CN108445108A - A kind of discrimination method of honeysuckle and Honeysuckle flower - Google Patents
A kind of discrimination method of honeysuckle and Honeysuckle flower Download PDFInfo
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Abstract
The present invention relates to chemical analysis technology field, more particularly to the discrimination method of a kind of honeysuckle and Honeysuckle flower：Measuring samples extracting solution is detected using liquid chromatograph and DAD detectors.The method has many advantages, such as easy, efficient, economic, environmental protection, is not necessarily to any control substance of plant drug or high poison organic chemical reagent.This method can be used not only for Chinese medicine, can be used for the honeysuckle true and false in preparation containing honeysuckle or mixes dummy check, have very high application value.
Description
Technical field
The present invention relates to chemical analysis technology field, more particularly to the discrimination method of a kind of honeysuckle and Honeysuckle flower.
Background technology
Honeysuckle and Honeysuckle flower are clinically used antipyretic and antidotal type Chinese medicine, existing version in 2015《Chinese Pharmacopoeia》Provide its medicine
The flower for being dry flower with position or just opening.Because the nature and flavor of honeysuckle and Honeysuckle flower and channel tropism, function and cure mainly with usage with
Dosage is completely the same, and character has higher similitude, but the price of honeysuckle is far above Honeysuckle flower, the silver therefore market is gone up a hill
Flower falsely use for honeysuckle the phenomenon that it is more serious.Come from the statistical data of the unqualified situation of the prepared slices of Chinese crude drugs in all parts of the country in recent years
It sees, honeysuckle disqualification rate is up to 30%, and major ABO incompatibility lattice reason is exactly to carry out partly or entirely mixing puppet to honeysuckle（It is main pseudo-
Product are Honeysuckle flower）Use is sold in laggard marketing, therefore is badly in need of establishing a kind of discrimination method of honeysuckle and Honeysuckle flower.
The main difference chemical composition that the Honeysuckle flower being currently known is different from honeysuckle is dipsacoside and grey felt
Hairy honeysuckle saponin(e second.Honeysuckle and Honeysuckle flower are carried out to differentiate that the main method of research has microscopic identification method, thin-layered chromatography, liquid
Phase chromatography-evaporative light method, PCR bioanalysis etc..
As disclosed a kind of quick high separation liquid chromatographic detection honeysuckle and Honeysuckle flower medicinal material in CN105606734A
Method, by liquid chromatogram evaporative light scattering detector detect saponins compound achieve the purpose that discriminating.Liquid chromatogram-steaming
Luminescence method needs seldom to be equipped with using laboratory and expensive evaporation photodetector method is tested, it is also necessary to using expensive
Control substance of plant drug.
CN103290127A discloses a kind of technology differentiating honeysuckle and Honeysuckle flower using PCR method.PCR biology rules
Instrument, a variety of chemical reagent and control substance of plant drug harsher, and that need to use costliness are required experimental condition.
Microscopic identification method and thin layer differential method are because honeysuckle and Honeysuckle flower have the similar diagnostic characteristics of height, and being difficult will
The two distinguishes.Therefore it is badly in need of establishing a kind of easy, economic honeysuckle and Honeysuckle flower discrimination method.
Invention content
In order to solve the above honeysuckle in the prior art and Honeysuckle flower differentiate present in distinguish difficulty is big, identification result is bad,
The problems such as method operation difficulty is big, of high cost, this application provides a kind of easy, economic honeysuckle and Honeysuckle flower discriminating sides
Method, is not necessarily to any control substance of plant drug, it is specified that chromatography main peak is with reference to peak, according to the rules in relative retention time ranges chromatographic peak whether there is or not
Method to differentiate honeysuckle and Honeysuckle flower.
