CN105699500A - A method of measuring contents of seven components in a Huoxiangzhengqi dripping pill through ultra-high performance liquid chromatography - Google Patents
A method of measuring contents of seven components in a Huoxiangzhengqi dripping pill through ultra-high performance liquid chromatography Download PDFInfo
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Abstract
The invention relates to a method of measuring contents of seven components in a Huoxiangzhengqi dripping pill through ultra-high performance liquid chromatography. The method can achieve one time of chromatographic analysis in 20 min. Chromatographic peaks of the seven components are good in degree of separation. The RSD of precision and the RSD of repeatability of the method are less than 2.0%. The method is stable in 24 h at room temperature. For each of the components, a linearity range is wide and a linearity relation is good (with r being not more less than 0.9995). The average sample recovery rate is 97.5-101.4%. RSDs are less than 2.0%. The method is simple and convenient in operation and accurate and reliable in measuring results, and can be used for quality control of the Huoxiangzhengqi dripping pill.
Description
Technical field:
The present invention relates to the detection method of a kind of ingredient, the method of 7 kinds of component contents in ageratum drop pill is measured particularly to a kind of ultra-performance liquid chromatography, described method includes preparing ageratum drop pill need testing solution, it is injected into Ultra Performance Liquid Chromatography instrument, adopt gradient elution, obtain the content of Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol and 7 kind compositions of atisine chloride atractydin。
Background technology
Ageratum drop pill is the exclusive kind that Tianjin Tasly Pharmaceutical Co., Ltd produces, said preparation include patchouli oil, Rhizoma Atractylodis, Pericarpium Citri Reticulatae, Cortex Magnoliae Officinalis etc. 10 taste medical material composition compound preparation, there is removing dampness of inducing sweat, regulate the flow of vital energy and in effect, the dusk weight that is used for having a headache, abdominal distention, vomiting are had loose bowels, common cold of gastrointestinal type etc.。Modern Chinese medicine as the research and development of sky scholar's power represents dosage form, has the characteristics such as medicine stability height, not easily hydrolysis oxidation, rapid-action, free from extraneous odour, mouthfeel are good, easy to carry。
The quality standard of existing ageratum drop pill is to adopt hplc simultaneous determination Hesperidin, magnolol and honokiol, as detection and the content's index controlling Pericarpium Citri Reticulatae and Cortex Magnoliae Officinalis, existing patent applied for (number of patent application is CN201110269071.8)。In order to the stricter quality controlling said preparation and improve quality inspection standard, present invention application ultra-performance liquid chromatography (UPLC) measures Hesperidin in ageratum drop pill, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol and 7 kinds of component contents of atisine chloride atractydin simultaneously。This method can complete a chromatography in 20min, and 7 kinds of determined composition chromatographic peaks all have good separating degree, and method precision, repeated RSD are respectively less than 2.0%；Stable in 24h at ambient temperature；Each composition all has the wider range of linearity and good linear relationship (r >=0.9995)；Mean sample recovery rate 97.5-101.4%, RSD are respectively less than 2.0%。The method of the present invention is easy and simple to handle, and measurement result accurately and reliably, can be used for the quality control of ageratum drop pill。
Summary of the invention
The present invention relates to a kind of ultra-performance liquid chromatography and measure the method for 7 kinds of component contents in ageratum drop pill, the method comprises the following steps:
Step 1: the preparation of need testing solution: ageratum drop pill is dissolved in methanol, filters, obtains filtrate
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Described Ultra Performance Liquid Chromatography instrument, chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.1-0.3% aqueous formic acid,
