qiqing Baidu granules lack content determination items.
Patent document CN201210257529, entitled "quality control method of Qiqing Baidu granules", provides a method for simultaneously determining the contents of baicalin and emodin by using HPLC chromatography, but the detection method can only simultaneously determine the contents of baicalin and emodin, and due to the limitations of HPLC instruments, the detection period is long, the efficiency is low, the amount of used reagents is large, and the cost is high.
The laboratory arranges partial approval products and spot check products, and adopts ultra-high performance liquid chromatography-diode array detection method to perform content determination on the main components of baicalin, emodin, physcion and chrysophanol in the prescription of Qiqing toxin-vanquishing granules. The method improves the separation effect by optimizing chromatographic conditions such as mobile phase gradient elution and the like, can be used as a qualitative and quantitative confirmation method, and provides a test basis for the next step of establishing pharmacopoeia standards.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles, improves the separation effect by optimizing chromatographic conditions such as mobile phase gradient elution and the like, and can be used as a qualitative and quantitative confirmation method.
In order to realize the purpose, the invention provides a method for simultaneously measuring baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles, which comprises the following steps:
(1) drawing a standard curve: preparing a plurality of groups of standard working solutions, wherein solutes of the standard working solutions are baicalin, emodin, physcion and chrysophanol, and the solute concentrations of each group of the standard working solutions are different; then testing each group of standard working solution by using an ultra-high performance liquid chromatograph, and recording the peak areas corresponding to the baicalin, the emodin, the physcion and the chrysophanol in each group of standard working solution; respectively drawing linear regression equation curves of the baicalin, the emodin, the physcion and the chrysophanol by taking the peak areas of the baicalin, the emodin, the physcion and the chrysophanol as vertical coordinates and taking the concentrations of the baicalin, the emodin, the physcion and the chrysophanol as horizontal coordinates, and respectively calculating to obtain linear regression equations of the baicalin, the emodin, the physcion and the chrysophanol;
(2) determination of baicalin, emodin, physcion and chrysophanol in the Qiqing toxin-vanquishing granules: grinding the Qiqing detoxifying particles, adding methanol to prepare a test solution, testing by using an ultra-high performance liquid chromatograph, respectively recording peak areas corresponding to baicalin, emodin, physcion and chrysophanol, substituting the peak areas into respective linear regression equations obtained in the step (1), and calculating to obtain the contents of the baicalin, the emodin, the physcion and the chrysophanol.
When the ultra-high performance liquid chromatograph is used for testing, the mobile phase A is methanol, and the mobile phase B is a phosphoric acid solution with the mass concentration of 0.4%;
the elution mode when the ultra-high performance liquid chromatograph is used for testing is gradient elution, and the gradient elution conditions are as follows:
0min, wherein the initial volume ratio of the mobile phase A to the mobile phase B is 20: 80;
changing the volume ratio of the mobile phases A and B from 20:80 to 55:45 in 0-1 min;
1-4.5min, the volume ratio of the mobile phase A to the mobile phase B is kept at 55: 45;
4.5-5.5min, the volume ratio of the mobile phase A to the mobile phase B is changed from 55:45 to 80: 20;
5.5-9.8min, keeping the volume ratio of the mobile phase A to the mobile phase B at 80: 20;
9.8-10min, changing the volume ratio of the mobile phase A to the mobile phase B from 80:20 to 85: 15;
keeping the volume ratio of the mobile phase A to the mobile phase B at 85:15 for 10-12.5 min;
12.5-13min, changing the volume ratio of the mobile phase A to the mobile phase B from 85:15 to 20: 80;
the volume ratio of the mobile phase A to the mobile phase B is kept at 20:80 for 13-14 min.
