CN110031564A - The quality determining method of natural plants anticoccidial feed addictive based on HPLC finger-print - Google Patents
The quality determining method of natural plants anticoccidial feed addictive based on HPLC finger-print Download PDFInfo
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Abstract
The present invention provides a kind of quality determining method of natural plants anticoccidial feed addictive based on HPLC finger-print, the preparation including test solution, chlorogenic acid, caffeic acid, the preparation of 3 kinds of reference substance solutions of aurantiamarin and HPLC detection.The present invention has demarcated 18 shared peaks altogether, wherein pointing out 3 known peaks.The present invention identifies natural plants anticoccidial feedstuff additive product using the method for establishing HPLC finger-print for the first time, this method establish natural plants anticoccidial feed addictive HPLC finger-print with compare the similarity of map 90% or more, its feature of energy Efficient Characterization, is conducive to overall monitor product quality.The technology of the present invention content is high, avoids the unicity and one-sidedness of quality control；This method is easy to operate simultaneously, reproducible, with a high credibility, promotes and applies convenient for processing of crude drugs, sale enterprise and quality inspection unit.
Description
Technical field
The present invention relates to high performance liquid chromatography detection technologies, specifically, being related to a kind of day based on HPLC finger-print
The quality determining method of right Genes For Plant Tolerance coccidiosis feed additive.
Background technique
In poultry cultivation, globidiosis is to seriously endanger one of the disease of aquaculture, and the incidence of the disease is high, is distributed model
It encloses extensively, case fatality rate is up to 40%~80%.Like that green feel well is a kind of safety height of Beijing Ai Lv Biotechnology Co., Ltd production
Effect, noresidue are not likely to produce the anticoccidial feed addictive of drug resistance (referring to ZL201410832060.X, denomination of invention: one kind
Natural plants anticoccidial feed addictive and its application).The feed addictive can effectively press down the spore for worm of killing, the first generation and
Two generation schizonts reduce its disease incidence and diseased region are avoided to continue to expand；Immunity of organism and premunition are improved simultaneously, reduce disease
Protocorm worm infects body；Protection and the impaired gastrointestinal mucosa of reparation, promote the absorption of nutriment, have and press down worm of killing, increase
Strong immunity repairs gastrointestinal tract triple function, and Product Green environmental protection is suitble to widely popularize in animal husbandry field.
Finger-print refer in certain natural plants or plant extracts common to, there is certain characteristic class or number
The chromatography of constituents or the map of spectrum.Like it is green feel well as natural plant extracts compound product, at this stage, natural plants have
The effect ingredient overwhelming majority does not have in specific situation, and HPLC finger-print is aobvious for the quality of natural plants or plant extracts
It obtains increasingly important.Therefore, the HPLC finger-print for establishing natural plants anticoccidial feed addictive, the quality inspection for product
It is significant.
Summary of the invention
The matter of the object of the present invention is to provide a kind of natural plants anticoccidial feed addictive based on HPLC finger-print
Quantity measuring method.
Present inventive concept is as follows: in natural plants anticoccidial feed addictive (love green refreshing product), containing Astragalus Root P.E,
Artemisinin, Radix Dichroae extract, Herba Agrimoniae extract, eucommia ulmoides extracts, dandelion extract, atractylodes chinensis, mulberry leaf mention
Object, aloe extract, dried orange peel extracts, haw thorn extract, licorice etc. are taken, according to product anticoccidial mechanism, wherein having
Imitating ingredient is mainly aurantiamarin (crude drug dried orange peel), caffeic acid (crude drug dandelion), chlorogenic acid (crude drug dandelion), aloe
It is glycosides (crude drug aloe), Astragaloside IV (crude drug Radix Astragali), qinghaosu (crude drug sweet wormwood), orixine (crude drug Changshan), sweet
Oxalic acid (crude drug Radix Glycyrrhizae) etc. is obtained natural after sample to be tested pre-treatment and mobile phase of high performance liquid chromatography gradient elution
18 shared peaks of Genes For Plant Tolerance coccidiosis feed additive HPLC finger-print, wherein 3 known peaks correspond respectively to chlorogenic acid, coffee
Acid, aurantiamarin, remaining 15 peak is characterized fingerprint peaks.
