CN104155383A - Detection method of dandelion and viola philippica granules - Google Patents
Detection method of dandelion and viola philippica granules Download PDFInfo
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- CN104155383A CN104155383A CN201410427483.3A CN201410427483A CN104155383A CN 104155383 A CN104155383 A CN 104155383A CN 201410427483 A CN201410427483 A CN 201410427483A CN 104155383 A CN104155383 A CN 104155383A
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Abstract
The invention provides a detection method of caffeic acid and aesculetin at the same time in a dandelion and viola philippica granules or an intermediate extract thereof. Through multiple experiment and research, chromatographic conditions of HPLC in detection of the two ingredients at the same time are screened and explored and a content determining method by which the two ingredients in the dandelion and viola philippica granules or the intermediate extract thereof can be detected at the same time. Under the chromatographic conditions, chromatographic peaks of the two ingredients can be separated successfully, interferences of impurity peaks are avoided, a retain time is proper and peak shapes are excellent. By means of the method, an object of successfully determining the contents of the two ingredients can be achieved and a basis of quality researching of a preparation is established. The detection method also provides a reference method of quantitative researching of a preparation containing the caffeic acid and the aesculetin at the same time.
Description
Technical field
The present invention relates to the detection method of blue or green Pu particle.
Background technology
Veterinary clinic facts have proved, thermic inversion is one of important symptom of pig physiological function multilated, the normal body temperature of pig is 38～40 DEG C, if exceed continuously more than 40 DEG C, it is heating, the high heat of continuity, large to body damage for a long time, first makes body kalabolism accelerate, nutriment expends too much, digestive function entanglement, causes body to be become thin, and the power of keeping out declines, can make again the central nervous system blood circulation system of unifying damage, cause that disease pig spirit is depressed, so that stupor, or the serious consequence such as in heart failure.A large amount of modern studies show, a large amount of Chinese medicine all has clearing heat and detoxicating, antibacterial, antiviral effect, there is own unique advantage at some febrile diseases for the treatment of, blue or green Pu particle is by Chinese violet, the herbal medicine compound granular agent that the medicinal materials such as dandelion are made, have clearing heat and detoxicating, effect of cool blood detumescence, be mainly used in the treatment of pig fever symptom, One's spirits are drooping to pig, hyperthermia, be short of breath, thirsty greedy drink, poor appetite, the short Huang of urine, generate heat persistent, the rubescent clinical disease that waits of whole body all has good result for the treatment of, cure rate reaches more than 70%, there is very strong researching value.
Dandelion and Chinese violet are the main Chinese medicinal materials in blue or green Pu particle prescription, one of main effective constituent that caffeic acid is dandelion, have antibacterial significantly, antiviral, central excitation, removing toxic substances, the effects such as blood coagulation; Aesculetin is one of main effective constituent of Chinese violet, there is the effects such as obvious anti-inflammatory, antibacterial, analgesia, anti-oxidant, antitumor and immunological regulation, therefore, this granule selects aesculetin and caffeic acid as assay index components, adopts high performance liquid chromatography to measure research to two kinds of compositions in granule.
This class preparation early-stage Study shows, the high performance liquid chromatography content assaying method that adopts respectively Chinese violet medicinal material or dandelion medicinal material is when in blue or green Pu particle, two kinds of compositions of aesculetin and caffeic acid are measured simultaneously, the chromatographic peak of two kinds of compositions all interferes with each other, degree of separation is poor, impurity component serious interference, peak area difference is large, and assay is difficulty comparatively.And in current document, also reported aesculetin and caffeinic detection method of content in other products, but these methods are used gradient elution more, and its mobile phase program is comparatively complicated.
Summary of the invention
The object of the present invention is to provide a kind of isocratic elution condition that adopts to method that in blue or green Pu particle or its intermediate extract, caffeic acid and aesculetin detect simultaneously.
