CN107505425A - A kind of method for building up of gold ring piece multi objective quantitative finger print atlas - Google Patents
A kind of method for building up of gold ring piece multi objective quantitative finger print atlas Download PDFInfo
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Abstract
The invention discloses a kind of method for building up of gold ring piece multi objective quantitative finger print atlas, the chromatographic condition of the multi objective quantitative finger print atlas of high performance liquid chromatography foundation gold ring piece is obtained by optimization method, the chemical information characteristic peak of the total chemical material in gold ring piece extract is detected using high performance liquid chromatography ESI TOF-MS, RPLC joint PDAD evaporation photodetector has carried out assay to 5 kinds of main components in gold ring piece simultaneously, the efficient liquid-phase chromatograph finger print atlas of gold ring piece is established by the analysis of multiple batches of sample, as gold ring piece multi objective quantitative finger print atlas；Wherein, in the chromatographic condition, the chromatographic column used is Agilent SB C18(250mm × 4.6mm, 5 μm), for the mobile phase used for acetonitrile 0.2% (volume) acetic acid aqueous solution system, the collection wavelength of finger-print is 280nm.
Description
Technical field
The present invention relates to chemical composition in gold ring piece to carry out analysis and identification, assay and quality control, and in particular to one
The method for building up of kind gold ring piece multi objective quantitative finger print atlas.
Background technology
Successive dynasties Chinese medical discrimination thinks that anemopyretic laryngalgia (acpuei pharyngitis) is that the heresy of wind-heat is violated caused by lung is defended, and wind and heat in the lung meridian, is followed
Through upper criminal, knot throat of fighting, gently then micro- foul wind swallows hypodynia, heavy then generate heat, and pharyngalgia is violent, dysphagia, chilly, generates heat, whole body is not
Suitable, treating this disease should be with wind and heat dispersing, based on the relieving sore throat and acesodyne that detoxifies.Gold ring piece is by glutinous rehmannia, borax (forging), radix scrophulariae, artificial ox
The extract of the kinds of traditional Chinese medicines material such as Huang, the tuber of dwarf lilyturf, borneol, peppermint, pearl powder, red sage root adds auxiliary material such as dextrin, sucrose, citric acid, thin
Film coating pre-mixing agent etc., the wherein Film coated tablets as made of Modern preparations technology, peppermint have clearing heat and promoting fluid, open sound relieving sore-throat
The effect of；Borneol has the effect of fire of dispelling, clearing lung-heat heat；Pearl powder can detoxify, therefore gold ring piece can be used for chronic pharyngitis, chronic larynx
Inflammation, abscess of throat, hoarseness and aphonia, itched with the dry throat after sound, larynx, sounding is laborious, plays the diseases such as sound difficulty.At present, Chinese Pharmacopoeia
In still without gold ring piece quality control standard.Existing Chinese medicinal tablet quality standard lack index in the tablet recipe that rung to gold into
The quantitative determination divided, it is impossible to overall method of quality control is provided, can not meet the quality control requirement of the modernization of Chinese medicine.
The content of the invention
In order to solve the deficiencies in the prior art, an object of the present invention is to provide a kind of quantitative fingerprint of gold ring piece multi objective
The method for building up of collection of illustrative plates, gold ring piece extract chromatographic peak is referred to by high performance liquid chromatography ESI TOF-MS
Recognize, 20 shared peaks can determine by multiple batches of sample analysis, by high performance liquid chromatography combine PDAD-
Evaporation photodetector has carried out assay to 5 kinds of main components in gold ring piece simultaneously, by being measured to containing for multiple shared peaks
Surely similarity analysis is combined, establishes a kind of multi objective quantitative finger print atlas, the quality evaluation water to gold ring piece can be greatly improved
It is flat.
