CN109541068A - A kind of multiple young mixture component detection method - Google Patents
A kind of multiple young mixture component detection method Download PDFInfo
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- CN109541068A CN109541068A CN201811534482.3A CN201811534482A CN109541068A CN 109541068 A CN109541068 A CN 109541068A CN 201811534482 A CN201811534482 A CN 201811534482A CN 109541068 A CN109541068 A CN 109541068A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to Pharmaceutical Analysis field, in particular to a kind of multiple young mixture component detection method.This method comprises: the multiple young mixture test solution of preparation and the standard solution containing Rosmarinic acid and harpagoside, are detected using HPLC, the content of Rosmarinic acid and harpagoside in multiple young mixture is obtained.Method of quality control provided by the invention has good precision, linear relationship, stability, repeatability, and the rate of recovery is high, provides foundation for the quality of Rosmarinic acid and harpagoside control in multiple young mixture, provides better guarantee for the treatment of patient.
Description
Technical field
The present invention relates to the detections of Rosmarinic acid and harpagoside in Pharmaceutical Analysis field more particularly to a kind of multiple young mixture
Method.
Background technique
Multiple children's mixture, by Prunella vulgaris, vinegar turtle shell, Radix Rehmanniae, the root bark of Chinese wolf-berry, rhizoma alismatis, radix scrophulariae, radix gentianae, cortex moutan, chrysanthemum, wheat
Bud, oyster, thallus laminariae prescription composition are that (approval of Bureau of Drugs Supervision, Beijing, authentication code are capital medicine to preparation in the institute of BJ Children's Hospital
Word Z20053679 processed), for treating youngest daughter's swollen breasts, vaginal fluid increases, colporrhagia, or with irritable, mouth
It is very thirsty, feverishness in palms and soles, the diseases such as night sweat.Tongue nature is partially red, and tongue fur is few, and arteries and veins is thin or counts accurately；Young girl's sex premature.
Multiple young mixture effective component is monitored currently, there is no, accurately reflects the report of its quality level, thus can not
Production process and product quality are effectively controlled, cannot preferably guarantee its clinical efficacy.Therefore, the quality of multiple young mixture is established
Control method has very important significance.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of multiple young mixture component detection methods.This method has good
Precision, linear relationship, stability, repeatability, the rate of recovery is high.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme:
The present invention provides a kind of multiple young mixture component detection methods, comprising:
The multiple young mixture test solution of preparation and the standard solution containing Rosmarinic acid and harpagoside, using HPLC into
Row detection obtains the content of Rosmarinic acid and harpagoside in multiple young mixture by external standard method；
The chromatographic condition of the HPLC detection are as follows:
Using acetonitrile as mobile phase A, using phosphoric acid solution as Mobile phase B, gradient elution, the gradient elution program are as follows:
Multiple children's mixture is by Prunella vulgaris, vinegar turtle shell, Radix Rehmanniae, the root bark of Chinese wolf-berry, rhizoma alismatis, radix scrophulariae, radix gentianae, cortex moutan, chrysanthemum, wheat
Bud, oyster, thallus laminariae prescription composition are that (approval of Bureau of Drugs Supervision, Beijing, authentication code are capital medicine to preparation in the institute of BJ Children's Hospital
Word Z20053679 processed), youngest daughter's swollen breasts are used for, vaginal fluid to increase, colporrhagia, or with irritable, dry
Yearningly, feverishness in palms and soles, the diseases such as night sweat.Tongue nature is partially red, and tongue fur is few, and arteries and veins is thin or counts accurately；Above-mentioned patient is shown in young girl's sex premature.
By the discovery that studies for a long period of time, rationally in the multiple young mixture of control Rosmarinic acid and harpagoside quality condition, can be with
Guarantee the clinical efficacy of multiple young mixture.Method of quality control provided by the invention can quickly and accurately measure multiple young mixture simultaneously
Middle Rosmarinic acid and harpagoside content have good precision, linear relationship, stability, repeatability, and the rate of recovery is high, can
Using the effective ways as key active ingredients Rosmarinic acid and harpagoside quality in the multiple young mixture of evaluation.
In some embodiments, the wavelength of HPLC detection are as follows: 0~37min330nm, 37~82min210nm.
In some embodiments, the volume fraction of phosphoric acid is 0.025%-0.035% in phosphoric acid solution.
