CN104849364B - Canzhiling oral solution fingerprint map building method, fingerprint map and application thereof - Google Patents
Canzhiling oral solution fingerprint map building method, fingerprint map and application thereof Download PDFInfo
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Abstract
The invention discloses a canzhiling oral solution fingerprint map building method. The map includes the following steps of preparation of a test solution, preparation of a reference solution, testing through a high performance liquid chromatograph and processing of data and a map. The invention further discloses a Canzhiling oral solution fingerprint map and a method for utilizing the fingerprint map to control quality of a Canzhiling oral solution. The Canzhiling oral solution fingerprint map building method is simple in operation, stable, reliable, high in accuracy and high in separation degree, the fingerprint map is high in stability and reproducibility and large in information quantity, and the fingerprint map is adopted as a quality control means for the Canzhiling oral solution, so that one-sidedness caused by judging of overall quality of a preparation by testing one or two chemical ingredients is avoided, and probability of artificial processing in order to enabling quality to be up to standards is lowered; samples of multiple batches are analyzed systematically, so that quality of the Canzhiling oral solution can be evaluated more comprehensively and scientifically, and product quality and efficacy are guaranteed.
Description
Technical field
The present invention relates to Chinese medicine fingerprint analysis method, particularly join the foundation side of branch tuckahoe oral liquid HPLC finger printing
Method and the finger printing of the ginseng branch tuckahoe oral liquid being obtained by this method.
Background technology
Ginseng branch tuckahoe oral liquid is by Radix Codonopsis, Ramulus Cinnamomi, the Radix Paeoniae Alba, Radix Glycyrrhizae (processed with honey), Poria, Rhizoma Zingiberiss, Radix Polygalae (system, stir-fry), stone Chang
Pu, Os Draconis and ten kinds of medical materials of the Concha Ostreae compound Chinese medicinal preparation through manufacturing meticulously, have been demonstrated that it contains multiple biological activities
Material, has good QI invigorating warming YANG, a curative effect that resolving sputum is calmed the nerves, for treating the motive that light moderate stages alzheimer's disease manifested not
Sequitur, is mainly shown as forgetful, cardiopalmus, dizziness, insomnia, few gas complaint, pale tongue, apathetic expression, spiritlessness and weakness, Weak pulse
Deng disease.In ginseng branch tuckahoe oral liquid, Radix Codonopsis act as bright, reinforcing the heart gas of calming the nerves；Radix Polygalae, Rhizoma Acori Graminei, Poria etc. then play logical thinking, shake the heart
Sun, the effect of the turbid expectorant of change.Alzheimer disease patient's memory, intelligence and viability and cognition can be effectively improved for clinic
Function.Show through pharmaceutical research, ginseng branch tuckahoe oral liquid can highly desirable improve all of segment pathology of senile dementia
Property change, the Spatial memory ability of alzheimer model of a syndrome rat can be improved, improve its Cortical neurons fiber and thicken
With the phenomenon increasing, and the symptom that its Hippocampus cones comes off can be treated.
Join branch tuckahoe oral liquid as a kind of compound Chinese medicinal preparation, prescription medical material more (being made up of 10 taste medical materials), composition is non-
Often complicated various." ginseng branch tuckahoe oral liquid standard " (State Food and Drug Administration's standard) is built using liquid chromatography
Peoniflorin and the content assaying method of two compositions of cinnamic acid are found.But ginseng branch tuckahoe oral liquid is compound preparation, pass through merely inspection
Survey one, two kind of index components quantitatively to control its drug quality clearly incomplete, nor embody compound Chinese medicinal preparation
Integral Characteristic.Therefore, a kind of simple and easy to do, accuracy height is set up in exploitation, and repeatability is good and can reflect multiple as much as possible
Multicomponent fingerprint atlas detection method contained by square preparation is very necessary for the quality control of ginseng branch tuckahoe oral liquid and evaluation.