To achieve these goals, the technical scheme is that：
A kind of method for building up of gold ring piece multi objective quantitative finger print atlas, RP-HPLC color is obtained by optimization method
The chromatographic condition of chemical composition in spectrum analysis gold ring piece extract, using RPLC-electron spray flight time matter
Spectrum detects the characteristic peak of a variety of total chemical materials in multiple batch gold ring piece extracts, is detected by the chromatographic condition of acquisition
The chromatographic peak of a variety of total chemical materials in the multiple batch gold ring piece extract, according to the multiple batches gold ring piece extractions of detection
The chromatographic peak and assay of a variety of total chemical materials of thing establish the efficient liquid-phase chromatograph finger print atlas of gold ring piece, are gold
Ring piece multi objective quantitative finger print atlas；Wherein, in the chromatographic condition, the chromatographic column used is Agilent SB C18(250mm
× 4.6mm, 5 μm), the mobile phase used is acetonitrile -0.2% (volume) acetic acid aqueous solution system, the collection wavelength of finger-print
For 280nm.
First passage high performance liquid chromatography of the present invention-ESI TOF-MS detects being total in multiple batch gold ring pieces
There is chemical substance, and based on these total chemical materials optimization chromatographic condition, the fingerprint of the high performance liquid chromatography of acquisition gold ring piece
Collection of illustrative plates, the quality standard to improving gold ring piece, ensure that its safe and reasonable medication provides methods and techniques support.
But during the finger-print of the high performance liquid chromatography of gold ring piece is obtained in detection, following difficulty be present in it：
1. gold medal rings, piece is made up of kinds of traditional Chinese medicines material compounding, and its chemical composition is more, thus the separating effect of chemical composition is difficult to control,
Easily there is the problem of detection is inaccurate；2. chromatographic peak conditions of streaking occurs in gold medal ring piece in separation process, so as to cause to detect
The problem of unstable；3. the Detection wavelength scope of detector is in 190~400nm, the gold ring piece that different Detection wavelengths detects
The information of chemical composition is different, it is difficult to determines which wavelength can more fully detect the chemical composition in gold ring piece.
First, present invention selection Agilent SB C18(250mm × 4.6mm, 5 μm) chromatographic column, under the same conditions,
The chromatographic column to gold ring piece separating effect than other chromatographic columns good separating effect, can farthest by gold ring piece in more
More chemical compositions are separated.
Secondly, when the present invention selection acetic acid aqueous solution of acetonitrile -0.2% system is as mobile phase obtained chromatographic peak peak shape compared with
Well and stably, solve the chromatographic peak conditions of streaking that gold ring piece occurs in separation process, make testing result more stable.
3rd, Detection wavelength of the invention is 280nm, the chemistry that the Detection wavelength can reflect in gold ring piece comprehensively into
Point, and baseline is more steady, while can make have preferable separating degree between each chromatographic peak.
The second object of the present invention is to provide the gold ring piece multi objective quantitative finger print atlas that a kind of above method is established.This refers to
Line collection of illustrative plates can carry out quantitative detection to gold ring piece Multiple components, can instruct the overall quality control of gold ring piece, meet gold ring
The quality control requirement of the modernization of piece.
Because method for building up provided by the invention or finger-print can monitor the oeverall quality situation of gold ring piece, thus this
The third purpose of invention is to provide a kind of above-mentioned method for building up or above-mentioned gold ring piece multi objective quantitative finger print atlas in detection gold ring
Application in tablet quality control and evaluation.
Because the purpose that the present invention provides gold ring piece multi objective quantitative finger print atlas is that Instructing manufacture has unified standard
Gold ring piece, thus the fourth object of the present invention is to provide a kind of above-mentioned method for building up or the above-mentioned quantitative fingerprint of gold ring piece multi objective
Application of the collection of illustrative plates in monitoring produces gold ring piece.
Beneficial effects of the present invention are：
1st, the method for building up of gold ring piece finger-print of the invention, its precision test determined, replica test, stably
Property experiment similarity evaluation all show that this method testing result is accurate, stability is high.
2nd, the method for building up of gold ring piece finger-print of the invention to active ingredient can qualitative and can quantify, be applicable to
The quality control of the raw medicinal material of monitoring gold ring piece, the quality and production technology of intermediate semi-finished product, it is applied widely.
