CN106290677B - The method for building up and its finger-print of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print - Google Patents
The method for building up and its finger-print of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print 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
- G01N30/02—Column chromatography
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- 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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- 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
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Abstract
The invention discloses a kind of method for building up and its finger-print of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print, belong to FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print establishes field.The present invention discloses a kind of method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print first, comprises the following steps：FENGLIAOXING FENGSHI DIEDA YAOJIU is diluted, filtered, collects filtrate as need testing solution；Need testing solution is subjected to efficient liquid phase chromatographic analysis, obtains HPLC finger-prints；Shared peak is determined according to the relative retention time of chromatographic peak in HPLC finger-prints；Characteristic fingerprint peak is selected from shared peak, obtains FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print.The precision of FENGLIAOXING FENGSHI DIEDA YAOJIU fingerprint of the present invention and reproducible, reliable and stable, FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print high sensitivity, the stability established are good, can be used in the quality control of FENGLIAOXING FENGSHI DIEDA YAOJIU.
Description
Technical field
The present invention relates to the method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print, further relates to Feng that methods described is established
Property rheumatism-traumatic medicinal liquor finger-print, belong to FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print establish field.
Background technology
FENGLIAOXING FENGSHI DIEDA YAOJIU is by the taste Chinese medicine white wine of ergcibe obtusifolia, Chinese ephedra, cassia twig, Fructus Aurantii, dried orange peel, sealwort etc. 27
Dipping forms, it is orally available, can external application, there is the effect of dispelling wind and eliminating dampness, promoting blood circulation and stopping pain.It is numb in every limb for wind-cold-dampness arthralgia, waist-leg
Ache；The disease such as injury from falling down, the stagnant swelling and pain of the stasis of blood.The quality standard of FENGLIAOXING FENGSHI DIEDA YAOJIU is recorded in from nineteen ninety version
《Chinese Pharmacopoeia》,《Chinese Pharmacopoeia》Quality standard under 2015 editions FENGLIAOXING FENGSHI DIEDA YAOJIU items covers character (eastern Liang
Henbane lactone, ephedrine hydrochloride, magnolol, honokiol, cinnaldehydrum, dehydro-α-curcumene, Paeonol reference substance and cassia twig, the banksia rose
Control medicinal material) thin layer differentiates, amount of alcohol, total solid, the test rating such as inspection of quantity of methyl alcohol, it is difficult to reflection Chinese medicine compound prescription system comprehensively
Comprehensive control of the globality and inherent quality of agent.
Traditional Chinese medicine fingerprint technology turns into the discriminating herbal species generally acknowledged both at home and abroad and evaluates the most effective of traditional Chinese medicine quality
Means.Finger-print is the understanding based on centering medicinal substances group's mass action, and Chinese medicine is obtained by means of technologies such as wave spectrum and chromatograms
The spectrum or chromatogram of chemical composition, it is to realize to differentiate the feasible of Chinese medicine authenticity, evaluation quality uniformity and product stability
Pattern.Finger-print includes the analysis to principal component and non-principal component, and the chemical composition information of reflection (is embodied in phase
To retention time and relative peak area) there is high degree of specificity and selectivity, it can more fully hereinafter reflect Chinese medicine complex mixts
The integral status of various chemical composition amount distributions in system.Chromatographic fingerprinting is due to can clearly reflect master contained in prescription
Composition situation, more characteristic and fingerprint are wanted, therefore various chromatographic processes are in the finger-print quality controling research of prescription
There is more common application.Therefore, a kind of finger-print of FENGLIAOXING FENGSHI DIEDA YAOJIU is established, will be Feng's property rheumatic bone-setting
The quality evaluation of medicinal liquor, scientific basis is provided for its production and quality control.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of foundation side of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print
Method, this method is simple and easy to do, reliable and stable, the FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print high sensitivity established, stability
It is good, it can be used in the quality control of FENGLIAOXING FENGSHI DIEDA YAOJIU.
In order to solve the above technical problems, the technical solution used in the present invention is：
The invention discloses a kind of method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print, comprise the following steps：(1)
FENGLIAOXING FENGSHI DIEDA YAOJIU is diluted, filtered, collects filtrate as need testing solution；(2) need testing solution is carried out efficient
Liquid-phase chromatographic analysis, obtain HPLC finger-prints；(3) determined altogether according to the relative retention time of chromatographic peak in HPLC finger-prints
There is peak；(4) characteristic fingerprint peak is selected from shared peak, obtains FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print.