Content of the invention
For above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a seed ginseng branch tuckahoe oral liquid fingerprint map construction
Method and its finger printing.The present invention passes through the research to ginseng branch tuckahoe oral liquid finger printing, there is provided a kind of easy and simple to handle, steady
Fixed, precision is high and favorable reproducibility, can be used for joining the quality testing of branch tuckahoe oral liquid and the method evaluated, compensate for existing quality
The deficiency of control method, the Quality Control Technology making ginseng branch tuckahoe oral liquid is more sophisticated, science.
For achieving the above object, the present invention adopts following technical proposals：
One seed ginseng branch tuckahoe oral liquid fingerprint map construction method, comprises the following steps：
(1) preparation of need testing solution：Take ginseng branch tuckahoe oral liquid, add methanol, supersound extraction, it is little that 0.5-1 is placed in cold preservation
When, recover to room temperature, filter, take subsequent filtrate, obtain final product need testing solution；
(2) preparation of reference substance solution：Take drying under reduced pressure to the cinnamic acid of constant weight, peoniflorin, liquirtin, peony lactone
Glycosides, ammonium glycyrrhizinate reference substance, are separately added into methanol and make reference substance solution；
(3) measure：Precision measures need testing solution and reference substance solution respectively, and injection high performance liquid chromatograph measures, note
Record chromatogram；
(4) with finger printing software, gained collection of illustrative plates is processed, obtain final product ginseng branch tuckahoe oral liquid finger printing；
In step (3), the liquid phase chromatogram condition of mensure is：Chromatographic column is phenomenex synergi Hydro-RP color
Spectrum post (4 μ, 250 × 4.6mm；I.e. packing material size is 4 μm, and column length is 250mm, and column internal diameter is 4.6mm)；Mobile phase A is 0.3%
Phosphate aqueous solution (volume fraction), Mobile phase B is acetonitrile；Gradient elution；Flow rate of mobile phase is 0.8ml/min；Column temperature is 30 DEG C；
Detection wavelength is 230nm.
In step (3), during gradient elution, the change of mobile phase A and Mobile phase B turns to：0-5min, mobile phase A 95%-
95%, Mobile phase B 5%-5%；5-10min, mobile phase A 95%-92%, Mobile phase B 5%-8%；10-20min, mobile phase
A92%-80%, Mobile phase B 8%-20%；20-25min, mobile phase A 80%-77.5%, Mobile phase B 20%-22.5%；
25-40min, mobile phase A 77.5%-67%, Mobile phase B 22.5%-33%；40-50min, mobile phase A 67%-60%, stream
Dynamic phase B 33%-40%；50-60min, mobile phase A 60%-50%, Mobile phase B 40%-50%；60-80min, mobile phase
A50%-15%, Mobile phase B 50%-85%；80-90min, mobile phase A 15%-5%, Mobile phase B 85%-95%；90-
100min, mobile phase A 5%-95%, Mobile phase B 95%-5%.(being volumn concentration).
Specifically, in step (1), the preparation method of need testing solution is：Precision measure 1.25ml ginseng branch tuckahoe oral liquid in
In 25ml volumetric flask, plus methanol constant volume, to scale, supersound extraction 5min, is placed 0.5 hour in 4 DEG C of refrigerators, is recovered to room temperature,
Through 0.22 μm of organic system membrane filtration, subsequent filtrate is taken to obtain final product need testing solution.
In step (2), the preparation method of reference substance solution is：
Precision weighs cinnamic acid standard substance 0.0103g, puts in 100mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains
To Cortex Cinnamomi acid solution, concentration is 0.103mg/mL；
Precision weighs peoniflorin standard substance 0.0302g, puts in 10mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains
To peoniflorin solution, concentration is 3.02mg/mL；
Precision weighs liquirtin standard substance 0.0204g, puts in 25mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains
To liquirtin solution, concentration is 0.816mg/mL；
Precision weighs lactone glucoside of Radix Paeoniae standard substance 0.0101g, puts in 10mL volumetric flask, plus methanol dissolves and is diluted to quarter
Degree, obtains lactone glucoside of Radix Paeoniae solution, and concentration is 1.01mg/mL；
Precision weighs ammonium glycyrrhizinate standard substance 0.0198g, puts in 10mL volumetric flask, plus methanol dissolves and is diluted to scale,
Obtain ammonium glycyrrhizinate solution, concentration is 1.98mg/mL.