3rd, the method for building up of gold ring piece finger-print of the invention, it more completely can effectively judge the quality of gold ring piece, have
Beneficial to overall monitor product quality and the true and false of discriminating product.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its illustrate be used for explain the application, do not form the improper restriction to the application.
Fig. 1 is RP-HPLC (HPLC-DAD) collection of illustrative plates of the gold ring piece of detection；
Fig. 2 is the gold ring piece multi objective quantitative finger print atlas obtained.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the application.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
Agilent SB C described herein18It is 250mm that (250mm × 4.6mm, 5 μm), which refers to length, and internal diameter is
4.6mm, aperture are 5 μm of Agilent SB C18Chromatographic column.
Optimization method described herein is the optimization method commonly used in general analysis chemistry, including standard curve and inspection
The detection of rising limit, precision test, replica test, the analysis of stabilometer recovery of standard addition etc..
(volume) the acetic acid aqueous solution system of acetonitrile -0.2% described herein is as mobile phase, and wherein acetonitrile is as A
Phase, 0.2% (volume) acetic acid aqueous solution is as B phases.
As background technology is introduced, the quality control standard in the presence of gold ring piece lacks, can not provided in the prior art
The method of quality control of entirety, it can not meet the deficiencies of quality control requirement of the modernization of Chinese medicine, in order to solve technology as above
Problem, present applicant proposes a kind of method for building up of gold ring piece multi objective quantitative finger print atlas.
A kind of a kind of exemplary embodiment of the application, there is provided foundation side of gold ring piece multi objective quantitative finger print atlas
Method, the chromatographic condition of chemical composition in rp-hplc analysis gold ring piece extract is obtained by optimization method, is used
RPLC-ESI TOF-MS detects a variety of total chemical things in multiple batch gold ring piece extracts
The characteristic peak of matter, a variety of total chemical materials in the multiple batch gold ring piece extract are detected by the chromatographic condition of acquisition
Chromatographic peak, gold is established according to the chromatographic peak for a variety of total chemical materials for detecting multiple batch gold ring piece extracts and assay
The efficient liquid-phase chromatograph finger print atlas of ring piece, it is gold ring piece multi objective quantitative finger print atlas；Wherein, in the chromatographic condition,
The chromatographic column used is Agilent SB C18(250mm × 4.6mm, 5 μm), the mobile phase used is acetonitrile -0.2% (volume)
Acetic acid aqueous solution system, the collection wavelength of finger-print is 280nm.
First passage high performance liquid chromatography of the present invention-ESI TOF-MS detects being total in multiple batch gold ring pieces
There is chemical substance, and based on these total chemical materials optimization chromatographic condition, the fingerprint of the high performance liquid chromatography of acquisition gold ring piece
Collection of illustrative plates, the quality standard to improving gold ring piece, ensure that its safe and reasonable medication provides methods and techniques support.
But during the finger-print of the high performance liquid chromatography of gold ring piece is obtained in detection, following difficulty be present in it：
1. gold medal rings, piece is made up of kinds of traditional Chinese medicines material compounding, and its chemical composition is more, thus the separating effect of chemical composition is difficult to control,
Easily there is the problem of detection is inaccurate；2. chromatographic peak conditions of streaking occurs in gold medal ring piece in separation process, so as to cause to detect
The problem of unstable；3. the Detection wavelength scope of detector is in 190~400nm, the gold ring piece that different Detection wavelengths detects
The information of chemical composition is different, it is difficult to determines which wavelength can more fully detect the chemical composition in gold ring piece.
First, present invention selection Agilent SB C18(250mm × 4.6mm, 5 μm) chromatographic column, under the same conditions,
The chromatographic column to gold ring piece separating effect than other chromatographic columns good separating effect, can farthest by gold ring piece in more
More chemical compositions are separated.
Secondly, when the present invention selection acetic acid aqueous solution of acetonitrile -0.2% system is as mobile phase obtained chromatographic peak peak type compared with
Well and stably, solve the chromatographic peak conditions of streaking that gold ring piece occurs in separation process, make testing result more stable.