What the present invention was obtained through the following steps：
A kind of discrimination method of honeysuckle and Honeysuckle flower, includes the following steps：
（1）It is prepared by measuring samples：Measuring samples are extracted using organic solvent, obtain extracting solution；
（2）Extracting solution is detected using liquid chromatograph and DAD detectors, and chromatographic condition is as follows：
Chromatographic column Agilent ZORBAX Extend- C18 chromatographic columns, 4.6 × 250mm, 5 μm；15-40 DEG C of column temperature；Flow velocity
0.9-1.10mL/min；Using acetonitrile as mobile phase A, using 0.5% phosphoric acid solution as Mobile phase B, gradient elution, 0~5 min, 5%
Mobile phase A, 5~20 min, 5 → 9% mobile phase As, 20~64 min, 9 → 21% mobile phase As, 64~80 min, 21 → 30%
Mobile phase A, 80~95 min, 30 → 80% mobile phase As, 95~106 min, 80 → 5% mobile phase As；DAD detectors（Wavelength
201nm）；
（3）With main peak（Chlorogenic acid peak）With reference to peak, within the scope of relative retention time 4.310-4.355, to there is 1 chromatography
Peak is then sentenced in the measuring samples and contains Honeysuckle flower.
It is found through lot of experiments, the standing high performance liquid chromatograph in general drug inspection laboratory and often can be utilized
The UV detector of rule realizes the discriminating to honeysuckle and Honeysuckle flower.Further experimental study discovery, the gold and silver of different batches
Main peak in the liquid chromatogram of flower and Honeysuckle flower is chlorogenic acid peak, the largeflower-like honeysuckle flower saponin in Honeysuckle flower liquid chromatogram
There is also an otherness unknown material X chromatographic peaks between second and dipsacoside chromatographic peak, therefore can be with computational rules chromatostrip
Known main peak under part（Chlorogenic acid peak）With largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside when retaining relatively
Between, by checking whether contain ash proprietary in Honeysuckle flower in judgement sample to the presence or absence of chromatographic peak at relative retention time
Three kinds of felt hairy honeysuckle saponin(e second, unknown material X and dipsacoside chemical substances exist, and then can differentiate honeysuckle and Shan Yin
Flower.
The discrimination method, preferred steps（3）In with main peak（Chlorogenic acid peak）For with reference to peak, in opposite retain
Between in 4.33 ± 5% ranges, there is 1~3 chromatographic peak, then sentence in the measuring samples and contain Honeysuckle flower.
The discrimination method, preferred steps（3）In with main peak（Chlorogenic acid peak）For with reference to peak, in relative retention time
In 4.11~4.55 ranges, there is 1~3 chromatographic peak, then sentence in the measuring samples and contain Honeysuckle flower.
The discrimination method, preferred steps（2）Middle chromatographic condition is as follows：Chromatographic column Agilent ZORBAX Extend-
C18 chromatographic columns, 4.6 × 250mm, 5 μm；25 DEG C of column temperature；Flow velocity 1.0mL/min.
The discrimination method, preferred steps（1）In take measuring samples, using 50% methanol, ultrasonic extraction 30 minutes surpasses
Acoustical power 250W, frequency 40kHz, filtration, obtain extracting solution.
The discrimination method, the preferably number of theoretical plate at chlorogenic acid peak are not less than 10 000.
Beneficial effects of the present invention：
1, in Honeysuckle flower control medicinal material chromatogram, also occur between largeflower-like honeysuckle flower saponin second and dipsacoside chromatographic peak
Unknown material X chromatographic peaks, the corresponding chromatographic peak of the ingredient only occurs in Honeysuckle flower and Honeysuckle flower is different from the special of honeysuckle
Attribute chemical composition can be used for differentiating honeysuckle and Honeysuckle flower；
2, this method is a kind of only with most simple UV detector, easier without evaporative light or EFI fog detector, method
It is easy to operate；
3, this method does not need any reference material, and no matter honeysuckle or Honeysuckle flower are under the chromatographic condition, and main peak is all green original
Sour peak, thus directly define the main peak be with reference to peak, according to the presence or absence of chromatographic peak at relative retention time come judge be honeysuckle and
Honeysuckle flower saves use or the high poison organic chemical reagent of expensive control substance of plant drug, makes easier method, efficient, economy, ring
It protects；
4, this method can be used not only for Chinese medicine, can be used for the honeysuckle true and false in preparation containing honeysuckle or mixes pseudo- inspection
It tests, there is very high application value.