Particularly, the invention provides the method that in blue or green Pu particle, caffeic acid and aesculetin detect simultaneously, it comprises following operation steps:
(1) preparation of need testing solution: get the powder after blue or green Pu particle porphyrize, accurately weighed, extract and prepare need testing solution with methyl alcohol;
(2) preparation of reference substance solution: get aesculetin reference substance, caffeic acid reference substance, prepare reference substance solution taking methyl alcohol as solvent;
(3) get respectively need testing solution and reference substance solution and inject high performance liquid chromatograph, calculate caffeic acid and content of Aesculetin in blue or green Pu particle with external standard method;
Wherein, blue or green Pu particle prepares by the following method:
Get folium isatidis 6～10 weight portions, dandelion 6～10 weight portions, Chinese violet 2～4 weight portions, Radix Glycyrrhizae 1～2 weight portion; Above four traditional Chinese medicine material, boiling, merges decocting liquid, concentrated, adds cane sugar powder and starch is appropriate, mixes granulation.
The present invention's research shows:
(1) adopt Agilent-ODS (4.6 × 150mm, 5 μ are m) when shorter chromatogram column is tested, due to chromatogram column length restriction, two kinds of compositions of aesculetin and caffeic acid cannot separate at all; And employing Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m) grow chromatographic column and test, and two kinds of compositions of aesculetin and caffeic acid can separate substantially, therefore, (4.6 × 250mm, 5 μ are isometric chromatographic column m) to select Shimadzu WondaCract ODS-2.
(2) although the present invention has selected comparatively suitable chromatographic column, and its separating effect still people is poor, exist impurity to disturb, still need and carry out further experimental study.Research discovery, because caffeic acid content in preparation is extremely low, sample concentration is low, and under 353nm wavelength condition, peak area is very little, and after aesculetin absorption peak, has serious hangover.For take into account make two kinds of compositions all there is good absorption peak, final Selective determination wavelength is 326nm.
(3), after defining chromatographic column model and wavelength, although aesculetin can be separated with caffeic acid, degree of separation is still poor.The present invention finds, after column temperature is regulated, can improve the degree of separation of two chromatographic peaks: two chromatographic peaks can reach baseline separation in the time that column temperature condition is 25 DEG C, 30 DEG C, and separate better with other impurity component, is better than 35 DEG C.
Therefore, synthesise various factor, the present invention has finally drafted following chromatographic condition:
Chromatographic column be enlightening horse Diamonsil II (4.6 × 250mm, 5 μ m) or Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution=20:80 as mobile phase; Detection wavelength is 326nm; Column temperature is 25-30 DEG C.
If realize better the separation of each composition, preferably column temperature is 25 DEG C.
Further, in step (1), extracting mode is ultrasonic.
Further, ultrasonic power 200W, frequency 32kHz, 30 minutes.
Further, in step (1), the mass volume ratio of powder and methyl alcohol is 3:25g/ml.
Further, in the preparation method of blue or green Pu particle, the concrete technology of decocting is: boiling secondary, add for the first time 18 times of water gagings, and add for the second time 15 times of water gagings, decoct 1 hour at every turn.
Further, in the preparation method of blue or green Pu particle, decocting liquid is concentrated into 80 DEG C, and to record relative density be 1.30～1.35.
Further, the consumption proportion of four traditional Chinese medicine material is as follows: folium isatidis 8 weight portions, dandelion 8 weight portions, Chinese violet 3 weight portions, Radix Glycyrrhizae 1 weight portion.
The present invention also provides the method that in blue or green Pu particle intermediate extract, caffeic acid and aesculetin detect simultaneously, and it comprises following operation steps:
(1) preparation of need testing solution: get blue or green Pu particle intermediate extract, remove moisture content, accurately weighed, extract and prepare need testing solution with methyl alcohol;
(2) preparation of reference substance solution: get aesculetin reference substance, caffeic acid reference substance, prepare reference substance solution taking methyl alcohol as solvent;
(3) get respectively need testing solution and reference substance solution and inject high performance liquid chromatograph, calculate caffeic acid and content of Aesculetin in blue or green Pu particle with external standard method, wherein, chromatographic condition is as follows:
Chromatographic column be enlightening horse Diamonsil II (4.6 × 250mm, 5 μ m) or Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution=20:80 as mobile phase; Detection wavelength is 326nm; Column temperature is 25 DEG C;
Wherein, blue or green Pu particle intermediate extract prepares by the following method:
Get folium isatidis 6～10 weight portions, dandelion 6～10 weight portions, Chinese violet 2～4 weight portions, Radix Glycyrrhizae 1～2 weight portion; Above four traditional Chinese medicine material, boiling, merges decocting liquid, obtains blue or green Pu particle intermediate extract.
