CN110487941A - The method for building up and its finger-print of Taohong Siwu Tang preparation finger - Google Patents
The method for building up and its finger-print of Taohong Siwu Tang preparation finger Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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Abstract
The invention belongs to drug detection technique fields, are specifically related to the method for building up and its finger-print of a kind of Taohong Siwu Tang preparation finger.Prepare Taohong Siwu Tang preparation testing sample solution, accurate testing sample solution of drawing injects high performance liquid chromatograph respectively, record finger-print, and the finger-print integrated signal of acquisition is imported into similarity evaluation, select in the chromatogram of different batches Taohong Siwu Tang preparation existing for chromatographic peak as shared peak；The standard control finger-print of Taohong Siwu Tang preparation is generated with mean value calculation method；Calculate the relative retention time at each shared peak.The finger-print established using the method for building up of Taohong Siwu Tang preparation finger of the present invention, can effectively be characterized the quality of Taohong Siwu Tang preparation, be conducive to that work is instructed to produce, overall monitor drug quality；The method of the present invention has method simplicity, stablizes, precision height, high repeatability and other advantages.
Description
Technical field
The invention belongs to drug detection technique fields, are specifically related to a kind of foundation of Taohong Siwu Tang preparation finger
Method and its finger-print.
Background technique
Taohong Siwu Tang comes from " Yizong Jinjian gynaecology heart method gist menstruation regulating door " written by Qing Dynasty Wu Qian, by basic Fang Si
Object soup (Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rehmannia glutinosa, Radix Paeoniae Alba) plus peach kernel, safflower are formed, and are had the effect of promoting blood circulation, nourishing blood for regulating menstruation, are cured mainly
Woman in menstrual period belongs to hemostasis in advance person.Recent study shows that Taohong Siwu Tang has anti-inflammatory, analgesia, improves microcirculation, promotes blood
Pipe generates, improves body non-specific immune function, promote union, treat femoral head ischemic necrosis, delay hepatic fibrosis
Change process and other effects.
In Chinese medicine tradition medication, decoction is still occupied an leading position, have absorb rapidly, that production is quick, drug effect is powerful is excellent
The problems such as point, but because its stability is poor, not easy to maintain, the dose of patient is big, not portable limits its application.Granule exists
Take it is the most consistent with traditional decoction in form, on the basis of maintaining decoction advantage, have it is easy to carry, property is stable,
It is easy to save, is suitable for the advantages that industrial mass production.
Since the quality of Chinese medicine is very different, complicated component, existing analysis method are difficult to react medicinal material matter comprehensively in addition
It measures, content substantially only controls one or two index ingredient in the drug standards of compound Chinese medicinal preparation currently on the market,
It is difficult to preferably control drug quality.Traditional Chinese medicine fingerprint is a kind of synthesis, quantifiable identification of means, during it is built upon
On the basis of medicine chemical component system research, be mainly used for evaluating the authenticity of Chinese medicine and Chinese materia medica preparation semi-manufactured goods quality,
Optimality and stability.Chinese medicine and its preparation are multi-component complex system, therefore evaluate its quality should be using being adapted therewith
, the detection method of abundant authentication information can be provided, establishing traditional Chinese medicine fingerprint more will comprehensively reflect Chinese medicine and its system
The type and quantity of contained chemical component in agent, and then whole description and evaluation are carried out to drug quality.Therefore Chinese medicine system is established
Agent finger-print promotes the modernization of Chinese medicine to be of great significance for improving traditional Chinese medicine quality.
" the Taohong Siwu Tang characteristic spectrum of Different Extraction Method studies [J] Yunnan University of Traditional Chinese Medicine journal, and 2017 (3) " are adopted
Map research is done to the Different Extraction Method of Taohong Siwu Tang with wavelength switching method, establishes the HPLC characteristic pattern of Taohong Siwu Tang
Spectrum, shares 22 shared peaks, and pointed out to the chemical structure at wherein 5 shared peaks, according to reported in literature, galla turcica
Acid, Paeoniflorin are mostly derived from the Radix Paeoniae Alba in prescription, and 5 hydroxymethyl furfural is mostly derived from the glutinous rehmannia in prescription, Sydroxy carthamin
A is mostly derived from the safflower in prescription, and ferulic acid is mostly derived from Radix Angelicae Sinensis and Rhizoma Chuanxiong in prescription.But it is not pointed out in prescription in text
The characteristic component of peach kernel and the assay ingredient of glutinous rehmannia, and 4,5,18, No. 21 in finger-print disclosed in text are altogether
There is peak, peak area is larger, is the representative component in Taohong Siwu Tang, but its chemical structure is not pointed out in text, moreover, fingerprint
Map will also calculate similarity compared to characteristic spectrum, give between each component other than the structural information for providing characteristic peak
Amount relationship, the product information provided is more comprehensively.
It is disclosed in patent " a kind of detection method of the pharmaceutical preparation of Taohong Siwu Tang of CN 201710780204- " pink
The fingerprint spectrum method of Siwu Tang shares 11 common characteristic peaks, and is referred to the chemical structure at wherein 5 shared peaks
Recognize, respectively hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, ferulic acid, acteoside, Ligustilide, but does not point out prescription in text
In peach kernel characteristic component, and as a compound Chinese medicinal preparation by 6 taste Chinese medicinal compositions, 11 common characteristic peaks are given
Chemical information out slightly shows thin.Therefore, method provided by the prior art cannot full and accurate embodiment each group patent medicine comprehensively letter
Breath, it is comprehensive as the method not enough science for instructing industrial production to control drug quality.
Summary of the invention
Refer to the technical problem to be solved by the present invention is overcoming the deficiencies of the prior art and provide a kind of Taohong Siwu Tang preparation
The method for building up and its finger-print of line map.The fingerprint image established using the method for building up of finger-print of the present invention
Spectrum, can characterize whole medicinal material information in Taohong Siwu Tang preparation comprehensively, more comprehensively embody the quality of drug；For referring to
Industrial production is led, can preferably guarantee product quality.
The method for building up of Taohong Siwu Tang preparation finger of the present invention, comprising the following steps:
(1) preparation of testing sample solution
It takes Taohong Siwu Tang preparation finely ground, the methanol aqueous solution of concentration expressed in percentage by volume 30% is added and carries out ultrasonic dissolution,
The methanol aqueous solution for adding concentration expressed in percentage by volume 30% supplies the weight of less loss, shakes up, and miillpore filter filtration takes filtrate to obtain the final product
Testing sample solution；
(2) preparation of reference solution
Take gallic acid, tryptophan, chlorogenic acid, amarogentin, hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, ferulic acid, Mao Rui
Flower glucosides and Ligustilide, add methanol to dissolve, solution of every 1ml containing each 20 μ g of reference substance are made, as reference solution；
(3) accurate respectively to draw reference solution and each 5-10 μ l of testing sample solution, inject high performance liquid chromatograph, note
Finger-print in record 65-75 minutes；
(4) by the finger-print integrated signal of sample to be tested import Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica is similar
Degree evaluation system A editions " software；Select in the finger-print of different batches Taohong Siwu Tang preparation existing chromatographic peak as being total to
There is peak；Similarity result is calculated with mean value calculation method, and generates standard control finger-print；Calculate the opposite guarantor at each shared peak
Stay time and each shared peak relative retention time and the ratio referring to peak relative retention time.