Mobile phase preferred acetonitrile-0.2% aqueous formic acid
Gradient elution program is as shown in table 1；
Table 1 gradient elution program
| Time (min) | Acetonitrile (%) |
| 0-6 | 10-35 |
| 6-9 | 35-50 |
| 9-14 | 50-60 |
| 14-16 | 60-75 |
| 16-18 | 75 |
| 18-19 | 75-10 |
| 19-20 | 10 |
Flow velocity is 0.2～0.6mL/min, it is preferred to 0.4mL/min；
Ultraviolet detection wavelength: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Hesperidin is 284nm；Magnolol, magnolol are 290nm；Atisine chloride atractydin is 336nm；；
Preferred dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；
Column temperature: 25 DEG C～35 DEG C, it is preferred to 30 DEG C；
Sample size: 1 μ L～10 μ L, it is preferred to 2 μ L。
Wherein, step 1: the preparation of need testing solution；Method is as follows: take 0.5-1.5g ageratum drop pill, accurately weighed, accurate addition methanol 10-30mL, weighed weight, supersound process 15-25min, put to room temperature, supply the weight of less loss with methanol, filter with 0.2-0.3 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Preferably, step 1: the preparation of need testing solution；Method is as follows: take about 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 20mL, weighed weight, ultrasonic (500W, 40kHz) processes 20min, puts to room temperature, supply the weight of less loss with methanol, filter with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Wherein, in step 3, calculating need testing solution, the content of Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin, uses the standard control collection of illustrative plates of preparation in advance；The preparation of described standard control collection of illustrative plates, method is as follows: to weigh Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin reference substance appropriate for precision respectively, add methanol dissolve and constant volume, prepared mass concentration respectively Hesperidin 0.6000mg/mL, glycyrrhizic acid 0.2024mg/mL, imperatorin 0.0806mg/mL, honokiol 0.4032mg/mL, isoimperatorin 0.0230mg/mL, magnolol 0.3016mg/mL, atisine chloride atractydin 0.0416mg/mL mixing reference substance storing solution。Precision measures mixing reference substance storing solution 1.00mL, puts in 10mL measuring bottle, with methanol dilution to scale, shakes up, obtains mixing reference substance solution。Mixing reference substance solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；It is standard control collection of illustrative plates。
The core of the present invention is in that the selection of chromatographic condition, how to solve to measure the chromatographic condition of the content of 7 kinds of effective ingredient simultaneously, is a major challenge, and the present invention has carried out substantial amounts of screening operation for this, finally gives the chromatographic condition of the present invention。It addition, the preparation of need testing solution has been also carried out screening by the present invention, the result suitability is fabulous。
Instrument selected by the present invention is as follows with material:
ACCURICYUPLCHCLASS system (includes binary supertension solvent system, auto injection constant temperature sample management device, TUV detector, Empower2 chromatographic work station, waters company of the U.S.)；MettlerToledoXS105 analytical balance (MettlerToledo company of Switzerland)；KQ-500DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；MilliporeSimplicity ultrapure water system。
Reference substance Hesperidin (lot number: 110721-201115), imperatorin (lot number: 110826-201214), honokiol (lot number: 110730-201313), isoimperatorin (lot number: 110827-201109), magnolol (lot number: 110729-200412.), atisine chloride atractydin (lot number: 11924-201303) are all purchased from National Institute for Food and Drugs Control, for assay, glycyrrhizic acid (lot number: 17239-64, purity 98.0%) it is purchased from NACALAITESOUE, INC。Methanol (chromatographically pure, Merck company of the U.S.), acetonitrile (chromatographically pure, Merck company of the U.S.), formic acid (chromatographically pure, Fluke company of Switzerland), glacial acetic acid (chromatographically pure, Concord, Tianjin company limited), phosphoric acid (chromatographically pure, Tianjin recovery fine chemistry industry institute)。