The detector of the ultra-high performance liquid chromatograph is a diode array detector, and the detection wavelength is 278 nm;
the chromatographic column is an ACQUITY UPLCTMHSS T3 chromatographic column with the specification of 2.1mm multiplied by 100mm and the diameter of the filler of 1.8 mu m;
preferably, the column temperature of the high performance liquid chromatography column is 30-40 ℃, the sample injection amount is 5 mu l, and the flow rate is 0.30 mL/min.
Further preferably, the column temperature of the high performance liquid chromatography column is 35 ℃. The invention selects the column temperature of 30 ℃, 35 ℃ and 40 ℃, and the comparison shows that the effect at 35 ℃ is the best, firstly, the separation degree of three target compounds is better compared with 40 ℃, and the system pressure is relatively lower compared with 30 ℃, so the column temperature is determined to be the best temperature when being 35 ℃.
The preparation method of the test solution in the step (2) comprises the following steps:
grinding the Qiqing Baidu granules, precisely weighing 0.5g of fine powder, placing the fine powder in a 25ml volumetric flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 20 minutes, cooling, adding methanol to a constant volume, shaking up uniformly, standing or centrifuging, and filtering supernate with a 0.22 mu m filter membrane to obtain the test solution.
The preparation method of the standard working solution in the step (1) comprises the following steps:
(a) preparation of standard stock solutions: weighing baicalin control 10.64 mg, adding methanol to 50mL, and making into 200 μ g/mL solution as standard stock solution; adding methanol into emodin 11.33mg to 100 mL to prepare a solution of 100 mug/mL as a standard stock solution; adding 10.48mg of chrysophanol into 100 mL of methanol to prepare a solution of 100 mu g/mL as a standard stock solution; adding 10.16mg of physcion into 200 mL of methanol to prepare a solution of 50 mu g/mL as a standard stock solution;
(b) preparation of standard working solution: mixing appropriate amount of standard stock solutions of baicalin, emodin, chrysophanol and physcion prepared in step (a), diluting with mobile phase of initial proportion, and preparing into standard working solution containing baicalin 50ug/ml and emodin, chrysophanol and physcion 12.5 ug/ml;
diluting into standard working solution containing baicalin 20ug/ml and emodin, chrysophanol and physcion 5 ug/ml;
diluting into standard working solution containing baicalin 10ug/ml and emodin, chrysophanol and physcion 2.5 ug/ml;
diluting into standard working solution containing baicalin 5ug/ml, emodin, chrysophanol, and physcion all 1.25 ug/ml;
diluting into standard working solution containing baicalin 2.5ug/ml and emodin, chrysophanol and physcion 0.625 ug/ml.
The invention has the beneficial effects that: according to the invention, the UPLC-PDA is used for optimizing chromatographic conditions such as column temperature, mobile phase gradient elution and the like, so that the separation effect of baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles is improved, and the detection limit and the quantitative limit of the method are reduced; the method is economical, rapid, sensitive and good in reproducibility, and is suitable for qualitative and quantitative detection of baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles; the detection method of the invention needs 14 minutes for operating one needle, saves about 40 minutes compared with the common liquid chromatogram time, uses about 4.2ml of mobile phase for operating one needle, saves about 50ml of mobile phase compared with the common high performance liquid chromatograph, not only reduces reagent use, energy consumption and power consumption in a laboratory, but also reduces waste liquid discharge, and accords with the principle of green energy conservation and emission reduction.
Drawings
FIG. 1 is a chromatogram of comparison samples of 50. mu.g/mL baicalin, 12.5. mu.g/mL emodin, physcion and chrysophanol according to the embodiment of the present invention.
Fig. 2 is a chromatogram of a batch sample 2173 of the qiqing baidu particle according to an embodiment of the present invention.
Fig. 3 is a chromatogram of a batch sample 3040 of a Qiqing Baidu particle according to an embodiment of the present invention.
FIG. 4 is a chromatogram of a sample 0369 for spot-checking Qiqing toxin-vanquishing particles in an embodiment of the invention.