In step (3), precision measures need testing solution and the volume of reference substance solution is 10 μ L respectively.
It should be noted that the construction method of the finger printing of the present invention screens through scientific experimentss and obtains, and
Non- is that the conventional of this area selects, the present invention mainly preparation method to need testing solution, chromatographic condition, Detection wavelength and analysis
The conditions such as time have been carried out preferably.
Wherein：1) investigation of need testing solution compound method：Test compares Extraction solvent extension rate to sample solution
The impact of Detection results, such as 40 times of dilution, 20 times and 10 times, result dilutes 20 times of Detection results using methanol as Extraction solvent
It is preferred.
2) selection of chromatographic condition and optimization：Different phosphate buffer proportioning (0.1%, 0.2% and in mobile phase are investigated
0.3%) impact to chromatograph separating effect.Result of the test shows：In mobile phase A, phosphate buffer solution proportioning is color when 0.3%
Preferably, steadily, peak shape is symmetrical for baseline, and preferably, therefore mobile phase is defined as separating degree for spectrum separating effect：A phase (0.3% phosphoric acid
Water) B phase (acetonitrile).After adjustment mobile phase different time eluting ratio, the retention time of each chromatographic peak is moderate, and baseline is put down
Surely, it is difficult to drift about, improve the separating degree of chromatogram simultaneously, effectively prevent the conditions of streaking of chromatogram, be conducive to chromatograph to refer to
The detection of stricture of vagina collection of illustrative plates and analysis.
3) selection of Detection wavelength：The each ripple being scanned in 190～400nm using photodiode array detector (PDAD)
Long lower chromatogram is compared analysis, and set 8 wavelength detected simultaneously (inclusion 220nm, 230nm, 235nm,
237nm, 240nm, 245nm, 254nm and 290nm), testing result shows：At 230nm, not only baseline is steady for the chromatogram of detection,
Chromatographic peak is more, contains much information, and preferably, peak area is also larger for the separating effect of each chromatographic peak.Therefore, select 230nm as detection
Wavelength.Investigated the chromatogram under other wavelength according to the requirement of tentative standard, its result display ginseng branch tuckahoe oral liquid exists simultaneously
Chromatographic fingerprinting under different wave length has preferable similarity.
4) selection of analysis time：Have recorded the chromatogram of 2h when selecting the elution time of finger printing.Result shows
Substantially obvious chromatographic peak is not had to occur after 85min, simultaneously in order to look after the diversity of batch sample it is ensured that all batch samples
The characteristic peak of product can be detected, and therefore selects 100min as analysis time.
5) selection of column temperature：Test four different column temperatures (such as 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C) to finger printing detection
The impact of result.When result display column temperature is 30 DEG C, chromatographic peak retention time is suitable, and steadily, each chromatographic peak separating degree is relatively for baseline
Good, peak shape is symmetrical, therefore selects column temperature to be 30 DEG C.
6) selection of flow velocity：Test four flow velocitys (0.7ml/min, 0.8ml/min, 0.9ml/min and 1.0ml/min)
Impact to finger printing testing result.Result shows：When flow velocity is 0.8ml/min, separating effect is optimal, and each chromatographic peak retains
Time is suitable, and separating degree is good, and steadily, peak shape is symmetrical for baseline, therefore selects flow velocity to be 0.8ml/min.
The ginseng branch tuckahoe oral liquid finger printing that ginseng branch tuckahoe oral liquid fingerprint map construction method obtains as previously mentioned, this fingerprint
Total peak in collection of illustrative plates is 11.