Wherein, the condition of step (2) described efficient liquid phase chromatographic analysis includes：Agilent high performance liquid chromatographs；Chromatogram
Post is Kromasil C18 posts (specification be 4.6 × 250mm, 5 μm) or Ecosil C18 posts (specification be 4.6 × 250mm, 5 μm)；
Mobile phase is selected from：By volume fraction, methanol (A, i.e. mobile phase A, similarly hereinafter) -0.1% acetic acid：0.1% phosphoric acid=1：1 it is water-soluble
Liquid (B, i.e. Mobile phase B, similarly hereinafter), the formic acid (B) of methanol (A) -0.1%, the acetic acid (B) of methanol (A) -0.2%, acetonitrile (A) -0.1%
Any one in phosphoric acid (B) or the phosphoric acid (B) of methanol (A) -0.1%；Gradient elution；Detection wavelength is 210-360nm；Flow velocity
1ml/min；30 DEG C of column temperature；The μ l of sample size 5.
As the preferred technical solution of the present invention, the mobile phase is：Count by volume, the acetic acid of methanol (A) -0.1%：
0.1% phosphoric acid=1：1 aqueous solution (B).The step of gradient elution, includes：It is according to volume fraction, 0-20min, A
12%；20-30min, A are risen progressively to 25% by 12%；30-50min, A are risen progressively to 38% by 25%；50-60min, A 38%；
60-75min, A are risen progressively to 60% by 38%；75-95min, A are risen progressively to 90% by 60%；90-100min, A 90%.This hair
The bright Detection wavelength is 210nm and 300nm；Preferably, the Detection wavelength is：0-10min is 300nm, and 10-20min is
210nm, 20-100min 300nm.
The present invention is with scopolin, scopolactone, chlorogenic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, cinnaldehydrum, new
Aurantiamarin, aurantiin are the HPLC finger-prints that object of reference peak determines FENGLIAOXING FENGSHI DIEDA YAOJIU.The inventive method passes through
Compare the HPLC finger-prints of 10 batches of need testing solutions, according to the relative retention time of each chromatographic peak in chromatogram, it is determined that 15
Individual shared peak is characterized fingerprint peakses；Relative retention time of the 15 shared peaks using chlorogenic acid as reference be respectively：
The average relative retention time in No. 1 peak is 0.265, RSD (relative standard deviation) ＜ 1%, it is preferred that RSD is
0.37%, relative peak area average value is 277.484；The average relative retention time in No. 2 peaks is 0.494, RSD ＜ 1%, preferably
, RSD 0.74%, relative peak area average value is 203.291；The average relative retention time in No. 3 peaks is 0.525, RSD ＜
1%, it is preferred that RSD 0.61%, relative peak area average value are 617.653；The average relative retention times in No. 4 peaks are
0.960, RSD ＜ 1%, it is preferred that RSD 0.23%, relative peak area average value are 288.293；No. 5 peaks are averagely relative to be retained
Time is 1.000, RSD ＜ 1%, it is preferred that RSD 0.2%, relative peak area average value are 797.696；The average phase in No. 6 peaks
It is 1.078, RSD ＜ 1% to retention time, it is preferred that RSD 0.17%, relative peak area average value are 276.629；No. 7 peaks
Average relative retention time is 1.368, RSD ＜ 1%, it is preferred that RSD 0.15%, relative peak area average value are
307.821；The average relative retention time in No. 8 peaks is 1.621, RSD ＜ 1%, it is preferred that RSD 0.15%, relative peak area is put down
Average is 212.11；The average relative retention time in No. 9 peaks is 1.691, RSD ＜ 1%, it is preferred that RSD 0.16%, with respect to peak
Area average is 740.134；The average relative retention times in No. 10 peaks are 1.713, RSD ＜ 1%, it is preferred that RSD is
0.17%, relative peak area average value is 496.374；The average relative retention time in No. 11 peaks is 1.745, RSD ＜ 1%, preferably
, RSD 0.18%, relative peak area average value is 92.442；The average relative retention time in No. 12 peaks is 1.824, RSD ＜
1%, it is preferred that RSD 0.2%, relative peak area average value are 370.97；The average relative retention time in No. 13 peaks is 2.000,
RSD ＜ 1%, it is preferred that RSD 0.28%, relative peak area average value are 290.672；The average relative retention time in No. 14 peaks
For 2.047, RSD ＜ 1%, it is preferred that RSD 0.09%, relative peak area average value are 1228.019；No. 15 peaks are averagely relative
Retention time is 2.081, RSD ＜ 1%, it is preferred that RSD 0.14%, relative peak area average value are 194.893.
The present invention is compared by retention time, uv-spectrogram and reference substance control, and 8 chromatograms are identified from shared peak
Peak, respectively ephedrine hydrochloride (2, i.e., shared peak numbering, similarly hereinafter), pseudoephedrine hydrochloride (3), scopolin (4), chlorogenic acid
(5), scopolactone (7), aurantiin (11), neohesperidin (12) and cinnaldehydrum (14).