FIG. 5 is a chromatogram of a sample 0536 for the selective examination of QIQINGBADU granule in accordance with an embodiment of the present invention.
FIG. 6 is a chromatogram of sample 0237 from a sample of QIQINGBAIDU granule.
FIG. 7 is a spectrum of baicalin.
FIG. 8 is a spectrum of emodin.
FIG. 9 is a spectrum of physcion.
Fig. 10 is a spectrum of chrysophanol.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1 detection procedure
The embodiment of the invention provides a detection step for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles by using UPLC-PDA, which comprises the following steps:
(1) drawing a standard curve: preparing a plurality of groups of standard working solutions, wherein solutes of the standard working solutions are baicalin, emodin, physcion and chrysophanol, and the solute concentrations of each group of the standard working solutions are different; then testing each group of standard working solution by using an ultra-high performance liquid chromatograph, and recording the peak areas corresponding to the baicalin, the emodin, the physcion and the chrysophanol in each group of standard working solution; respectively drawing linear regression equation curves of the baicalin, the emodin, the physcion and the chrysophanol by taking the peak areas of the baicalin, the emodin, the physcion and the chrysophanol as vertical coordinates and taking the concentrations of the baicalin, the emodin, the physcion and the chrysophanol as horizontal coordinates, and respectively calculating to obtain linear regression equations of the baicalin, the emodin, the physcion and the chrysophanol;
(2) determination of baicalin, emodin, physcion and chrysophanol in the Qiqing toxin-vanquishing granules: grinding the Qiqing detoxifying particles, adding methanol to prepare a test solution, testing by using an ultra-high performance liquid chromatograph, respectively recording peak areas corresponding to baicalin, emodin, physcion and chrysophanol, substituting the peak areas into respective linear regression equations obtained in the step (1), and calculating to obtain the contents of the baicalin, the emodin, the physcion and the chrysophanol.
Wherein,
the preparation method of the standard working solution in the step (1) comprises the following steps:
(a) preparation of standard stock solutions: weighing baicalin control 10.64 mg, adding methanol to 50mL, and making into 200 μ g/mL solution as standard stock solution; adding methanol into emodin 11.33mg to 100 mL to prepare a solution of 100 mug/mL as a standard stock solution; adding 10.48mg of chrysophanol into 100 mL of methanol to prepare a solution of 100 mu g/mL as a standard stock solution; adding 10.16mg of physcion into 200 mL of methanol to prepare a solution of 50 mu g/mL as a standard stock solution;
(b) preparation of standard working solution: mixing appropriate amount of standard stock solutions of baicalin, emodin, chrysophanol and physcion prepared in step (a), diluting with mobile phase of initial proportion, and preparing into standard working solution containing baicalin 50ug/ml and emodin, chrysophanol and physcion 12.5 ug/ml;
diluting into standard working solution containing baicalin 20ug/ml and emodin, chrysophanol and physcion 5 ug/ml;
diluting into standard working solution containing baicalin 10ug/ml and emodin, chrysophanol and physcion 2.5 ug/ml;
diluting into standard working solution containing baicalin 5ug/ml, emodin, chrysophanol, and physcion all 1.25 ug/ml;
diluting into standard working solution containing baicalin 2.5ug/ml and emodin, chrysophanol and physcion 0.625 ug/ml.
The preparation method of the test solution in the step (2) comprises the following steps:
grinding the Qiqing Baidu granules, precisely weighing 0.5g of fine powder, placing the fine powder in a 25ml volumetric flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 20 minutes, cooling, adding methanol to a constant volume, shaking up uniformly, standing or centrifuging, and filtering supernate with a 0.22 mu m filter membrane to obtain the test solution.