3rd, Detection wavelength of the invention is 280nm, the chemistry that the Detection wavelength can reflect in gold ring piece comprehensively into
Point, and baseline is more steady, while can make have preferable separating degree between each chromatographic peak.
In order to obtain the main chemical compositions of gold ring piece, the extracting method of existing gold ring piece extract, which is divided into, to be heated to reflux
Two methods of extraction and ultrasonic extraction, the application is preferable, and the preparation method of the gold ring piece extract uses ultrasonic extraction.
Using ultrasonic extraction, operation is simple and extraction efficiency is higher.
In order to obtain more preferable extraction effect, the application is it is further preferred that extractant uses 50% (volume) methanol.Adopt
During by the use of 50% (volume) methanol as Extraction solvent, chromatographic peak is more, and chromatogram peak-to-peak signal is stronger, and baseline is more steady.
In order that the finger-print obtained possesses more preferable reappearance, the application is preferable, the chromatographic column in chromatographic condition
Temperature is 25 DEG C.When column temperature is that each composition separating degree and the symmetry of chromatographic peak are preferable in 25 DEG C of gained collection of illustrative plates.
In order to preferably obtain finger-print, the application is preferable, and chromatographic condition is：The volume flow 0.8mL/ of mobile phase
Min, evaporative light gas flow rate 1.6mL/min, 80 DEG C of temperature, sample size are 30 μ L, and gradient elution program is 0~5min, 8%A；
5~15min, 8%~12%A；15~20min, 12%~13%A；20~25min, 13%~15%A；25~30min,
15%~16%A；30~50min, 16%~18%A；50~60min, 18%~20%A；60~65min, 20%~20%
A；65~70min, 20%~25%A；70~85min, 25%~25%A；85~90min, 25%~26%A；91~
110min, 60%~100%A.Wherein, " % " all in the technical characteristic represents volumn concentration.
In order that the information of finger-print is more accurate, while reduces the cost of establishing of finger-print, the application is preferable, institute
It is 14 batches to state multiple batches.
In order to improve the degree of accuracy of finger-print, the application is preferable, and a variety of total chemical materials are 20 kinds.Protecting
Under the conditions of demonstrate,proving instrument accuracy grade, it is most species that existing instrument can detect to detect to 20 kinds, so as to improve finger-print
The degree of accuracy.
In order to obtain the chromatographic condition of the finger-print of the high performance liquid chromatography of the gold ring piece of foundation, the application is preferable,
Select the detection object of tanshin polyphenolic acid B, harpagoside, cholic acid, acteoside, Tanshinone I as the optimization method.According to existing
There are technical conditions, five kinds of chemical compositions that gold ring piece contains are only capable of detecting and obtain, using five kinds of chemical compositions as detection
Object can increase the accuracy of optimization.
It is further preferred that chromatostrip is carried out to the tanshin polyphenolic acid B, harpagoside, cholic acid, acteoside, Tanshinone I
Detection of the optimization method of part including directrix curve and detection limit, precision test, replica test, stabilometer mark-on return
The assay of yield analysis and sample.
In order to which the tanshin polyphenolic acid B in the piece that rung to gold, harpagoside, cholic acid, acteoside, Tanshinone I carry out quantitative analysis,
The application still more preferably, the tanshin polyphenolic acid B that is obtained according to detection, harpagoside, cholic acid, acteoside, Tanshinone I
Analysis regression equation, the range of linearity and test limit, the quantitative limit such as following table that standard curve and detection limit obtain,
Present invention also provides the gold ring piece multi objective quantitative finger print atlas that a kind of above method is established.The finger-print energy
It is enough that quantitative detection is carried out to gold ring piece, the overall method of quality control of gold ring piece can be instructed, meets the modernization of gold ring piece
Quality control requirement.
The method for building up or finger-print provided due to the application can monitor the oeverall quality situation of gold ring piece, thus this
Application additionally provides a kind of above-mentioned method for building up or above-mentioned gold ring piece multi objective quantitative finger print atlas in detection gold ring tablet quality control
Application in system and evaluation.