In the method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print of the present invention, step (1) is described to be diluted to：Use methanol
FENGLIAOXING FENGSHI DIEDA YAOJIU is diluted 1-3 times, preferably dilutes 1 times.Step (1) is described to be filtered into 0.45 μm of micropore filter
Film.
Condition of the invention to the efficient liquid phase chromatographic analysis in FENGLIAOXING FENGSHI DIEDA YAOJIU fingerprint,
It is optimized including Detection wavelength, chromatographic column and mobile phase.The optimum results of Detection wavelength show, with ultraviolet DAD detectors
Need testing solution is detected, appearance is most abundant under the conditions of 300nm, hence it is evident that better than in 246nm, 274nm, 340nm or
Appearance situation under the conditions of 360nm；But the pseudo- fiber crops of the index components ephedrine hydrochloride and hydrochloric acid of the larger Chinese ephedra of medicinal liquor Chinese medicine dosage
Yellow alkali appearance at 210nm.Therefore, present invention determine that 210 and 300nm is as Detection wavelength.
Selection for chromatographic column, the present invention compare the chromatographic column of 3 kinds of different brands models, from chromatographic peak peak shape, divide
Compare from degree etc., preferably Ecosil C18 posts (4.6 × 250mm, 5 μm) or Kromasil C18 posts (4.6 × 250mm,
5 μm) it is experiment chromatographic column.
The optimum results of mobile phase show, respectively with the formic acid of methanol -0.1%, the acetic acid of methanol -0.2%, the phosphorus of acetonitrile -0.1%
As a result acid or the phosphoric acid of methanol -0.1% show that chromatographic peak is undesirable as mobile phase；And select methanol-(0.1% acetic acid：
0.1% phosphoric acid=1：1) when being used as flow phase system, eluting power is strong, and chromatogram contains much information, and chromatographic peak profile is good, separating effect
Good, baseline is steady；In mobile phase, phosphoric acid increase ultraviolet absorption value, be advantageous to the detection of ephedrine；Acetic acid have to other peaks compared with
Good separating effect.Therefore, present invention selection methanol-(0.1% acetic acid：0.1% phosphoric acid=1：1) it is used as flow phase system.
Precision test shows, according to the method for building up of finger-print of the present invention, takes FENGLIAOXING FENGSHI DIEDA YAOJIU parallel
Result is simultaneously imported " similarity evaluation " and carries out chromatogram peak match by sample introduction 6 times, and as a result 15 altogether
There are the relative retention time RSD equal ＜ 5% of equal ＜ 1%, relative peak area RSD at peak, show that precision is good.Stability test table
It is bright, FENGLIAOXING FENGSHI DIEDA YAOJIU is taken respectively at the equal ＜ of relative retention time RSD of 0-24h sample introductions, as a result 15 shared peaks
The equal ＜ 5% of 1%, relative peak area RSD, shows that the inventive method has good stability.Replica test shows, the property rheumatism by Feng
6 parts of difference sample introductions of medical liquor for treating injury need testing solution, as a result equal ＜ 1% of the relative retention time RSD at 15 shared peaks, with respect to peak face
The product equal ＜ 5% of RSD, show that the inventive method repeatability is good.
The present invention further discloses the FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print that methods described is established, it is preferred that institute
FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print is stated as shown in Figure of description Fig. 3.Present invention Feng for being established property rheumatic bone-setting
Medicinal liquor finger-print can be applied to the quality control or quality evaluation of FENGLIAOXING FENGSHI DIEDA YAOJIU.The similarity of finger-print
Evaluation result shows that for the similarity of 10 batches of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-prints substantially more than 0.9, precision is good.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention establishes the finger-print of FENGLIAOXING FENGSHI DIEDA YAOJIU, this method letter using HPLC-UV atlas analysis
Easy row, precision is good, reproducible, reliable and stable.The FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print that the present invention is established
High sensitivity, stability are good, are a kind of effective FENGLIAOXING FENGSHI DIEDA YAOJIU quality evaluating methods, will being Feng, property rheumatism be fallen
The production and quality control of laxative wine provide scientific basis.
