CN107102087A - A kind of method of a variety of organic acid contents in chromatography of ions detection coptis - Google Patents
A kind of method of a variety of organic acid contents in chromatography of ions detection coptis Download PDFInfo
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- CN107102087A CN107102087A CN201710507238.7A CN201710507238A CN107102087A CN 107102087 A CN107102087 A CN 107102087A CN 201710507238 A CN201710507238 A CN 201710507238A CN 107102087 A CN107102087 A CN 107102087A
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- acid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Abstract
The invention provides a kind of method that chromatography of ions detects a variety of organic acid contents, step is as follows：A, prepare need testing solution；B, preparation reference substance solution；C, respectively by need testing solution and reference substance solution injection ion chromatograph detection；D, calculated according to testing result and obtain organic acid content in the coptis.The inventive method can determine a variety of organic acid contents in the coptis simultaneously, accurately and reliably, easy quick, provide and effectively refer to for the clinical practice of the coptis.
Description
Technical field
The present invention relates to the method for a variety of organic acid contents in a kind of chromatography of ions detection coptis.
Background technology
The coptis is ranunculaceae plant coptis Coptis chinensis Franch., triangle leaf coptis Coptis
Deltoidea C.Y.Chenget Hsiao or cloud connect Coptis teeta wall. dry rhizome, practise respectively for three kinds and claim " taste
Even ", " refined company " and " cloud company ".Main product Sichuan, Hubei, Hunan, Shaanxi, Gansu etc., which are saved, also cultivation.It is bitter, cold in nature, the thoughts of returning home,
Spleen, stomach, liver, courage, large intestine channel.With heat-clearing and damp-drying drug, effect of purging intense heat and detonicating.It is mainly used in damp and hot feeling of fullness, vomiting acid regurgitation, rushes down
Dysentery, jaundice, unconsciousness due to high fever, heart-fire hyperactivity, dysphoria and insomnia, blood-head, hot eyes, toothache, carbuncle swells furunculosis, control eczema, wet sore, duct outside
Suppurate.
The coptis contains the plurality of active ingredients such as Flavonoid substances, amino acid, organic acid, and organic acid is the important substance of the coptis
Composition, research shows, organic acid composition has the extensive bioactivity such as antitumor, antibacterial, ascorbic acid, reducing blood lipid, can be with
It is used as the index of quality control.
At present, separation and detection of the document report to organic acid in the coptis are had, method mainly has thin-layered chromatography, hair
Cons electrophoresis method and high performance liquid chromatography, but the kinds of organic acids determined only relates to vanillic acid, forulic acid etc. and has no more than 4 kinds
Machine acid, species is less.It is various in view of the Determination of Organic Acids in Rhizoma Coptidis, quality standard is used as using the content of a small number of organic acids
Index reliability it is not high.But it there is no at present disposable to the chinic acid in the coptis, acetic acid, tartaric acid, oxalic acid, citric acid etc. five
Above organic acid is planted, the report determined simultaneously is carried out.
The content of the invention
In order to more effectively control Rhizoma Coptidis quality, have it is an object of the invention to provide a variety of in a kind of coptis simultaneously
The method of machine acid content.
The invention provides a kind of method that chromatography of ions detects a variety of organic acid contents, step is as follows：
A, prepare need testing solution：
Golden Thread is taken, extracting in water, filtering obtains need testing solution；
B, preparation reference substance solution：Prepare the hybrid standard working solution of organic acid；
C, respectively by need testing solution and reference substance solution injection ion chromatograph detection, chromatographic condition is as follows：
Chromatographic column：AS11-HC chromatographic columns；
Mobile phase：KOH solution；
Flow velocity：0.9~1.1mL/min；
Gradient program is：
Column temperature：30℃；
D, calculated according to testing result and obtain organic acid content in the coptis.
Wherein, the organic acid is chinic acid, acetic acid, tartaric acid, oxalic acid, citric acid, malic acid, butanedioic acid, formic acid, crow
Head acid in two or more.
Further, the organic acid is chinic acid, acetic acid, tartaric acid, oxalic acid and citric acid；
Wherein, the organic acid also includes formic acid and aconitic acid；And/or, the organic acid also includes malic acid and amber
Acid.