Because the purpose that the application provides gold ring piece multi objective quantitative finger print atlas is that Instructing manufacture has unified standard
Gold ring piece, thus present invention also provides a kind of above-mentioned method for building up or it is above-mentioned gold ring piece multi objective quantitative finger print atlas to supervise
Application in control production gold ring piece.
In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes the technical scheme of the application in detail.
Embodiment
Instrument and material
The high performance liquid chromatographs of Agilent 1260 (equipped with quaternary pump, automatic sampler, column oven, DAD detectors,
ELSD detectors), Agilent G6520 type level Four bar time-of-flight mass spectrometry instrument (be furnished with ESI sources, Qualitative
Analysis mass spectral analyses software) Agilent companies of the U.S..
Acetonitrile, chromatographically pure, Tianjin Ou Pusen companies；Acetic acid, chromatographically pure, Tianjin Ke Miou companies；Wahaha Pure Water, Hangzhoupro
State Wahaha Group Co., Ltd.Other reagents are that analysis is pure.5 kinds of compound standard product (acteosides
(P29M7F12159), tanshin polyphenolic acid B (140927), harpagoside (H11M3X1), cholic acid (KN1117BA14)), purchased from Shang Haiyuan
Leaf bio tech ltd, Tanshinone I (make) HPLC quality measurements fraction by oneself >=98% for laboratory.The present embodiment is adopted
14 batches of gold ring pieces (table 1) are purchased from difference pharmacy of Jinan City.
The gold medal of table 1 ring piece sample source
The preparation of 1 reference substance solution
1.0mg tanshin polyphenolic acid Bs, harpagoside, cholic acid, acteoside, Tanshinone I reference substance are accurately weighed respectively, are placed in
In 1mL volumetric flasks, with 50% methanol constant volume to scale, the reference substance storing liquid that each reference substance is 1.0mg/mL is made into, crosses 0.22 μ
M miillpore filters.
The preparation of 2 sample solutions
Gold ring tablet is rolled into crushing, precision weighs 1.0g, adds 50% methanol solution 10mL, ultrasonic 20min, takes
Clear liquid, need testing solution is used as after crossing 0.22 μm of miillpore filter.
3 reverse-phase chromatographies-ESI TOF-MS condition
Agilent SB C18Chromatographic column (2.5mm × 4.6mm, 5 μm), mobile phase acetonitrile (A) and 0.2% acetic acid aqueous solution
(B), volume flow 0.8mL/min, 25 DEG C of room temperature, Detection wavelength 280nm；Evaporative light gas flow rate 1.6mL/min, temperature 80
DEG C, sample size is 30 μ L, and gradient elution program is 0~5min, 8%A；5~15min, 8%~12%A；15~20min, 12%
~13%A；20~25min, 13%~15%A；25~30min, 15%~16%A；30~50min, 16%~18%A；50
~60min, 18%~20%A；60~65min, 20%~20%A；65~70min, 20%~25%A；70~85min,
25%~25%A；85~90min, 25%~26%A；91~110min, 60%~100%A.Golden the anti-phase of piece of ringing of acquisition
Efficient liquid phase (HPLC-DAD) collection of illustrative plates is as shown in Figure 1.
4ESI-TOF/MS differentiates
To further confirm that the chemical information of characteristic peak in gold ring piece, ESI-TOF/MS is employed in 20 shared peaks
6 chromatographic peaks are pointed out, and by being compareed with reference substance and consulting literatures data, it is cohosh to be identified respectively as No. 7 peaks
Glycosides, No. 12 peaks are tanshin polyphenolic acid B, No. 14 peaks are harpagoside, No. 17 peaks are Tanshinone I, No. 18 peaks are Catalpol, No. 20 peaks are courage
Acid.It the results are shown in Table 2：
The HPLC-MS psycho sedatives results of 20 compounds in the gold medal of table 2 ring piece extract
5 fingerprint spectrum methods are investigated
5.1 standard curves and detection limit
5 kinds of hybrid standard storing solutions of certain volume are drawn, are made into the hybrid standard liquid of various concentrations；By " 3 anti-phase colors
Chromatographic condition in spectrum-ESI TOF-MS condition " enters to acteoside, tanshin polyphenolic acid B, harpagoside, Tanshinone I
Row HPLC-DAD is determined, and because cholic acid UV absorption is relatively low, HPLC-ELSD measure has been used to the assay of cholic acid, is determined
The peak area of each n-compound, with each n-compound concentration (μ g/mL) for abscissa, using peak area as ordinate, draw
Standard curve, and try to achieve regression equation.It the results are shown in Table 3.