In order to achieve the object of the present invention, the present invention provides a kind of natural plants anticoccidial feed addictive HPLC finger-print
Method for building up and its finger-print, be using chlorogenic acid, caffeic acid, aurantiamarin this 3 kinds of ingredients as characteristic component as reference,
Using its finger-print of high effective liquid chromatography for measuring.The method includes preparing test sample, reference substance solution, gradient elution with
And finger-print is established, control map generates, characteristic peak is pointed out, similarity calculation.
The quality determining method of natural plants anticoccidial feed addictive provided by the invention based on HPLC finger-print,
Specifically includes the following steps:
1) preparation of test solution
Natural plants anticoccidial feed addictive sample is taken, is dissolved with methanol, is ultrasonically treated, filtration is collected filtrate, will be filtered
Liquid is evaporated, and methanol is added into residue and redissolves and with after filtering with microporous membrane, as test solution；
2) preparation of 3 kinds of reference substance solutions
Using methanol as solvent, chlorogenic acid reference substance solution, caffeic acid reference substance solution and aurantiamarin reference substance are prepared respectively
Solution, concentration are respectively 0.5~1.5mg/ml, 0.2~0.8mg/ml and 0.5~1.5mg/ml, and preferred concentration is respectively 1mg/
Ml, 0.2~0.8mg/ml and 0.5~1.5mg/ml；
3) HPLC is detected
Chromatographic condition: AgilentSB-C18 chromatographic column, specification 4.6mm × 250mm, 5 μm；Detection wavelength 322-375nm；
30-45 DEG C of column temperature；Sample volume: 5-15 μ l；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid solution, mobile phase A and Mobile phase B
The sum of concentration is 100%, carries out gradient elution；Flow velocity 0.5-1mlmin-1；
Elution requirement:
0-10min, mobile phase A: 10-15%, Mobile phase B: 90-85%；
10-20min, mobile phase A: 15-18%, Mobile phase B: 85-82%；
20-30min, mobile phase A: 18-20%, Mobile phase B: 82-80%；
30-40min, mobile phase A: 20-30%, Mobile phase B: 80-70%；
40-45min, mobile phase A: 30-10%, Mobile phase B: 70-90%；
45-47min, mobile phase A: 10%, Mobile phase B: 90%；
Test solution is injected into high performance liquid chromatograph, obtains HPLC finger-print；It is molten with 3 kinds of reference substances respectively simultaneously
Liquid sample introduction, determine chlorogenic acid, caffeic acid and aurantiamarin goes out peak position；Measurement result are as follows: amount to 18 shared peaks, wherein 3,5,
No. 11 peaks, respectively chlorogenic acid, caffeic acid, aurantiamarin, and No. 3 peaks are internal reference peak, remaining 15 peak is characterized fingerprint peaks；Respectively
The relative retention time at a peak is respectively 3.108,3.988,10.029,10.655,12.672,15.343,18.867,
22.379,24.182,28.575,35.540,37.646,38.315,40.767,41.486,41.899,42.551,43.573,
Average relative peak area than be respectively 2320.555,643.841,9545.936,633.801,4930.843,719.967,
1044.565、5439.596、1193.835、812.337、703.755、6692.912、10358.90、8624.709、
436.598、833.476、2554.748、7052.612。
Natural plants anticoccidial feed addictive sample 2g is taken in step 1), is dissolved with methanol 10-30ml, and 60W, 45Hz are super
Sonication 15-30min；Residue is added methanol 1-3ml and redissolves, as test solution.
In the present invention, the natural plants anticoccidial feed addictive is made by the raw material of following parts by weight: astragalus extraction
50-100 parts of object, 50-150 parts of Artemisinin, 50-150 parts of Radix Dichroae extract, 50-100 parts of Herba Agrimoniae extract, Cortex Eucommiae mention
Take 50-100 parts of object, 50-100 parts of dandelion extract, 50-100 parts of atractylodes chinensis, 50-100 parts of mulberry-leaf extract, aloe
50-100 parts of extract, 50-100 parts of dried orange peel extracts, 50g-100 parts of haw thorn extract, 50-100 parts of licorice.