Wherein, in step a, the condition of the extracting in water is：The water of 50 times of amounts, ultrasound are added in Golden Thread
30min, is then placed 30 minutes.
Wherein, in step a, the condition of the filtering is：By the sample suction filtration after the processing that adds water, RP posts are crossed, then mistake
0.2um miillpore filters.
Wherein, in step c, the ion chromatograph model ICS-2100.
Wherein, the flow velocity of the chromatographic condition is 1.0mL/min.
Wherein, the electric conductivity detector detection cell temperature of the chromatographic condition is 35 DEG C.
Wherein, the need testing solution and the sample size of reference substance solution are 25 μ L.
The present invention has been successfully established while determining a variety of organic in the coptis by the screening to extracting method and chromatographic condition
The ion chromatographic method of acid content, this method accurately and reliably, it is easy quick, to improve the quality control level of the coptis, promoting medicine
The exploitation of material are laid a good foundation.Meanwhile, method of the invention can also accurately detect in different base source coptis kinds 5 kinds with
Upper Determination of Organic Acids, effective reference is provided for the clinical practice of the coptis.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other diversified forms can also be made, replaces or changes.
The embodiment of form, remakes further specifically to the above of the invention by the following examples
It is bright.But the scope that this should not be interpreted as to above-mentioned theme of the invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
The various organic acid standard diagrams of Fig. 1
Embodiment
It is described further below with embodiment, but the present invention is not limited to these embodiments.
Experiment reagent used in the present invention, can be bought by commercial system.
Wherein, ion chromatograph：Model ICS-2100, producer is U.S.'s thermoelectricity.
The inventive method of embodiment 1 determines organic acid content in the coptis
1st, standard liquid is prepared
Chinic acid, acetic acid, formic acid, tartaric acid, oxalic acid, citric acid, aconitic acid hybrid standard stock solution:Weigh respectively each
Organic acid 50mg is in 50mL volumetric flasks, and be dissolved in water constant volume, mixes, in 4 DEG C of preservations, wherein the mass concentration of each organic acid is
1000μg/mL。
Chinic acid, acetic acid, formic acid, tartaric acid, malic acid, butanedioic acid, oxalic acid, citric acid and aconitic acid blended standard curve
Working solution：Mixed standard solution (3.4.1) 0.50mL, 1.00mL, 2.00mL, 5.00mL, 10.00mL is drawn respectively in 25mL to hold
In measuring bottle, scale is settled to ultra-pure water, is mixed, in 4 DEG C of preservations.
2nd, sample treatment
First a small amount of sample is ground with grater, discarded, broken remainder is regrinded, fully mixes, is used as sample to be tested.Claim
The coptis sample of 2g ± 0.0020 or so is taken, is placed in 250mL volumetric flasks, the accurate 100mL ultra-pure waters that add extract sample, shake
Even, ultrasonic extraction 30min, suction filtration after placing 30 minutes crosses RP posts, crosses 0.2um miillpore filters, places to be measured after 30min.
The concrete operations for crossing RP posts are as follows：RP posts are activated with 10ml methanol first, 15ml water are then crossed, after activation half an hour
Directly upper sample extracting solution, discards above 3ml, collects efflux below, you can.
3. chromatography of ions
1) chromatographic condition is as follows：
Chromatographic column：AS11-HC
Mobile phase：KOH solution (exempting from chemical reagent) external water mode
Flow velocity：1.0mL/min.
Detector：Electric conductivity detector, detection cell temperature：35℃.
Column temperature：30℃.
Sample size is 25 μ L.
2) Specification Curve of Increasing：
Standard series working solution is injected separately into high performance liquid chromatograph, corresponding peak height or peak area is determined.To mark
The concentration of quasi- working solution is abscissa, using chromatogram peak height or peak area as ordinate, draws standard curve.
3) measure of sample solution
Sample solution is injected in ion chromatograph, peak height or peak area is obtained, is obtained according to standard curve in prepare liquid
The concentration of organic acid.