35 kinds of standard items HPLC analyses regression equations of table, the range of linearity and test limit, quantitative limit measurement result
5.2 precision test
Lot number is taken to draw the μ L of need testing solution 30 for gold ring piece 1511001-1 need testing solutions, precision, continuously repeat sample introduction
6 times, the peak area and retention time of wherein 5 characteristic peaks are measured, calculates its relative standard deviation (RSD), its characteristic peak respectively
Retention time RSD is 0.11%, 0.12%, 0.24%, 0.33%, 0.49%, respectively less than 1.0% successively, and peak area RSD is successively
It is 0.73%, 1.35%, 1.07%, 1.38%, 1.34%, respectively less than 5.0%, as a result meets the technical requirements of finger-print,
Show that instrument precision is good.
5.3 replica test
6 parts of need testing solution (lot number is gold ring piece 1511001-1) is taken, by the processing in " preparation of 1 reference substance solution "
Need testing solution is made in method, is surveyed according to the chromatographic condition sample introduction in " 3 reverse-phase chromatographies-ESI TOF-MS condition "
It is fixed, the peak area and retention time of 5 characteristic peaks are measured, and calculate its relative standard deviation.As a result 5 characteristic peak retention times
RSD is 0.85%, 0.17%, 0.15%, 0.35%, 0.53%, respectively less than 1.0% successively, and peak area RSD is successively
1.90%th, 2.38%, 1.71%, 2.30%, 1.56%, respectively less than 5.0%, the results showed that method repeatability is good, meets finger
The technical requirements of line collection of illustrative plates.
5.4 stability test
Need testing solution (lot number is gold ring piece 1511001-1) is taken according to " 3 reverse-phase chromatographies-ESI TOF-MS
Chromatographic condition in condition ", analyzed respectively in 0,2,4,8,12,24h sample introductions, record chromatogram.As a result when 5 characteristic peaks retain
Between RSD be 0.69%, 0.89%, 0.58%, 0.64%, 0.99%, respectively less than 1% successively, peak area RSD is successively
3.08%th, 2.44%, 2.64%, 3.19%, 2.01%, respectively less than 5%, sample solution 24h internalizations at ambient temperature are shown
Have good stability, and meets the technical requirements of finger-print.
5.5 recovery of standard addition
Need testing solution is made by the method for " preparation of 1 reference substance solution ", it is accurate respectively to add acteoside, red phenol
Sour B, harpagoside, Tanshinone I, cholic acid reference substance (concentration is 1.0mg/mL) in right amount, are determined respectively with above-mentioned chromatographic condition
The amount of each composition and to calculate the rate of recovery be 95.10%, 94.98%, 94.23%, 95.09% and 94.82% successively, average recovery
Rate RSD is 2.98%, 2.81%, 1.89%, 2.13% and 2.69%.
The assay of 6 samples：
14 batches of gold ring piece samples are taken, by the method for " preparations of 2 sample solutions " 14 parts of test solutions of each parallel preparation.By " 3
Chromatographic condition in reverse-phase chromatography-ESI TOF-MS condition " is to acteoside, tanshin polyphenolic acid B, harpagoside, pellet
Join ketone I and carry out HPLC-DAD measure, because cholic acid UV absorption is relatively low, HPLC-ELSD surveys have been used to the assay of cholic acid
It is fixed, chromatographic peak area is recorded, peak area is brought into regression equation, calculates each component content, the results are shown in Table 4.