In certain embodiments, the volume fraction of phosphoric acid is 0.03% in phosphoric acid solution.
In certain embodiments, the chromatographic column of HPLC detection is octadecyl silane chromatographic column.
In certain embodiments, the flow rate of mobile phase of HPLC detection is 1.2mL/min.
In certain embodiments, the column temperature of HPLC detection is 30 DEG C.
In some embodiments, multiple young mixture is oral solution, multiple children's mixture test solution the preparation method comprises the following steps:
Multiple young mixture is taken, 50% ethyl alcohol is added, centrifugation takes supernatant, filters, and takes subsequent filtrate, obtains the multiple young mixture
Test solution.
In a specific embodiment, multiple young mixture test solution the preparation method comprises the following steps:
It is accurate to measure multiple young mixture oral solution 10mL, it sets in 50mL brown volumetric flask, precision 50% ethyl alcohol of addition to scale,
Centrifugation, takes supernatant, filters, takes subsequent filtrate.
In some embodiments, detection method provided by the invention further includes the steps that preparing standard solution, specifically
Ground, prepare standard solution the preparation method comprises the following steps: takes Rosmarinic acid, harpagoside reference substance, respectively plus 50% ethyl alcohol, fan is made
Repeatedly fragrant acid mother liquor and harpagoside mother liquor, measure Rosmarinic acid mother liquor and harpagoside mother liquor respectively, and it is (mixed to obtain standard solution
Mark solution).
In a specific embodiment, standard solution the preparation method comprises the following steps: precision weighs Rosmarinic acid, harpagoside pair
According to product, add 50% ethyl alcohol, the mother liquor of Rosmarinic acid containing 0.4mg/mL is respectively prepared, 0.5mg/mL harpagoside mother liquor is accurate respectively
Rosmarinic acid mother liquor 1.2mL is drawn, harpagoside mother liquor 2.5mL is set in 10mL brown volumetric flask, and 50% ethyl alcohol is added to be settled to quarter
Degree obtains standard solution (mixed mark solution).
In the standard solution, Rosmarinic acid concentration is 0.048mg/mL, harpagoside concentration 0.125mg/mL.
In some embodiments, further include the steps that preparing negative controls solution, the preparation method comprises the following steps: in multiple young mixture
On the basis of, preparation lacks the oral solution of Prunella vulgaris and radix scrophulariae, and 50% ethyl alcohol is added, and centrifugation takes supernatant, filters, takes continuous filter
Liquid obtains the negative controls solution.
Detection method provided by the invention can quickly and accurately measure Rosmarinic acid and harpagoside in multiple young mixture simultaneously
Content, this method have good precision, linear relationship, stability, repeatability, and the rate of recovery is high, can be used as the multiple children of evaluation
The effective ways of key active ingredients Rosmarinic acid and harpagoside quality in mixture.
Specific embodiment
The invention discloses a kind of multiple young mixture component detection method, those skilled in the art can use for reference present disclosure,
It is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications carry out those skilled in the art
Say it is it will be apparent that they are considered as being included in the present invention.Method and application of the invention has passed through preferred embodiment
It is described, related personnel can obviously not depart from the content of present invention, in spirit and scope to method described herein and answer
With being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
To the explanation of the disclosed embodiments, enable those skilled in the art to implement or use the present invention.To this
A variety of modifications of a little embodiments will be readily apparent to those skilled in the art, as defined herein general
Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not
It can be intended to be limited to the embodiments shown herein, and be to fit to consistent with the principles and novel features disclosed in this article
Widest scope.
The test material that the present invention uses is all common commercially available product, can all be bought in market.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
The preparation of standard solution: precision weighs Rosmarinic acid, harpagoside reference substance, adds 50% ethyl alcohol, is respectively prepared
The mother liquor of Rosmarinic acid containing 0.4mg/mL, 0.5mg/mL harpagoside mother liquor is accurate respectively to draw Rosmarinic acid mother liquor 1.2mL, breathes out
Bar Russia glycosides mother liquor 2.5mL is set in 10mL brown volumetric flask, and 50% ethyl alcohol is added to be settled to scale, and obtaining standard solution, (mixed mark is molten
Liquid).