Ageratum drop pill (Tianjin Tasly Pharmaceutical Co., Ltd produces 11 batches, lot number respectively 2014C08,131128,131201,131202,131203,131204,131205,131206,131207,131208,131209)；Ageratum drop pill is commercially available or application application number is that the method in CN03145667.7 prepares gained。
Currently preferred experimental technique, chromatographic condition and solvent extraction process, obtain through screening, and screening process is as follows:
The preparation of mixing reference substance solution
To weigh Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin reference substance appropriate for precision respectively, add methanol dissolve and constant volume, prepared mass concentration respectively Hesperidin 0.6000mg/mL, glycyrrhizic acid 0.2024mg/mL, imperatorin 0.0806mg/mL, honokiol 0.4032mg/mL, isoimperatorin 0.0230mg/mL, magnolol 0.3016mg/mL, atisine chloride atractydin 0.0416mg/mL mixing reference substance storing solution。Precision measures mixing reference substance storing solution 1.00mL, puts in 10mL measuring bottle, with methanol dilution to scale, shakes up, obtains mixing reference substance solution。
1,7 kinds of component content mensuration methodologies of ageratum drop pill are investigated
(1) system suitability
Taking need testing solution, chromatographic condition is: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；Mobile phase is acetonitrile-0.2% aqueous formic acid, and gradient elution program is as shown in table 1；Flow velocity is 0.4mL/min；Detection wavelength: passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；Column temperature: 30 DEG C；Sample size: 2 μ L。Sample introduction, 7 kinds of determined compositions all can reach baseline separation as a result, is all higher than 1.5 with the separating degree of adjacent chromatographic peak, calculates the theoretical number of plates all more than 20000 with each composition chromatographic peak, and peak shape is better。UPLC chromatogram is shown in Fig. 1
(2) linear relationship is investigated
Accurate draw mixing reference substance storing solution 0.25,0.50,1.00,2.50,5.00,10.00mL, put in 10mL measuring bottle respectively, by methanol constant volume to scale, shake up, obtain the mixing reference substance solution of 6 different quality solubility。Measure according to the chromatographic condition sample introduction under system suitability item, with peak area (Y), its concentration (X) is set up regression equation, by reference substance solution stepwise dilution, determine detection limit with 3 times of signal to noise ratios, 10 times of signal to noise ratios determine quantitative limit, and result is in Table 2。
Table 2 regression equation, the range of linearity, detection limit and quantitative limit
(3) Precision Experiment
Take mixing reference substance storing solution and be respectively configured the reference substance solution of basic, normal, high three concentration, according to the chromatographic condition under system suitability item, continuous sample introduction 6 times, METHOD FOR CONTINUOUS DETERMINATION three days, calculate withinday precision and day to day precision respectively according to measurement result, result is in Table 3。
Table 3 precision measurement result
(4) repeated experiment
Take sample (lot number 2014C08), 6 parts of need testing solutions are prepared according to the method for step 2, it is measured according to the chromatographic condition under system suitability item, calculate the amount of each composition, result Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin RSD respectively 1.7%, 1.4%, 1.7%, 1.5%, 1.3%, 1.5%, 1.9%, it was shown that method repeatability is good。
(5) stability experiment
Take the need testing solution of sample (lot number 2014C08), after preparation 0,2,4,8,12h, 24h be according to the chromatographic condition sample introduction under system suitability item, result Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin peak area RSD respectively 0.7%, 0.8%, 0.8%, 0.4%, 0.9%, 0.5%, 0.8%, it was shown that need testing solution is stable in 24h。
(6) response rate experiment
Precision weighs the ageratum drop pill (lot number 2014C08) totally 9 parts measured, every part of 0.5g, and precision adds basic, normal, high mixing reference substance stock solution in right amount respectively, prepares into need testing solution by step 2 method is parallel。Measure according to the chromatographic condition sample introduction under system suitability item, calculate the response rate and the RSD of each composition, result Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin average recovery rate respectively 100.1%, 101.4%, 97.5%, 101.6%, 99.6%, 100.5%, 98.7%, RSD respectively 1.3%, 1.4%, 1.1%, 1.1%, 1.2%, 1.5%, 1.2%。