In the specific embodiment of the present invention, the natural plants anticoccidial feed based on HPLC finger-print
The quality determining method of additive the following steps are included:
The preparation of S1, test solution
Natural plants anticoccidial feed addictive sample 2g is taken, is dissolved with methanol 20ml, 60W, 45Hz ultrasonic treatment
20min；Then it filters, collects filtrate, filtrate is evaporated, methanol 2ml is added into residue and redissolves, as test solution；
The preparation of S2,3 kinds of reference substance solutions
Using methanol as solvent, chlorogenic acid reference substance solution, caffeic acid reference substance solution and aurantiamarin reference substance are prepared respectively
Solution, concentration are respectively 1mg/ml, 0.5mg/ml and 1mg/ml；
S3, HPLC detection
Chromatographic condition: AgilentSB-C18 chromatographic column, specification 4.6mm × 250mm, 5 μm；Detection wavelength 355nm；Column temperature
40℃；Sample volume: 10 μ l；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid solution, the sum of mobile phase A and Mobile phase B concentration
It is 100%, carries out gradient elution；Flow velocity 0.8mlmin-1；
Elution requirement:
0-10min, mobile phase A: 10-15%, Mobile phase B: 90-85%；
10-20min, mobile phase A: 15-18%, Mobile phase B: 85-82%；
20-30min, mobile phase A: 18-20%, Mobile phase B: 82-80%；
30-40min, mobile phase A: 20-30%, Mobile phase B: 80-70%；
40-45min, mobile phase A: 30-10%, Mobile phase B: 70-90%；
45-47min, mobile phase A: 10%, Mobile phase B: 90%.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) present invention identifies natural plants anticoccidial feed addictive using the method for establishing HPLC finger-print for the first time
Product, the natural plants anticoccidial feed addictive HPLC finger-print that this method is established exist with the similarity for compareing map
90% or more, its feature of energy Efficient Characterization is conducive to overall monitor product quality.
(2) the natural plants anticoccidial feed addictive HPLC finger-print that the present invention establishes has been demarcated 18 altogether and has been shared
Peak, wherein 3 known peaks are pointed out, it is with high content of technology, avoid the unicity and one-sidedness of quality control；This method operation simultaneously
Simply, reproducible, it is with a high credibility, it is promoted and applied convenient for processing of crude drugs, sale enterprise and quality inspection unit.
Detailed description of the invention
Fig. 1-Fig. 4 is to investigate-0.1% phosphoric acid of methanol ,-0.1% phosphoric acid of acetonitrile, acetonitrile-respectively in the embodiment of the present invention 1
The result of four kinds of 0.2% phosphoric acid, acetonitrile-water difference flow phase systems.
Fig. 5-Fig. 7 is to investigate 30 DEG C of different column temperatures, 40 DEG C, 45 DEG C of result in the embodiment of the present invention 1 respectively.
Fig. 8-Figure 10 is the result for investigating different Detection wavelength 322nm, 355nm, 375nm in the embodiment of the present invention 1 respectively.
Figure 11 is the HPLC finger-print testing result of 10 batches of green refreshing products of love in the embodiment of the present invention 4.
Figure 12 shows 18 shared peaks that green refreshing product calibration is liked in the embodiment of the present invention 2 and 4.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Instrument is respectively as follows: 1260 high performance liquid chromatograph of Agilent (quaternary pump, DAD detection in following embodiment
Device, autosampler, on-line degassing)；Double frequency numerical control ultrasonic cleaner (KQ-500VD type)；Electric drying oven with forced convection (WGL-
45B).Agents useful for same are as follows: methanol, chromatographically pure (Fisher company, the U.S.)；Acetonitrile, chromatographically pure (Fisher company, the U.S.)；Phosphoric acid,
Chromatographically pure (Aladdin reagent)；Ultrapure water (ultrapure water machine: U.S. Ai Kepu).
Reference substance: chlorogenic acid (lot number: Y24J7K16726)；Caffeic acid (lot number: Y17D6C7672)；Aurantiamarin (lot number:
M03J8S39169).Above-mentioned reference substance is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.
Sample: being processed by Hebei Ai Lv bioengineering Co., Ltd under Ai Lv group, obtains 10 batches of green refreshing samples of love
Product (lot number is shown in Table 1).
1 sample lot number of table
It is the green refreshing product of love of HBAL20181215 that lot number is used in embodiment 1-3, is liked in embodiment 4 using 10 batches
Green refreshing product.
The foundation of 1 HPLC finger-print of embodiment and the selection and optimization of chromatographic condition
It is complicated with content at being grouped as due to liking that green refreshing product is natural plant extracts compound product, it is final determining
Before chromatographic condition, a large amount of preliminary experiments need to be carried out, to determine optimal conditions.
