CN115754040A - Method for simultaneously determining multiple active ingredients in oriental wormwood golden flower powder - Google Patents
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Abstract
The invention belongs to the field of detection of effective components in veterinary drugs, and provides a method for simultaneously determining multiple active components in oriental wormwood golden flower powder, which comprises the following steps: carrying out ultrasonic extraction on a capillary artemisia golden flower powder sample by adopting an extracting agent, centrifuging and filtering to obtain an extracting solution; performing qualitative and quantitative detection on multiple main components simultaneously by adopting ultra-high performance liquid chromatography, and performing multichannel chromatographic detection and 3D scanning spectrogram; the chromatographic column is Waters UPLC HSS T 3 A chromatographic column; gradient elution: methanol-1 mol/L ammonium acetate solution (99: 1) is used as a mobile phase A, water-1 mol/L ammonium acetate solution (99: 1) is used as a mobile phase B, and the gradient elution program is optimized and explored. The method is simple, convenient and quick, and has good separation effectThe method has the advantages of high determination precision, good result reproducibility, easy observation, strong specificity and good chromatographic peak shape, not only meets the requirements of Chinese animal pharmacopoeia, but also saves time and reagents.
Description
Technical Field
The invention belongs to the field of detection of active ingredients in veterinary drugs, and relates to a method for simultaneously determining multiple active ingredients in virgate wormwood herb and golden flower powder.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The herba artemisiae scopariae and flos chrysanthemi powder is a traditional Chinese veterinary medicine prescription preparation and is collected in the quality standard of traditional Chinese medicine herba artemisiae scopariae and flos chrysanthemi powder in the veterinary medicine quality standard (2017 edition).
[ prescription ]: 70g of oriental wormwood, 50g of honeysuckle, 60g of scutellaria baicalensis, 40g of golden cypress, 40g of radix bupleuri, 60g of gentian, 60g of divaricate saposhnikovia root, 60g of schizonepeta, 40g of liquorice and 120g of isatis root.
[ PREPARATION METHOD ]: pulverizing the above 10 materials, sieving, and mixing.
[ PROPERTIES ] the product is a light yellow powder; fragrant and slightly bitter.
[ IDENTIFICATION ] this product was taken and observed under a microscope: t-shaped non-glandular hair, with stem and single cell arm, two arms of unequal length, wall thickness, 1-2 stem cells. The fiber is light yellow, fusiform, thick in wall and fine in pore and groove. The fiber bundle is bright yellow, the peripheral cells contain calcium oxalate cubic crystals to form crystal fibers, and the wall containing the crystal cells is lignified and thickened. The oil pipe contains light yellow or yellow brown strip secretion with the diameter of 8-25 mu m. The outer skin layer has a fusiform cell surface, and each cell is divided into a plurality of small cells by transverse walls. The oil pipe contains gold yellow secretion with the diameter of about 17-60 mu m. Non-glandular hair 1-6 cells, mostly with wall warts. The wood fiber is mostly bundled, is faint yellow and is mostly broken, has the diameter of 14 to 25 mu m, and is slightly lignified, and has obvious pits and grooves. The parenchyma cells around the fiber bundle contain calcium oxalate crystals to form crystal fibers. The pollen grains are similar to circles, the diameter is about 76 mu m, and the outer wall of the pollen grains is provided with thorn-shaped sculptures with 3 germination holes.
(2) Taking 2g of the product, adding 20mL of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, and taking the filtrate as a test solution. Adding methanol into chlorogenic acid control to obtain 1mg solution per 1mL, and making into control solution. The two solutions were pipetted at 10. Mu.L each, spotted on the same silica gel H thin layer plate, developed with the upper solution of butyl acetate-formic acid-water (7. In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the control solution.
[ FUNCTIONS ] can clear away heat and toxic materials, dispel wind, and dissipate heat.
[ INDICATIONS ] can treat affection of exogenous wind-heat and sore throat.