Example 2 elution method
The embodiment of the invention provides a method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing detoxifying particles by using UPLC-PDA, wherein the elution method is gradient elution, a mobile phase A is methanol, and a mobile phase B is a phosphoric acid solution with the mass concentration of 0.4%; gradient elution conditions were as follows:
0min, wherein the initial volume ratio of the mobile phase A to the mobile phase B is 20: 80;
changing the volume ratio of the mobile phases A and B from 20:80 to 55:45 in 0-1 min;
1-4.5min, the volume ratio of the mobile phase A to the mobile phase B is kept at 55: 45;
4.5-5.5min, the volume ratio of the mobile phase A to the mobile phase B is changed from 55:45 to 80: 20;
5.5-9.8min, keeping the volume ratio of the mobile phase A to the mobile phase B at 80: 20;
9.8-10min, changing the volume ratio of the mobile phase A to the mobile phase B from 80:20 to 85: 15;
keeping the volume ratio of the mobile phase A to the mobile phase B at 85:15 for 10-12.5 min;
12.5-13min, changing the volume ratio of the mobile phase A to the mobile phase B from 85:15 to 20: 80;
the volume ratio of the mobile phase A to the mobile phase B is kept at 20:80 for 13-14 min.
The elution method of example 2 can be preferably used in the ultra high performance liquid chromatography test of example 1.
Example 3 chromatographic conditions
The embodiment of the invention provides a chromatographic condition for a method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing detoxifying particles by using UPLC-PDA, wherein a detector of an ultra-high performance liquid chromatograph is a diode array detector, and the detection wavelength is 278 nm;
the chromatographic column is ACQUITY UPLCTMHSS T3 chromatographic column with specification of 2.1mm × 100mm and filler diameter of 1.8 μm;
the column temperature of the HPLC column was 35 deg.C, the amount of sample was 5. mu.l, and the flow rate was 0.30 mL/min.
Example 4 detection method
The embodiment of the invention provides a method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing detoxifying particles, which adopts the detection steps provided in the embodiment 1, the elution method provided in the embodiment 2 and the chromatographic conditions provided in the embodiment 3.
Experimental example 1 method Linear examination and addition recovery test results
1 Instrument and reagent
1.1 instrumental analytical balance: 0.00001g of sensory quality, 0.0001g of sensory quality; an instrument Waters AcquityTM Ultra performance LC Ultra performance liquid chromatograph;
1.2 medicine and reagent baicalin, batch No. 110715-containing 201016 with the content of 94.0 percent, emodin, physcion, chrysophanol, and China food and drug testing research institute. The methanol and the phosphoric acid are both chromatographically pure; ultrapure water.
2 method
2.1 chromatographic conditions the conditions provided in example 3 were used.
2.2 elution method the method provided in example 2 was used.
2.3 Standard working solutions and test solutions were prepared using the method provided in example 1.
3 method Linear investigation and addition recovery test results
3.1 specificity test
Preparing standard working solution containing 50 mug/mL of baicalin, 12.5 mug/mL of emodin, physcion and chrysophanol according to the preparation method provided by the embodiment 1, and performing on-machine detection to obtain a chromatogram shown in a figure 1, wherein the retention time of the baicalin, the emodin, the chrysophanol and the physcion is respectively 2.9min, 8.2min, 8.9 min and 10.4min, and four peaks are well separated.
3.2 Standard Curve
Under the chromatographic conditions provided in example 3, by using the gradient elution method provided in example 2, ions can be effectively separated, the standard curve equation is Y = 61900X +13200 in the range of 2.5-50 μ g/mL, Y is the baicalin peak area, X is the concentration of baicalin, and the correlation coefficient R2 is 0.9999, so that the linearity is good. The standard curve equations of emodin, physcion and chrysophanol in the range of 0.625-12.5 mug/mL are respectively y =69000x-1290, y =28000x-13000 and y =29300x-5580, the correlation coefficient R2 is 0.9999, and the linearity is good.
3.3 repeatability test
Preparing standard working solution containing baicalin 20ug/ml, emodin, chrysophanol, and physcion 5ug/ml, repeating for 6 times, sampling 10 μ L each, and calculating peak areas RSD of baicalin, emodin, chrysophanol, and physcion to be 0.75%, 0.56%, 0.49%, and 0.52%, respectively.