Further, in blue or green Pu particle intermediate extract preparation process, the concrete technology of decocting is: boiling secondary, add for the first time 18 times of water gagings, and add for the second time 15 times of water gagings, decoct 1 hour at every turn.
Further, the consumption proportion of four traditional Chinese medicine material is as follows: folium isatidis 8 weight portions, dandelion 8 weight portions, Chinese violet 3 weight portions, Radix Glycyrrhizae 1 weight portion.
The present invention studies by test of many times, and the HPLC chromatographic condition of simultaneously measuring two kinds of compositions is screened and explored, and has obtained a kind of content assaying method that can simultaneously detect two kinds of compositions in blue or green Pu particle or its extract.Although chromatographic condition provided by the invention, do not adopt complicated gradient elution program, but, by parameters such as particular detection wavelength, column temperature, chromatographic column models, two kinds of composition chromatographic peaks can be separated smoothly, impurity peaks is noiseless, and retention time is suitable, peak shape is good, can reach the object of two kinds of component contents of smooth mensuration, lay a good foundation for the quality research of preparation, also can be other quantitative examinatioies that simultaneously contain aesculetin and two kinds of component preparations of caffeic acid method of offering reference.
Brief description of the drawings
Fig. 1: mobile phase is acetonitrile-0.6% glacial acetic acid aqueous solution (8:92); Chromatographic column be Agilent-ODS (4.6 × 150mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm
Fig. 2: mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Chromatographic column be Agilent-ODS (4.6 × 150mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm
Fig. 3: mobile phase is acetonitrile-0.6% glacial acetic acid aqueous solution (8:92); Chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm
Fig. 4: mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm
Fig. 5: mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m), 35 DEG C of column temperatures, wavelength 326nm
Fig. 6 Shimadzu WondaCract ODS-2 post, methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80), 30 DEG C of column temperatures
Fig. 7 Shimadzu WondaCract ODS-2 post, methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80), 25 DEG C of column temperatures
Fig. 8 reference substance collection of illustrative plates
Embodiment
The detection method of the blue or green Pu particle of embodiment 1 the present invention
The preparation method of blue or green Pu particle of the present invention is as follows:
Prescription: 1 part, 3 portions of Radix Glycyrrhizaes of 8 portions of Chinese violets of 8 portions of dandelions of folium isatidis
Technique: above four traditional Chinese medicine material, boiling secondary, adds 18 times of water gagings for the first time, add for the second time 15 times of water gagings, each decoction 1 hour, collecting decoction, filters, it is 1.30～1.35 (80 DEG C) that filtrate is concentrated into relative density, add cane sugar powder and starch is appropriate, mix granulation, dry, to obtain final product.
Detection method is as follows:
Chromatographic condition and system suitability experiment Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ are m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) as mobile phase; Detection wavelength is 326nm; Column temperature is 25 DEG C.Number of theoretical plate calculates and should be not less than 3000 by aesculetin peak.
Aesculetin reference substance is got in the preparation of reference substance solution, caffeic acid reference substance is appropriate, accurately weighed, adds methyl alcohol and makes the mixed solution of every 1ml containing aesculetin 80 μ g, caffeic acid 16 μ g, to obtain final product.
The about 3g of powder after blue or green Pu particle porphyrize is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.
Determination method respectively accurate absorption is mixed reference substance solution and the each 10 μ l of need testing solution, and injection liquid chromatography, measures, and to obtain final product.
The every 1g of this product contains Chinese violet with aesculetin (C
9h
6o
4) meter, must not lack 0.45mg, contain dandelion with caffeic acid (C
9h
8o
4) meter, must not lack 0.08mg.