The identification items comprise microscopic identification and thin-layer identification. The thin-layer identification is used for detecting the honeysuckle (chlorogenic acid). Thin layer chromatography is very cumbersome to operate.
In the thin-layer chromatography detection method of the national standard method, the operation is complicated, a plurality of steps such as unfolding, airing, inspecting and checking results are needed, the influence of environmental temperature, humidity and manual operation on spots is large, such as edge effect and the like, the spots are often unclear, the result is difficult to judge, time and reagents are wasted, and the toxicity of a developing agent for chromatography is found in actual inspection work, such as: butyl acetate, formic acid, methanol and the like have large damage to experimenters.
Disclosure of Invention
In order to solve the problems, the invention provides a method for simultaneously measuring a plurality of active ingredients in virgate wormwood golden flower powder.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for simultaneously measuring multiple active ingredients in oriental wormwood and golden flower powder, which comprises the following steps:
carrying out ultrasonic extraction on a capillary artemisia golden flower powder sample by using an extracting agent, carrying out solid-liquid separation, and collecting an extracting solution;
performing on-machine determination of adenosine, (R, S) goitrin, chlorogenic acid, caffeic acid, liquiritin, glycyrrhizic acid, gentiopicroside, baicalin and berberine hydrochloride in the extractive solution by ultra-high performance liquid chromatography.
The invention has the advantages of
(1) In the invention, the capillary artemisia and golden flower powder is simultaneously treated by liquid chromatography: adenosine and (R, S) in isatis root are reported in spring, chlorogenic acid and caffeic acid in honeysuckle, liquiritin and glycyrrhizic acid in liquorice, gentiopicroside in gentian, baicalin in scutellaria baicalensis and berberine hydrochloride in phellodendron are measured once, and the method is simple, convenient and quick, good in separation effect, high in measurement precision, good in result reproducibility, easy to observe, strong in specificity and good in chromatographic peak shape, not only meets the requirements of Chinese veterinary pharmacopoeia, but also saves time and reagents.
(2) The determination method of the invention can select 30-70% ethanol as an extraction solvent to extract the test sample, has small toxicity, moderate peak area response value of the compound, simple and convenient sample treatment, reagent saving, time saving, small toxicity, safe and environment-friendly operation and small harm to human and environment.
(3) The determination method of the invention uses a high performance liquid chromatograph, presents results in the mode of chromatogram and spectrogram, is convenient and rapid, has intuitive results, is easy to judge, has low detection limit, greatly shortens the detection time, and can finish the detection in about 2 hours from the sample processing to the on-machine detection product determination result; the original standard of the thin-layer detection method only identifies the chlorogenic acid as an index compound, and one sample needs about 2-3 hours, so that the detection efficiency, the detection sensitivity and the detection effect are greatly improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention, and together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 is a spectrum of 9 mixed control samples according to example 1 of the present invention; wherein the arrangement sequence is from top to bottom: adenosine 1.99min; chlorogenic acid 2.56min; (R, S) Guerin for 2.71min; caffeic acid for 3.12min; liquiritin 3.52min; gentiopicroside 4.59min; baicalin 6.13min; berberine hydrochloride for 6.65min; glycyrrhizic acid for 8.93min.