Wherein:
Preferably, the preparation of testing sample solution described in step (1) is: taking the Taohong Siwu Tang granule of different batches
Sample, it is finely ground, it takes 0.8g Taohong Siwu Tang granule sample to be placed in stuffed conical flask respectively, concentration expressed in percentage by volume 30% is added
Methanol aqueous solution 25ml, close plug, weighed weight, and being ultrasonically treated after ultrasonic treatment, adds concentration expressed in percentage by volume
30% methanol aqueous solution supplies the weight of less loss, shakes up, and miillpore filter (aperture is 0.45 μm) filtration takes filtrate up to be measured
Sample solution.
The chromatographic condition of determining fingerprint pattern described in step (3) are as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；
Column temperature: 25-35 DEG C；
Flow velocity: 0.8-1.2ml/min；
Sample volume is 5-10 μ l；
Ultraviolet detection wavelength: 210-250nm, it is preferred that ultraviolet detection wavelength is one in 210nm, 220nm or 250nm
Kind.
Mobile phase is any one in following (1), (2), (3) or (4):
(1) 0.02% phosphate aqueous solution of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution；
(2) 0.05% aqueous formic acid of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution；
(3) 0.1% aqueous formic acid of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution；
(4) 0.02% trifluoroacetic acid aqueous solution of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution.
Type of elution uses gradient elution, and the process of gradient elution is any one in following (1), (2) or (3):
(1) 0~5min, 2% Mobile phase B of concentration expressed in percentage by volume, 5~15min, concentration expressed in percentage by volume 2%-20% mobile phase
B, 15~35min, concentration expressed in percentage by volume 20%-28% Mobile phase B, 35~60min, concentration expressed in percentage by volume 28%-80% flowing
Phase B, 60~65min, 80% Mobile phase B of concentration expressed in percentage by volume, 65~66min, concentration expressed in percentage by volume 80%-2% Mobile phase B,
66~75min, 2% Mobile phase B of concentration expressed in percentage by volume；
(2) 0~5min, 3% Mobile phase B of concentration expressed in percentage by volume, 5~15min, concentration expressed in percentage by volume 3%-16% mobile phase
B, 15~20min, concentration expressed in percentage by volume 16%-25% Mobile phase B, 20~30min, 25% Mobile phase B of concentration expressed in percentage by volume, 30
~40min, concentration expressed in percentage by volume 25%-40% Mobile phase B, 40~50min, concentration expressed in percentage by volume 40%-80% Mobile phase B,
50~60min, 80% Mobile phase B of concentration expressed in percentage by volume, 60~61min, concentration expressed in percentage by volume 80%-3% Mobile phase B, 61~
70min, 3% Mobile phase B of concentration expressed in percentage by volume；
(3) 0~5min, 3% Mobile phase B of concentration expressed in percentage by volume, 5~15min, concentration expressed in percentage by volume 3%-15% mobile phase
B, 15~20min, concentration expressed in percentage by volume 15%-25% Mobile phase B, 20~30min, 25% Mobile phase B of concentration expressed in percentage by volume, 30
~40min, concentration expressed in percentage by volume 25%-40% Mobile phase B, 40~55min, concentration expressed in percentage by volume 40%-80% Mobile phase B,
50~60min, 80% Mobile phase B of concentration expressed in percentage by volume, 60~61min, concentration expressed in percentage by volume 80%-3% Mobile phase B, 61~
70min, 3% Mobile phase B of concentration expressed in percentage by volume.
The preparation method of Taohong Siwu Tang granule described in step (1) is: taking 9 parts of radix rehmanniae recen (wine is washed), Rhizoma Chuanxiong 3
Part, 12 parts of Radix Angelicae Sinensis (wine is washed), 3.78 parts of peach kernel (mud is ground in peeling), 4.5 parts of Radix Paeoniae Alba (parched with wine) and 3 parts of safflower (wine is washed), above six
Taste, add water to cook it is secondary, 1 hour every time, for the first time plus the water of whole 12 times of weight of medicine materical crude slice, second plus whole medicine materical crude slice weight 10
Water again, decocting liquid filtration, filtrate merge, and filtrate decompression is concentrated into thick paste, and thick paste is dried under reduced pressure to solid, crushes, is added in right amount
Auxiliary material, granulation, whole grain is to get Taohong Siwu Tang granule.
The finger-print include 28 shared peaks, each shared peak relative retention time with referring to peak relative retention time
Ratio is respectively as follows:
No. 1 peak relative retention time RRT is 0.16；No. 2 peak relative retention time RRT are 0.22；The opposite reservation at No. 3 peaks
Time RRT is 0.24；No. 4 peak relative retention time RRT are 0.27；No. 5 peak relative retention time RRT are 0.33；No. 6 peaks are opposite
Retention time RRT is 0.36；No. 7 peak relative retention time RRT are 0.38；No. 8 peak relative retention time RRT are 0.48；No. 9 peaks
Relative retention time RRT is 0.54；No. 10 peak relative retention time RRT are 0.57；No. 11 peak relative retention time RRT are
0.58；No. 12 peak relative retention time RRT are 0.65；No. 13 peak relative retention time RRT are 0.66；When No. 14 peaks retain relatively
Between RRT be 0.72；No. 15 peak relative retention time RRT are 0.76；No. 16 peak relative retention time RRT are 0.79；No. 17 peak phases
It is 0.81 to retention time RRT；No. 18 peak relative retention time RRT are 0.83；No. 19 peak relative retention time RRT are 0.88；
No. 20 peak relative retention time RRT are 1.00；No. 21 peak relative retention time RRT are 1.16；No. 22 peak relative retention time RRT
It is 1.19；No. 23 peak relative retention time RRT are 1.23；No. 24 peak relative retention time RRT are 1.26；No. 25 peaks are opposite to be retained
Time RRT is 1.36；No. 26 peak relative retention time RRT are 1.43；No. 27 peak relative retention time RRT are 1.49；No. 28 peaks
Relative retention time RRT is 1.53.Wherein: No. 20 peaks S are referring to peak.
No. 6 peaks are gallic acid in shared peak, and No. 10 peaks are tryptophan, and No. 14 peaks are amarogentin, and No. 15 peaks are green original
Acid, No. 19 peaks are hydroxyl radical carthamin yellow carthamus A, and No. 20 peaks are Paeoniflorin, and No. 21 peaks are ferulic acid, and No. 24 peaks are acteoside,
No. 28 peaks are Ligustilide.
The present invention establishes the finger-print of Taohong Siwu Tang preparation using high performance liquid chromatography, shares 28 shared peaks,
And the chemical structure at wherein 9 shared peaks has been pointed out, whole medicinal material information in Taohong Siwu Tang preparation can be characterized comprehensively, more
It can preferably guarantee product quality for instructing industrial production for the quality for comprehensively embodying drug.
Compared with the prior art, the present invention has the following beneficial effects:
(1) finger-print of Taohong Siwu Tang preparation of the present invention shares 28 shared peaks, and wherein peak has been determined
The chemical structure at the biggish 9 shared peaks of area ratio, wherein the spy comprising characteristic component amarogentin and glutinous rehmannia derived from peach kernel
Ingredient acteoside is levied, finger-print provided by the present invention can embody pink four in a map under Same Wavelength
The medicinal material information of object soup whole more comprehensively embodies the quality of drug, and the method relative to wavelength switching, institute of the present invention
The method of offer is more simple and easy to do.