Present invention also offers the method for a seed ginseng branch tuckahoe oral liquid quality control, comprise the following steps：
(1) structure of ginseng branch tuckahoe oral liquid finger printing to be measured：Take ginseng branch tuckahoe oral liquid to be measured, build as stated above
The finger printing of ginseng branch tuckahoe oral liquid to be measured；
(2) finger printing of ginseng branch tuckahoe oral liquid to be measured is carried out similarity system design with reference sample finger printing, to comment
The quality of valency ginseng to be measured branch tuckahoe oral liquid, computational methods are correlation coefficient process, and the oral liquid quality that similarity is not less than 0.900 is closed
Lattice.If similarity is less than 0.900, show that batch sample mass discrepancy is larger.
In step (2), the construction method of described reference sample finger printing is：Calculate each sample and other samples first
Similarity, then select the sample maximum with other sample similarity sums to be reference sample, its finger printing is with reference to sample
Product finger printing.
Beneficial effects of the present invention：
(1) present invention adopts phosphate aqueous solution-acetonitrile system, establishes ginseng branch tuckahoe oral liquid using the method for gradient elution
Finger printing, the method is simple to operate, reliable and stable, and precision is high, and separating degree is good, the stability of finger printing and repeatability
Preferably, and contain much information.
(2) because finger printing is not configured to measure the Precise levels of certain composition, and it is intended to fully reflect chemical composition
Information, therefore, the present invention select be measured at 230nm wavelength, its appearance is more, and the information of reflection is more complete, respectively
Individual peak absorption value is good, and baseline is steady.
(3) present invention adopts the quality control method as ginseng branch tuckahoe oral liquid for the finger printing of ginseng branch tuckahoe oral liquid, both
Avoid and judge the one-sidedness of preparation total quality because only measuring one, two chemical compositions, decrease as requisite quality again
The artificial probability processing, by carrying out systematic analysiss to the sample of multiple batches, more comprehensive, scientifical can evaluate ginseng branch Siberian cocklebur mouth
Take the quality of liquid, so that the quality of product and curative effect are guaranteed.
(4) present invention to carry out the comparison of finger printing using reference sample finger printing, it is to avoid each batch sample is to right
According to the impact of finger printing, the quality separating capacity of different batches sample is strengthened, performance is better than reference fingerprint, judge knot
Fruit more tallies with the actual situation.
Brief description
Fig. 1 is each batch ginseng branch tuckahoe oral liquid HPLC finger printing and reference fingerprint；
Fig. 2 is ginseng branch tuckahoe oral liquid reference substance mixed liquor finger printing；
Fig. 3 is the similarity sum (correlation coefficient process) of each batch sample and other batch sample；
Fig. 4 is the similarity (correlation coefficient process) of each batch sample and reference sample finger printing.
Specific embodiment
With reference to embodiment, the present invention is further illustrated it should explanation, and the description below is merely to solve
Release the present invention, its content is not defined.
Embodiment 1：The structure of ginseng branch tuckahoe oral liquid finger printing and Method validation
Concretely comprise the following steps：
(1) preparation of need testing solution：Precision measures 1.25ml ginseng branch tuckahoe oral liquid in 25ml volumetric flask, plus methanol is fixed
Hold to scale, supersound extraction 5min, place 0.5 hour in 4 DEG C of refrigerators, recover to room temperature, through 0.22 μm of organic system filter membrane mistake
Filter, obtains final product the need testing solution of 20 times of dilution, obtains final product.
(2) preparation of reference substance solution：
Precision weighs cinnamic acid standard substance (lot number：110786-200503) 0.0103g, puts in 100mL volumetric flask, plus first
Alcohol dissolves and is diluted to scale, obtains Cortex Cinnamomi acid solution, and concentration is 0.103mg/mL.
Precision weighs peoniflorin standard substance (lot number：MUST-13030401) 0.0302g, puts in 10mL volumetric flask, plus methanol
Dissolve and be diluted to scale, obtain peoniflorin solution, concentration is 3.02mg/mL.