FIG. 2 is a mixed control chromatogram of Artemisia capillaris Thunb and Datura flower powder of 240nm, with a sample size of 1 μ L in example 1;
FIG. 3 shows a chromatogram of a 1 sample of the capillary artemisia golden camellia powder of example 1 of the present invention at 240nm, with a sample size of 1 μ L;
FIG. 4 is a chromatogram of a capillary artemisia golden camellia powder sample 2 of example 1 of the present invention at 240nm, with a sample size of 1 μ L;
FIG. 5 is a mixed control chromatogram of Artemisia capillaris Thunb and Jinhua powder of 275nm, with a sample size of 1 μ L in example 1;
FIG. 6 shows a chromatogram of 1 sample of the capillary artemisia golden camellia powder of example 1 of the present invention at 275nm, with a sample size of 1 μ L;
FIG. 7 is a chromatogram of a capillary artemisia golden camellia powder sample 2 of example 1 of the present invention at 275nm, with a sample size of 1 μ L;
FIG. 8 shows a mixed control chromatogram of Artemisia capillaris Thunb and Datura flower powder of 264nm, with a sample size of 1 μ L in example 1;
FIG. 9 shows a chromatogram of 1 sample of Artemisia capillaris Thunb golden flower powder of example 1 of the present invention at 264nm, with a sample size of 1 μ L;
FIG. 10 shows a chromatogram of a capillary artemisia golden camellia powder sample 2 at 264nm, with a sample size of 1 μ L, according to example 1 of the present invention;
FIG. 11 is a mixed control chromatogram of Artemisia capillaris Thunb and golden flower powder of 252nm, with a sample size of 1 μ L in example 1 of the present invention;
FIG. 12 shows a chromatogram of 1 of a herba Artemisiae Scopariae flos Daturae Metelis powder sample of example 1 of the present invention at 252nm, with a sample size of 1 μ L;
FIG. 13 shows a chromatogram of a capillary artemisia golden camellia powder sample 2 of example 1 at 252nm, with a sample size of 1 μ L;
FIG. 14 is a mixed control chromatogram of herba Artemisiae Scopariae and flos Daturae Metelis powder of 324nm, with a sample size of 1 μ L in example 1 of the present invention;
FIG. 15 shows a chromatogram of 1 sample of the herba Artemisiae Scopariae flos Daturae Metelis powder of example 1 of the present invention at 324nm, with a sample size of 1 μ L;
FIG. 16 shows a chromatogram of 324nm of a sample of the Yinchenhua san in example 1, with a sample size of 1 μ L.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A method for simultaneously measuring multiple active ingredients in herba Artemisiae Scopariae and flos Daturae Metelis powder comprises the following steps: carrying out ultrasonic extraction on a capillary artemisia golden flower powder sample by using an extracting agent, centrifuging and filtering to obtain an extracting solution; detecting adenosine and (R, S) goitrin in radix Isatidis, chlorogenic acid and caffeic acid in flos Lonicerae, liquiritin and glycyrrhizic acid in Glycyrrhrizae radix, gentiopicroside in radix Gentianae, baicalin in Scutellariae radix, and berberine hydrochloride in cortex Phellodendri simultaneously by ultra high performance liquid chromatography;
in some embodiments, the detection wavelength of the ultra performance liquid chromatography is initially selected for multi-wavelength detection, is finally optimized to 240nm, and is subjected to 3D scanning of a spectrogram; the chromatographic column is Waters UPLC HSS T 3 A chromatographic column; methanol-1 mol/L ammonium acetate solution (99: 1) is used as a mobile phase A, and water-1 mol/L ammonium acetate solution (99: 1) is used as a mobile phase B; flow rate: 0.45mL/min; sample introduction amount: 0.5-2 μ L;
wherein, the preparation method of the 1mol/L ammonium acetate solution comprises the following steps: 38.5g of ammonium acetate is taken, water is added to 500mL, and the pH value is adjusted to 5.0 by glacial acetic acid.
In some embodiments, the gradient elution procedure is: 0-0.25min, and the volume fraction of the mobile phase A is maintained at 2%;0.25-12.25min, the volume fraction of the mobile phase A is changed from 2% to 99%;12.25-13.00min, and the volume fraction of the mobile phase A is maintained at 99%;13-13.01min, the volume fraction of the mobile phase A is changed from 99% to 2%;13.01-15.5min, and the volume fraction of the mobile phase A is maintained at 2%.