4. interpretation of result
The content of organic acid is calculated as follows in sample：
In formula：
Constituent content is tested in X---- samples, unit is grams per kilogram (g/kg)；
The tested component solution concentration that C---- is obtained from standard working curve, unit is micrograms per millilitre (μ g/ml)；
The final constant volume of V---- sample solutions, unit is milliliter (ml)；
The quality of final sample representated by M---- sample solutions, unit is gram (g).
Beneficial effects of the present invention are illustrated below by way of test example.
The technological parameter screening of the inventive method of test example 1
First, the comparison of sample Different treatments
It is as follows to removing the effect that rhizoma extracting liquid larger molecular organicses are disturbed, method that experiment compares C18, RP post：System
During standby coptis need testing solution, by the way that respectively through C18 and RP posts, rhizoma extracting liquid is collected into eluent, compare elution front and rear
The area of chromatography of ions Interference Peaks, draws clearance of each pillar to rhizoma extracting liquid organic matter.
It the results are shown in Table 1.
Table 1 C18, RP post is to removing the result that rhizoma extracting liquid larger molecular organicses are disturbed
It can be seen that, RP posts remove the best results of rhizoma extracting liquid larger molecular organicses, up to 93%, are significantly better than C18 posts.
2nd, the separating effect of different chromatographic columns compares
Experiment compares two kinds of chromatographic columns (AS 11-HC and AS-15) separating effect, as a result shows, As-15 chromatographic columns
It is not so good as As11-HC in the separating effect of chinic acid, acetic acid and formic acid, separating degree is poor, does not reach baseline separation；And AS 11-HC
There is good separating effect to the organic acid of the present invention compared to AS-15.
Measurement result of the inventive method of test example 2 to the different base sources coptis
Take refined company (picking up from Emei, Sichuan Province), cloud to connect (picking up from Yunnan Fugong), taste respectively and connect (picking up from strata in Shizhu, Chongqing), according to reality
The method for applying example 1 determines respective Determination of Organic Acids.It the results are shown in Table 2.
The testing result of the different coptis organic acids of 2 pairs of table
| Refined company (mg/g) | Yun Lian (mg/g) | Taste connects (mg/g) | |
| Chinic acid | 4.00 | 27.83 | 2.90 |
| Acetic acid | 1.03 | 0.18 | 0.78 |
| Malic acid+butanedioic acid | 9.93 | 15.25 | 9.82 |
| Tartaric acid | 0.76 | 0.46 | 0.96 |
| Oxalic acid | 1.25 | 0.34 | 0.64 |
| Citric acid | 1.40 | 1.53 | 1.19 |
| Formic acid | Do not detect | Do not detect | Do not detect |
| Aconitic acid | Do not detect | Do not detect | Do not detect |
It can be seen that, the inventive method can determine various organic acid contents in the different Ji Yuan coptis.
The Method validation of the inventive method of test example 3
1st, the investigation of linear relationship
The organic acid mixed standard solution for including the following range of linearity is prepared, ion color is carried out according to the method for embodiment 1
Analysis of spectrum, and mapped with each ion concentration and peak area, the working curve of each ion is obtained, 3 are the results are shown in Table.
The range of linearity of the different organic acids of table 3
It can be seen that, each reference substance linear relationship in the range of respective mass concentration is good, illustrates the inventive method range of linearity
Extensively, the degree of accuracy is high.
2nd, the investigation of test limit
10 standard liquids close to blank are prepared, according to the method for embodiment 1, ion chromatography are carried out respectively, with 3
Times signal-to-noise ratio computation goes out the qualitative detection limit (being represented with concentration) of each organic acid, and 10 times of signal to noise ratio are the quantitative detection of each organic acid
Limit (concentration).By the sample weighting amount and constant volume of embodiment 1, detection limit (being represented with content) is calculated.
The test limit of the different organic acids of table 4
It can be seen that, the inventive method can detect the organic acid of various low concentrations, and sensitivity is high.
3rd, the investigation of the rate of recovery
Example is linked as with cloud, adding each organic acids of three concentration levels in the sample respectively, (sample of 1. additions 50% is dense
Degree；2. the sample concentration of addition 100%；3. the sample concentration of addition 150%), detect, obtain according to the method for embodiment 1
Rate of recovery scope is shown in Table 5.
The rate of recovery of the organic acid of table 5 (example is linked as with cloud)
It can be seen that, using the inventive method, sample recovery rate is higher, illustrates that the inventive method degree of accuracy is high..