Assay (the μ gg of 5 kinds of active ingredient in the gold medal of table 4 ring piece-1, n=10)
Pass through to 14 batches gold ring tablet in active ingredient carry out assay, it can be seen that wherein five kinds effectively into
Content of danshinolic acid B highest in point, average value is 598.96 μ gg-1, harpagoside content is minimum, and average value is 11.75 μ gg-1, content is followed successively by cholic acid, Tanshinone I, acteoside from high to low in other three kinds of active ingredients.
The foundation of 7 finger-prints
14 batches of different batches gold ring pieces are taken, it is molten to be prepared into test sample by the processing method of " preparations of 2 sample solutions "
Liquid, analyzed by the chromatographic condition sample introduction in " 3 reverse-phase chromatographies-ESI TOF-MS condition ", record chromatogram, establish gold
The HPLC finger-prints of ring piece.Under the analysis condition that this research institute establishes, separating degree is preferable in each sample and stablizes what is occurred
Peak has 20, as shown in Figure 2.
8 similarity evaluations：
By " similarity evaluation " (《Chinese Pharmacopoeia》2004A versions) ring piece to 14 batches of gold
Finger-print carries out similarity analysis.Chromatographic work station data are imported into traditional Chinese medicine fingerprint Similarity Measure software, choosing first
Fixed above-mentioned 20 shared peaks carry out Peak tracking, using common pattern as reference fingerprint, the phase for 14 batches of gold ring pieces
Evaluated like degree, the results are shown in Table 5.
The similarity evaluation result of table 5
Discuss
The selection of extraction conditions
The main chemical compositions in two methods of heating and refluxing extraction and ultrasonic extraction extraction gold ring piece are respectively adopted, with color
Spectral peak quantity and peak area are as evaluation index.As a result showing, operation is simple for ultrasonic extraction, and extraction efficiency is higher,
So choose extracting method of the ultrasonic extraction as active component in gold ring piece.
Investigation compares the extraction effect of different solvents (50% methanol, methanol, 50% ethanol, ethanol, water), as a result
Show, using factors such as chromatographic peak, peak area, baselines as index, during using 50% methanol as Extraction solvent, chromatographic peak compared with
More, chromatogram peak-to-peak signal is stronger, and baseline is more steady, therefore final choice uses 50% methanol as Extraction solvent.
The influence of extraction time (10,20,40min) has further been investigated, has as a result found sample composition in 20min
Reach abundant extraction, to improve efficiency, select 20min as final extraction time.
Chromatographic condition optimizes
The chromatographic column of different brands is investigated, respectively Kromasil C18(250mm × 4.6mm, 5 μm),
Agilent SB C18(250mm × 4.6mm, 5 μm), Agilent XDB C18(250mm × 4.6mm, 5 μm).As a result show
Agilent SB C18(250mm × 4.6mm, 5 μm) is preferable to the separating effect of sample, thus selection the type chromatographic column for
Gold ring piece fingerprint map analyzing post.Gold ring piece is compound preparation, and contained chemical composition is more, has investigated methanol-water solution and second
The separating effect of nitrile-aqueous systems, the results showed that, using acetonitrile aqueous systems as mobile phase, during gradient elution, gold ring piece extract
In the separation of each composition it is preferable.For the chromatographic peak conditions of streaking occurred in chromatographic separation process, different proportion is added in aqueous phase
Weak acid (0.1% formic acid, 0.2% formic acid, 0.1% acetic acid, 0.2% acetic acid) improve peak type.As a result display using acetonitrile-
The chromatographic peak peak shape obtained when 0.2% acetic acid aqueous solution system is as mobile phase is preferably and stably.When column temperature is that 25 DEG C of gained are schemed
Each composition separating degree and the symmetry of chromatographic peak are preferable in spectrum, it is contemplated that laboratory temperature and method reappearance, select post
Temperature is used for subsequent analysis for 25 DEG C.
190~400nm full wavelength scanners are carried out to sample using PDA detectors, and reciprocity absorption figure is analyzed, and is carried
The wavelength 280nm that absworption peak is more is taken, at this wavelength, the chemical composition in gold ring piece can be than more comprehensively reflection, and base
Line is more steady, and has preferable separating degree between each chromatographic peak, therefore selects collection ripples of the 280nm as gold ring piece finger-print
It is long.