2, the screening of test sample extraction conditions
(1) selection of Extraction solvent
What primarily look at is 100% methanol, 50% methanol-water, water, 50% ethanol, 100% ethanol。Taking about 1.0g ageratum drop pill, accurately weighed, the above-mentioned solution 20mL of accurate addition, weighed weight, ultrasonic (500W, 40kHz) processes 20min, puts to room temperature, supplies weight, filters with 0.22 μm of microporous filter membrane, takes subsequent filtrate UPLC and analyzes。According to whether effective ingredient extracts completely, selecting methanol extraction, the later stage has investigated 80%, and 60%, 40%, the extraction effect of 20% methanol, same procedure processes, found that when adopting 100% methanol extraction, 7 Component peak area are maximum, and baseline is more flat, therefore select methanol as Extraction solvent。Chromatogram is Fig. 2 such as。
(2) optimization of extracting mode
Determine methanol as Extraction solvent after, investigated backflow (30,60,90min), ultrasonic (10,20,30,40,50min) extraction effect of two ways, result is as shown in Figure 3 and Figure 4。Result shows that both extraction effects are without significant difference, and because supersound extraction is simple, and ultrasonic energy avoids the high temperature impact on unstable compound, finally determines supersound extraction 20min。
(3) investigation of Extraction solvent consumption
Investigating Extraction solvent consumption (1:5,1:10,1:20 and 1:40, w/v), draw through comparing, finally determine employing 1.0g sample 20mL methanol supersound extraction 20min, 7 kinds of compositions can extract completely。
3, the selection of chromatographic condition
(1) mobile phase is investigated
Having investigated the flow phase system that methanol-water, acetonitrile-water are different respectively, result methanol-water separation case is not so good as acetonitrile-water, and adopts methanol-water system pressure relatively big, selects acetonitrile-water system, and result is as shown in Figure 5；Investigating formic acid, acetic acid and the phosphoric acid impact on peak shape, as shown in Figure 6, during result employing acetonitrile-0.2% formic acid water, 7 composition peak shapes are better, separate more satisfactory for result。
(2) selection of gradient condition
Arranging different gradient elution program with acetonitrile-0.2% formic acid water for mobile phase, as shown in table 4, as shown in table 5, as shown in table 6, as shown in table 7, method 5 is as shown in table 8 for method 4 for method 3 for method 2 for method 1。
Table 4 method 1
| Time (min) | Acetonitrile (%) |
| 0-6 | 10-35 |
| 6-10 | 35-60 |
| 10-17 | 60-75 |
| 17-18 | 75 |
| 18-19 | 75-10 |
| 19-20 | 10 |
Table 5 method 2
| Time (min) | Acetonitrile (%) |
| 0-5 | 5-30 |
| 5-10 | 30-60 |
| 10-15 | 60-70 |
| 15-17 | 70-75 |
| 17-18 | 75 |
| 18-19 | 75-5 |
| 19-20 | 5 |
Table 6 method 3:
| Time (min) | Acetonitrile (%) |
| 0-5 | 15-35 |
| 5-8 | 35-50 |
| 8-12 | 50-60 |
| 12-14 | 60 |
| 14-20 | 60-75 |
| 20-21 | 75 |
| 21-22 | 75-15 |
| 22-23 | 15 |
Table 7 method 4:
| Time (min) | Acetonitrile (%) |
| 0-6 | 10-35 |
| 6-9 | 35-50 |
| 9-14 | 50-60 |
| 14-16 | 60-75 |
| 16-18 | 75 |
| 18-19 | 75-10 |
| 19-20 | 10 |
Table 8 method 5:
| Time (min) | Acetonitrile (%) |
| 0-8 | 10-35 |
| 8-11 | 35-50 |
| 11-12 | 50 |
| 12-18 | 50-60 |
| 18-22 | 60-75 |
| 22-23 | 75 |
| 23-24 | 75-10 |
| 24-25 | 10 |
Comparing the peak shape of 7 compositions of distinct methods and the separation case at peak, result is as shown in Figure 7。Experiments show that the gradient elution program of employing method 4,7 composition chromatographic peak separating degree is good, peak shape is symmetrical, analysis time is shorter。
(3) investigation of wavelength is detected