Description of the drawings
The HPLC chromatogram of Fig. 1 mixed reference substance solutions, 1 chlorogenic acid of peak；2 largeflower-like honeysuckle flower saponin second of peak；4 teasel of peak
Saponin(e second,
The HPLC chromatogram of Fig. 2 honeysuckle control medicinal materials,
The HPLC chromatogram of Fig. 3 Honeysuckle flower control medicinal materials, 1 chlorogenic acid of peak；2 largeflower-like honeysuckle flower saponin second of peak；4 teasel of peak
Saponin(e second,
The HPLC chromatogram of Fig. 4 samples 4,
The HPLC chromatogram of Fig. 5 samples 6.
Specific implementation mode
With reference to specific embodiment, invention is further explained：
Embodiment 1
1. instrument and reagent
2695 high performance liquid chromatographs of Waters, 2448 DAD detectors of Waters；Sartorius CP225D electronic balances；
BK-600C ultrasonic washing instruments（Bark ultrasonic device Co., Ltd）；Agilent ZORBAX Extend- C18 chromatographic columns
(4.6 × 250mm, 5 μm).
Chlorogenic acid reference substance（Content is 96.2%, lot number 110753-201415）, largeflower-like honeysuckle flower saponin second reference substance（Batch
Number：111814-201303）, dipsacoside reference substance（Lot number：111813-201202）, honeysuckle control medicinal material（Lot number
121060-201107）, Honeysuckle flower control medicinal material（Lot number 121595-201202）It is provided by Zhong Jian institutes, honeysuckle and Honeysuckle flower
It is commercial samples；Acetonitrile is chromatographically pure.
2. method and result
The preparation of 2.1 solution
2.1.1 the preparation of test solution
Take this product powder（Cross No. four sieves）About 0.20g, it is accurately weighed, it sets in conical flask, 50% methanol 20mL, ultrasound is added in precision
Processing（Power 250W, frequency 40kHz）It 30 minutes, lets cool, shakes up, filter, take subsequent filtrate as test solution.
2.1.2 the preparation of control medicinal material solution
Extracting honeysuckle control medicinal material and Honeysuckle flower control medicinal material each about 0.20g, it is accurately weighed, according to the preparation side of test solution
Control medicinal material solution is made in method respectively.
2.1.3 the preparation of mixed reference substance solution
It is appropriate that precision weighs chlorogenic acid, largeflower-like honeysuckle flower saponin second and three kinds of reference substances of dipsacoside, and 50% methanol is added to be made
Per mL mixed reference substance solutions containing 0.2178mg, 0.2128mg and 0.2305mg respectively.
2.2 chromatographic conditions and system suitability
Using following chromatographic condition：Chromatographic column Agilent ZORBAX Extend- C18 chromatographic columns (4.6 × 250mm, 5 μm)；
25 DEG C of column temperature；Flow velocity 1.0mL/min；Using acetonitrile as mobile phase A, using 0.5% phosphoric acid solution as Mobile phase B, gradient elution, 0~5
Min, 5% A, 5~20 min, 5 → 9% A, 20~64 min, 9 → 21%A, 64~80 min, 21 → 30% A, 80~95
Min, 30 → 80% A, 95~106 min, 80 → 5% A；DAD detectors（Wavelength 201nm）.
The selection of 2.1 Detection wavelengths
Because largeflower-like honeysuckle flower saponin second and dipsacoside do not have a very strong UV absorption, therefore general Study more options evaporative light
Detector is detected.It is found through experiment that ultraviolet end absorbing wavelength 201nm is selected to be detected, largeflower-like honeysuckle flower saponin second,
Unknown material X and dipsacoside have apparent chromatographic peak to occur, while chlorogenic acid also has stronger suction at that wavelength
It receives, therefore selects 201nm as Detection wavelength.