(2) finger-print of Taohong Siwu Tang preparation of the present invention, can more fully react drug quality, work as drug
When quality occurs problem or adverse reaction occurs, can more accurately confirm which taste medicinal material there is a problem in prescription, to guidance
Industrial production control drug quality is more significant, and finger-print information is more comprehensively, more full and accurate, can preferably guarantee product matter
Amount.
(3) method for building up of the finger-print of Taohong Siwu Tang preparation of the present invention has easy, stable, accurate
The advantages of degree height, favorable reproducibility.
Detailed description of the invention
Fig. 1 is 18010201-18010216 batches of sample finger-prints and standard control finger-prints；
Fig. 2 is reference solution finger-print；
Fig. 3 is 18010201 batches of test article fingerprints；
Fig. 4 is 18010201 stability experiment finger-prints；
Fig. 5 is 18010201 Precision Experiment finger-prints；
Fig. 6 is 18010201 repeated experiment finger-prints；
In figure: 1, gallic acid；2, tryptophan；3, amarogentin；4, chlorogenic acid；5, hydroxyl radical carthamin yellow carthamus A；6, Chinese herbaceous peony
Glycosides；7, ferulic acid B；8, acteoside A；9, Ligustilide；S1,18010201 crowdes；S2,18010202 crowdes；S318010203
Batch,；S4,18010204 crowdes；S5,18010205 crowdes；S6,18010206 crowdes；S7,18010207 crowdes；S8,18010208 crowdes；S9,
18010209 batches；S10,18010210 crowdes；S11,18010211 crowdes；S12,18010212 crowdes；S13,18010213 crowdes；S14,
18010214 batches；S15,18010215 crowdes；S16,18010216 crowdes；R, reference fingerprint.
Specific embodiment
The invention will be further described with reference to embodiments.
1, the preparation of Taohong Siwu Tang preparation
Prescription medicine totally 16 batches are bought from national main Chinese medicinal materials producing region and Genuine producing area respectively, place of production information is shown in Table 1.
Taohong Siwu Tang preparation of the present invention the preparation method is as follows: taking radix rehmanniae recen (wine is washed) 9g, Rhizoma Chuanxiong 3g, Radix Angelicae Sinensis
(wine is washed) 12g, peach kernel 3.78g, Radix Paeoniae Alba (parched with wine) 4.5g, safflower (wine is washed) 3g, the above Six-element adds water to cook secondary, and 1 is small every time
When, add the water of whole 12 times of weight of medicine materical crude slice for the first time, for the second time plus the water of whole 10 times of weight of medicine materical crude slice, decocting liquid filter, and filtrate is closed
And filtrate decompression is concentrated into thick paste, thick paste is dried under reduced pressure to solid, crushes, and appropriate amount of auxiliary materials is added, granulation, whole grain is to get pink
Siwu Tang granule.
Medicinal material is taken to prepare Taohong Siwu Tang particle by the number of medicinal material listed by table 1, sampling method is to take Radix Paeoniae Alba No. 1, safflower 1
Number, peach kernel 1, glutinous rehmannia No. 1, No. 1 Radix Angelicae Sinensis 1, Rhizoma Chuanxiong medicinal material prepare Taohong Siwu Tang particle, lot number 18010201；It takes white
No. 2 Chinese herbaceous peony 2, safflower 2, peach kernel 2, glutinous rehmannia No. 2, Radix Angelicae Sinensis 2, Rhizoma Chuanxiong medicinal materials prepare Taohong Siwu Tang particle, and lot number is
18010202, and so on.
The Taohong Siwu Tang particle of preparation, lot number 18010201-18010216.18010201 batches of numbers are S1,
18010202 batches of numbers are S2, and so on, 18010216 batches of numbers are S16.
Embodiment 1
The method for building up of Taohong Siwu Tang preparation finger described in embodiment 1, comprising the following steps:
1, instrument and reagent
High performance liquid chromatograph (Agilent 1260), assay balance (METTLER TOLEDO XS/205DU), ultrasound are clear
Washing machine (KQ-5200E).
Reagent: methanol is chromatographically pure, and water is ultrapure water；Other reagents are that analysis is pure.Gallic acid, color ammonia in object of reference
During acid, amarogentin, chlorogenic acid, hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, ferulic acid, acteoside, Ligustilide are purchased from
State's food and medicine examines and determine research institute.
For reagent: Taohong Siwu Tang particle (lot number: 18010201-18010216)
2, chromatographic condition: using octadecylsilane chemically bonded silica as filler (Agilent 5TC-C18,4.6*250mm, 5
μm), using 0.02% phosphate aqueous solution as mobile phase A, using methanol as Mobile phase B, condition of gradient elution is 0~5min, volume hundred
Divide 3% Mobile phase B of concentration, 5~15min, concentration expressed in percentage by volume 3%-16% Mobile phase B, 15~20min, concentration expressed in percentage by volume
16%-25% Mobile phase B, 20~30min, 25% Mobile phase B of concentration expressed in percentage by volume, 30~40min, concentration expressed in percentage by volume
25%-40% Mobile phase B, 40~50min, concentration expressed in percentage by volume 40%-80% Mobile phase B, 50~60min, volume basis are dense
Spend 80% Mobile phase B, 60~61min, concentration expressed in percentage by volume 80%-3% Mobile phase B, 61~70min, concentration expressed in percentage by volume 3%
Mobile phase B.
Flow velocity is 1.0ml/min；35 DEG C of column temperature；Detection wavelength is 220nm.Number of theoretical plate should not be low by the calculating of Paeoniflorin peak
In 3000.
3, the preparation of reference solution: take gallic acid, tryptophan, chlorogenic acid, amarogentin, hydroxyl radical carthamin yellow carthamus A,
Paeoniflorin, ferulic acid, acteoside and Ligustilide, add methanol to dissolve, and solution of every 1ml containing each 20 μ g of reference substance is made,
As reference solution.
4, the preparation of testing sample solution: taking Taohong Siwu Tang particle, finely ground, takes about 0.8g, accurately weighed, sets tool plug cone
In shape bottle, 30% methanol 25ml of concentration expressed in percentage by volume, close plug, weighed weight, with 30% methanol benefit after ultrasonic treatment is added in precision
The weight of sufficient less loss shakes up filtration, take subsequent filtrate to get.
5, measuring method: it is accurate respectively to draw reference solution and each 10 μ l of test solution, inject high performance liquid chromatograph
In, measurement records 70 minutes chromatograms.
Obtained finger-print includes 28 shared peaks, each peak relative retention time are as follows:
No. 1 peak 5.821min；No. 2 peak 7.876min；No. 3 peak 8.592min；No. 4 peak 9.367min；No. 5 peaks
11.764min；No. 6 peak 12.890min；No. 7 peak 13.497min；No. 8 peaks 17.126；No. 9 peaks 19.113；No. 10 peaks
20.122min；No. 11 peak 20.527min；No. 12 peak 22.968min；No. 13 peak 23.485min；No. 14 peak 25.564min；15
Number peak 26.739min；No. 16 peak 27.844min；No. 17 peak 28.445min；No. 18 peak 29.456min；No. 19 peaks
31.171min；No. 20 peak 35.329min；No. 21 peak 40.999min；No. 22 peak 42.096min；No. 23 peak 43.378min；24
Number peak 44.442min；No. 25 peak 48.220min；No. 26 peak 50.417min；No. 27 peak 52.546min；No. 28 peaks
54.059min.No. 20 peaks are referring to peak.