7 kinds of compositions are carried out ultraviolet full wavelength scanner, it is determined that the maximum absorption wavelength of each composition, Hesperidin 284nm, glycyrrhizic acid 251nm, imperatorin 249nm, honokiol 292nm, isoimperatorin 250nm, magnolol 290nm, atisine chloride atractydin 336nm。What this research adopted is dual pathways TUV detector, for ensureing that each composition can detect at maximum absorption wavelength, adopts following wavelength program: passage A: glycyrrhizic acid, imperatorin, isoimperatorin 250nm；Passage B:0-10min, Hesperidin 284nm；10.01-15min, honokiol, magnolol 290nm；15.01-20min, atisine chloride atractydin 336nm。
The uv-spectrogram of glycyrrhizic acid, imperatorin, isoimperatorin, Hesperidin, magnolol, honokiol and atisine chloride atractydin is respectively as shown in Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12, Figure 13 and Figure 14。
(4) comparison of different chromatographic columns
Investigate AcquityUPLCBEHC18 post (100mm × 2.1mm respectively, 1.7 μm), AcquityUPLCHSSC18 post (100mm × 2.1mm, 1.8 μm) and AcquityUPLCHSST3 post (100mm × 2.1mm, 1.8 μm) at the separating effect of same chromatographic condition, result is as shown in figure 15。The separating effect using 7 compositions of BEHC18 post under same chromatographic condition is best, and peak shape is symmetrical, and baseline is steady。Finally determine use BEHC18 post。Chromatogram is Figure 15 such as
(5) selection of column temperature
Having investigated column temperature is 25 DEG C, 30 DEG C, the 35 DEG C impacts on chromatographic isolation, and at three temperature, chromatographic column is similar to the separating effect at peak, raise with temperature, go out peak retention time somewhat to shift to an earlier date, for convenience of conventional laboratory conditions, select 30 DEG C of chromatographic column column temperatures as final optimization pass。Result is as shown in figure 16。
Four, beneficial effect
1, the present invention adopts ultra-performance liquid chromatography to measure 7 kinds of compositions simultaneously, reduces the resources occupation rate of test instrument, be greatly saved manpower and time cost compared with quality standard before。
2, the detection method of the ageratum drop pill that the present invention sets up is obtained after being screened by a large amount of scientific experimentss, Extraction solvent during by test sample is processed, the screening of extracting method, and the screening of chromatographic condition so that the method specificity of the present invention is better, economical and practical, quickly effective。
3, assay method analysis time short (chromatography can be completed in 20 minutes) of the present invention, sensitivity, precision height, favorable reproducibility, can be used for the routine analysis of ageratum drop pill product stability and quality control, the quality of thoroughly evaluating ageratum drop pill。
4, in order to compared with prior art, Comparison study file (Liu Yongli, Zhao Zhenxia, Li Dongmei etc. ultra-performance liquid chromatography measures 7 kinds of component contents [J] in HUOXIANG ZHENGQI SHUI simultaneously. China Medicine University's journal, 2013,44 (3): 249-252) the 7 kinds of compositions in chromatographic condition detection ageratum drop pill in。Experimental result is as follows: Figure 17 is ageratum drop pill chromatogram under documents chromatographic condition, Figure 18 is that under chromatographic condition of the present invention, the method for ageratum drop pill chromatogram and the present invention compares, and in ageratum drop pill, 7 kinds of composition separating degrees under documents and condition of the present invention are as shown in table 9。
7 kinds of composition separating degrees in ageratum drop pill under table 9 documents and condition of the present invention
| Numbering | Component | When documents | Under condition of the present invention |
| Separating degree | Separating degree | ||
| 1 | Hesperidin | 5.09 | 3.24 |
| 2 | Glycyrrhizic acid | 1.09 | 1.53 |
| 3 | Imperatorin | 2.66 | 3.80 |
| 4 | Honokiol | 2.60 | 1.90 |
| 5 | Isoimperatorin | 3.00 | 2.01 |
| 6 | Magnolol | 3.43 | 2.00 |
| 7 | Atisine chloride atractydin | 4.84 | 4.80 |
Two kinds of methods are contrasted, it is possible to obtain as drawn a conclusion by above experimental result:
In 7 kinds of compositions of documents method, glycyrrhizic acid separating degree is 1.09, and less than 1.5, peak is not completely separable；And the baseline of sample is not as steady；7 kinds of composition separating degrees of the method for the present invention are all higher than 1.5, can be completely separable, and baseline is steady。So the chromatographic condition that the present invention adopts improves separating power with gradient elution program compared with documents, peak shape is not trailed, and sensitivity is also with raising。