Precision weighs liquirtin standard substance (lot number：111610-200604) 0.0204g, puts in 25mL volumetric flask, plus methanol
Dissolve and be diluted to scale, obtain liquirtin solution, concentration is 0.816mg/mL.
Precision weighs lactone glucoside of Radix Paeoniae standard substance (lot number：MUST-14031214) 0.0101g, puts in 10mL volumetric flask, plus
Methanol dissolves and is diluted to scale, obtains lactone glucoside of Radix Paeoniae solution, and concentration is 1.01mg/mL.
Precision weighs ammonium glycyrrhizinate standard substance (lot number：G07-110317) 0.0198g, puts in 10mL volumetric flask, plus methanol
Dissolve and be diluted to scale, obtain ammonium glycyrrhizinate solution, concentration is 1.98mg/mL.
(3) measure：Accurate absorption need testing solution and reference substance solution respectively, injection high performance liquid chromatograph measures, note
Record chromatogram, and carry out methodological study.
Chromatographic condition is：Chromatographic column is phenomenex synergi 4 μ, Hydro-RP chromatographic column (4 μ, 250 ×
4.6mm)；Mobile phase：0.3% phosphoric acid water-acetonitrile, mobile phase A is 0.3% phosphate aqueous solution, and Mobile phase B is acetonitrile, eluting side
Formula：Gradient elution；Detection wavelength 230nm, 30 DEG C of column temperature, flow velocity 0.8ml/min, sample size 10 μ l, theoretical cam curve presses Radix Paeoniae
Glycosides chromatographic peak calculates and is not less than 5000, is all higher than 1.0 with the separating degree of other main chromatographic peaks, all components are all in 100min
It has been detected.Wherein, flowing phase linear gradient is as shown in table 1：
Table 1 chromatogram flow phase condition of gradient elution
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 5 | 95 | 5 |
| 10 | 92 | 8 |
| 20 | 80 | 20 |
| 25 | 77.5 | 22.5 |
| 40 | 67 | 33 |
| 50 | 60 | 40 |
| 60 | 50 | 50 |
| 80 | 15 | 85 |
| 90 | 5 | 95 |
| 100 | 95 | 5 |
Methodological study mainly includes stability, repeatability and precision and investigates, and investigates method and result is as follows：
1. stability experiment takes same batch ginseng branch tuckahoe oral liquid sample, after pretreatment, under the chromatographic condition optimizing,
Respectively at 0h, 2h, 4h, 8h, 12h, 16h and 24h sample introduction is analyzed, and records chromatogram, the results are shown in Table 2.From table 2, each main
Chromatographic peak relative retention time and its relative peak area ratio no significant change, RSD is respectively 0.050%～0.31% He
2.0%～4.3%, illustrate that the composition of need testing solution is stable in 24h.
2. Precision Experiment takes same batch ginseng branch tuckahoe oral liquid sample, after pretreatment, under the chromatographic condition optimizing,
METHOD FOR CONTINUOUS DETERMINATION 6 times, records chromatogram, the results are shown in Table 2.It can be seen that, each main chromatographic peak relative retention time and its relative peak area
Ratio no significant change, RSD is respectively 0.01%～0.25% and 1.7%～4.6%, RSD ＜ 5.0%, and the essence of instrument is described
Density is good.
3. repeated experiment takes 5 parts with a batch of ginseng branch tuckahoe oral liquid sample, after pretreatment, in the chromatostrip optimizing
Under part, sample introduction analysis respectively, record chromatogram, the results are shown in Table 2.Each main chromatographic peak relative retention time and its relative peak area
Ratio no significant change, RSD is respectively 0.01%～1.2% and 0.8%～4.6%, RSD ＜ 5.0%, and this experimental technique is described
Repeatability preferably.