The detection method of the blue or green Pu particle of embodiment 2 the present invention
The preparation method of blue or green Pu particle of the present invention is as follows:
Prescription: 1.5 parts, 2 portions of Radix Glycyrrhizaes of 10 portions of Chinese violets of 10 portions of dandelions of folium isatidis
Technique: above four traditional Chinese medicine material, boiling secondary, adds 18 times of water gagings for the first time, add for the second time 15 times of water gagings, each decoction 1 hour, collecting decoction, filters, it is 1.30～1.35 (80 DEG C) that filtrate is concentrated into relative density, add cane sugar powder and starch is appropriate, mix granulation, dry, to obtain final product.
Detection method is as follows:
Chromatographic condition and system suitability experiment Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ are m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) as mobile phase; Detection wavelength is 326nm; Column temperature is 25 DEG C.Number of theoretical plate calculates and should be not less than 3000 by aesculetin peak.
Aesculetin reference substance is got in the preparation of reference substance solution, caffeic acid reference substance is appropriate, accurately weighed, adds methyl alcohol and makes the mixed solution of every 1ml containing aesculetin 80 μ g, caffeic acid 16 μ g, to obtain final product.
The about 3g of powder after blue or green Pu particle porphyrize is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.
Determination method respectively accurate absorption is mixed reference substance solution and the each 10 μ l of need testing solution, and injection liquid chromatography, measures, and to obtain final product.
The every 1g of this product contains Chinese violet with aesculetin (C
9h
6o
4) meter, must not lack 0.45mg, contain dandelion with caffeic acid (C
9h
8o
4) meter, must not lack 0.08mg.
The detection method of the blue or green Pu particle of embodiment 3 the present invention
The preparation method of blue or green Pu particle of the present invention is as follows:
Prescription: 2 parts, 4 portions of Radix Glycyrrhizaes of 6 portions of Chinese violets of 6 portions of dandelions of folium isatidis
Technique: above four traditional Chinese medicine material, boiling secondary, adds 18 times of water gagings for the first time, add for the second time 15 times of water gagings, each decoction 1 hour, collecting decoction, filters, it is 1.30～1.35 (80 DEG C) that filtrate is concentrated into relative density, add cane sugar powder and starch is appropriate, mix granulation, dry, to obtain final product.
Detection method is as follows:
Chromatographic condition and system suitability experiment Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ are m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) as mobile phase; Detection wavelength is 326nm; Column temperature is 25 DEG C.Number of theoretical plate calculates and should be not less than 3000 by aesculetin peak.
Aesculetin reference substance is got in the preparation of reference substance solution, caffeic acid reference substance is appropriate, accurately weighed, adds methyl alcohol and makes the mixed solution of every 1ml containing aesculetin 80 μ g, caffeic acid 16 μ g, to obtain final product.
The about 3g of powder after blue or green Pu particle porphyrize is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.
Determination method respectively accurate absorption is mixed reference substance solution and the each 10 μ l of need testing solution, and injection liquid chromatography, measures, and to obtain final product.
The every 1g of this product contains Chinese violet with aesculetin (C
9h
6o
4) meter, must not lack 0.45mg, contain dandelion with caffeic acid (C
9h
8o
4) meter, must not lack 0.08mg.
The detection method of the blue or green Pu particle of embodiment 4 the present invention intermediate extract
The preparation method of blue or green Pu particle intermediate extract of the present invention is as follows:
Prescription: 1 part, 3 portions of Radix Glycyrrhizaes of 8 portions of Chinese violets of 8 portions of dandelions of folium isatidis
Technique: above four traditional Chinese medicine material, boiling secondary, adds 18 times of water gagings for the first time, adds for the second time 15 times of water gagings, decocts 1 hour at every turn, and collecting decoction, obtains blue or green Pu particle intermediate extract.
Detection method is as follows:
Chromatographic condition and system suitability experiment Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ are m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) as mobile phase; Detection wavelength is 326nm; Column temperature is 25 DEG C.Number of theoretical plate calculates and should be not less than 3000 by aesculetin peak.
Aesculetin reference substance is got in the preparation of reference substance solution, caffeic acid reference substance is appropriate, accurately weighed, adds methyl alcohol and makes the mixed solution of every 1ml containing aesculetin 80 μ g, caffeic acid 16 μ g, to obtain final product.