The confirmation of 2.2 chromatographic peaks
Precision draws mixed reference substance solution, honeysuckle control medicinal material solution and each 10 μ l of Honeysuckle flower control medicinal material solution, injection
Liquid chromatograph is analyzed, and collection of illustrative plates is shown in Fig. 1~3.Chromatographic peak judgement is carried out according to retention time and DAD collection of illustrative plates relevant informations,
18.585min in Honeysuckle flower control medicinal material solution chromatogram（Peak 1）、79.500min（Peak 2）、80.901min（Peak 3）With
82.300min（Peak 4）Place's chromatographic peak is followed successively by chlorogenic acid, largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside.Gold
18.821min in honeysuckle flower control medicinal material solution chromatogram（Peak 1）Place's chromatographic peak is chlorogenic acid.Honeysuckle control medicinal material solution and
The chromatography main peak of Honeysuckle flower control medicinal material solution is chlorogenic acid peak.
2.3 precision tests are investigated
Precision draws 10 μ l of Honeysuckle flower control medicinal material solution, and continuous sample introduction 6 times is composed with gained Honeysuckle flower control medicinal material solution mass-tone
Peak（Chlorogenic acid peak）With reference to peak, to calculate the opposite guarantor of largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside chromatographic peak
Stay the time.
1 precision test of table investigates result
The above results are it is found that largeflower-like honeysuckle flower saponin second, the opposite of 3 chromatographic peaks of unknown chromatographic peak X and dipsacoside are protected
It stays the RSD of time to be respectively less than 1%, prompts the precision test of this method good.
2.4 stability tests are investigated
Precision draws 10 μ l of Honeysuckle flower control medicinal material solution, respectively in 0,2,4,8,12 and sample introduction for 24 hours, with Honeysuckle flower control medicinal material
Solution mass-tone spectral peak（Chlorogenic acid peak）With reference to peak, to calculate largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside chromatography
The relative retention time at peak.
Result is investigated in 2 stability test of table
The above results it is found that largeflower-like honeysuckle flower saponin second, when retaining of 3 chromatographic peaks of unknown material X and dipsacoside relatively
Between RSD be respectively less than 1%, prompt the stability test of this method good.
2.5 repetitive tests are investigated
Precision weighs 6 parts, every part of 0.20g of Honeysuckle flower control medicinal material, and test sample is made according to above-mentioned test solution preparation method
Solution, difference 10 μ l of sample introduction, with Honeysuckle flower control medicinal material solution mass-tone spectral peak（Chlorogenic acid peak）With reference to peak, to calculate grey felt hair and bearing
Winter saponin(e second, the relative retention time of unknown material X and dipsacoside chromatographic peak.
3 repetitive test of table investigates result
The above results it is found that largeflower-like honeysuckle flower saponin second, when retaining of 3 chromatographic peaks of unknown material X and dipsacoside relatively
Between RSD be respectively less than 1%, prompt the repetitive test of this method good.
The investigation of 2.6 column temperatures
This method mainly carries out cultivar origin judgement by examining at relative retention time the presence or absence of chromatographic peak to sample, and column
Temperature can significantly affect the retention time of chromatographic peak, therefore should carry out column temperature investigation, to ensure the durability of this method.Precision is drawn
10 μ l of Honeysuckle flower control medicinal material solution, respectively in 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C sample introductions of column temperature, with Honeysuckle flower
Control medicinal material solution mass-tone spectral peak（Chlorogenic acid peak）With reference to peak, to calculate largeflower-like honeysuckle flower saponin second, unknown material X and teasel soap
The relative retention time of glycosides second chromatographic peak.
The experiment investigation result of 4 column temperature of table
| Title column temperature（℃） | 15 | 20 | 25 | 30 | 35 | 40 | It is average | RSD(%) |
| Peak 1（Chlorogenic acid） | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.00 | 0 |
| Peak 2（Largeflower-like honeysuckle flower saponin second） | 4.267 | 4.271 | 4.278 | 4.275 | 4.277 | 4.279 | 4.27 | 0.10 |
| Peak 3（Unknown material X） | 4.339 | 4.327 | 4.348 | 4.326 | 4.333 | 4.329 | 4.33 | 0.18 |
| Peak 4（Dipsacoside） | 4.428 | 4.439 | 4.429 | 4.441 | 4.407 | 4.449 | 4.43 | 0.30 |
The above results it is found that largeflower-like honeysuckle flower saponin second, when retaining of 3 chromatographic peaks of unknown material X and dipsacoside relatively
Between RSD be respectively less than 1%, prompt the stability test of this method good.