The preparation of test solution: it is accurate to measure multiple young mixture oral solution 10mL, it sets in 50mL brown volumetric flask, precision adds
Enter 50% ethyl alcohol to scale, centrifugation takes supernatant, filters, takes subsequent filtrate.
The preparation of negative controls solution: on the basis of multiple young mixture, preparation lacks the oral solution of Prunella vulgaris and radix scrophulariae,
50% ethyl alcohol is added, is centrifuged, takes supernatant, filters, takes subsequent filtrate, obtain the negative controls solution.
HPLC chromatogram condition are as follows:
Stationary phase: octadecyl silane
Mobile phase: using acetonitrile as mobile phase A, using 0.03% phosphoric acid solution as Mobile phase B gradient elution.
Elution program is shown in Table 1.
1 elution program of table
| Time (min) | A% (acetonitrile) | B% (0.03% phosphoric acid) |
| 0~10 | 3~10 | 97~90 |
| 10~20 | 10~20 | 90~80 |
| 20~40 | 20~30 | 80~70 |
| 40~55 | 30~40 | 70~60 |
| 55~60 | 40~80 | 60~20 |
| 60~80 | 80~85 | 20~15 |
| 80~83 | 85~3 | 15~97 |
Detection wavelength: 0~37min330nm, 37~82min210nm.
Detection temperature: 30 DEG C.
Elution speed: 1.2mL/min.
The test of 2 methodological study of embodiment
(1) specificity is tested
Accurate absorption reference substance solution, test solution, each 10uL of negative control solution inject liquid chromatograph respectively,
It is measured by 1 method of embodiment.Main peak separating effect is preferable as the result is shown, and the indexs such as symmetrical factor, separating degree meet regulation, reason
Reach 3000 or more by the number of plates, negative sample is noiseless.
(2) linear relationship investigates test
Accurately weighed Rosmarinic acid reference substance 8.07mg, sets in 20mL brown volumetric flask, adds 50% ethyl alcohol constant volume；Separately take Kazakhstan
Bar Russia glycosides reference substance 5.02mg, sets in 25mL volumetric flask, adds 50% ethyl alcohol constant volume.It is accurate respectively to draw Rosmarinic acid mother liquor
1.2mL, harpagoside mother liquor 2.5mL are set in 10mL brown volumetric flask, and 50% ethyl alcohol is added to be settled to scale, for mixed mark solution (mark
Quasi- product solution).2,10,25,30,35 μ L of sample introduction respectively is measured by 1 method of embodiment, records peak area, calculates standard curve.
It the results are shown in Table 2 and table 3.
Table 2
Table 3
The results show that Rosmarinic acid standard curve: Y=2996201.9827X-82630.7000 (r=0.9997), Ha Ba
Russia's glycosides standard curve: Y=1668790.1583X-44297.9909 (r=0.9997) is linear good.
(3) precision test
Mixed reference substance solution is taken, according to 1 chromatographic condition continuous sample introduction of embodiment 6 times, peak area is recorded, calculates RSD value.
It the results are shown in Table 4.
Table 4
| NO. | Sampling volume/μ L | Rosmarinic acid peak area | Harpagoside peak area |
| 1 | 10 | 287492 | 211213 |
| 2 | 10 | 289203 | 213306 |
| 3 | 10 | 286163 | 210682 |
| 4 | 10 | 285834 | 209912 |
| 5 | 10 | 287453 | 206874 |
| 6 | 10 | 288790 | 207549 |
| Average value | 287489 | 209922 | |
| STDE | 1233.4912 | 2183.9287 | |
| RSD | 0.43 | 1.04 |
The results show that Rosmarinic acid peak area RSD in mixed mark is 0.43%, harpagoside peak area RSD in mixed mark
It is 1.04%, shows that instrument precision is good.
(4) repeatability measurement test
6 parts of test solutions are prepared in parallel according to the preparation method of 1 test solution of embodiment, according to 1 condition of embodiment
It is measured, records peak area and calculates content, the results are shown in Table 5.
5 repetitive test result of table
The results show that rosmarinic acid contents RSD is 0.33%, harpagoside content RSD is 0.85%, and repeatability is good.
(5) stability test
Prepare test solution according to 1 method of embodiment, and by 1 chromatographic condition of embodiment respectively 0,2,4,8,12h into
Sample measurement records peak area and calculates content, the results are shown in Table 6.