3.4 precision test
Preparing standard working solution containing baicalin 20ug/ml, emodin, chrysophanol, and physcion 5ug/ml, injecting 10 μ L, repeating for 6 times, and calculating peak areas RSD of baicalin, emodin, chrysophanol, and physcion to be 0.35%, 0.26%, 0.17%, and 0.23%, respectively.
3.5 stability test
Preparing standard working solution containing baicalin 20ug/ml, emodin, chrysophanol and physcion 5ug/ml, standing at room temperature, 4 deg.C and-20 deg.C for 3 hr and 6 hr respectively, re-dissolving, sampling 10 μ L each, and calculating the peak areas RSD of baicalin, emodin, chrysophanol and physcion to be 0.45%, 0.29% and 0.37%, respectively.
3.6 detection and quantitation limits of the method
Baicalin is 1ug/ml, and the signal-to-noise ratio (S/N) of emodin, chrysophanol and physcion is more than 3 and is determined as the detection limit of an instrument method; baicalin is 2.5ug/ml, and the signal-to-noise ratio (S/N) of emodin, chrysophanol and physcion is more than 10, and is determined as the limit of the instrument method;
the signal-to-noise ratio (S/N) > 3 determines the detection limit of the method, and the detection limit of the result method is 1 mu g/mL; S/N is more than 10, the quantitative limit of the method is determined, and the quantitative limit of the result method is 2 mug/mL; since the drug concentration is typically many times higher, the detection and quantification limits are sufficient to meet the requirements.
The method establishes ultra-high performance liquid chromatography for measuring baicalin, emodin, physcion and chrysophanol in the Qiqing toxin-vanquishing particles, adopts an external standard method to extract and dilute a sample for measurement, and has strong specificity, high sensitivity and low detection limit. Through methodology investigation, the method is completely suitable for qualitative and quantitative detection of main components in the Qiqing toxin-vanquishing particles.
Experimental example 2 detection of sample
Seven clear toxin-vanquishing particles which are reported by two enterprises and 3 batches of samples which are sampled and inspected are adopted, and the detection steps provided by the embodiment 1, the elution method provided by the embodiment 2 and the chromatographic conditions provided by the embodiment 3 are adopted for machine detection.
The obtained chromatogram is shown in fig. 2-6, and is identified by referring to the peak shape variation trend and maximum absorption wavelength of the spectrograms of scutelloside, emodin, physcion and chrysophanol in the reference substances of fig. 7-10. Baicalin, emodin and physcion can be quantitatively detected by a batch sample chromatogram, and the chrysophanol content is low and can be ignored. The content of physcion in the sample for spot inspection is very low, and the content of baicalin and emodin is higher. Therefore, the effective components of the sample for spot inspection are low relative to the approved products, and certain problems exist in the quality, so that the method is necessary to be formulated for detecting the samples in the market.