Brief summary
This research is rung for gold, and the existing quality standard of piece is single, can not meet the problem of actual production Quality Control, using HPLC-
A variety of chemical compositions in the golden ring piece of the quick analysis and identification of DAD-ESI-TOF/MS methods, the more of gold ring piece are established on this basis
Index quantification finger-print, and similarity analysis is combined, for its quality control and evaluation study.This research is to improving gold ring piece
Quality standard, ensure its safe and reasonable medication provide methods and techniques support.
The preferred embodiment of the application is the foregoing is only, is not limited to the application, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of method for building up of gold ring piece multi objective quantitative finger print atlas, it is characterized in that, anti-phase height is obtained by optimization method
The chromatographic condition of chemical composition, is flown using RPLC-electron spray in effect liquid phase chromatogram analysis gold ring piece extract
Row time mass spectrum detects the characteristic peak of a variety of total chemical materials in multiple batch gold ring piece extracts, passes through the chromatogram of acquisition
Condition detects the chromatographic peak of a variety of total chemical materials in the multiple batch gold ring piece extract, golden according to multiple batches are detected
The chromatographic peak and assay of a variety of total chemical materials of ring piece extract establish the high performance liquid chromatography fingerprint image of gold ring piece
Spectrum, it is gold ring piece multi objective quantitative finger print atlas；Wherein, in the chromatographic condition, the chromatographic column used is Agilent SB
C18Chromatographic column, its specification be length be 250mm, internal diameter 4.6mm, aperture be 5 μm, the mobile phase used is acetonitrile -0.2%
(volume) acetic acid aqueous solution system, the collection wavelength of finger-print is 280nm.
2. method for building up as claimed in claim 1, it is characterized in that, the extracting method of the gold ring piece extract is carried using ultrasound
Follow the example of.
3. method for building up as claimed in claim 2, it is characterized in that, extractant uses 50% (volume) methanol.
4. method for building up as claimed in claim 1, it is characterized in that, chromatographic condition is：The volume flow 0.8mL/ of mobile phase
Min, evaporative light gas flow rate 1.6mL/min, 80 DEG C of temperature, sample size are 30 μ L, and gradient elution program is 0~5min, 8%A；
5~15min, 8%~12%A；15~20min, 12%~13%A；20~25min, 13%~15%A；25~30min,
15%~16%A；30~50min, 16%~18%A；50~60min, 18%~20%A；60~65min, 20%~20%
A；65~70min, 20%~25%A；70~85min, 25%~25%A；85~90min, 25%~26%A；91~
110min, 60%~100%A.
5. method for building up as claimed in claim 1, it is characterized in that, the selection gold ring piece efficient liquid-phase chromatograph finger print atlas
Shared peak is 20 kinds.
6. method for building up as claimed in claim 1, it is characterized in that, using RPLC joint diode array inspection
Survey device-evaporation photodetector and assay, respectively tanshin polyphenolic acid B, Ha Bae have been carried out to 5 kinds of main components in gold ring piece simultaneously
Glycosides, cholic acid, acteoside, Tanshinone I.
7. method for building up as claimed in claim 6, it is characterized in that, the tanshin polyphenolic acid B that is obtained according to detection, harpagoside, cholic acid,
It is analysis regression equation, the range of linearity and test limit that acteoside, the standard curve of Tanshinone I and detection limit obtain, quantitative
Limit such as following table,
8. the gold ring piece multi objective quantitative finger print atlas that a kind of any described method for building up of claim 1~7 is established.
9. the quantitative fingerprint of gold ring piece multi objective described in a kind of any described method for building up of claim 1~7 or claim 7
Application of the collection of illustrative plates in gold ring tablet quality control and evaluation.
10. the quantitative fingerprint of gold ring piece multi objective described in a kind of any described method for building up of claim 1~7 or claim 7
Application of the collection of illustrative plates in monitoring produces gold ring piece.
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