Branch tuckahoe oral liquid fingerprint spectrum method the result joined by table 2
(4) with finger printing software, gained collection of illustrative plates is processed, obtain final product ginseng branch tuckahoe oral liquid finger printing.Wherein, no
With the ginseng branch tuckahoe oral liquid sample of batch and the finger printing of reference substance as shown in figure 1, finger printing such as Fig. 2 institute of reference substance
Show.
With Fig. 1 ginseng branch tuckahoe oral liquid finger printing compare visible, need identification and quantitation 5 kinds of compositions separate well, color
Spectral peak peak shape is symmetrical, and baseline is steady.The retention time of lactone glucoside of Radix Paeoniae, peoniflorin, liquirtin, cinnamic acid and ammonium glycyrrhizinate is respectively
For：25.2,26.2,30.4,47.2 and 55.7min.
Embodiment 2：Carry out joining the quality control of branch tuckahoe oral liquid using finger printing
Carry out joining the quality control of branch tuckahoe oral liquid using finger printing, concrete grammar comprises the following steps：
(1) structure of ginseng branch tuckahoe oral liquid finger printing to be measured：Take the ginseng branch tuckahoe oral liquid sample that 11 batches are to be measured, by reality
The method applying example 1 builds the finger printing of ginseng branch tuckahoe oral liquid to be measured；
(2) using " similarity evaluation " software to 11 batches of ginseng branch tuckahoe oral liquid samples
HPLC finger printing chromatographic peak is mated.Select S1 (numbering is 1 sample) as with reference to collection of illustrative plates, the comparison fingerprint image of generation
Compose as R.Between the finger printing of each batch sample and reference fingerprint and each sample, the similarity result of finger printing is shown in
Table 3.
3 11 batches of table join the Similarity value (correlation coefficient process) of branch tuckahoe oral liquid finger printing
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | S11 | R | |
| S1 | 1.000 | 0.985 | 0.980 | 0.986 | 0.991 | 0.994 | 0.991 | 0.991 | 0.887 | 0.901 | 0.893 | 0.991 |
| S2 | 0.985 | 1.000 | 0.993 | 0.979 | 0.991 | 0.994 | 0.993 | 0.993 | 0.871 | 0.883 | 0.889 | 0.989 |
| S3 | 0.980 | 0.993 | 1.000 | 0.988 | 0.989 | 0.988 | 0.987 | 0.987 | 0.876 | 0.888 | 0.893 | 0.989 |
| S4 | 0.986 | 0.979 | 0.988 | 1.000 | 0.985 | 0.981 | 0.980 | 0.980 | 0.889 | 0.901 | 0.892 | 0.987 |
| S5 | 0.991 | 0.991 | 0.989 | 0.985 | 1.000 | 0.993 | 0.991 | 0.991 | 0.881 | 0.893 | 0.890 | 0.991 |
| S6 | 0.994 | 0.994 | 0.988 | 0.981 | 0.993 | 1.000 | 0.999 | 0.999 | 0.881 | 0.893 | 0.890 | 0.992 |
| S7 | 0.991 | 0.993 | 0.987 | 0.980 | 0.991 | 0.999 | 1.000 | 0.999 | 0.881 | 0.893 | 0.885 | 0.991 |
| S8 | 0.991 | 0.993 | 0.987 | 0.980 | 0.991 | 0.999 | 0.999 | 1.000 | 0.882 | 0.895 | 0.885 | 0.991 |
| S9 | 0.887 | 0.871 | 0.876 | 0.889 | 0.881 | 0.881 | 0.881 | 0.882 | 1.000 | 0.999 | 0.847 | 0.923 |
| S10 | 0.901 | 0.883 | 0.888 | 0.901 | 0.893 | 0.893 | 0.893 | 0.895 | 0.999 | 1.000 | 0.858 | 0.934 |
| S11 | 0.893 | 0.889 | 0.893 | 0.892 | 0.890 | 0.890 | 0.885 | 0.885 | 0.847 | 0.858 | 1.000 | 0.919 |
| R | 0.991 | 0.989 | 0.989 | 0.987 | 0.991 | 0.992 | 0.991 | 0.991 | 0.923 | 0.934 | 0.919 | 1.000 |
As can be seen from Table 3, each batch sample and the similarity of R, all more than 0.900, illustrate that ginseng branch tuckahoe oral liquid is each
The chemical composition of batch sample is generally more close.But number be 1～8 sample larger with the similarity of R, 0.987 with
On, and the sample Similarity value of 9-11 less (0.919～0.934).From the Preliminary Analysis Results of similarity, 1～8 lot sample
Product can be divided into a class, and 9～11 batch sample are another kind of.According to the information of each batch sample, 1～8 batch sample
For the batch sample producing for 2013～2014, and 9,10 batches are the sample of 2010, and 11 batches are the sample of 2012 years.