Accompanying drawing explanation
The chromatogram of Fig. 1 mixing reference substance: passage A (A), passage B (B)；
The chromatogram of ageratum drop pill: passage A (C), passage B (D)。
1 Hesperidin, 2 glycyrrhizic acids, 3 imperatorin, 4 honokiols,
5 isoimperatorin, 6 magnolol, 7 atisine chloride atractydin
The selection of Fig. 2 test sample Extraction solvent
S1:100% methanol extraction；S2:50% methanol extraction；S3: water extraction；S4:50% ethanol extraction；
S5:100% ethanol extraction；S6:80% methanol-water extracts；S7:60% methanol-water extracts；
S8:40% methanol-water extracts；S9:20% methanol-water extracts
Fig. 3 difference return time extraction chromatography figure
Fig. 4 difference ultrasonic time chromatogram
Fig. 5 methanol-water, acetonitrile water system chromatogram
Fig. 6 adds the chromatogram after different acid
The chromatogram that 7 compositions of the different elution program of Fig. 7 five kinds separate
The uv-spectrogram of Fig. 8 glycyrrhizic acid
The uv-spectrogram of Fig. 9 imperatorin
The uv-spectrogram of Figure 10 isoimperatorin
The uv-spectrogram of Figure 11 Hesperidin
The uv-spectrogram of Figure 12 magnolol
The uv-spectrogram of Figure 13 honokiol
The uv-spectrogram of Figure 14 atisine chloride atractydin
The chromatogram of Figure 15 difference chromatographic column condition
Chromatogram when Figure 16 difference column temperature
Ageratum drop pill chromatogram under Figure 17 documents chromatographic condition
Ageratum drop pill chromatogram under Figure 18 chromatographic condition of the present invention: passage A (C), passage B (D)
1 Hesperidin, 2 glycyrrhizic acids, 3 imperatorin, 4 honokiols,
5 isoimperatorin, 6 magnolol, 7 atisine chloride atractydin
Detailed description of the invention
1, embodiment 1
Step 1: the preparation of need testing solution
Take about 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 20mL, weighed weight, ultrasonic (500W, 40kHz) processes 20min, puts to room temperature, supply the weight of less loss with methanol, filter with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Step 2: chromatographic condition
Chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；Mobile phase is acetonitrile-0.2% aqueous formic acid, and gradient elution program is as shown in table 1；Flow velocity is 0.4mL/min；Detection wavelength: passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；Column temperature: 30 DEG C；Sample size: 2 μ L。
Step 3: the mensuration of 7 kinds of component contents in ageratum drop pill
Taking the ageratum drop pill of 11 different batches, prepare need testing solution according to the method for step 1, analyze according to the lower chromatographic condition sample introduction of step 2, calculate with external standard method, measurement result is in Table 9。
7 kinds of composition measurement results in table 9 ageratum drop pill
Embodiment 2
Step 1: the preparation of need testing solution；Method is as follows: takes 0.5g ageratum drop pill, accurate addition methanol 10mL, weighed weight, supersound process 15min, puts to room temperature, supply the weight of less loss with methanol, filter with 0.2 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.1% aqueous formic acid,
Gradient elution program
Flow velocity is 0.2mL/min；
Ultraviolet detection wavelength: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Hesperidin is 284nm；Magnolol, magnolol are 290nm；Atisine chloride atractydin is 336nm；；
Preferred dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；
Column temperature: 25 DEG C；
Sample size: 1 μ L。
Embodiment 3
Step 1: the preparation of need testing solution；Method is as follows: take 1.5g ageratum drop pill, accurately weighed, accurate addition methanol 30mL, weighed weight, and supersound process 25min is put to room temperature, supplied the weight of less loss with methanol, filters with 0.3 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.3% aqueous formic acid,
Gradient elution program
Flow velocity is 0.6mL/min；
Ultraviolet detection wavelength: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Hesperidin is 284nm；Magnolol, magnolol are 290nm；Atisine chloride atractydin is 336nm；；