Blue or green Pu particle intermediate extract is got in the preparation of need testing solution, concentrated, dry, get the powder after dry product porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 30 minutes, lets cool, weighed weight again, supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method respectively accurate absorption is mixed reference substance solution and the each 10 μ l of need testing solution, and injection liquid chromatography, measures, and to obtain final product.
The investigation of embodiment 5 detection methods of the present invention
1 instrument, reagent and reference substance
Agilent 1260infinity high performance liquid chromatograph (OpenLAB CDS Data Processing in Chromatography Workstation); KQ5200DE ultrasonic cleaner; CPA225D type electronic analytical balance (100,000/);
Blue or green Pu particle, methyl alcohol are chromatographically pure (fisher company), and water is water for injection, and other reagent is analyzes pure (Chengdu chemical reagent factory).Before using, all reagent all filters through 0.45 μ m miillpore filter.
Aesculetin reference substance (is provided by National Institute for Food and Drugs Control, lot number: 110741-200506), caffeic acid reference substance (being provided lot number by National Institute for Food and Drugs Control: 110885-200102), before using at 60 DEG C drying under reduced pressure 4 hours.
2 chromatographic conditions
The selection of 2.1 chromatographic columns and mobile phase
Adopt methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80), acetonitrile-0.6% glacial acetic acid aqueous solution (8:92) as mobile phase, to Agilent-ODS (4.6 × 150mm, 5 μ m), (4.6 × 250mm, 5 μ m) the sample composition separation case of two kinds of chromatographic columns have carried out contrast test to Shimadzu WondaCract ODS-2.The results are shown in Figure 1～Fig. 4.
Fig. 1: mobile phase is acetonitrile-0.6% glacial acetic acid aqueous solution (8:92); Chromatographic column be Agilent-ODS (4.6 × 150mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm.
Fig. 2: mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Chromatographic column be Agilent-ODS (4.6 × 150mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm.
Fig. 3: mobile phase is acetonitrile-0.6% glacial acetic acid aqueous solution (8:92); Chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm.
Fig. 4: mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m), 35 DEG C of column temperatures, wavelength 353nm.
Result shows, adopt Agilent-ODS (4.6 × 150mm, 5 μ are m) when shorter chromatogram column is tested, due to chromatogram column length restriction, two kinds of compositions of aesculetin and caffeic acid cannot separate at all; And employing Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m) grow chromatographic column and test, and two kinds of compositions of aesculetin and caffeic acid can separate substantially, but inferior separating effect exists impurity to disturb, and still needs and carries out further experimental study.
Meanwhile, by contrast, methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) is relatively better as mobile phase separating effect, therefore finally select methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) as mobile phase.
2.2 measure the selection of wavelength
Get aesculetin reference substance, add methyl alcohol and make every 1ml approximately containing the solution of 0.038mg, using methyl alcohol as blank, at 200～500nm length scanning, have absorption maximum at 353nm place; Separately get caffeic acid reference substance, add methyl alcohol and make every 1ml approximately containing the solution of 0.015mg, using methyl alcohol as blank, at 200～500nm length scanning, have absorption maximum at 326nm place; Meanwhile, to (353nm and 326nm) under different wave length, the separation of sample and peak area have carried out contrast test, the results are shown in Figure 4, Fig. 5.
Fig. 5: mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m), 35 DEG C of column temperatures, wavelength 326nm.
Consider, because caffeic acid content in preparation is extremely low, sample concentration is low, and under 353nm wavelength condition, peak area is very little, and after aesculetin absorption peak, has serious hangover.For take into account make two kinds of compositions all there is good absorption peak, final Selective determination wavelength is 326nm.But aesculetin and caffeinic degree of separation are still poor, still need other conditions are further studied.
The selection of 2.3 column temperature conditions
Adopting methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80) is mobile phase, and (4.6 × 250mm, m) chromatographic column of 5 μ are carried out trial test respectively at 25 DEG C, 30 DEG C of column temperatures to Shimadzu WondaCract ODS-2, and result is as Fig. 6, Fig. 7.