It to be probably these batch sample time of making the product longer to lead to certain in oral liquid that the similarity of 9～11 batch sample and R is relatively low
Caused by a little compositions or content change.Because reference fingerprint is the average collection of illustrative plates using 11 batch sample finger printing
To represent, therefore each batch sample finger printing all has a certain impact to the R ultimately generating.It can be seen that, R is to different batches sample
Quality separating capacity weaker.
In order to avoid the impact to reference fingerprint for each batch sample, the present invention proposes to be based on reference sample finger printing
To carry out the comparison of finger printing.The construction method of reference sample finger printing is：Calculate each sample and other samples first
Similarity, then select the sample maximum with other sample similarity sums to be reference sample.According to result of calculation, the 6th batch
Secondary sample and similarity sum maximum (see Fig. 3) of other batch sample, therefore select the finger printing conduct of the 6th batch sample
Reference sample finger printing.Similarity evaluation is carried out to each batch sample based on this sample, result is shown in Fig. 4.
From fig. 4, it can be seen that the similarity of 1～8 batch sample and reference sample finger printing is all more than 0.981, and 9～11
The similarity of batch sample is 0.881～0.893, less than 0.900.Illustrate that the quality of these batch sample is relatively low, concordance is not
Good.(it is shown in Table 3 data compared with reference fingerprint), the quality separating capacity to different batches sample for the reference sample finger printing
Strengthened, performance is better than reference fingerprint, judged result more tallies with the actual situation.Because this sample finger printing is true
Measure finger printing, do not affected by each batch sample finger printing, and with other each batch sample fingerprint similarities
Sum is maximum, therefore has preferable representativeness.
Claims (5)
1. a seed ginseng branch tuckahoe oral liquid fingerprint map construction method is it is characterised in that comprise the following steps：
(1) preparation of need testing solution：Take ginseng branch tuckahoe oral liquid, add methanol, supersound extraction, 0.5-1 hour is placed in cold preservation, extensive
Again to room temperature, filter, take subsequent filtrate, obtain final product need testing solution；
(2) preparation of reference substance solution：Take drying under reduced pressure to the cinnamic acid of constant weight, peoniflorin, liquirtin, lactone glucoside of Radix Paeoniae, sweet
Ammonium oxalate reference substance, is separately added into methanol and makes reference substance solution；
(3) measure：Precision measures need testing solution and reference substance solution respectively, and injection high performance liquid chromatograph measures, and records color
Spectrogram；
(4) with finger printing software, gained collection of illustrative plates is processed, obtain final product ginseng branch tuckahoe oral liquid finger printing；
In step (3), the liquid phase chromatogram condition of mensure is：Chromatographic column is phenomenex synergi Hydro-RP chromatographic column；
Mobile phase A is 0.3% phosphate aqueous solution, and Mobile phase B is acetonitrile；Gradient elution；Flow rate of mobile phase is 0.8ml/min；Column temperature is
30℃；Detection wavelength is 230nm；
In step (3), during gradient elution, the change of mobile phase A and Mobile phase B turns to：0-5min, mobile phase A 95%-
95%, Mobile phase B 5%-5%；5-10min, mobile phase A 95%-92%, Mobile phase B 5%-8%；10-20min, mobile phase
A92%-80%, Mobile phase B 8%-20%；20-25min, mobile phase A 80%-77.5%, Mobile phase B 20%-22.5%；
25-40min, mobile phase A 77.5%-67%, Mobile phase B 22.5%-33%；40-50min, mobile phase A 67%-60%, stream
Dynamic phase B 33%-40%；50-60min, mobile phase A 60%-50%, Mobile phase B 40%-50%；60-80min, mobile phase
A50%-15%, Mobile phase B 50%-85%；80-90min, mobile phase A 15%-5%, Mobile phase B 85%-95%；90-
100min, mobile phase A 5%-95%, Mobile phase B 95%-5%.