1, the optimization of mobile phase
Like the chemical component complicated composition of green refreshing product and most of compositional polarities are larger, each group swarming when Isocratic clution
Separating effect is bad, can not be each at swarming in effective district sorting sample, therefore gradient elution is selected to carry out chromatographic isolation.This hair
It is bright to have investigated the different flowings such as -0.1% phosphoric acid of methanol, -0.1% phosphoric acid of acetonitrile, -0.2% phosphoric acid of acetonitrile and acetonitrile-water respectively
Phase system, chromatogram are shown in Fig. 1-Fig. 4 respectively, by comparing the chromatogram obtained under four kinds of different flow phase systems, acetonitrile-
The map baseline that the flow phase system of 0.1% phosphoric acid obtains is steady, each peak is evenly distributed, separating effect is preferable, therefore selects second
- 0.1% phosphoric acid of nitrile is as mobile phase.
2, the optimization of column temperature
Adjust respectively 30 DEG C of column temperature, 40 DEG C, 45 DEG C sample is measured, obtained under three kinds of different temperatures by comparing
Chromatogram (Fig. 5-Fig. 7), each peak separating degree of the map that column temperature is obtained at 40 DEG C is good, therefore column temperature is optimized for 40 DEG C.
3, the optimization of Detection wavelength
The test sample under 322nm, 355nm, 375nm wavelength respectively obtains under three kinds of different Detection wavelengths by comparing
Chromatogram (Fig. 8-Figure 10), as seen from the figure, chromatogram peak shape under 355nm wavelength is stablized, and each peak is evenly distributed, separating effect
It is good, therefore preferably Detection wavelength is 355nm.
Embodiment 2 is measured based on HPLC establishes the finger-print for liking green refreshing product
The method for building up of HPLC finger-print is as follows:
1. the preparation of test solution
The green refreshing product 2g of love is taken, it is accurately weighed, it is dissolved with methanol 20ml, 60W, 45Hz are ultrasonically treated 20min；Then it filters
It crosses, collects filtrate, filtrate is evaporated, methanol 2ml is added into residue and redissolves, as test solution.
2. the preparation of reference substance solution
Chlorogenic acid: taking chlorogenic acid reference substance 5mg, accurately weighed, is dissolved with methanol, be settled in 5ml volumetric flask to get
The chlorogenic acid reference substance solution of 1mg/ml.
Caffeic acid: taking caffeic acid reference substance 2.5mg, accurately weighed, is dissolved with methanol, be settled in 5ml volumetric flask to get
The aurantiamarin reference substance solution of 0.5mg/ml.
Aurantiamarin: taking aurantiamarin reference substance 5mg, accurately weighed, is dissolved with methanol, be settled in 5ml volumetric flask to get
The aurantiamarin reference substance solution of 1mg/ml.
3. HPLC is detected
Each 10 μ l of above-mentioned test solution and control solution is implanted sequentially high performance liquid chromatograph, by high-efficient liquid phase color
Spectrometry is measured, and is recorded the chromatogram in 47min, is handled with finger-print software, obtain the fingerprint for liking green refreshing product
Map.
4. high-efficient liquid phase chromatogram condition specifically:
Chromatographic column: AgilentSB-C18 (4.6mm × 250mm, 5 μm)；
Mobile phase: mobile phase A: acetonitrile；Mobile phase B: 0.1% phosphoric acid solution
Flow velocity 0.8mlmin-1；
Detection wavelength 355nm；
Column temperature: 40 DEG C.
5. during gradient elution, the variation ratio of mobile phase A and Mobile phase B are as follows:
0-10min, mobile phase A: 10-15%, Mobile phase B: 90-85%；
10-20min, mobile phase A: 15-18%, Mobile phase B: 85-82%；
20-30min, mobile phase A: 18-20%, Mobile phase B: 82-80%；
30-40min, mobile phase A: 20-30%, Mobile phase B: 80-70%；
40-45min, mobile phase A: 30-10%, Mobile phase B: 70-90%；
45-47min, mobile phase A: 10%, Mobile phase B: 90%.
Measurement result is as shown in figure 12, amount to 18 shared peaks, wherein 3,5, No. 11 peaks, respectively chlorogenic acid, caffeic acid,
Aurantiamarin, and No. 3 peaks are internal reference peak, remaining 15 peak is characterized fingerprint peaks；The relative retention time at each peak is respectively
3.108、3.988、10.029、10.655、12.672、15.343、18.867、22.379、24.182、28.575、35.540、
37.646,38.315,40.767,41.486,41.899,42.551,43.573, average relative peak area ratio is respectively
2320.555、643.841、9545.936、633.801、4930.843、719.967、1044.565、5439.596、
1193.835、812.337、703.755、6692.912、10358.90、8624.709、436.598、833.476、2554.748、
7052.612。
The methodological study of 3 HPLC finger-print of embodiment
Methodological study is carried out to the HPLC finger-print established in embodiment 2, specific as follows:
Using chlorogenic acid as internal reference peak, with its peak area for 1, calculates and like other peaks in green refreshing product HPLC finger-print
Relative peak area calculates relative standard deviation RSD value, investigates to methodology.