Preferred dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；
Column temperature: 35 DEG C；
Sample size: 10 μ L。
Embodiment 4
Step 1: the preparation of need testing solution；Method is as follows: take 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 30mL, weighed weight, and supersound process 20min is put to room temperature, supplied the weight of less loss with methanol, filters with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Chromatographic condition: chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.2% aqueous formic acid,
Gradient elution program
Flow velocity is 0.4mL/min；
Ultraviolet detection wavelength: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Hesperidin is 284nm；Magnolol, magnolol are 290nm；Atisine chloride atractydin is 336nm；；
Column temperature: 30 DEG C；
Sample size: 2 μ L。
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Embodiment 5
Step 1: the preparation of need testing solution；Method is as follows: take 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 30mL, weighed weight, and supersound process 20min is put to room temperature, supplied the weight of less loss with methanol, filters with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Chromatographic condition: chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.2% aqueous formic acid,
Gradient elution program
Flow velocity is 0.4mL/min；
Dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；
Column temperature: 30 DEG C；
Sample size: 4 μ L。
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Embodiment 6
Step 1: the preparation of need testing solution；Method is as follows: take 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 30mL, weighed weight, and supersound process 15min is put to room temperature, supplied the weight of less loss with methanol, filters with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Chromatographic condition: chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.2% aqueous formic acid,
Gradient elution program
Flow velocity is 0.2mL/min；
Dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；
Column temperature: 25 DEG C；
Sample size: 4 μ L。
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Embodiment 7
Step 1: the preparation of need testing solution；Method is as follows: taking 1.0g ageratum drop pill, accurate addition methanol 30mL, weighed weight, methanol constant volume, to scale, supersound process 25min, is put to room temperature, supplied the weight of less loss with methanol, filters with 0.22 μm of microporous filter membrane, takes subsequent filtrate as need testing solution。
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Chromatographic condition: chromatographic column: ACQUITYUPLCBEHC18 chromatographic column (100mm × 2.1mm, 1.7 μm)；
Mobile phase is acetonitrile-0.2% aqueous formic acid,
Gradient elution program
Flow velocity is 0.6mL/min；
Dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；
Column temperature: 35 DEG C；
Sample size: 10 μ L。
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
Claims (9)
1. a ultra-performance liquid chromatography measures the method for 7 kinds of component contents in ageratum drop pill, it is characterised in that said method comprising the steps of:
Step 1: the preparation of need testing solution: ageratum drop pill is dissolved in methanol, filters, obtains filtrate；
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
2. method according to claim 1, it is characterised in that the chromatographic condition of described step 2 Ultra Performance Liquid Chromatography instrument is as follows:
Chromatographic column: C18 chromatographic column；
Mobile phase is acetonitrile-0.1-0.3% aqueous formic acid；
Gradient elution program
Flow velocity is 0.2～0.6mL/min；
UV-detector sets wavelength: glycyrrhizic acid, imperatorin, isoimperatorin are as 250nm；Hesperidin is 284nm；Honokiol, magnolol are 290nm；Atisine chloride atractydin is 336nm；
Column temperature: 25 DEG C～35 DEG C；
Sample size: 1 μ L～10 μ L。
3. method according to claim 2, it is characterised in that described detector is dual pathways UV-detector, passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm。
4. method according to claim 2, it is characterised in that the chromatographic condition of described Ultra Performance Liquid Chromatography instrument is as follows:
Mobile phase is acetonitrile-0.2% aqueous formic acid；
Flow velocity is 0.4mL/min；