Each peak relative retention time is respectively as follows: with referring to peak relative retention time ratio
No. 1 peak relative retention time RRT is 0.16；No. 2 peak relative retention time RRT are 0.22；The opposite reservation at No. 3 peaks
Time RRT is 0.24；No. 4 peak relative retention time RRT are 0.27；No. 5 peak relative retention time RRT are 0.33；No. 6 peaks are opposite
Retention time RRT is 0.36；No. 7 peak relative retention time RRT are 0.38；No. 8 peak relative retention time RRT are 0.48；No. 9 peaks
Relative retention time RRT is 0.54；No. 10 peak relative retention time RRT are 0.57；No. 11 peak relative retention time RRT are
0.58；No. 12 peak relative retention time RRT are 0.65；No. 13 peak relative retention time RRT are 0.66；When No. 14 peaks retain relatively
Between RRT be 0.72；No. 15 peak relative retention time RRT are 0.76；No. 16 peak relative retention time RRT are 0.79；No. 17 peak phases
It is 0.81 to retention time RRT；No. 18 peak relative retention time RRT are 0.83；No. 19 peak relative retention time RRT are 0.88；
No. 20 peak relative retention time RRT are 1.00；No. 21 peak relative retention time RRT are 1.16；No. 22 peak relative retention time RRT
It is 1.19；No. 23 peak relative retention time RRT are 1.23；No. 24 peak relative retention time RRT are 1.26；No. 25 peaks are opposite to be retained
Time RRT is 1.36；No. 26 peak relative retention time RRT are 1.43；No. 27 peak relative retention time RRT are 1.49；No. 28 peaks
Relative retention time RRT is 1.53.Wherein: No. 20 peaks S are the chromatographic peaks of object of reference.Characteristic spectrum described in embodiment 1 is shown in attached
Fig. 1.
Wherein: No. 6 peaks are gallic acid in shared peak, and No. 10 peaks are tryptophan, and No. 14 peaks are amarogentin, and No. 15 peaks are
Chlorogenic acid, No. 19 peaks are hydroxyl radical carthamin yellow carthamus A, and No. 20 peaks are Paeoniflorin, and No. 21 peaks are ferulic acid, and No. 24 peaks are verbascose
Glycosides, No. 28 peaks are Ligustilide.See attached drawing 2.
The corresponding No. 6 shared peaks in No. 1 peak in Fig. 2, are gallic acid；The corresponding No. 10 shared peaks in No. 2 peaks, are tryptophan；No. 3 peaks
Corresponding No. 14 shared peaks are amarogentin；The corresponding No. 15 shared peaks in No. 4 peaks, are chlorogenic acid；The corresponding No. 19 shared peaks in No. 5 peaks are
Hydroxyl radical carthamin yellow carthamus A；The corresponding No. 20 shared peaks in No. 6 peaks, are Paeoniflorin；The corresponding No. 21 shared peaks in No. 7 peaks, are ferulic acid；No. 8
No. 24 shared peaks are coped at peak, are acteoside；The corresponding 28 shared peaks in No. 9 peaks, are Ligustilide.
Integrated signal is imported to " similarity evaluation A editions " software of Chinese Pharmacopoeia committee, with
S1 is to calculate to obtain similarity result referring to spectrogram by mean value method, and generate standard control finger-print (RFP), see Fig. 1, phase
It the results are shown in Table 2 like degree.Similarity is substandard product less than 0.90, carries out quality evaluation to 16 batches of samples.
18010201 batches of numbers are S1, and 18010202 batches of numbers are S2, and so on, 18010216 batches of numbers are S16.
2 16 batches of sample finger-prints of table are compared with standard control fingerprint similarity
| Number | Similarity |
| S1 | 0.987 |
| S2 | 0.986 |
| S3 | 0.968 |
| S4 | 0.963 |
| S5 | 0.971 |
| S6 | 0.972 |
| S7 | 0.926 |
| S8 | 0.984 |
| S9 | 0.978 |
| S10 | 0.987 |
| S11 | 0.986 |
| S12 | 0.968 |
| S13 | 0.963 |
| S14 | 0.972 |
| S15 | 0.926 |
| S16 | 0.984 |
The above result shows that 18010201-18010216 batches of samples are qualified products.
Embodiment 2
The method for building up of Taohong Siwu Tang preparation finger as described in example 2, comprising the following steps:
1. instrument and reagent
High performance liquid chromatograph (Agilent 1260), assay balance (METTLER TOLEDO XS/205DU), ultrasound are clear
Washing machine (KQ-5200E).
Reagent: methanol is chromatographically pure, and water is ultrapure water；Other reagents are that analysis is pure.Gallic acid, color ammonia in object of reference
During acid, amarogentin, chlorogenic acid, hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, ferulic acid, acteoside, Ligustilide are purchased from
State's food and medicine examines and determine research institute.
For reagent: Taohong Siwu Tang particle (lot number: 18010201-18010216).
2. chromatographic condition: using octadecylsilane chemically bonded silica as filler (Agilent ZORBAX SB-C18,4.6 ×
250mm, 5 μm), using 0.05% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, condition of gradient elution is 0~5min,
2% Mobile phase B, 5~15min, 2%-20% Mobile phase B, 15~35min, 20%-28% Mobile phase B, 35~60min,
28%-80% Mobile phase B, 60~65min, 80% Mobile phase B, 65~66min, 80%-2% Mobile phase B, 66~75min,
2% Mobile phase B.Flow velocity is 0.8ml per minute；25 DEG C of column temperature；Detection wavelength is 210nm.Number of theoretical plate is calculated by Paeoniflorin peak
3000 should be not less than.
3. the preparation of reference solution: take gallic acid, tryptophan, chlorogenic acid, amarogentin, hydroxyl radical carthamin yellow carthamus A,
Paeoniflorin, ferulic acid, acteoside and Ligustilide, add methanol to dissolve, and solution of every 1ml containing each 20 μ g of reference substance is made,
As reference solution.
4. the preparation of test solution: Taohong Siwu Tang particle is taken, it is finely ground, about 0.8g is taken, it is accurately weighed, set tool plug taper
In bottle, 30% methanol 25ml, close plug is added in precision, and weighed weight is supplied the weight of less loss after ultrasonic treatment with 30% methanol, shaken
Even filtration, take subsequent filtrate to get.
5. measuring method: it is accurate respectively to draw reference solution and each 8 μ l of test solution, inject high performance liquid chromatograph
In, measurement records 75 minutes chromatograms.