Brief description of the drawings
Fig. 1 is FENGLIAOXING FENGSHI DIEDA YAOJIU and the finger-print of reference substance solution；Wherein, the unit of abscissa is min；
Fig. 2 is the HPLC finger-prints of FENGLIAOXING FENGSHI DIEDA YAOJIU test sample；Wherein, S1-S10 is 10 batches of test samples；
The unit of abscissa is min；
Fig. 3 is the finger-print of FENGLIAOXING FENGSHI DIEDA YAOJIU；Wherein, 1-15 is characterized fingerprint peakses；The unit of abscissa
For min；
Fig. 4 is appearance situations of the 300nm as Detection wavelength；
Fig. 5 is appearance situations of the 246nm as Detection wavelength；
Fig. 6 is appearance situations of the 274nm as Detection wavelength；
Fig. 7 is appearance situations of the 340nm as Detection wavelength；
Fig. 8 is appearance situations of the 360nm as Detection wavelength；
Fig. 9 is appearance situations of the 210nm as Detection wavelength；
Figure 10 is the chromatographic peak that the formic acid of methanol -0.1% is mobile phase；
Figure 11 is the chromatographic peak that the acetic acid of methanol -0.2% is mobile phase；
Figure 12 is the chromatographic peak that the phosphoric acid of acetonitrile -0.1% is mobile phase；
Figure 13 is the chromatographic peak that the phosphoric acid of methanol -0.1% is mobile phase；
Figure 14 is methanol-(0.1% acetic acid:0.1% phosphoric acid=1:1) it is the chromatographic peak of mobile phase.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any restrictions are not formed to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1st, instrument and reagent
1.1 instrument
Agilent high performance liquid chromatographs (Agilent company of the U.S., the series of model 1260), electronic balance (plum Teller-support
Benefit, XS105DU).
1.2 reagent
Reference substance scopolactone (110768-200504), cinnaldehydrum (110710-201418), chlorogenic acid (110753-
201314), neohesperidin (11857-201102), ephedrine hydrochloride (171241-201508), pseudoephedrine hydrochloride (171237-
201208), aurantiin (110722-201312) is provided by Nat'l Pharmaceutical ＆ Biological Products Control Institute；Scopolin (purity >=
98%, lot number Y24S6Y17687) provided by Shanghai Yuan Ye bio tech ltd.
FENGLIAOXING FENGSHI DIEDA YAOJIU (production of Chinese medicines group Feng property (Foshan) pharmaceutcal corporation, Ltd, 500mL/ bottles).First
Alcohol is chromatographically pure, and water is ultra-pure water, and other reagents are that analysis is pure.
The foundation of the FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print of embodiment 1
1st, experimental method
The foundation of 1.1 FENGLIAOXING FENGSHI DIEDA YAOJIU finger-prints
1.1.1 chromatographic condition
Kromasil C18 posts (4.6 × 250mm, 5 μm)；Mobile phase is the acetic acid of (counting by volume) methanol (A) -0.1%：
0.1% phosphoric acid=1：1 aqueous solution (B)；Gradient elution (counts) (0~20min, A 12% by volume；20~30min, A by
12% rises progressively to 25%；30~50min, A are risen progressively to 38% by 25%；50~60min, A 38%；60~75min, A by
38% rises progressively to 60%；75~95min, A are risen progressively to 90% by 60%；90~100min, A 90%)；Detection wavelength (0-
10min is 300nm, 10-20min 210nm, 20-100min 300nm)；Flow velocity 1ml/min；30 DEG C of column temperature；The μ of sample size 5
l。
1.1.2 the preparation of need testing solution
Precision measures FENGLIAOXING FENGSHI DIEDA YAOJIU 10ml, puts in 20ml measuring bottles, adds methanol dilution to be shaken up, mistake to scale
0.45 μm of miillpore filter, takes subsequent filtrate, produces.
1.1.3 the preparation of reference substance solution
Precision weighs scopolactone, cinnaldehydrum, chlorogenic acid, neohesperidin, ephedrine hydrochloride, pseudoephedrine hydrochloride, shaddock
Skin glycosides, scopolin reference substance are appropriate, add the dissolving of 80% methanol aqueous solution that scopolactone (40 μ g/ml), cinnaldehydrum (10 is made
μ g/ml), chlorogenic acid (40 μ g/ml), neohesperidin (80 μ g/ml), ephedrine hydrochloride (40 μ g/ml), pseudoephedrine hydrochloride (40 μ
G/ml), aurantiin (80 μ g/ml), scopolin (212 μ g/ml) solution, are produced.
1.2 methodological study
1.2.1 precision test
FENGLIAOXING FENGSHI DIEDA YAOJIU is taken, need testing solution is prepared according to 1.1.2 method, according to 1.1.1 chromatostrip
Part, parallel sample introduction 6 times, and result is imported into " similarity evaluation " 2004A versions, carry out chromatographic peak
Matching.
1.2.2 stability test
FENGLIAOXING FENGSHI DIEDA YAOJIU is taken, need testing solution is prepared according to 1.1.2 method, according to 1.1.1 chromatostrip
Part, respectively at 0,2,4,8,12,24h sample introduction, investigate the stability of the inventive method.