6 stability test result of table
The results show that rosmarinic acid contents RSD is 7.23%, harpagoside content RSD is 8.72%, is had good stability.
(6) it is loaded recovery test
Precision weighs sample powder 5g, sets in 50ml volumetric flask, adds 50% ethyl alcohol, weighs 6 parts in parallel, accurate respectively to be added
The harpagoside solution 1.1mL of Rosmarinic acid solution 0.3mL, 0.2mg/mL of 0.4mg/mL are prepared by 1 preparation method of embodiment
Test solution is measured by 1 chromatographic condition of embodiment, is recorded peak area and is calculated the content rate of recovery, the results are shown in Table 7 and table 8.
7 rate of recovery experimental result of table
8 rate of recovery experimental result of table
The results show that the sample recovery rate of Rosmarinic acid is 99.28%, RSD 0.74%, the sample-adding recycling of harpagoside
Rate is 101.09%, RSD 0.49%, and sample recovery rate is good.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of multiple young mixture component detection method characterized by comprising
The multiple young mixture test solution of preparation and the standard solution containing Rosmarinic acid and harpagoside, are examined using HPLC
It surveys, the content of Rosmarinic acid and harpagoside in multiple young mixture is obtained by external standard method；
The chromatographic condition of the HPLC detection are as follows:
Using acetonitrile as mobile phase A, using phosphoric acid solution as Mobile phase B, gradient elution, the gradient elution program are as follows:
2. detection method according to claim 1, which is characterized in that the wavelength of the HPLC detection are as follows: 0~
37min330nm, 37~82min210nm.
3. detection method according to claim 1, which is characterized in that the volume fraction of phosphoric acid is in the phosphoric acid solution
0.025%-0.035%.
4. detection method according to claim 1, which is characterized in that the flow rate of mobile phase of the HPLC detection is 1.2mL/
min。
5. detection method according to claim 1, which is characterized in that the chromatographic column of the HPLC detection is octadecyl key
Close silica gel chromatographic column.
6. detection method according to claim 1, which is characterized in that the column temperature of the HPLC detection is 30 DEG C.
7. detection method according to claim 1, which is characterized in that the preparation method of the multiple young mixture test solution
Are as follows:
Multiple young mixture is taken, 50% ethyl alcohol is added, centrifugation takes supernatant, filters, and takes subsequent filtrate, obtains the multiple young mixture for examination
Product solution.
8. detection method according to claim 1, which is characterized in that further include the steps that preparing standard solution, it is described
In standard solution, Rosmarinic acid concentration is 0.048mg/mL, harpagoside concentration 0.125mg/mL.
9. detection method according to claim 1, which is characterized in that further include the steps that preparing negative controls solution.
10. detection method according to claim 9, which is characterized in that the negative controls solution manufacturing method are as follows: multiple
On the basis of young mixture, preparation lacks the oral solution of Prunella vulgaris and radix scrophulariae, and 50% ethyl alcohol is added, and centrifugation takes supernatant, filters,
Subsequent filtrate is taken, the negative controls solution is obtained.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103257188A (en) * | 2013-01-05 | 2013-08-21 | 中山大学 | Construction method for compound thrombus clearing preparation bioactivity chromatography finger print |
| CN104524359A (en) * | 2014-12-31 | 2015-04-22 | 吉林省德商药业有限公司 | Traditional Chinese medicine composition and preparing method and application thereof |
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2018
- 2018-12-14 CN CN201811534482.3A patent/CN109541068A/en active Pending
Patent Citations (2)
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| CN103257188A (en) * | 2013-01-05 | 2013-08-21 | 中山大学 | Construction method for compound thrombus clearing preparation bioactivity chromatography finger print |
| CN104524359A (en) * | 2014-12-31 | 2015-04-22 | 吉林省德商药业有限公司 | Traditional Chinese medicine composition and preparing method and application thereof |
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| Title |
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| BAGHDIKIAN, B 等: "Extraction by solvent using microwave and ultrasound-assisted techniques followed by HPLC analysis of Harpagoside from Harpagophytum procumbens and comparison with conventional solvent extraction methods", 《COMPTES RENDUS CHIMIE》 * |
| 宋旭红 等: "不同配方肥对玄参产量及品质的影响研究", 《时珍国医国药》 * |
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