Obtained finger-print includes 28 shared peaks, each peak relative retention time are as follows:
No. 1 peak 5.075min；No. 2 peak 6.028min；No. 3 peak 6.284min；No. 4 peak 7.716min；No. 5 peaks
9.006min；No. 6 peak 10.066min；No. 7 peak 12.205min；No. 8 peaks 12.319；No. 9 peaks 18.790；No. 10 peaks
18.875min；No. 11 peak 22.366min；No. 12 peak 24.867min；No. 13 peak 26.345min；No. 14 peak 27.662min；15
Number peak 28.265min；No. 16 peak 28.848min；No. 17 peak 29.545min；No. 18 peak 31.201min；No. 19 peaks
34.586min；No. 20 peak 35.209min；No. 21 peak 41.990min；No. 22 peak 43.165min；No. 23 peak 44.028min；24
Number peak 48.425min；No. 25 peak 50.448min；No. 26 peak 50.882min；No. 27 peak 57.589min；No. 28 peaks
60.325min.No. 20 peaks are referring to peak.
Each peak relative retention time is respectively as follows: with referring to peak relative retention time ratio
No. 1 peak relative retention time RRT is 0.14；No. 2 peak relative retention time RRT are 0.17；The opposite reservation at No. 3 peaks
Time RRT is 0.18；No. 4 peak relative retention time RRT are 0.22；No. 5 peak relative retention time RRT are 0.26；No. 6 peaks are opposite
Retention time RRT is 0.29；No. 7 peak relative retention time RRT are 0.35；No. 8 peak relative retention time RRT are 0.35；No. 9 peaks
Relative retention time RRT is 0.53；No. 10 peak relative retention time RRT are 0.54；No. 11 peak relative retention time RRT are
0.64；No. 12 peak relative retention time RRT are 0.71；No. 13 peak relative retention time RRT are 0.75；When No. 14 peaks retain relatively
Between RRT be 0.79；No. 15 peak relative retention time RRT are 0.80；No. 16 peak relative retention time RRT are 0.82；No. 17 peak phases
It is 0.84 to retention time RRT；No. 18 peak relative retention time RRT are 0.89；No. 19 peak relative retention time RRT are 0.98；
No. 20 peak relative retention time RRT are 1.00；No. 21 peak relative retention time RRT are 1.19；No. 22 peak relative retention time RRT
It is 1.23；No. 23 peak relative retention time RRT are 1.25；No. 24 peak relative retention time RRT are 1.38；No. 25 peaks are opposite to be retained
Time RRT is 1.43；No. 26 peak relative retention time RRT are 1.45；No. 27 peak relative retention time RRT are 1.64；No. 28 peaks
Relative retention time RRT is 1.71.Wherein: No. 20 peaks S are the chromatographic peaks of object of reference.
Wherein: No. 7 peaks are gallic acid in shared peak, and No. 10 peaks are tryptophan, and No. 12 peaks are amarogentin, and No. 13 peaks are
Chlorogenic acid, No. 18 peaks are hydroxyl radical carthamin yellow carthamus A, and No. 20 peaks are Paeoniflorin, and No. 21 peaks are ferulic acid, and No. 23 peaks are verbascose
Glycosides, No. 28 peaks are Ligustilide.
6. similarity result: integrated signal is imported to " the chromatographic fingerprints of Chinese materia medica similarity evaluation system of Chinese Pharmacopoeia committee
System A editions " software is to calculate to obtain similarity result by mean value method, similarity result is shown in Table 3 referring to spectrogram with S1.Similarity is small
It is substandard product in 0.90, quality evaluation is carried out to 16 batches of samples.
18010201 batches of numbers are S1, and 18010202 batches of numbers are S2, and so on, 18010216 batches of numbers are S16.
3 16 batches of sample finger-prints of table are compared with standard control fingerprint similarity
| Number | Similarity |
| S1 | 0.983 |
| S2 | 0.979 |
| S3 | 0.975 |
| S4 | 0.977 |
| S5 | 0.982 |
| S6 | 0.982 |
| S7 | 0.973 |
| S8 | 0.983 |
| S9 | 0.981 |
| S10 | 0.950 |
| S11 | 0.982 |
| S12 | 0.988 |
| S13 | 0.983 |
| S14 | 0.966 |
| S15 | 0.980 |
| S16 | 0.981 |
The above result shows that 18010201-18010216 batches of samples are qualified products.
Embodiment 3
The method for building up of Taohong Siwu Tang preparation finger described in embodiment 3, comprising the following steps:
1. instrument and reagent
High performance liquid chromatograph (Agilent 1260), assay balance (METTLER TOLEDO XS/205DU), ultrasound are clear
Washing machine (KQ-5200E).
Reagent: methanol is chromatographically pure, and water is ultrapure water；Other reagents are that analysis is pure.Gallic acid, color ammonia in object of reference
During acid, amarogentin, chlorogenic acid, hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, ferulic acid, acteoside, Ligustilide are purchased from
State's food and medicine examines and determine research institute.
For reagent: Taohong Siwu Tang particle (lot number: 18010201-18010216).
2. chromatographic condition: using octadecylsilane chemically bonded silica as filler (Cosmosil 5C18-AR-II, 4.6 ×
250mm, 5 μm), using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, condition of gradient elution is 0~5min,
3%B, 5~15min, 3%-16% Mobile phase B, 15~20min, 16%-25% Mobile phase B, 20~30min, 25% mobile phase
B, 30~40min, 25%-40% Mobile phase B, 40~50min, 40%-80% Mobile phase B, 50~60min, 80% mobile phase
B, 60~61min, 80%-3% Mobile phase B, 61~70min, 3% Mobile phase B.Flow velocity is 1.2ml/min per minute；Column temperature 35
℃；Detection wavelength is 250nm.Number of theoretical plate is calculated by Paeoniflorin peak should be not less than 3000.
3. the preparation of reference solution: take gallic acid, tryptophan, chlorogenic acid, amarogentin, hydroxyl radical carthamin yellow carthamus A,
Paeoniflorin, ferulic acid, acteoside and Ligustilide, add methanol to dissolve, and every 1ml methanol is made containing the molten of each 20 μ g of reference substance
Liquid, as reference solution.
4. the preparation of test solution: Taohong Siwu Tang particle is taken, it is finely ground, about 0.8g is taken, it is accurately weighed, set tool plug taper
In bottle, 30% methanol 25ml, close plug is added in precision, and weighed weight is supplied the weight of less loss after ultrasonic treatment with 30% methanol, shaken
Even filtration, take subsequent filtrate to get.
5. measuring method: it is accurate respectively to draw reference solution and each 10 μ l of test solution, inject high performance liquid chromatograph
In, measurement records 70 minutes chromatograms.
Obtained finger-print includes 28 shared peaks, each peak relative retention time are as follows:
No. 1 peak 5.678min；No. 2 peak 7.536min；No. 3 peak 8.063min；No. 4 peak 9.078min；No. 5 peaks
11.536min；No. 6 peak 12.590min；No. 7 peak 13.197min；No. 8 peaks 16.923；No. 9 peaks 19.113；No. 10 peaks
19.853min；No. 11 peak 20.227min；No. 12 peak 22.668min；No. 13 peak 23.185min；No. 14 peak 25.278min；15
Number peak 26.459min；No. 16 peak 27.522min；No. 17 peak 28.145min；No. 18 peak 29.156min；No. 19 peaks
30.636min；No. 20 peak 35.029min；No. 21 peak 40.796min；No. 22 peak 41.036min；No. 23 peak 43.358min；24
Number peak 44.402min；No. 25 peak 47.920min；No. 26 peak 50.117min；No. 27 peak 52.036min；No. 28 peaks
54.369min.No. 20 peaks are referring to peak.