1.2.3 replica test
FENGLIAOXING FENGSHI DIEDA YAOJIU is taken, 6 parts of need testing solution is prepared according to 1.1.2 methods are parallel, according to 1.1.1 color
Spectral condition, distinguish sample introduction, investigate the repeatability of the inventive method.
2nd, experimental result
The foundation and analysis of 2.1 finger-prints
2.1.1 the foundation of finger-print
FENGLIAOXING FENGSHI DIEDA YAOJIU test sample of the present invention and the finger-print of reference substance solution are shown in Fig. 1.
The present invention is measured by above-mentioned 1.1.1 chromatographic condition, obtains FENGLIAOXING FENGSHI DIEDA YAOJIU S1-S10 for examination
The HPLC finger-prints (Fig. 2) of product, according to the relative retention time of each chromatographic peak in chromatogram, it is determined that shared peak, and choose 15
Individual shared peak is characterized fingerprint peakses (Fig. 3).Because collection of illustrative plates Content of Chlorogenic Acid separating degree is good, appearance time is moderate, and peak area is larger and steady
It is fixed, therefore as chromatogram with reference to peak S.
Relative retention time of the 15 shared peaks using chlorogenic acid as reference is shown in Table 1.
The relative retention time and relative peak area at the 15 shared peaks of S1-S10 test sample HPLC finger-prints of table 1
The present invention is compared by retention time, uv-spectrogram and reference substance control, and 8 chromatograms are identified from shared peak
Peak, respectively ephedrine hydrochloride (2, i.e., shared peak numbering, similarly hereinafter), pseudoephedrine hydrochloride (3), scopolin (4), chlorogenic acid
(5), scopolactone (7), aurantiin (11), neohesperidin (12) and cinnaldehydrum (14).
2.1.2 the similarity evaluation of finger-print
Using " chromatographic fingerprints of Chinese materia medica similarity evaluation software " 2004A versions (Chinese Pharmacopoeia Commission) to 10 crowdes of (S1-
S10) FENGLIAOXING FENGSHI DIEDA YAOJIU carries out similarity evaluation, the results are shown in Table 2.
The similarity evaluation of 2 10 batches of FENGLIAOXING FENGSHI DIEDA YAOJIUs of table
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Reference fingerprint | |
| S1 | 1.000 | 0.930 | 0.980 | 0.940 | 0.985 | 0.929 | 0.956 | 0.974 | 0.919 | 0.955 | 0.988 |
| S2 | 0.930 | 1.000 | 0.913 | 0.928 | 0.942 | 0.955 | 0.877 | 0.912 | 0.930 | 0.897 | 0.945 |
| S3 | 0.980 | 0.913 | 1.000 | 0.905 | 0.978 | 0.905 | 0.984 | 0.971 | 0.918 | 0.972 | 0.988 |
| S4 | 0.940 | 0.928 | 0.905 | 1.000 | 0.960 | 0.969 | 0.855 | 0.919 | 0.911 | 0.893 | 0.944 |
| S5 | 0.985 | 0.942 | 0.978 | 0.960 | 1.000 | 0.952 | 0.951 | 0.971 | 0.948 | 0.960 | 0.992 |
| S6 | 0.929 | 0.955 | 0.905 | 0.969 | 0.952 | 1.000 | 0.856 | 0.901 | 0.947 | 0.881 | 0.941 |
| S7 | 0.956 | 0.877 | 0.984 | 0.855 | 0.951 | 0.856 | 1.000 | 0.969 | 0.883 | 0.981 | 0.973 |
| S8 | 0.974 | 0.912 | 0.971 | 0.919 | 0.971 | 0.901 | 0.969 | 1.000 | 0.908 | 0.986 | 0.987 |
| S9 | 0.919 | 0.930 | 0.918 | 0.911 | 0.948 | 0.947 | 0.883 | 0.908 | 1.000 | 0.910 | 0.945 |
| S10 | 0.955 | 0.897 | 0.972 | 0.893 | 0.960 | 0.881 | 0.981 | 0.986 | 0.910 | 1.000 | 0.983 |
| Reference fingerprint | 0.988 | 0.945 | 0.988 | 0.944 | 0.992 | 0.941 | 0.973 | 0.987 | 0.945 | 0.983 | 1.000 |
As a result show, for the similarity of 10 batches of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-prints substantially more than 0.9, precision is good
It is good.
2.2 methodological study results
2.2.1 precision test
The present invention takes the parallel sample introduction of FENGLIAOXING FENGSHI DIEDA YAOJIU 6 times, and result is imported into " chromatographic fingerprints of Chinese materia medica phase
Like degree evaluation system " 2004A versions, chromatogram peak match is carried out, the results are shown in Table 3.
As a result (table 3, S1-S6 are 6 data of sample introduction, are peak area data) 15 shared peaks (numbering 1-15) are shown
The relative retention time RSD equal ＜ 5% of equal ＜ 1%, relative peak area RSD, show instrument stabilizer, and precision is good.