Result shows, in the time that column temperature condition is 25 DEG C, 30 DEG C, two chromatographic peaks can reach baseline separation, and separates better with other impurity component, consider, and be to ensure separating effect and analysis speed, finally selecting column temperature condition is 25 DEG C.
2.4 brief summaries:
After deliberation determine chromatographic condition as follows: chromatographic column be Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m); Detection wavelength is 326nm; Mobile phase is methyl alcohol-0.6% glacial acetic acid aqueous solution (20:80); Flow velocity is 1.0ml/min; Column temperature is 25 DEG C.
The preparation of 3 reference substance solution
Get aesculetin reference substance, caffeic acid reference substance is appropriate, accurately weighed, adds methyl alcohol and makes every 1ml containing the mixed solution of aesculetin 80 μ g, caffeic acid 16 μ g, to obtain final product.Fig. 8 is shown in by reference substance collection of illustrative plates.
4 need testing solution preparation method researchs
Be soluble in the character of methyl alcohol according to composition aesculetin to be measured and caffeic acid, select methyl alcohol to extract as solvent; This granule is concentrated rear granulation of medicinal material extract, therefore, can adopt the method for ultrasonic processing to study.Concrete research is as follows.
4.1 extraction times were investigated
The ultrasonic processing time is studied, and concrete grammar is as follows: it is appropriate to get this product, after porphyrize, gets three parts, powder, every part of about 3g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 10 minutes, 20 minutes, 30 minutes, 40 minutes, lets cool respectively, weighed weight again, supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, respectively as need testing solution.With above-mentioned chromatographic condition, accurate reference substance solution and the need testing solution 10 μ l of drawing respectively, injection liquid chromatography, records peak area value, calculates aesculetin and caffeic acid content with every 1g finished product.Result shows, two kinds of component contents increased with ultrasonic extraction time, in the time of ultrasonic processing 20 minutes, approach and extracted completely, and be 20 minutes therefore select the ultrasonic extraction time of sample powder.
4.2 brief summaries:
According to above research, determine that the preparation method of need testing solution is: get the about 3g of powder after this product porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 20 minutes, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
5 negative interference tests
Get respectively the scarce dandelion negative sample of preparing by sample preparation technique, lack Chinese violet negative sample, lack the two negative samples of dandelion and Chinese violet, by need testing solution, preparation method prepares need testing solution, with above-mentioned chromatographic condition, and sample introduction respectively.Result shows, in aesculetin and caffeic acid chromatogram relevant position, each negative sample all noiseless peak occurs, proves that this sample size assay method specificity is stronger.
The investigation of 6 ranges of linearity
Precision takes caffeic acid reference substance 20mg, puts in 250ml volumetric flask, adds methyl alcohol and dissolves and be diluted to scale, fully dissolves; Separately get aesculetin reference substance 20mg, put in 50ml volumetric flask, add above-mentioned caffeic acid reference substance and dissolve and be diluted to scale; Get respectively this mixing reference substance solution 1.25ml, 2.5ml, 5ml, 10ml, 20.0ml in 25ml volumetric flask, methanol constant volume is to scale.Must be respectively 0.02mg/ml, 0.04mg/ml, 0.08mg/ml, 0.16mg/ml, 0.32mg/ml containing aesculetin concentration, be respectively the mixing reference substance solution of 0.004mg/ml, 0.008mg/ml, 0.016mg/ml, 0.032mg/ml, 0.064mg/ml containing caffeic acid concentration.Draw respectively each concentration and mix reference substance solution 10 μ l, sample introduction, records peak area value.
With aesculetin peak area value (A), reference substance concentration (C) is returned, obtain aesculetin typical curve equation and be: y=29829.884x-10.140, R
2=0.9999.From equation, aesculetin sample introduction concentration is between 0.02mg/ml-0.32mg/ml, and aesculetin sample introduction concentration and peak area linear relationship are good.
With caffeic acid peak area value (A), reference substance concentration (C) is returned, obtain caffeic acid typical curve equation and be: y=53817.627x-10.482, R
2=0.9999.From equation, caffeic acid sample introduction concentration is between 0.004mg/ml-0.064mg/ml, and sample introduction concentration and peak area linear relationship are good.