Each peak relative retention time is respectively as follows: with referring to peak relative retention time ratio
No. 1 peak relative retention time RRT is 0.16；No. 2 peak relative retention time RRT are 0.22；The opposite reservation at No. 3 peaks
Time RRT is 0.23；No. 4 peak relative retention time RRT are 0.27；No. 5 peak relative retention time RRT are 0.33；No. 6 peaks are opposite
Retention time RRT is 0.36；No. 7 peak relative retention time RRT are 0.38；No. 8 peak relative retention time RRT are 0.48；No. 9 peaks
Relative retention time RRT is 0.55；No. 10 peak relative retention time RRT are 0.57；No. 11 peak relative retention time RRT are
0.58；No. 12 peak relative retention time RRT are 0.65；No. 13 peak relative retention time RRT are 0.66；When No. 14 peaks retain relatively
Between RRT be 0.72；No. 15 peak relative retention time RRT are 0.76；No. 16 peak relative retention time RRT are 0.79；No. 17 peak phases
It is 0.80 to retention time RRT；No. 18 peak relative retention time RRT are 0.83；No. 19 peak relative retention time RRT are 0.87；
No. 20 peak relative retention time RRT are 1.00；No. 21 peak relative retention time RRT are 1.16；No. 22 peak relative retention time RRT
It is 1.17；No. 23 peak relative retention time RRT are 1.24；No. 24 peak relative retention time RRT are 1.27；No. 25 peaks are opposite to be retained
Time RRT is 1.37；No. 26 peak relative retention time RRT are 1.43；No. 27 peak relative retention time RRT are 1.49；No. 28 peaks
Relative retention time RRT is 1.55.Wherein: No. 20 peaks S are the chromatographic peaks of object of reference.
Wherein: No. 6 peaks are gallic acid in shared peak, and No. 10 peaks are tryptophan, and No. 14 peaks are amarogentin, and No. 15 peaks are
Chlorogenic acid, No. 19 peaks are hydroxyl radical carthamin yellow carthamus A, and No. 20 peaks are Paeoniflorin, and No. 21 peaks are ferulic acid, and No. 24 peaks are verbascose
Glycosides, No. 28 peaks are Ligustilide.
6. similarity result: integrated signal is imported to " the chromatographic fingerprints of Chinese materia medica similarity evaluation system of Chinese Pharmacopoeia committee
System A editions " software is to calculate to obtain similarity result by mean value method, similarity result is shown in Table 4 referring to spectrogram with S1.Similarity is small
It is substandard product in 0.90, quality evaluation is carried out to 16 batches of samples.
18010201 batches of numbers are S1, and 18010202 batches of numbers are S2, and so on, 18010216 batches of numbers are S16.
4 16 batches of sample finger-prints of table are compared with standard control fingerprint similarity
| Number | Similarity |
| S1 | 0.972 |
| S2 | 0.987 |
| S3 | 0.960 |
| S4 | 0.983 |
| S5 | 0.968 |
| S6 | 0.964 |
| S7 | 0.975 |
| S8 | 0.973 |
| S9 | 0.970 |
| S10 | 0.975 |
| S11 | 0.959 |
| S12 | 0.984 |
| S13 | 0.969 |
| S14 | 0.922 |
| S15 | 0.980 |
| S16 | 0.978 |
The above result shows that 18010201-18010216 batches of samples are qualified products.
Embodiment 4
The method for building up of Taohong Siwu Tang preparation finger as described in example 4, comprising the following steps:
1. instrument and reagent
High performance liquid chromatograph (Agilent 1260), assay balance (METTLER TOLEDO XS/205DU), ultrasound are clear
Washing machine (KQ-5200E).
Reagent: methanol is chromatographically pure, and water is ultrapure water；Other reagents are that analysis is pure.Gallic acid, color ammonia in object of reference
Acid, amarogentin, chlorogenic acid, Sydroxy carthamin, Paeoniflorin, ferulic acid, acteoside, Ligustilide are purchased from China
Food and medicine examines and determine research institute.
Reagent: Taohong Siwu Tang particle (lot number: 18010201-18010216).
2. chromatographic condition: using octadecylsilane chemically bonded silica as filler (Agilent 5TC-C18,4.6 × 250mm,
5 μm), using 0.02% trifluoroacetic acid aqueous solution as mobile phase A, using methanol as Mobile phase B, condition of gradient elution is 0~5min,
3% Mobile phase B, 5~15min, 3%-15% Mobile phase B, 15~20min, 15%-25% Mobile phase B, 20~30min, 25%
Mobile phase B, 30~40min, 25%-40% Mobile phase B, 40~50min, 40%-80% Mobile phase B, 50~60min, 80%
Mobile phase B, 60~61min, 80%-3% Mobile phase B, 61~70min, 3% Mobile phase B.Flow velocity is 1.0ml/min per minute；
35 DEG C of column temperature；Detection wavelength is 220nm.Number of theoretical plate is calculated by Paeoniflorin peak should be not less than 3000.
3. the preparation of reference solution: take gallic acid, tryptophan, chlorogenic acid, amarogentin, hydroxyl radical carthamin yellow carthamus A,
Paeoniflorin, ferulic acid, acteoside and Ligustilide, add methanol to dissolve, and solution of every 1ml containing each 20 μ g of reference substance is made,
As reference solution.
4. the preparation of test solution: Taohong Siwu Tang particle is taken, it is finely ground, about 0.8g is taken, it is accurately weighed, set tool plug taper
In bottle, 30% methanol 25ml, close plug is added in precision, and weighed weight is supplied the weight of less loss after ultrasonic treatment with 30% methanol, shaken
Even filtration, take subsequent filtrate to get.
5. measuring method: it is accurate respectively to draw reference solution and each 5 μ l of test solution, inject high performance liquid chromatograph
In, measurement records 70 minutes chromatograms.
Obtained finger-print includes 28 shared peaks, each peak relative retention time are as follows:
No. 1 peak 5.268min；No. 2 peak 7.036min；No. 3 peak 7.563min；No. 4 peak 8.578min；No. 5 peaks
11.636min；No. 6 peak 12.031min；No. 7 peak 12.697min；No. 8 peaks 16.636；No. 9 peaks 18.613；No. 10 peaks
19.365min；No. 11 peak 20.072min；No. 12 peak 22.168min；No. 13 peak 22.685min；No. 14 peak 24.768min；15
Number peak 25.959min；No. 16 peak 27.024min；No. 17 peak 27.645min；No. 18 peak 28.636min；No. 19 peaks
30.179min；No. 20 peak 35.339min；No. 21 peak 40.296min；No. 22 peak 40.536min；No. 23 peak 42.858min；24
Number peak 43.956min；No. 25 peak 47.63min；No. 26 peak 49.617min；No. 27 peak 51.798min；No. 28 peak 53.863min.
No. 20 peaks are referring to peak.