The Precision test result of table 3
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | Retention time RSD (%) | Peak area RSD (%) |
| 1 | 192.374 | 193.291 | 193.069 | 191.852 | 193.184 | 194.433 | 0.33 | 0.46 |
| 2 | 93.589 | 100.384 | 104.209 | 99.920 | 107.373 | 104.518 | 0.27 | 4.76 |
| 3 | 139.954 | 150.301 | 145.430 | 151.469 | 137.297 | 151.620 | 0.77 | 4.25 |
| 4 | 375.944 | 382.082 | 390.045 | 418.230 | 403.351 | 419.630 | 0.62 | 4.64 |
| 5 | 107.001 | 106.143 | 115.885 | 115.482 | 111.398 | 112.104 | 0.27 | 3.69 |
| 6 | 482.051 | 484.085 | 482.500 | 485.005 | 488.054 | 488.189 | 0.21 | 0.55 |
| 7 | 143.608 | 144.500 | 144.181 | 143.352 | 146.476 | 146.642 | 0.14 | 0.99 |
| 8 | 173.101 | 173.284 | 173.290 | 173.633 | 173.321 | 180.898 | 0.09 | 1.77 |
| 9 | 124.141 | 128.842 | 129.468 | 129.745 | 131.670 | 131.288 | 0.04 | 2.09 |
| 10 | 354.968 | 356.834 | 356.662 | 357.539 | 358.435 | 360.101 | 0.03 | 0.49 |
| 11 | 288.593 | 291.035 | 291.139 | 291.644 | 293.448 | 294.252 | 0.03 | 0.69 |
| 12 | 351.148 | 354.587 | 352.411 | 353.153 | 356.274 | 356.665 | 0.06 | 0.62 |
| 13 | 238.089 | 238.203 | 240.456 | 243.033 | 245.486 | 247.332 | 0.08 | 1.59 |
| 14 | 669.048 | 674.082 | 669.737 | 676.427 | 680.011 | 689.067 | 0.07 | 1.10 |
| 15 | 180.151 | 182.024 | 182.786 | 182.736 | 183.487 | 186.028 | 0.07 | 1.05 |
2.2.2 stability test
The present invention takes FENGLIAOXING FENGSHI DIEDA YAOJIU, respectively at 0,2,4,8,12,24h sample introduction (S1-S6), as a result (table 4,
S1-S6 is 6 data of sample introduction, is peak area data) the equal ＜ of relative retention time RSD of 15 shared peaks (numbering 1-15)
The equal ＜ 5% of 1%, relative peak area RSD, show that need testing solution is stable in 24h, the inventive method has good stability.
The stability test result of table 4
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | Retention time RSD (%) | Peak area RSD (%) |
| 1 | 192.374 | 193.291 | 193.069 | 191.852 | 193.184 | 194.433 | 0.33 | 0.46 |
| 2 | 93.589 | 100.384 | 104.209 | 99.920 | 107.373 | 104.518 | 0.27 | 4.76 |
| 3 | 139.954 | 150.301 | 145.430 | 151.469 | 137.297 | 151.620 | 0.77 | 4.25 |
| 4 | 375.944 | 382.082 | 390.045 | 418.230 | 403.351 | 419.630 | 0.62 | 4.64 |
| 5 | 107.001 | 106.143 | 115.885 | 115.482 | 111.398 | 112.104 | 0.27 | 3.69 |
| 6 | 482.051 | 484.085 | 482.500 | 485.005 | 488.054 | 488.189 | 0.21 | 0.55 |
| 7 | 143.608 | 144.500 | 144.181 | 143.352 | 146.476 | 146.642 | 0.14 | 0.99 |
| 8 | 173.101 | 173.284 | 173.290 | 173.633 | 173.321 | 180.898 | 0.09 | 1.77 |
| 9 | 124.141 | 128.842 | 129.468 | 129.745 | 131.670 | 131.288 | 0.04 | 2.09 |
| 10 | 354.968 | 356.834 | 356.662 | 357.539 | 358.435 | 360.101 | 0.03 | 0.49 |
| 11 | 288.593 | 291.035 | 291.139 | 291.644 | 293.448 | 294.252 | 0.03 | 0.69 |
| 12 | 351.148 | 354.587 | 352.411 | 353.153 | 356.274 | 356.665 | 0.06 | 0.62 |
| 13 | 238.089 | 238.203 | 240.456 | 243.033 | 245.486 | 247.332 | 0.08 | 1.59 |
| 14 | 669.048 | 674.082 | 669.737 | 676.427 | 680.011 | 689.067 | 0.07 | 1.10 |
| 15 | 180.151 | 182.024 | 182.786 | 182.736 | 183.487 | 186.028 | 0.07 | 1.05 |
2.2.3 replica test
The present invention takes FENGLIAOXING FENGSHI DIEDA YAOJIU, parallel to prepare 6 parts of difference sample introductions of need testing solution, as a result (table 5, S1-
S6 is the peak area data of sample introduction 6 times) the equal ＜ 1% of relative retention time RSD of 15 shared peaks (numbering 1-15), with respect to peak
The equal ＜ 5% of area RSD, show that the inventive method repeatability is good.