1, precision is investigated
The green refreshing product test solution of love is taken, continuous sample introduction 6 times, HPLC map is measured, when calculating the reservation at each shared peak
Between and relative peak area RSD value.
The results show that the RSD value of retention time is between 0.1%~0.5%, the RSD of relative peak area 1.2%~
Between 2.5%, RSD value shows that instrument performance is good less than 3%, meets and establishes finger-print requirement.
2, reproducibility is investigated
Green 6 parts of the refreshing product of love is taken, it is accurately weighed, it is prepared by the preparation method of test liquid, measures HPLC map, calculated each total
There are the retention time at peak and the RSD value of relative peak area.
The results show that the RSD value of retention time, between 0.1%~0.5%, the RSD value of relative peak area is 0.56%
Between~2.99%, RSD value shows that the reproducibility of sample preparation methods is good, meets and establish finger-print requirement less than 3%.
3, study on the stability
Take the green refreshing product test solution of freshly prepd love, place at room temperature, respectively at 0,2,4,8,12, detect for 24 hours
Finger-print calculates the retention time at each shared peak and the RSD value of relative peak area.
The results show that the RSD value of retention time is 0.2%~0.58%, the RSD value of relative peak area 0.23%~
2.48%, RSD value show sample solution interior for 24 hours basicly stable less than 3%.
Embodiment 4 likes the HPLC finger-print detection of green refreshing product
The present embodiment likes that green refreshing product is measured analysis to S1~S10 batch according to 1 providing method of embodiment, is referred to
Line map.
The chromatographic data of 10 batches of green refreshing products of love is imported into " similarity evaluation "
In (2012.130723 version) software, chromatographic peak correction matching is carried out, automatically generates the green refreshing product reference fingerprint (figure of love
11), if No. 3 peaks (chlorogenic acid) are to calculate each chromatographic peak relative retention time and relative peak area (table 2) referring to peak (s).
According to 10 batches of green refreshing product HPLC fingerprint map analyzings of love as a result, the green refreshing product of love demarcates 18 shared peaks, share
The peak area at peak accounts for the 91% of total peak area, and selected shared peak can more comprehensively reflect chemical component information in sample,
In have 3 known peaks, i.e., 3,5, No. 11 peaks, respectively chlorogenic acid, caffeic acid, aurantiamarin, other are unknown peak (Figure 12).
Using common pattern as reference, counted by " traditional Chinese medicine fingerprint similarity calculation software " (2012.130723 version)
Calculate the similarity (table 3) of 10 batches of green refreshing product HPLC finger-prints of love, it can be seen that the similarity of 10 batches of green refreshing products of love exists
80% or more, compared with compareing map (R), similarity 90% or more,
18 common characteristic peaks are demarcated in obtained finger-print altogether, wherein pointing out 3 known peaks, it is known that peak is respectively as follows:
Chlorogenic acid (No. 3 peaks), caffeic acid (No. 5 peaks) and aurantiamarin (No. 11 peaks).By to each day for liking green refreshing product and its composition
Right plant extracts carries out correlation research, 3 known peaks can be corresponded respectively to dandelion extract and dried orange peel extracts.