Each peak relative retention time is respectively as follows: with referring to peak relative retention time ratio
No. 1 peak relative retention time RRT is 0.15；No. 2 peak relative retention time RRT are 0.20；The opposite reservation at No. 3 peaks
Time RRT is 0.21；No. 4 peak relative retention time RRT are 0.24；No. 5 peak relative retention time RRT are 0.33；No. 6 peaks are opposite
Retention time RRT is 0.34；No. 7 peak relative retention time RRT are 0.36；No. 8 peak relative retention time RRT are 0.47；No. 9 peaks
Relative retention time RRT is 0.53；No. 10 peak relative retention time RRT are 0.55；No. 11 peak relative retention time RRT are
0.57；No. 12 peak relative retention time RRT are 0.63；No. 13 peak relative retention time RRT are 0.64；When No. 14 peaks retain relatively
Between RRT be 0.70；No. 15 peak relative retention time RRT are 0.73；No. 16 peak relative retention time RRT are 0.76；No. 17 peak phases
It is 0.78 to retention time RRT；No. 18 peak relative retention time RRT are 0.81；No. 19 peak relative retention time RRT are 0.85；
No. 20 peak relative retention time RRT are 1.00；No. 21 peak relative retention time RRT are 1.14；No. 22 peak relative retention time RRT
It is 1.15；No. 23 peak relative retention time RRT are 1.21；No. 24 peak relative retention time RRT are 1.24；No. 25 peaks are opposite to be retained
Time RRT is 1.35；No. 26 peak relative retention time RRT are 1.40；No. 27 peak relative retention time RRT are 1.47；No. 28 peaks
Relative retention time RRT is 1.52.Wherein: No. 20 peaks S are the chromatographic peaks of object of reference.
Wherein: No. 6 peaks are gallic acid in shared peak, and No. 10 peaks are tryptophan, and No. 14 peaks are amarogentin, and No. 15 peaks are
Chlorogenic acid, No. 19 peaks are hydroxyl radical carthamin yellow carthamus A, and No. 20 peaks are Paeoniflorin, and No. 21 peaks are ferulic acid, and No. 24 peaks are verbascose
Glycosides, No. 28 peaks are Ligustilide.
6. similarity result: integrated signal is imported to " the chromatographic fingerprints of Chinese materia medica similarity evaluation system of Chinese Pharmacopoeia committee
System A editions " software is to calculate to obtain similarity result by mean value method, similarity result is shown in Table 5 referring to spectrogram with S1.Similarity is small
It is substandard product in 0.90, quality evaluation is carried out to 16 batches of samples.
18010201 batches of numbers are S1, and 18010202 batches of numbers are S2, and so on, 18010216 batches of numbers are S16.
5 16 batches of sample finger-prints of table are compared with standard control fingerprint similarity
| Number | Similarity |
| S1 | 0.987 |
| S2 | 0.972 |
| S3 | 0.990 |
| S4 | 0.964 |
| S5 | 0.983 |
| S6 | 0.971 |
| S7 | 0.977 |
| S8 | 0.972 |
| S9 | 0.980 |
| S10 | 0.963 |
| S11 | 0.976 |
| S12 | 0.979 |
| S13 | 0.963 |
| S14 | 0.968 |
| S15 | 0.981 |
| S16 | 0.968 |
The above result shows that 18010201-18010216 batches of samples are qualified products.
Taohong Siwu Tang preparation finger methodological study
1. stability test
Test solution (lot number 18010201) is taken, test solution is prepared by the method in embodiment 1 and is examined
It surveys, respectively at 0,2h, 4h, 8h, 12h, for 24 hours, sample introduction is analyzed by 48h, investigates the relative retention time of main chromatographic peak and with respect to peak
The consistency of area, the results are shown in Table 6, Fig. 4.
6 stability similarity of table
Test solution is placed at room temperature for 48 hours, and fingerprint similarity is all larger than 0.95, shows test solution 48
Hour internal stability is good.
2. Precision Experiment
Test solution (lot number 18010201) is taken, test solution is prepared by the method in embodiment 1 and is detected,
Continuous sample introduction 6 times, the relative retention time of main chromatographic peak and the consistency of relative peak area are investigated, the results are shown in Table 7, Fig. 5.
7 precision similarity of table
| Number | Similarity |
| S1 | 1.000 |
| S2 | 1.000 |
| S3 | 1.000 |
| S4 | 1.000 |
| S5 | 1.000 |
| S6 | 1.000 |
Test solution continuous sample introduction 6 times, fingerprint similarity is all larger than 0.95, shows that method precision is good.
3. repeated experiment
6 parts of test solution (lot number 18010201) is taken, prepare test solution by the method in embodiment 1 and is carried out
Detection, investigates the relative retention time of main chromatographic peak and the consistency of relative peak area, the results are shown in Table 8, Fig. 6.
8 stability similarity of table
| Number | Similarity |
| S1 | 1.000 |
| S2 | 0.999 |
| S3 | 1.000 |
| S4 | 0.999 |
| S5 | 1.000 |
| S6 | 1.000 |
6 parts of test solution fingerprint similarities are all larger than 0.95, show that method repeatability is good.
Claims (9)
1. a kind of method for building up of Taohong Siwu Tang preparation finger, it is characterised in that: the following steps are included:
(1) preparation of testing sample solution
It takes Taohong Siwu Tang preparation finely ground, the methanol aqueous solution of concentration expressed in percentage by volume 30% is added and carries out ultrasonic dissolution, then plus
The methanol aqueous solution for entering concentration expressed in percentage by volume 30% supplies the weight of less loss, shakes up, and miillpore filter filtration takes filtrate up to be measured
Sample solution；
(2) preparation of reference solution
Take gallic acid, tryptophan, chlorogenic acid, amarogentin, hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, ferulic acid, verbascose
Glycosides and Ligustilide, add methanol to dissolve, and solution of every 1ml containing each 20 μ g of reference substance are made, as reference solution；
(3) accurate respectively to draw reference solution and each 5-10 μ l of testing sample solution, inject high performance liquid chromatograph, record
Finger-print in 65-75 minutes；
(4) by the finger-print integrated signal of sample to be tested import Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity is commented
Valence system A editions " software；Select in the finger-print of different batches Taohong Siwu Tang preparation existing chromatographic peak as sharing
Peak；Similarity result is calculated with mean value calculation method, and generates standard control finger-print；Calculate the opposite reservation at each shared peak
Time and each shared peak relative retention time and the ratio referring to peak relative retention time.
2. the method for building up of Taohong Siwu Tang preparation finger according to claim 1, it is characterised in that: step (1)
The preparation of the testing sample solution is: the Taohong Siwu Tang granule sample of different batches is taken, it is finely ground, and 0.8g peach is taken respectively
Red Siwu Tang granule sample is placed in stuffed conical flask, the methanol aqueous solution 25ml of addition concentration expressed in percentage by volume 30%, close plug,
Weighed weight, and be ultrasonically treated, after ultrasonic treatment, the methanol aqueous solution for adding concentration expressed in percentage by volume 30% supplies less loss
Weight, shake up, miillpore filter filtration, take filtrate up to testing sample solution.
3. the method for building up of Taohong Siwu Tang preparation finger according to claim 1, it is characterised in that: step (3)
Described in determining fingerprint pattern chromatographic condition are as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；
Column temperature: 25-35 DEG C；
Flow velocity: 0.8-1.2ml/min；
Sample volume is 5-10 μ l；
Ultraviolet detection wavelength: 210-250nm.
4. the method for building up of Taohong Siwu Tang preparation finger according to claim 3, it is characterised in that: ultraviolet detection
Wavelength is one of 210nm, 220nm or 250nm.