In some embodiments, the ultra performance liquid chromatography detects wavelength selection: 240nm, 252nm, 265nm, 275nm, 324nm, due to: 240nm, (R, S) is reported to have higher response in springy color spectrum; the chromatographic response of 252nm adenosine and glycyrrhizic acid is high; the 265nm berberine hydrochloride has higher response; 275nm gentiopicroside and baicalin have high chromatographic response; the chromatographic response of 324nm chlorogenic acid, caffeic acid and liquiritin is high; in order to search for the common detection wavelength, the invention checks the respective spectrogram and the situation of the wave crest and the wave trough, at 240nm, 9 compounds have response, and the chromatographic response is better than other 4 wavelengths.
In some embodiments, the extraction agents screened are ethanol or methanol solutions of varying concentrations.
In some embodiments, the substances contained in the capillary artemisia golden flower powder are numerous and complicated, so that the interference on the substances to be detected is serious, and the direct detection is difficult. Repeated tests show that ethanol and methanol solutions can completely and selectively extract 9 substances to be detected in the capillary artemisia eurotium golden flower powder sample, but the methanol has high toxicity, so that 30-70% ethanol aqueous solution is finally selected.
In some embodiments, the volume ratio of the oriental wormwood golden flower powder sample to the extracting agent is 1.
In some embodiments, the time for ultrasonic extraction is 20-30min. The ultrasonic extraction mode can effectively improve the extraction efficiency and the extraction effect. The ultrasonic power is 40KHz.
In some embodiments, the centrifugation is performed at a speed of 8000-15000rpm/min for a time of 10-20min; further preferably, the rotation speed of the centrifugation is 10000rpm/min, and the centrifugation time is 13-16min.
In some embodiments, the detector of the ultra performance liquid chromatography is a photodiode array detector, the column temperature is 38-42 ℃, the flow rate of the mobile phase is 0.44-0.46 mL/min, preferably 0.45mL/min, and the sample injection amount is 0.5-2 μ L.
In some embodiments, the column temperature is 40 ℃ and the sample size is 1. Mu.L.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1
And (3) testing the sample: the samples to be tested in this example were provided by the bio-pharmaceuticals of shandong, n.p. from the south of china, inc.
Phosphoric acid is of high grade purity, ethanol and methanol are of chromatographic purity, and the reference substance is mainly purchased from Chinese veterinary medicine inspection institute, chinese food and drug testing research institute and Chinese biological product research institute.
The apparatus used; BP211D analytical balance (sidoris, germany); waters Acquity TM Ultra high performance liquid chromatograph (Waters corporation, usa), PDA detector, chromatography column: waters UPLC HSS T 3 Column chromatography (2.1 mm. Times.100mm, 1.8. Mu.m).
A method for simultaneously determining 9 effective components in herba Artemisiae Scopariae and flos Lonicerae powder comprises determining adenosine, chlorogenic acid, (R, S) goitrin, caffeic acid, liquiritin, gentiopicroside, baicalin, berberine hydrochloride, and glycyrrhizic acid.
The determination method comprises the following steps:
a. preparation of control stock solutions and Mixed control stock solutions
Respectively dissolving adenosine, chlorogenic acid, (R, S) goitrin, caffeic acid, liquiritin, gentiopicroside, baicalin, berberine hydrochloride, and glycyrrhizic acid reference substances in ethanol or methanol solution with appropriate concentration, and fixing volume to obtain respective reference stock solutions; the control stock solutions were mixed in the appropriate volumes and were made up to 10mL volumetric flasks with ethanol to give mixed control stock solutions, as shown in table 1 below.