The replica test result of table 5
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | Retention time RSD (%) | Peak area RSD (%) |
| 1 | 195.046 | 191.010 | 191.779 | 190.940 | 192.281 | 191.012 | 0.22 | 0.82 |
| 2 | 106.056 | 97.714 | 96.706 | 104.481 | 106.177 | 106.653 | 0.16 | 4.40 |
| 3 | 184.365 | 162.533 | 176.416 | 167.341 | 165.031 | 168.082 | 0.31 | 4.81 |
| 4 | 436.650 | 448.090 | 435.733 | 442.124 | 425.000 | 389.637 | 0.35 | 4.89 |
| 5 | 154.575 | 147.569 | 149.428 | 147.764 | 148.062 | 147.076 | 0.13 | 1.88 |
| 6 | 512.442 | 484.259 | 492.630 | 484.762 | 489.996 | 485.369 | 0.10 | 2.19 |
| 7 | 149.017 | 135.944 | 138.233 | 141.282 | 143.137 | 141.838 | 0.07 | 3.17 |
| 8 | 179.635 | 172.963 | 173.297 | 172.152 | 172.977 | 172.099 | 0.06 | 1.65 |
| 9 | 136.853 | 130.139 | 133.249 | 131.357 | 132.167 | 131.793 | 0.05 | 1.75 |
| 10 | 374.508 | 352.893 | 360.368 | 355.031 | 358.385 | 355.302 | 0.05 | 2.19 |
| 11 | 305.819 | 288.558 | 294.755 | 289.073 | 293.395 | 290.916 | 0.05 | 2.17 |
| 12 | 370.906 | 349.328 | 355.070 | 351.884 | 355.312 | 350.402 | 0.07 | 2.23 |
| 13 | 256.257 | 238.998 | 242.693 | 236.845 | 242.585 | 240.112 | 0.11 | 2.84 |
| 14 | 712.222 | 675.358 | 683.473 | 675.477 | 676.044 | 676.027 | 0.13 | 2.14 |
| 15 | 182.448 | 180.514 | 182.165 | 179.258 | 181.691 | 180.354 | 0.08 | 0.68 |
The optimization of the FENGLIAOXING FENGSHI DIEDA YAOJIU fingerprint of experimental example 1
1st, the selection of Detection wavelength
The present invention is entered with ultraviolet DAD detectors to need testing solution (preparing need testing solution according to 1.1.2 in embodiment 1)
Row detection, appearance is most abundant under the conditions of as a result (Fig. 4) is shown in 300nm, hence it is evident that better than in 246nm, 274nm, 340nm, 360nm
Under the conditions of appearance situation (Fig. 5-Fig. 8)；But the index components ephedrine hydrochloride and hydrochloric acid of the larger Chinese ephedra of medicinal liquor Chinese medicine dosage
Pseudoephedrine will at 210nm appearance (Fig. 9).
Therefore, present invention determine that 210 and 300nm is as Detection wavelength.
2nd, the selection of chromatographic column
The present invention compares the chromatographic column of 3 different brands models, compares from chromatographic peak peak shape, separating degree etc., excellent
It is experiment chromatographic column to have selected Ecosil C18 posts (4.6 × 250mm, 5 μm), Kromasil C18 posts (4.6 × 250mm, 5 μm).
3rd, the selection of mobile phase
The present invention have selected (volume hundred respectively under conditions of the preparation of need testing solution and chromatographic condition are with embodiment 1
Point ratio) formic acid of methanol -0.1%, the acetic acid of methanol -0.2%, the phosphoric acid of acetonitrile -0.1%, the phosphoric acid of methanol -0.1% enters as mobile phase
Row experiment, as a result show that chromatographic peak is undesirable (Figure 10-Figure 13)；And select methanol-(0.1% acetic acid：0.1% phosphoric acid=1：
1) when being used as flow phase system, eluting power is strong, and chromatogram contains much information, and chromatographic peak profile is good, good separating effect, and baseline is steady
(Figure 14, table 6)；In mobile phase, phosphoric acid increase ultraviolet absorption value, be advantageous to the detection of ephedrine；Acetic acid has preferably to other peaks
Separating effect.
