CN106290677A - The method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing and finger printing thereof - Google Patents
The method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing and finger printing thereof 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
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Abstract
The invention discloses method for building up and the finger printing thereof of a kind of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing, belong to FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing sets up field.First the present invention discloses the method for building up of a kind of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing, comprises the following steps: FENGLIAOXING FENGSHI DIEDA YAOJIU diluted, and filters, and collects filtrate as need testing solution；Need testing solution is carried out efficient liquid phase chromatographic analysis, obtains HPLC finger printing；Total peak is determined according to the relative retention time of chromatographic peak in HPLC finger printing；From total peak, select characteristic fingerprint peak, obtain FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing.The precision of FENGLIAOXING FENGSHI DIEDA YAOJIU fingerprint of the present invention and reproducible, reliable and stable, the FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing set up is highly sensitive, good stability, it is possible to for 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 printing, further relate to Feng that described method is set up
Property rheumatism-traumatic medicinal liquor finger printing, belong to FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing set up field.
Background technology
FENGLIAOXING FENGSHI DIEDA YAOJIU is by 27 taste Chinese crude drug Chinese liquor such as Caulis Erycibes, Herba Ephedrae, Ramulus Cinnamomi, Fructus Aurantii, Pericarpium Citri Reticulatae, Rhizoma Polygonatis
Dipping forms, orally available, can external, there is expelling wind and removing dampness, effect of promoting blood circulation and stopping pain.For anemofrigid-damp arthralgia, numb hand and foot, waist-leg
Ache；Injury from falling down, the stasis of blood is stagnant the disease such as to swell and ache.The quality standard of FENGLIAOXING FENGSHI DIEDA YAOJIU records in from nineteen ninety version
" Chinese Pharmacopoeia ", the quality standard under " Chinese Pharmacopoeia " 2015 editions FENGLIAOXING FENGSHI DIEDA YAOJIU items contains character (east Liang
Henbane lactone, ephedrine hydrochloride, magnolol, honokiol, cinnamic aldehyde, dehydrocostuslactone, paeonol reference substance and Ramulus Cinnamomi, the Radix Aucklandiae
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 system comprehensively
The globality of agent and comprehensive control of inherent quality.
Chinese medicine fingerprint technology has become the discriminating herbal species generally acknowledged both at home and abroad and has evaluated the most effective of Chinese medicine quality
Means.Finger printing is understanding based on centering medicinal substances group's mass action, obtains Chinese medicine by means of the technology such as wave spectrum and chromatograph
The spectrum of chemical composition or chromatogram, be to realize differentiating the feasible of Chinese medicine verity, evaluation quality concordance and product stability
Pattern.Finger printing includes principal component and the analysis of 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, can more fully hereinafter reflect Chinese medicine complex mixts
The integral status of various chemical composition amounts distribution in system.Chromatographic fingerprinting is owing to can clearly reflect master contained in prescription
Wanting composition situation, more characteristic and fingerprint, the most various chromatographic processes are in the finger printing quality controling research of prescription
There is more common application.Therefore, set up the finger printing of a kind of FENGLIAOXING FENGSHI DIEDA YAOJIU, will for Feng property rheumatic bone-setting
The quality evaluation of medicated wine, produces for it and quality control provides scientific basis.
Summary of the invention
The technical problem to be solved is to provide the foundation side of a kind of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing
Method, the method is simple and easy to do, reliable and stable, and the FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing set up is highly sensitive, stability
Good, it is possible to for the quality control of FENGLIAOXING FENGSHI DIEDA YAOJIU.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
The invention discloses the method for building up of a kind of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing, comprise the following steps: (1)
FENGLIAOXING FENGSHI DIEDA YAOJIU is diluted, filters, collect filtrate as need testing solution；(2) need testing solution is carried out efficiently
Liquid-phase chromatographic analysis, obtains HPLC finger printing；(3) determine altogether according to the relative retention time of chromatographic peak in HPLC finger printing
There is peak；(4) from total peak, select characteristic fingerprint peak, obtain FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing.
Wherein, the condition of step (2) described efficient liquid phase chromatographic analysis includes: Agilent high performance liquid chromatograph；Chromatograph
Post is Kromasil C18 post (specification is 4.6 × 250mm, 5 μm) or Ecosil C18 post (specification is 4.6 × 250mm, 5 μm)；
Flowing is selected from mutually: by volume mark meter, methanol (A, i.e. mobile phase A, lower same)-0.1% acetic acid: 0.1% phosphoric acid=1:1's is water-soluble
Liquid (B, i.e. Mobile phase B, lower same), methanol (A)-0.1% formic acid (B), methanol (A)-0.2% acetic acid (B), acetonitrile (A)-0.1%
Any one in phosphoric acid (B) or methanol (A)-0.1% phosphoric acid (B)；Gradient elution；Detection wavelength is 210-360nm；Flow velocity
1ml/min；Column temperature 30 DEG C；Sample size 5 μ l.