4th, repeated investigation
Example is linked as with cloud, according to the method for embodiment 1,6 replications have been carried out respectively, the RSD of measurement result is calculated,
It the results are shown in Table 6.
The repeatability of the organic acid of table 6 (example is linked as with cloud)
It can be seen that, using the inventive method, the relative standard deviation of each organic acid is respectively less than 10%, illustrate the inventive method
It is repeated good.
To sum up, the present invention has been successfully established while determining many in the coptis by the screening to extracting method and chromatographic condition
The ion chromatographic method of kind of organic acid content, this method accurately and reliably, it is easy to be quick, for improve the coptis quality control level,
The exploitation of medicinal material are promoted to lay a good foundation.
Claims (10)
1. a kind of method that chromatography of ions detects a variety of organic acid contents, it is characterised in that：Step is as follows：
A, prepare need testing solution：
Golden Thread is taken, extracting in water, filtering obtains need testing solution；
B, preparation reference substance solution：Prepare the hybrid standard working solution of organic acid；
C, respectively by need testing solution and reference substance solution injection ion chromatograph detection, chromatographic condition is as follows：
Chromatographic column：AS11-HC chromatographic columns；
Mobile phase：KOH solution；
Flow velocity：0.9~1.1mL/min；
Gradient program is：
Column temperature：30℃；
D, calculated according to testing result and obtain organic acid content in the coptis.
2. according to the method described in claim 1, it is characterised in that：The organic acid be chinic acid, acetic acid, tartaric acid, oxalic acid,
Two or more in citric acid, malic acid, butanedioic acid, formic acid, aconitic acid.
3. method according to claim 2, it is characterised in that：The organic acid is chinic acid, acetic acid, tartaric acid, oxalic acid
And citric acid.
4. method according to claim 3, it is characterised in that：The organic acid also includes formic acid and aconitic acid；And/or,
The organic acid also includes malic acid and butanedioic acid.
5. according to the method described in claim 1, it is characterised in that：In step a, the condition of the extracting in water is：In the coptis
The water of 50 times of amounts is added in powder, then ultrasonic 30min places 30 minutes.
6. according to the method described in claim 1, it is characterised in that：In step a, the condition of the filtering is：To be added water processing
Sample suction filtration afterwards, crosses RP posts, then crosses 0.2um miillpore filters.
7. according to the method described in claim 1, it is characterised in that：In step c, the ion chromatograph model ICS-
2100。
8. according to the method described in claim 1, it is characterised in that：The flow velocity of the chromatographic condition is 1.0mL/min.
9. according to the method described in claim 1, it is characterised in that：The electric conductivity detector detection cell temperature of the chromatographic condition is
35℃。
10. according to the method described in claim 1, it is characterised in that：The sample size of the need testing solution and reference substance solution
For 25 μ L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710507238.7A CN107102087B (en) | 2017-06-28 | 2017-06-28 | Method for detecting contents of various organic acids in coptis chinensis by ion chromatography |
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| CN201710507238.7A CN107102087B (en) | 2017-06-28 | 2017-06-28 | Method for detecting contents of various organic acids in coptis chinensis by ion chromatography |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110954631A (en) * | 2019-12-28 | 2020-04-03 | 湖南九典制药股份有限公司 | Method for determining citrate and aconitic acid contents |
| CN115327021A (en) * | 2022-07-22 | 2022-11-11 | 上海柏狮生物科技有限公司 | Ion chromatography for determining ferric citrate and organic acid impurities thereof |
-
2017
- 2017-06-28 CN CN201710507238.7A patent/CN107102087B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110954631A (en) * | 2019-12-28 | 2020-04-03 | 湖南九典制药股份有限公司 | Method for determining citrate and aconitic acid contents |
| CN115327021A (en) * | 2022-07-22 | 2022-11-11 | 上海柏狮生物科技有限公司 | Ion chromatography for determining ferric citrate and organic acid impurities thereof |
| CN115327021B (en) * | 2022-07-22 | 2024-01-23 | 上海柏狮生物科技有限公司 | Ion chromatography for determining ferric citrate and organic acid impurities thereof |
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