Therefore, present invention selection methanol-(0.1% acetic acid：0.1% phosphoric acid=1：1) it is used as flow phase system.
The methanol of table 6-(0.1% acetic acid：0.1% phosphoric acid=1：1) the chromatogram testing result as mobile phase
Claims (6)
1. a kind of method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print, it is characterised in that comprise the following steps：(1) will
FENGLIAOXING FENGSHI DIEDA YAOJIU dilutes, filtering, collects filtrate as need testing solution；(2) need testing solution is subjected to efficient liquid
Analysis of hplc, obtain HPLC finger-prints；(3) determined according to the relative retention time of chromatographic peak in HPLC finger-prints shared
Peak；(4) characteristic fingerprint peak is selected from shared peak, obtains FENGLIAOXING FENGSHI DIEDA YAOJIU finger-print；
Wherein, the condition of step (2) described efficient liquid phase chromatographic analysis includes：
Chromatographic column is Kromasil C18 posts, and specification is 4.6 × 250mm, 5 μm, or, Ecosil C18 posts, specification is 4.6 ×
250mm, 5 μm；
Mobile phase is：Count by volume, the acetic acid of methanol (A) -0.1%：0.1% phosphoric acid=1：1 aqueous solution (B)；
Gradient elution；The step of gradient elution, includes：According to volume fraction, 0-20min, A 12%；20-30min, A
Risen progressively by 12% to 25%；30-50min, A are risen progressively to 38% by 25%；50-60min, A 38%；60-75min, A are by 38%
Rise progressively to 60%；75-95min, A are risen progressively to 90% by 60%；90-100min, A 90%；
Detection wavelength is：0-10min is 300nm, 10-20min 210nm, 20-100min 300nm.
2. according to the method for building up described in claim 1, it is characterised in that the bar of step (2) described efficient liquid phase chromatographic analysis
Part includes：30 DEG C of column temperature；The μ l of sample size 5.
3. according to the method for building up described in claim 1, it is characterised in that step (4) selects 15 shared peaks to be characterized fingerprint
Peak, relative retention time of the 15 shared peaks using chlorogenic acid as reference are respectively：
The average relative retention time in No. 1 peak is 0.265, RSD ＜ 1%；
The average relative retention time in No. 2 peaks is 0.494, RSD ＜ 1%；
The average relative retention time in No. 3 peaks is 0.525, RSD ＜ 1%；
The average relative retention time in No. 4 peaks is 0.960, RSD ＜ 1%；
The average relative retention time in No. 5 peaks is 1.000, RSD ＜ 1%；
The average relative retention time in No. 6 peaks is 1.078, RSD ＜ 1%；
The average relative retention time in No. 7 peaks is 1.368, RSD ＜ 1%；
The average relative retention time in No. 8 peaks is 1.621, RSD ＜ 1%；
The average relative retention time in No. 9 peaks is 1.691, RSD ＜ 1%；
The average relative retention time in No. 10 peaks is 1.713, RSD ＜ 1%；
The average relative retention time in No. 11 peaks is 1.745, RSD ＜ 1%；
The average relative retention time in No. 12 peaks is 1.824, RSD ＜ 1%；
The average relative retention time in No. 13 peaks is 2.000, RSD ＜ 1%；
The average relative retention time in No. 14 peaks is 2.047, RSD ＜ 1%；
The average relative retention time in No. 15 peaks is 2.081, RSD ＜ 1%.
4. according to the method for building up described in claim 3, it is characterised in that
The RSD at No. 1 peak is 0.37%；
The RSD at No. 2 peaks is 0.74%；
The RSD at No. 3 peaks is 0.61%；
The RSD at No. 4 peaks is 0.23%；
The RSD at No. 5 peaks is 0.2%；
The RSD at No. 6 peaks is 0.17%；
The RSD at No. 7 peaks is 0.15%；
The RSD at No. 8 peaks is 0.15%；
The RSD at No. 9 peaks is 0.16%；
The RSD at No. 10 peaks is 0.17%；
The RSD at No. 11 peaks is 0.18%；
The RSD at No. 12 peaks is 0.2%；
The RSD at No. 13 peaks is 0.28%；
The RSD at No. 14 peaks is 0.09%；
The RSD at No. 15 peaks is 0.14%.
5. according to the method for building up described in claim 1, it is characterised in that step (1) is described to be diluted to：With methanol by Feng Liaoxing
Rheumatism-traumatic medicinal liquor dilutes 1-3 times；
Step (1) is described to be filtered into 0.45 μm of miillpore filter.
6. according to the method for building up described in claim 5, it is characterised in that step (1) is described to be diluted to：With methanol by Feng Liaoxing
Rheumatism-traumatic medicinal liquor dilutes 1 times.
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