Column temperature: 30 DEG C；
Sample size: 2 μ L。
5. method according to claim 1, it is characterised in that wherein, step 1: the preparation of need testing solution；Method is as follows: take 0.5-1.5g ageratum drop pill, accurately weighed, accurate addition methanol 10-30mL, weighed weight, supersound process 15-25min, put to room temperature, supply the weight of less loss with methanol, filter with 0.2-0.3 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
6. method according to claim 1, it is characterized in that, wherein step 1: the preparation method of need testing solution is as follows: take about 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 20mL, weighed weight, 500W, 40kHz supersound process 20min, put to room temperature, supply the weight of less loss with methanol, filter with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution。
7. method according to claim 1, it is characterised in that wherein, in step 3, calculating need testing solution, Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin content method are for using standard control collection of illustrative plates。
8. method according to claim 7, it is characterized in that, the preparation of described standard control collection of illustrative plates, method is as follows: precision weighs Hesperidin respectively, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin reference substance is appropriate, add methanol to dissolve and constant volume, prepared mass concentration respectively Hesperidin 0.6000mg/mL, glycyrrhizic acid 0.2024mg/mL, imperatorin 0.0806mg/mL, honokiol 0.4032mg/mL, isoimperatorin 0.0230mg/mL, magnolol 0.3016mg/mL, the mixing reference substance storing solution of atisine chloride atractydin 0.0416mg/mL；Precision measures mixing reference substance storing solution 1.00mL, puts in 10mL measuring bottle, with methanol dilution to scale, shakes up, obtains mixing reference substance solution；Mixing reference substance solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram and be standard control collection of illustrative plates。
9. method according to claim 1, it is characterised in that
Step 1: the preparation of need testing solution
Take 1.0g ageratum drop pill, accurately weighed, accurate addition methanol 20mL, weighed weight, 500W, 40kHz supersound process 20min, put to room temperature, supply the weight of less loss with methanol, filter with 0.22 μm of microporous filter membrane, take subsequent filtrate as need testing solution；
Step 2: measure need testing solution with Ultra Performance Liquid Chromatography instrument: need testing solution is injected Ultra Performance Liquid Chromatography instrument, obtains chromatogram；
Chromatographic condition
Chromatographic column: C18 chromatographic column, specification 100mm × 2.1mm, 1.7 μm；Mobile phase is acetonitrile-0.2% aqueous formic acid, and flow velocity is 0.4mL/min；Detection wavelength: passage A: glycyrrhizic acid, imperatorin, isoimperatorin are 250nm；Passage B:0-10min, Hesperidin is 284nm；10.01-15min, honokiol, magnolol are 290nm；15.01-20min, atisine chloride atractydin is 336nm；Column temperature: 30 DEG C；Sample size: 2 μ L；
Step 3: according to the content of 7 kinds of compositions in chromatogram calculation need testing solution: calculating the content of 7 kinds of compositions in need testing solution with external standard method, 7 kinds of wherein said compositions are Hesperidin, glycyrrhizic acid, imperatorin, honokiol, isoimperatorin, magnolol, atisine chloride atractydin。
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| CN108051515A (en) * | 2017-12-01 | 2018-05-18 | 云南枝蒿制药有限公司 | A kind of method falls the method for quality control of extra large piece |
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| CN114200038A (en) * | 2021-11-23 | 2022-03-18 | 太极集团重庆涪陵制药厂有限公司 | Method for detecting compound content in wrinkled gianthyssop herb zhengqi oral liquid by liquid chromatography-mass spectrometry |
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| CN115326983A (en) * | 2022-08-26 | 2022-11-11 | 上海和黄药业有限公司 | Method for measuring contents of various components in vital qi tablet extract |
| CN116183739A (en) * | 2022-11-25 | 2023-05-30 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Determination method for analyzing fingerprint of Huoxiang Zhengqi soft capsule |
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