7 precision tests
Accurate absorption mixed reference substance solution (aesculetin concentration: 0.080mg/ml, caffeic acid concentration: 0.016mg/ml) 10 μ l, by above-mentioned chromatographic condition, repeats sample introduction 6 times, records chromatogram and peak area value.Result shows, two kinds of composition peak area RSD<1.5%, illustrate that instrument precision is good.
8 stability tests
Get the about 3g of powder after this product porphyrize, prepare need testing solution according to the preparation method of need testing solution, separately prepare mixing reference substance solution according to reference substance solution preparation method.Get respectively and mix reference substance solution (aesculetin concentration: 0.081mg/ml, caffeic acid concentration: 0.016mg/ml) and need testing solution, 0h, 2h, 4h, 6h, 8h, 10h sample introduction respectively after preparation, measure by above-mentioned chromatographic condition, each sample size 10 μ l, record chromatogram and peak area value.
Result shows, survey two kinds of composition reference substance solution and need testing solution is good at 10 hours internal stabilities.
9 reappearance tests
Get 6 parts, powder after this product porphyrize, every part of about 3g, prepares need testing solution by the preparation method of need testing solution respectively, separately prepares mixing reference substance solution according to reference substance solution preparation method.Get and mix reference substance solution and need testing solution, measure by above-mentioned chromatographic condition, each sample size 10 μ l, record chromatogram and peak area value, two kinds of component contents in calculation sample.
Result shows, in blue or green Pu particle, the average content of aesculetin is 0.615mg/g, RSD<1.5%, and caffeinic average content is 0.134mg/g, RSD<1.5%, the reappearance of the method for investigating is good.
10 application of sample recovery tests
Get the about 1.5g of sample powder after known content porphyrize, totally 6 parts, accurately weighed, put in tool plug conical flask, add respectively aesculetin reference substance solution (0.178mg/ml) and the each 5ml of caffeic acid reference substance solution (0.0344mg/ml), precision adds methyl alcohol 15ml, close plug, weighed weight, ultrasonic processing (power 200W, frequency 32kHz) 20 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, obtain need testing solution; Separately prepare mixing reference substance solution according to reference substance solution preparation method; Get and mix reference substance solution and need testing solution, measure by above-mentioned chromatographic condition, sample introduction 10 μ l, record peak area value and chromatogram respectively, calculate average recovery.
The test of table 1 average recovery
Result shows, aesculetin average recovery measurement result is all between 95-105%, and its mean value is that 100.08%, RSD is 0.98%; Caffeic acid average recovery measurement result is all between 95-105%, and its mean value is that 99.69%, RSD is 0.84%.Show that this method is reliable, accuracy is good, meets the requirement of assay.
The assay of 11 samples
The two kinds of component contents method for measuring simultaneously obtaining according to above-mentioned research is measured aesculetin and caffeinic content in 3 batches of blue or green Pu particle pilot products.
Table 2 three batch sample assay results
Result shows, survey in 3 batch samples, aesculetin average content is 0.606mg/g, caffeic acid average content is 0.134mg/g, press screened content assaying method, can measure two kinds of compositions in sample smoothly simultaneously, for the quality determining method of formulating sample is laid a good foundation.
Claims (10)
1. the method that in pair blue or green Pu particle, caffeic acid and aesculetin detect simultaneously, is characterized in that: it comprises following operation steps:
(1) preparation of need testing solution: get the powder after blue or green Pu particle porphyrize, accurately weighed, extract and prepare need testing solution with methyl alcohol;
(2) preparation of reference substance solution: get aesculetin reference substance, caffeic acid reference substance, prepare reference substance solution taking methyl alcohol as solvent;
(3) get respectively need testing solution and reference substance solution and inject high performance liquid chromatograph, calculate caffeic acid and content of Aesculetin in blue or green Pu particle with external standard method, wherein, chromatographic condition is as follows:
Chromatographic column be enlightening horse Diamonsil II (4.6 × 250mm, 5 μ m) or Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution=20:80 as mobile phase; Detection wavelength is 326nm; Column temperature is 25-30 DEG C;
Wherein, blue or green Pu particle prepares by the following method: get folium isatidis 6～10 weight portions, dandelion 6～10 weight portions, Chinese violet 2～4 weight portions, Radix Glycyrrhizae 1～2 weight portion; Above four traditional Chinese medicine material, boiling, merges decocting liquid, concentrated, adds cane sugar powder and starch is appropriate, mixes granulation.