2. ginseng branch tuckahoe oral liquid fingerprint map construction method as claimed in claim 1 is it is characterised in that in step (1), for examination
The preparation method of product solution is：Precision measures 1.25ml ginseng branch tuckahoe oral liquid in 25ml volumetric flask, plus methanol constant volume is to scale,
Supersound extraction 5min, places 0.5 hour in 4 DEG C, recovers to room temperature, through 0.22 μm of organic system membrane filtration, obtain final product test sample molten
Liquid.
3. ginseng branch tuckahoe oral liquid fingerprint map construction method as claimed in claim 1 is it is characterised in that in step (2), compare
The preparation method of product solution is：
Precision weighs cinnamic acid standard substance 0.0103g, puts in 100mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains meat
Cinnamic acid solution, concentration is 0.103mg/mL；
Precision weighs peoniflorin standard substance 0.0302g, puts in 10mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains Chinese herbaceous peony
Medicine glycosides solution, concentration is 3.02mg/mL；
Precision weighs liquirtin standard substance 0.0204g, puts in 25mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains sweet
Careless glycosides solution, concentration is 0.816mg/mL；
Precision weighs lactone glucoside of Radix Paeoniae standard substance 0.0101g, puts in 10mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains
To lactone glucoside of Radix Paeoniae solution, concentration is 1.01mg/mL；
Precision weighs ammonium glycyrrhizinate standard substance 0.0198g, puts in 10mL volumetric flask, plus methanol dissolves and is diluted to scale, obtains
Ammonium glycyrrhizinate solution, concentration is 1.98mg/mL.
4. ginseng branch tuckahoe oral liquid fingerprint map construction method as claimed in claim 1 is it is characterised in that in step (3), described
The packing material size of chromatographic column is 4 μm, and column length is 250mm, and column internal diameter is 4.6mm.
5. ginseng branch tuckahoe oral liquid fingerprint map construction method as claimed in claim 1 is it is characterised in that in step (3), distinguish
Precision measures need testing solution and the volume of reference substance solution is 10 μ L.
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| CN105467052B (en) * | 2015-11-18 | 2017-03-29 | 江苏康缘药业股份有限公司 | The black kidney tonifying piece component detection method of ginseng and fingerprint map construction method |
| CN108459129B (en) * | 2017-02-17 | 2020-06-05 | 华润三九医药股份有限公司 | Quality control method of radix Stephaniae Tetrandrae and Poria decoction composition |
| CN107632086B (en) * | 2017-09-12 | 2019-06-18 | 山东大学 | The construction method of one seed ginseng branch tuckahoe oral liquid finger-print and application |
| CN108107130B (en) * | 2017-12-22 | 2020-12-08 | 山东沃华医药科技股份有限公司 | Method for measuring fingerprint spectrum of Shenzhiling preparation |
| CN110057927B (en) * | 2018-12-28 | 2020-04-21 | 山东大学 | Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method |
| CN112782326B (en) * | 2020-12-30 | 2022-03-15 | 上海交通大学 | Method for simultaneously measuring fingerprint spectrum and multi-index component content of Weikangling capsule |
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