TABLE 1 content (mg/g) of four ingredients in Qiqing Baidu granule
| Emodin methyl ether | Baicalin | Emodin | Chrysophanol | |
| Submission 2173 | 0.291 | 1.258 | 3.179 | 0.33 |
| Newspaper 3040 | 0.544 | 3.703 | 5.969 | 0.022 |
| Spot check 237 | 0.001 | 1.216 | 0.008 | 0.003 |
| Spot check 353 | 0.003 | 0.117 | 0.024 | 0.001 |
| Spot check 369 | 0 | 0.312 | 0.01 | 0 |
According to the invention, 0.4% phosphoric acid and methanol are selected as mobile phases, an ultra-high performance liquid phase PDA detector is selected, gradient elution is carried out, the separation degrees of baicalin, emodin, physcion and chrysophanol meet the requirements, and the system adaptability is good. The time for operating one needle by using the ultra-high performance liquid chromatography is 14 minutes, the time is saved by 40 minutes compared with the common liquid chromatography, the flow rate is 0.30mL, the mobile phase is saved, the energy and power consumption of a laboratory are reduced, the discharge of waste liquid is also reduced, and the principle of green energy conservation and emission reduction is met.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
Claims (2)
1. A method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing toxin-vanquishing particles is characterized by comprising the following steps:
(1) drawing a standard curve: preparing a plurality of groups of standard working solutions, wherein solutes of the standard working solutions are baicalin, emodin, physcion and chrysophanol, and the solute concentrations of each group of the standard working solutions are different; then testing each group of standard working solution by using an ultra-high performance liquid chromatograph, and recording the peak areas corresponding to the baicalin, the emodin, the physcion and the chrysophanol in each group of standard working solution; respectively drawing linear regression equation curves of the baicalin, the emodin, the physcion and the chrysophanol by taking the peak areas of the baicalin, the emodin, the physcion and the chrysophanol as vertical coordinates and taking the concentrations of the baicalin, the emodin, the physcion and the chrysophanol as horizontal coordinates, and respectively calculating to obtain linear regression equations of the baicalin, the emodin, the physcion and the chrysophanol;
(2) determination of baicalin, emodin, physcion and chrysophanol in the Qiqing toxin-vanquishing granules: grinding the Qiqing toxin-vanquishing particles, adding methanol to prepare a test solution, testing by using an ultra-high performance liquid chromatograph, respectively recording peak areas corresponding to baicalin, emodin, physcion and chrysophanol, substituting the peak areas into respective linear regression equations obtained in the step (1), and calculating to obtain the contents of the baicalin, the emodin, the physcion and the chrysophanol;
when the ultra-high performance liquid chromatograph is used for testing, the mobile phase A is methanol, and the mobile phase B is a phosphoric acid solution with the mass concentration of 0.4%;
the elution mode when the ultra-high performance liquid chromatograph is used for testing is gradient elution, and the gradient elution conditions are as follows:
0min, wherein the initial volume ratio of the mobile phase A to the mobile phase B is 20: 80;
changing the volume ratio of the mobile phases A and B from 20:80 to 55:45 in 0-1 min;
1-4.5min, the volume ratio of the mobile phase A to the mobile phase B is kept at 55: 45;
4.5-5.5min, the volume ratio of the mobile phase A to the mobile phase B is changed from 55:45 to 80: 20;
5.5-9.8min, keeping the volume ratio of the mobile phase A to the mobile phase B at 80: 20;
9.8-10min, changing the volume ratio of the mobile phase A to the mobile phase B from 80:20 to 85: 15;
keeping the volume ratio of the mobile phase A to the mobile phase B at 85:15 for 10-12.5 min;
12.5-13min, changing the volume ratio of the mobile phase A to the mobile phase B from 85:15 to 20: 80;
13-14min, and keeping the volume ratio of the mobile phase A to the mobile phase B at 20: 80;
the detector of the ultra-high performance liquid chromatograph is a diode array detector, and the detection wavelength is 278 nm;
the chromatographic column is ACQUITY UPLCTMHSS T3 chromatographic column with specification of 2.1mm × 100mm and filler diameter of 1.8 μm;
the column temperature of the column was 35 ℃ and the amount of sample was 5. mu.l at a flow rate of 0.30 mL/min.
2. The method for simultaneously determining baicalin, emodin, physcion and chrysophanol in Qiqing detoxifying particles according to claim 1, wherein the preparation method of the test solution in the step (2) comprises the following steps:
grinding the Qiqing Baidu granules, precisely weighing 0.5g of fine powder, placing the fine powder in a 25ml volumetric flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 20 minutes, cooling, adding methanol to a constant volume, shaking up uniformly, standing or centrifuging, and filtering supernate with a 0.22 mu m filter membrane to obtain the test solution.
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