5. the method for building up of Taohong Siwu Tang preparation finger according to claim 3, it is characterised in that: mobile phase is
Any one in (1), (2), (3) or (4) below:
(1) 0.02% phosphate aqueous solution of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution；
(2) 0.05% aqueous formic acid of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution；
(3) 0.1% aqueous formic acid of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution；
(4) 0.02% trifluoroacetic acid aqueous solution of mobile phase A concentration expressed in percentage by volume-Mobile phase B methanol aqueous solution.
6. the method for building up of Taohong Siwu Tang preparation finger according to claim 3, it is characterised in that: type of elution
Using gradient elution, the process of gradient elution is any one in following (1), (2) or (3):
(1) 0~5min, 2% Mobile phase B of concentration expressed in percentage by volume, 5~15min, concentration expressed in percentage by volume 2%-20% Mobile phase B, 15
~35min, concentration expressed in percentage by volume 20%-28% Mobile phase B, 35~60min, concentration expressed in percentage by volume 28%-80% Mobile phase B,
60~65min, 80% Mobile phase B of concentration expressed in percentage by volume, 65~66min, concentration expressed in percentage by volume 80%-2% Mobile phase B, 66~
75min, 2% Mobile phase B of concentration expressed in percentage by volume；
(2) 0~5min, 3% Mobile phase B of concentration expressed in percentage by volume, 5~15min, concentration expressed in percentage by volume 3%-16% Mobile phase B, 15
~20min, concentration expressed in percentage by volume 16%-25% Mobile phase B, 20~30min, 25% Mobile phase B of concentration expressed in percentage by volume, 30~
40min, concentration expressed in percentage by volume 25%-40% Mobile phase B, 40~50min, concentration expressed in percentage by volume 40%-80% Mobile phase B, 50
~60min, 80% Mobile phase B of concentration expressed in percentage by volume, 60~61min, concentration expressed in percentage by volume 80%-3% Mobile phase B, 61~
70min, 3% Mobile phase B of concentration expressed in percentage by volume；
(3) 0~5min, 3% Mobile phase B of concentration expressed in percentage by volume, 5~15min, concentration expressed in percentage by volume 3%-15% Mobile phase B, 15
~20min, concentration expressed in percentage by volume 15%-25% Mobile phase B, 20~30min, 25% Mobile phase B of concentration expressed in percentage by volume, 30~
40min, concentration expressed in percentage by volume 25%-40% Mobile phase B, 40~55min, concentration expressed in percentage by volume 40%-80% Mobile phase B, 50
~60min, 80% Mobile phase B of concentration expressed in percentage by volume, 60~61min, concentration expressed in percentage by volume 80%-3% Mobile phase B, 61~
70min, 3% Mobile phase B of concentration expressed in percentage by volume.
7. the method for building up of Taohong Siwu Tang preparation finger according to claim 1, it is characterised in that: step (1)
Described in the preparation method of Taohong Siwu Tang granule be: take 9 parts of radix rehmanniae recen, 3 parts of Rhizoma Chuanxiong, 12 parts of Radix Angelicae Sinensis, 3.78 parts of peach kernel,
4.5 parts of Radix Paeoniae Alba and 3 parts of safflower, the above Six-element, add water to cook it is secondary, 1 hour every time, for the first time plus whole 12 times of weight of medicine materical crude slice
Water, for the second time plus the water of whole 10 times of weight of medicine materical crude slice, decocting liquid filtration, filtrate merge, and filtrate decompression is concentrated into thick paste, and thick paste depressurizes
Dry to solid, auxiliary material is added in crushing, is pelletized, whole grain is to get Taohong Siwu Tang granule.
8. a kind of Taohong Siwu Tang preparation finger described in claim 1, it is characterised in that: finger-print includes 28 total
There is peak, each shared peak relative retention time is respectively as follows: with referring to peak relative retention time ratio
No. 1 peak relative retention time RRT is 0.16；No. 2 peak relative retention time RRT are 0.22；The relative retention time at No. 3 peaks
RRT is 0.24；No. 4 peak relative retention time RRT are 0.27；No. 5 peak relative retention time RRT are 0.33；No. 6 peaks are opposite to be retained
Time RRT is 0.36；No. 7 peak relative retention time RRT are 0.38；No. 8 peak relative retention time RRT are 0.48；No. 9 peaks are opposite
Retention time RRT is 0.54；No. 10 peak relative retention time RRT are 0.57；No. 11 peak relative retention time RRT are 0.58；12
Number peak relative retention time RRT is 0.65；No. 13 peak relative retention time RRT are 0.66；No. 14 peak relative retention time RRT are
0.72；No. 15 peak relative retention time RRT are 0.76；No. 16 peak relative retention time RRT are 0.79；When No. 17 peaks retain relatively
Between RRT be 0.81；No. 18 peak relative retention time RRT are 0.83；No. 19 peak relative retention time RRT are 0.88；No. 20 peak phases
It is 1.00 to retention time RRT；No. 21 peak relative retention time RRT are 1.16；No. 22 peak relative retention time RRT are 1.19；
No. 23 peak relative retention time RRT are 1.23；No. 24 peak relative retention time RRT are 1.26；No. 25 peak relative retention time RRT
It is 1.36；No. 26 peak relative retention time RRT are 1.43；No. 27 peak relative retention time RRT are 1.49；No. 28 peaks are opposite to be retained
Time RRT is 1.53；Wherein: No. 20 peaks S are referring to peak.
9. Taohong Siwu Tang preparation finger according to claim 8, it is characterised in that: No. 6 peaks are not in shared peak
Gallate-based, No. 10 peaks are tryptophan, and No. 14 peaks are amarogentin, and No. 15 peaks are chlorogenic acid, and No. 19 peaks are Sydroxy carthamin
A, No. 20 peaks are Paeoniflorin, and No. 21 peaks are ferulic acid, and No. 24 peaks are acteoside, and No. 28 peaks are Ligustilide.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114636779A (en) * | 2022-03-29 | 2022-06-17 | 陕西科技大学 | Method for constructing sanhua decoction reference sample freeze-dried powder fingerprint spectrum and fingerprint spectrum thereof |
| CN114924021A (en) * | 2022-03-21 | 2022-08-19 | 海南康茂信医药科技有限公司 | Detection method of peach-red Siwu decoction formula |
| CN114636779B (en) * | 2022-03-29 | 2024-05-24 | 陕西盘龙药业集团股份有限公司 | Construction method of three-conversion soup reference sample freeze-dried powder fingerprint and fingerprint thereof |
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2019
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114924021A (en) * | 2022-03-21 | 2022-08-19 | 海南康茂信医药科技有限公司 | Detection method of peach-red Siwu decoction formula |
| CN114924021B (en) * | 2022-03-21 | 2023-12-08 | 海南康茂信医药科技有限公司 | Detection method of Taohong Siwu decoction prescription |
| CN114636779A (en) * | 2022-03-29 | 2022-06-17 | 陕西科技大学 | Method for constructing sanhua decoction reference sample freeze-dried powder fingerprint spectrum and fingerprint spectrum thereof |
| CN114636779B (en) * | 2022-03-29 | 2024-05-24 | 陕西盘龙药业集团股份有限公司 | Construction method of three-conversion soup reference sample freeze-dried powder fingerprint and fingerprint thereof |
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