The investigation of 2.7 flow velocitys
In view of the variation of flow velocity can influence the retention time of chromatographic peak, therefore flow velocity investigation should be carried out, precision draws Honeysuckle flower
10 μ l of control medicinal material solution are compareed respectively in flow velocity 0.90,0.95,1.00,1.05 and 1.10 mL/min sample introductions with Honeysuckle flower
Medicinal material solution mass-tone spectral peak（Chlorogenic acid peak）With reference to peak, to calculate largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside
The relative retention time of chromatographic peak.
The experiment investigation result of 5 flow velocity of table
The above results it is found that largeflower-like honeysuckle flower saponin second, when retaining of 3 chromatographic peaks of unknown material X and dipsacoside relatively
Between RSD be respectively less than 1%, prompt this method on the minor change of flow velocity substantially without influence.
The investigation of 2.8 different lot number chromatographic columns
Rule of thumb, the C18 chromatographic columns of different manufacturers type influence retention time and relative retention time very big, by opposite
Retention time determines chromatographic peak, answers producer and the model of stationary chromatographic column, otherwise can not ensure the C18 produced in whole producers
Chromatographic column can obtain satisfactory result, therefore not investigate durability of the different manufacturers chromatographic column to this method, and should investigate same
The chromatographic column of brand and model but different lot numbers has an impact this experimental result, therefore to the Agilent ZORBAX of different lot numbers
Extend- C18 chromatographic columns (4.6 × 250mm, 5 μm) are investigated.Precision draws 10 μ l of Honeysuckle flower control medicinal material solution, point
Not Cai Yong different lot numbers chromatographic column sample introduction, with Honeysuckle flower control medicinal material solution mass-tone spectral peak（Chlorogenic acid peak）For with reference to peak, meter
Calculate largeflower-like honeysuckle flower saponin second, the relative retention time of unknown material X and dipsacoside chromatographic peak.
The experiment investigation result of the different lot number chromatographic columns of table 6
The above results it is found that largeflower-like honeysuckle flower saponin second, when retaining of 3 chromatographic peaks of unknown material X and dipsacoside relatively
Between RSD be respectively less than 1%, prompt the Agilent ZORBAX Extend- C18 chromatographic columns of different lot numbers to this method result without
It influences.
2.9 result judgement methods
It is tested by honeysuckle to various sources and Honeysuckle flower, it is found that largeflower-like honeysuckle flower saponin second, unknown material X and river are continuous
Disconnected saponin(e second is the otherness chemical substance that Honeysuckle flower is different from honeysuckle, is reference peak with chlorogenic acid peak, then unknown material X phases
It is 4.31-4.35 to retention time.In view of the content of above-mentioned three kinds of ingredients in the Honeysuckle flower of different sources can have differences, with
And honeysuckle mixes the difference of pseudo- degree, can lead to above three chromatographic peak while detect there are certain difficulty, to ensure to examine
As a result accuracy, as long as therefore be further contemplated that three kinds of detection largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside at
Divide any one or can determine whether detection Honeysuckle flower for two or three.
Largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside relative retention time respectively may be about 4.28,4.33 and
4.43, relatively, unknown material X and the relative average debiation of the relative retention time of the two are less than 2% to three numerical value, and unknown
The relative retention value of object X is just close to the average value of largeflower-like honeysuckle flower saponin second and the relative retention value of dipsacoside.According to
The general limit specified value of relative retention time is 5%, to advanced optimize the discrimination method, it is specified that being protected in the opposite of unknown material X
It stays in ± 5% range of time 4.33, is i.e. occurs 1~3 chromatographic peak in 4.11~4.55 ranges, then sentence sample detection mountain silver
Flower.
The measurement of 2.10 samples
According to the experimental condition of above-mentioned determination, chromatographic condition and result judgement method, to 26 batches of honeysuckles of collection or Honeysuckle flower
Sample test, be to calculate largeflower-like honeysuckle flower saponin second, unknown material X and dipsacoside with reference to peak with chromatography main peak
Relative retention time, concrete outcome see the table below 7.