2. method according to claim 1, is characterized in that: in step (1), extracting mode is ultrasonic.
3. method according to claim 2, is characterized in that: ultrasonic power 200W, frequency 32kHz, 30 minutes.
4. method according to claim 1, is characterized in that: in step (1), the mass volume ratio of powder and methyl alcohol is 3:25g/ml.
5. method according to claim 1, is characterized in that: in the preparation method of blue or green Pu particle, the concrete technology of decocting is: boiling secondary, add for the first time 18 times of water gagings, and add for the second time 15 times of water gagings, decoct 1 hour at every turn.
6. method according to claim 1, is characterized in that: in the preparation method of blue or green Pu particle, decocting liquid is concentrated into 80 DEG C, and to record relative density be 1.30～1.35.
7. method according to claim 1, is characterized in that: the consumption proportion of four traditional Chinese medicine material is as follows: folium isatidis 8 weight portions, dandelion 8 weight portions, Chinese violet 3 weight portions, Radix Glycyrrhizae 1 weight portion.
8. the method that in pair blue or green Pu particle intermediate extract, caffeic acid and aesculetin detect simultaneously, is characterized in that: it comprises following operation steps:
(1) preparation of need testing solution: get blue or green Pu particle intermediate extract, remove moisture content, accurately weighed, extract and prepare need testing solution with methyl alcohol;
(2) preparation of reference substance solution: get aesculetin reference substance, caffeic acid reference substance, prepare reference substance solution taking methyl alcohol as solvent;
(3) get respectively need testing solution and reference substance solution and inject high performance liquid chromatograph, calculate caffeic acid and content of Aesculetin in blue or green Pu particle with external standard method, wherein, chromatographic condition is as follows:
Chromatographic column be enlightening horse Diamonsil II (4.6 × 250mm, 5 μ m) or Shimadzu WondaCract ODS-2 (4.6 × 250mm, 5 μ m); Taking methyl alcohol-0.6% glacial acetic acid aqueous solution=20:80 as mobile phase; Detection wavelength is 326nm; Column temperature is 25 DEG C;
Wherein, blue or green Pu particle intermediate extract prepares by the following method:
Get folium isatidis 6～10 weight portions, dandelion 6～10 weight portions, Chinese violet 2～4 weight portions, Radix Glycyrrhizae 1～2 weight portion; Above four traditional Chinese medicine material, boiling, merges decocting liquid, obtains blue or green Pu particle intermediate extract.
9. method according to claim 8, is characterized in that: in blue or green Pu particle intermediate extract preparation process, the concrete technology of decocting is: boiling secondary, add for the first time 18 times of water gagings, and add for the second time 15 times of water gagings, decoct 1 hour at every turn.
10. method according to claim 8, is characterized in that: the consumption proportion of four traditional Chinese medicine material is as follows: folium isatidis 8 weight portions, dandelion 8 weight portions, Chinese violet 3 weight portions, Radix Glycyrrhizae 1 weight portion.
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| CN109557211A (en) * | 2018-12-21 | 2019-04-02 | 亚宝药业四川制药有限公司 | A kind of method of quality control of the antipyretic oral solution of children's clearing |
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Address after: 611130 Wenjiang City, Chengdu Province, Chengdu cross strait science and Technology Industrial Park Road, No. 259 Jin Fu Road, No. Patentee after: Chengdu Qiankun animal pharmaceutical Limited by Share Ltd Address before: 611130 Wenjiang City, Chengdu Province, Chengdu cross strait science and Technology Industrial Park Road, No. 259 Jin Fu Road, No. Patentee before: Chengdu Qiankun Animal Pharmaceutical Co.,Ltd. |