Reference fingerprint provided by the invention can be used for identifying the green refreshing authenticity of products of love and detect its quality, if to
Sample finger-print can be determined as genuine piece 80% or more with reference fingerprint similarity, and similarity is greater than 90% and is
Qualified products.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (3)
1. the quality determining method of the natural plants anticoccidial feed addictive based on HPLC finger-print, which is characterized in that packet
Include following steps:
1) preparation of test solution
Natural plants anticoccidial feed addictive sample is taken, is dissolved with methanol, is ultrasonically treated, filtration collects filtrate, filtrate is steamed
It is dry, methanol is added into residue and redissolves and with after filtering with microporous membrane, as test solution；
2) preparation of 3 kinds of reference substance solutions
Using methanol as solvent, chlorogenic acid reference substance solution, caffeic acid reference substance solution and aurantiamarin reference substance solution are prepared respectively,
Concentration is respectively 0.5~1.5mg/ml, 0.2~0.8mg/ml and 0.5~1.5mg/ml；
3) HPLC is detected
Chromatographic condition: AgilentSB-C18 chromatographic column, specification 4.6mm × 250mm, 5 μm；Detection wavelength 322-375nm；Column temperature
30-45℃；Sample volume: 5-15 μ l；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid solution, mobile phase A and Mobile phase B concentration
The sum of be 100%, carry out gradient elution；Flow velocity 0.5-1mlmin-1；
Elution requirement:
0-10min, mobile phase A: 10-15%, Mobile phase B: 90-85%；
10-20min, mobile phase A: 15-18%, Mobile phase B: 85-82%；
20-30min, mobile phase A: 18-20%, Mobile phase B: 82-80%；
30-40min, mobile phase A: 20-30%, Mobile phase B: 80-70%；
40-45min, mobile phase A: 30-10%, Mobile phase B: 70-90%；
45-47min, mobile phase A: 10%, Mobile phase B: 90%；
Test solution is injected into high performance liquid chromatograph, obtains HPLC finger-print；Simultaneously respectively with 3 kinds of reference substance solutions into
Sample, determine chlorogenic acid, caffeic acid and aurantiamarin goes out peak position；Measurement result are as follows: amount to 18 shared peaks, wherein 3,5, No. 11
Peak, respectively chlorogenic acid, caffeic acid, aurantiamarin, and No. 3 peaks are internal reference peak, remaining 15 peak is characterized fingerprint peaks；Each peak
Relative retention time be respectively 3.108,3.988,10.029,10.655,12.672,15.343,18.867,22.379,
24.182,28.575,35.540,37.646,38.315,40.767,41.486,41.899,42.551,43.573, average phase
Be respectively 2320.555 to peak area ratio, 643.841,9545.936,633.801,4930.843,719.967,1044.565,
5439.596、1193.835、812.337、703.755、6692.912、10358.90、8624.709、436.598、833.476、
2554.748、7052.612。
2. the method according to claim 1, wherein taking natural plants anticoccidial feed addictive sample in step 1)
Product 2g is dissolved with methanol 10-30ml, and 60W, 45Hz are ultrasonically treated 15-30min；Residue is added methanol 1-3ml and redissolves, as confession
Test sample solution.
3. method according to claim 1 or 2, which comprises the following steps:
The preparation of S1, test solution
Natural plants anticoccidial feed addictive sample 2g is taken, is dissolved with methanol 20ml, 60W, 45Hz are ultrasonically treated 20min；So
After filter, collect filtrate, filtrate is evaporated, into residue be added methanol 2ml redissolve, as test solution；
The preparation of S2,3 kinds of reference substance solutions
Using methanol as solvent, chlorogenic acid reference substance solution, caffeic acid reference substance solution and aurantiamarin reference substance solution are prepared respectively,
Concentration is respectively 1mg/ml, 0.5mg/ml and 1mg/ml；
S3, HPLC detection
Chromatographic condition: AgilentSB-C18 chromatographic column, specification 4.6mm × 250mm, 5 μm；Detection wavelength 355nm；40 DEG C of column temperature；
Sample volume: 10 μ l；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid solution, the sum of mobile phase A and Mobile phase B concentration are
100%, carry out gradient elution；Flow velocity 0.8mlmin-1；
Elution requirement:
0-10min, mobile phase A: 10-15%, Mobile phase B: 90-85%；
10-20min, mobile phase A: 15-18%, Mobile phase B: 85-82%；
20-30min, mobile phase A: 18-20%, Mobile phase B: 82-80%；
30-40min, mobile phase A: 20-30%, Mobile phase B: 80-70%；
40-45min, mobile phase A: 30-10%, Mobile phase B: 70-90%；
45-47min, mobile phase A: 10%, Mobile phase B: 90%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110376092A (en) * | 2019-08-28 | 2019-10-25 | 西南医科大学 | A kind of compound haw thorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating method |
| CN113533582A (en) * | 2021-08-13 | 2021-10-22 | 河北爱绿生物工程有限公司 | UPLC fingerprint quality detection method of natural plant antibacterial growth-promoting feed additive and application thereof |
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| CN113533582A (en) * | 2021-08-13 | 2021-10-22 | 河北爱绿生物工程有限公司 | UPLC fingerprint quality detection method of natural plant antibacterial growth-promoting feed additive and application thereof |
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