TABLE 1 reference configuration information
b. Preparing a sample solution to be tested: precisely measuring 0.1-0.25 g of oriental wormwood golden flower powder sample, placing the sample in a 20mL volumetric flask, dissolving the sample by using a proper solvent, fixing the volume to a scale, ultrasonically extracting for 20min, measuring 5mL of the sample from the volume, centrifuging at 10000rpm/min for 15min, filtering, and measuring the filtrate by using liquid chromatography;
c. the used instrument is an ultra-high performance liquid chromatograph, the detector is a photodiode array detector, and the chromatographic column is Waters UPLC HSS T 3 A chromatographic column, wherein the column temperature is 40 ℃, 240nm is optimally selected as the detection wavelength of 9 active ingredients, gradient elution is carried out, a methanol-1 mol/L ammonium acetate solution (99: 1) is taken as a mobile phase A, and a water-1 mol/L ammonium acetate solution (99: 1) is taken as a mobile phase B; flow rate: 0.45mL/min; sample introduction amount: 0.5-2 μ L;
wherein, the preparation method of the 1mol/L ammonium acetate solution comprises the following steps: 38.5g of ammonium acetate is taken, water is added to 500mL, and the pH value is adjusted to 5.0 by glacial acetic acid.
The PDA detector is selected to collect multiple wavelengths simultaneously, channels of 240nm, 252nm, 265nm, 275nm, 324nm and the like are selected in a multi-channel scanning mode, and the detection wavelengths are compared, so that 9 compounds have good chromatographic peak response and good separation degree at 240 nm.
The mobile phase gradient elution procedure is shown in table 2:
TABLE 2
d. Comparison of different sample volumes: injecting 0.5 muL, 1 muL and 2 muL of the mixed contrast stock solution into a high performance liquid chromatograph, and performing gradient elution and detection under the chromatographic condition of the step c to qualitatively identify retention time and a spectrogram; when the sample volume is 0.5 muL, the sample volume is too small, glycyrrhizic acid response is low, integral is neglected, and the sample volume is optimally selected to be 1 muL.
TABLE 3 chromatographic data sheet of 9 kinds of mixed reference substances of herba Artemisiae Scopariae and flos Daturae Metelis (sample size 1 μ L;240nm channel)
TABLE 4 capillary artemisia golden flower powder sample 1 chromatogram data table (sample size 1 uL; 240nm channel) 50% ethanol treatment sample
TABLE 5 capillary artemisia, golden flower powder sample 2 chromatographic data sheet (sample size 1 uL; 240nm channel) 70% ethanol treated sample
TABLE 6 chromatographic data sheet of 9 kinds of mixed control of herba Artemisiae Scopariae and flos Daturae Metelis (sample size 1 μ L;265nm channel)
TABLE 7 capillary artemisia-golden flower powder sample 2 chromatogram data table (sample size 1 uL; 265nm channel) 70% ethanol treatment sample
TABLE 8 capillary artemisia and golden flower powder mixing control chromatogram data table (sample size 1 uL; 324nm channel)
TABLE 9 capillary artemisia, golden flower powder sample 2 chromatographic data sheet (sample size 1 uL; 324nm channel) 70% ethanol treated sample
TABLE 10 capillary artemisia, golden flower and golden flower powder mixing control chromatogram data table (sample size 1 uL; 252nm channel)
TABLE 11 capillary artemisia golden flower powder sample 1 chromatogram data table (sample size 1 uL; 252nm channel) 50% ethanol treatment sample
TABLE 12 capillary artemisia, golden flower powder sample 2 chromatogram data table (sample size 1 uL; 252nm channel) 70% ethanol treatment sample
The data of different manufacturers show that the detected number of chromatographic peaks and the peak area of the chromatographic peaks are different and have a relationship with the production process of an enterprise and the base source of the used medicinal materials. The medicinal materials purchased by different enterprises have different content of compounds due to the difference of growth environment, climate, harvesting time and processing, and the chromatographic inspection is very necessary to improve the detection efficiency and the production level.