The relative retention time measurement result of 7 sample of table
| Number | Indicate title | Source | Peak 1（Chlorogenic acid） | Peak 2（Largeflower-like honeysuckle flower saponin second） | Peak 3（Unknown material X） | Peak 4 (dipsacoside) | The chromatographic peak number occurred in 4.11~4.55 ranges |
| Sample 1 | Honeysuckle | Zhong Jian institutes | 1.000 | / | / | / | 0 |
| Sample 2 | Honeysuckle flower | Zhong Jian institutes | 1.000 | 4.278 | 4.348 | 4.429 | 3 |
| Sample 3 | Honeysuckle | Linyi | 1.000 | / | / | / | 0 |
| Sample 4 | Honeysuckle flower | Bozhou | 1.000 | 4.271 | 4.351 | 4.433 | 3 |
| Sample 5 | Honeysuckle | Heze | 1.000 | / | / | / | 0 |
| Sample 6 | Honeysuckle flower | Linyi | 1.000 | 4.282 | 4.338 | 4.437 | 3 |
| Sample 7 | Honeysuckle | Heze | 1.000 | / | / | / | 0 |
| Sample 8 | Honeysuckle flower | Yuzhou | 1.000 | 4.286 | 4.341 | 4.439 | 3 |
| Sample 9 | Honeysuckle | Qi Zhou | 1.000 | / | / | / | 0 |
| Sample 10 | Honeysuckle | Puning | 1.000 | / | / | / | 0 |
| Sample 11 | Honeysuckle | Bozhou | 1.000 | / | / | / | 0 |
| Sample 12 | Honeysuckle | Anguo | 1.000 | / | / | / | 0 |
| Sample 13 | Honeysuckle | It is peaceful | 1.000 | / | / | / | 0 |
| Sample 14 | Honeysuckle | Qi Zhou | 1.000 | 4.278 | 4.332 | 4.427 | 3 |
| Sample 15 | Honeysuckle flower | Camphor tree | 1.000 | 4.277 | 4.335 | 4.430 | 3 |
| Sample 16 | Honeysuckle flower | Xi'an | 1.000 | 4.288 | 4.342 | 4.438 | 3 |
| Sample 17 | Honeysuckle flower | It is peaceful | 1.000 | 4.283 | 4.346 | 4.435 | 3 |
| Sample 18 | Honeysuckle flower | Yulin | 1.000 | 4.287 | 4.337 | 4.437 | 3 |
| Sample 19 | Honeysuckle flower | Yuzhou | 1.000 | 4.285 | 4.336 | 4.436 | 3 |
| Sample 20 | Honeysuckle flower | The west of Gansu Province | 1.000 | 4.280 | 4.331 | 4.436 | 3 |
| Sample 21 | Honeysuckle flower | Heze | 1.000 | 4.269 | 4.337 | 4.429 | 3 |
| Sample 22 | Honeysuckle flower | Yulin | 1.000 | 4.273 | 4.333 | 4.442 | 3 |
| Sample 23 | Honeysuckle flower | Bozhou | 1.000 | 4.289 | 4.338 | 4.439 | 3 |
| Sample 24 | Honeysuckle flower | Camphor tree | 1.000 | 4.280 | 4.328 | 4.433 | 3 |
| Sample 25 | Honeysuckle flower | Camphor tree | 1.000 | 4.283 | 4.330 | 4.431 | 3 |
| Sample 26 | Honeysuckle flower | Xi'an | 1.000 | 4.276 | 4.327 | 4.428 | 3 |
（The chromatographic peak is not detected in "/" expression）
Upper table content also further demonstrates unknown peak X can be as the characteristic peak of Honeysuckle flower, to differentiate honeysuckle and Shan Yin
Flower.
3 discuss
3.1 rule of thumb it is found that the chromatographic column of different model and column length influence significantly retention time, to ensure inspection result
Accuracy, should select this method determine Agilent ZORBAX Extend- C18 chromatographic columns (4.6 × 250mm, 5 μm),
Therefore the chromatographic column of different model and column length are not investigated.