e. And (4) analyzing results: and (c) injecting the filtrate obtained in the step (b) into a high performance liquid chromatograph, performing gradient elution and detection under the chromatographic condition of the step (c), measuring the peak area of each target object in the filtrate, and performing qualitative determination by using retention time and a spectrogram. According to the chromatogram, in the chromatogram of the positive sample of the test sample, the same chromatographic peaks appear at the positions corresponding to the chromatograms of the 9 reference substances, as shown in figures 2-16, the peak shapes are good, the separation degree reaches the standard, and the target compound can be detected. And quantitative detection is carried out through the retention time and peak area parameters of the reference substance and the sample.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for simultaneously determining active ingredients in oriental wormwood and golden flower powder is characterized by comprising the following steps:
carrying out ultrasonic extraction on a capillary artemisia golden flower powder sample by using an extracting agent, carrying out solid-liquid separation, and collecting an extracting solution;
performing on-machine determination of adenosine, (R, S) goitrin, chlorogenic acid, caffeic acid, liquiritin, glycyrrhizic acid, gentiopicroside, baicalin and berberine hydrochloride in the extractive solution by ultra-high performance liquid chromatography.
2. The method for simultaneously measuring active ingredients in oriental wormwood golden flower powder according to claim 1, wherein the extracting agent is an ethanol aqueous solution with the concentration of 30% -70%.
3. The method for simultaneously measuring active ingredients in the virgate wormwood Jinhua powder as claimed in claim 1, wherein the volume ratio of the virgate wormwood Jinhua powder sample to the extraction agent is 1.
4. The method for simultaneously measuring active ingredients in oriental wormwood golden flower powder according to claim 1, wherein the ultrasonic extraction time is 20-30min, and the ultrasonic power is 40-50 KHz.
5. The method for simultaneously determining active ingredients in oriental wormwood golden flower powder according to claim 1, wherein the ultrahigh performance liquid chromatography is used for multi-wavelength multi-channel detection, the optimized detection wavelength is 240nm, and a spectrogram is scanned in 3D.
6. The method for simultaneously measuring active ingredients in oriental wormwood golden flower powder according to claim 1, wherein a chromatographic column is Waters UPLC HSS T 3 A chromatographic column;
or, the column temperature is 38-42 ℃, the flow rate of the mobile phase is 0.44-0.46 mL/min, and the sample injection amount is 0.5-2 muL;
alternatively, the column temperature was 40 ℃ and the flow rate of the mobile phase was 0.45mL/min.
7. The method for simultaneously measuring active ingredients in the virgate wormwood golden flower powder as claimed in claim 1, wherein the mobile phase A is a methanol-1 mol/L ammonium acetate solution, the volume ratio of the two is 99: 1, and the mobile phase B is a water-1 mol/L ammonium acetate solution, the volume ratio of the two is 99: 1.
8. The method for simultaneously measuring active ingredients in oriental wormwood golden flower powder according to claim 1, wherein the gradient elution procedure is as follows: 0-0.25min, and the volume fraction of the mobile phase A is maintained at 2%;0.25-12.25min, the volume fraction of the mobile phase A is changed from 2% to 99%;12.25-13.00min, and the volume fraction of the mobile phase A is maintained at 99%;13-13.01min, and changing the volume fraction of the mobile phase A from 99% to 2%;13.01-15.5min, and the volume fraction of the mobile phase A is maintained at 2%.
9. The method for simultaneously measuring active ingredients in virgate wormwood golden flower powder as claimed in claim 1, wherein the detector of the ultra-high performance liquid chromatography is a photodiode array detector, and is characterized by being qualitative through retention time and a spectrogram, and quantitative through retention time and peak area of a reference substance and a sample.
10. The method for simultaneously measuring the active ingredients in the oriental wormwood golden flower powder as claimed in claim 1, wherein solid-liquid separation adopts centrifugation and filtration, wherein the rotation speed of the centrifugation is 8000-15000rpm/min, and the centrifugation time is 10-20min;
or the rotation speed of the centrifugation is 10000rpm/min, and the centrifugation time is 13-16min.
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