Once honeysuckle and the Honeysuckle flower that multiple lot numbers of Liao Zhongjian institutes and market collection were used during 3.2 experiments, at this
Under chromatographic condition（Wavelength 201nm）, can guarantee that the mass-tone spectral peak of medicinal material solution is chlorogenic acid.To ensure the separation of each chromatographic peak
Degree, it should be ensured that the number of theoretical plate of main peak must not be less than 10 000 less, and otherwise this method cannot be used for examining, and should replace chromatographic column,
And re-start experiment.
3.3 in Honeysuckle flower control medicinal material chromatogram, between largeflower-like honeysuckle flower saponin second and dipsacoside chromatographic peak
Also there are unknown material X chromatographic peaks, the corresponding chromatographic peak of the ingredient only occurs in Honeysuckle flower and Honeysuckle flower is different from honeysuckle
Specificity chemical composition, it is sufficient to for as differentiate honeysuckle and Honeysuckle flower property material, but specifically ingredient it is unknown, it is still necessary to
Further research.
3.4 this method can be used for differentiating honeysuckle and Honeysuckle flower have many advantages, such as easy, efficient, economic, environmental protection, nothing
Need any control substance of plant drug or high poison organic chemical reagent.This method can be used not only for Chinese medicine, can be used for containing honeysuckle
The honeysuckle true and false in preparation mixes dummy check, has very high application value.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not limited by embodiment
System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, replacement, simplification should be
Equivalence replacement mode, is included within the scope of the present invention.
Claims (6)
1. the discrimination method of a kind of honeysuckle and Honeysuckle flower, it is characterised in that include the following steps：
（1）It is prepared by measuring samples：Measuring samples are extracted using organic solvent, obtain extracting solution；
（2）Extracting solution is detected using liquid chromatograph and DAD detectors, and chromatographic condition is as follows：
Chromatographic column Agilent ZORBAX Extend- C18 chromatographic columns, 4.6 × 250mm, 5 μm；15-40 DEG C of column temperature；Flow velocity
0.9-1.10mL/min；Using acetonitrile as mobile phase A, using 0.5% phosphoric acid solution as Mobile phase B, gradient elution, 0~5 min, 5%
Mobile phase A, 5~20 min, 5 → 9% mobile phase As, 20~64 min, 9 → 21% mobile phase As, 64~80 min, 21 → 30%
Mobile phase A, 80~95 min, 30 → 80% mobile phase As, 95~106 min, 80 → 5% mobile phase As；DAD detectors detect wave
Long 201nm；
（3）With main peak（Chlorogenic acid peak）With reference to peak, within the scope of relative retention time 4.310-4.355, to there is 1 chromatography
Peak is then sentenced in the measuring samples and contains Honeysuckle flower.
2. discrimination method according to claim 1, it is characterised in that step（3）In with chlorogenic acid peak be with reference to peak, in phase
To in 4.33 ± 5% range of relative retention time, there is 1~3 chromatographic peak, then sentencing in the measuring samples and contain Honeysuckle flower.
3. discrimination method according to claim 1, it is characterised in that step（3）In with main peak（Chlorogenic acid peak）For reference
There is 1~3 chromatographic peak, then sentences in the measuring samples and contain mountain in 4.11~4.55 range of opposite relative retention time in peak
Honeysuckle flower.
4. discrimination method according to claim 1, it is characterised in that step（2）Middle chromatographic condition is as follows：Chromatographic column
Agilent ZORBAX Extend- C18 chromatographic columns, 4.6 × 250mm, 5 μm；25 DEG C of column temperature；Flow velocity 1.0mL/min.
5. discrimination method according to claim 1, it is characterised in that step（1）In take measuring samples, using 50% methanol,
Ultrasonic extraction 30 minutes, ultrasonic power 250W, frequency 40kHz, filtration, obtains extracting solution.
6. discrimination method according to claim 1, it is characterised in that the number of theoretical plate at chlorogenic acid peak is not less than 10 000.
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| CN114137125A (en) * | 2021-12-06 | 2022-03-04 | 苏州市药品检验检测研究中心 | Method for rapidly checking adulteration of lonicera confusa in heat-clearing and detoxifying oral liquid |
| CN118050453A (en) * | 2023-12-28 | 2024-05-17 | 广西金嗓子有限责任公司 | Quality control method of preparation for preventing and treating throat and oral diseases |
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