As the preferred technical solution of the present invention, described flowing is mutually: count by volume, methanol (A)-0.1% acetic acid:
The aqueous solution (B) of 0.1% phosphoric acid=1:1.The step of described gradient elution includes: according to volume fraction, 0-20min, A are
12%；20-30min, A are risen progressively 25% by 12%；30-50min, A are risen progressively 38% by 25%；50-60min, A are 38%；
60-75min, A are risen progressively 60% by 38%；75-95min, A are risen progressively 90% by 60%；90-100min, A are 90%.This
Bright described detection wavelength is 210nm and 300nm；Preferably, described detection wavelength is: 0-10min is that 300nm, 10-20min are
210nm, 20-100min are 300nm.
The present invention is with scopolin, scopoletin, chlorogenic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, cinnamic aldehyde, new
Hesperidin, naringin are the HPLC finger printing that object of reference peak determines FENGLIAOXING FENGSHI DIEDA YAOJIU.The inventive method is passed through
The relatively HPLC finger printing of 10 batches of need testing solutions, according to the relative retention time of chromatographic peak each in chromatogram, it is determined that 15
Individual total peak is characterized fingerprint peaks；Described 15 the total peaks relative retention time with chlorogenic acid as reference is respectively as follows:
No. 1 peak average relative retention time is 0.265, RSD (relative standard deviation) ＜ 1%, it is preferred that RSD is
0.37%, relative peak area meansigma methods is 277.484；No. 2 peak average relative retention times are 0.494, RSD ＜ 1%, preferably
, RSD is 0.74%, and relative peak area meansigma methods is 203.291；No. 3 peak average relative retention times are 0.525, RSD ＜
1%, it is preferred that RSD is 0.61%, relative peak area meansigma methods is 617.653；No. 4 peak average relative retention times are
0.960, RSD ＜ 1%, it is preferred that RSD is 0.23%, relative peak area meansigma methods is 288.293；No. 5 peak average relatives retain
Time is 1.000, RSD ＜ 1%, it is preferred that RSD is 0.2%, and relative peak area meansigma methods is 797.696；No. 6 average phases in peak
Being 1.078 to retention time, RSD ＜ 1%, it is preferred that RSD is 0.17%, relative peak area meansigma methods is 276.629；No. 7 peaks
Average relative retention time is 1.368, RSD ＜ 1%, it is preferred that RSD is 0.15%, and relative peak area meansigma methods is
307.821；No. 8 peak average relative retention times are 1.621, RSD ＜ 1%, it is preferred that RSD is 0.15%, and relative peak area is put down
Average is 212.11；No. 9 peak average relative retention times are 1.691, RSD ＜ 1%, it is preferred that RSD is 0.16%, relative peak
Area average is 740.134；No. 10 peak average relative retention times are 1.713, RSD ＜ 1%, it is preferred that RSD is
0.17%, relative peak area meansigma methods is 496.374；No. 11 peak average relative retention times are 1.745, RSD ＜ 1%, preferably
, RSD is 0.18%, and relative peak area meansigma methods is 92.442；No. 12 peak average relative retention times are 1.824, RSD ＜
1%, it is preferred that RSD is 0.2%, relative peak area meansigma methods is 370.97；No. 13 peak average relative retention times are 2.000,
RSD ＜ 1%, it is preferred that RSD is 0.28%, relative peak area meansigma methods is 290.672；No. 14 peak average relative retention times
Being 2.047, RSD ＜ 1%, it is preferred that RSD is 0.09%, relative peak area meansigma methods is 1228.019；No. 15 peak average relatives
Retention time is 2.081, RSD ＜ 1%, it is preferred that RSD is 0.14%, and relative peak area meansigma methods is 194.893.
The present invention is compared by retention time, uv-spectrogram and reference substance comparison, identifies 8 chromatographs from total peak
Peak, respectively ephedrine hydrochloride (2, i.e. have peak numbering, lower with), pseudoephedrine hydrochloride (3), scopolin (4), chlorogenic acid
(5), scopoletin (7), naringin (11), neohesperidin (12) and cinnamic aldehyde (14).
In the method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing of the present invention, step is diluted to described in (1): use methanol
FENGLIAOXING FENGSHI DIEDA YAOJIU is diluted 1-3 times, is preferably dilution 1 times.0.45 μm micropore filter it was filtered into described in step (1)
Film.
The present invention condition to the efficient liquid phase chromatographic analysis in FENGLIAOXING FENGSHI DIEDA YAOJIU fingerprint,
It is optimized mutually with flowing including detection wavelength, chromatographic column.The optimum results of detection wavelength shows, with ultraviolet DAD detector
Need testing solution is detected, under the conditions of 300nm, goes out peak the abundantest, hence it is evident that be better than at 246nm, 274nm, 340nm or
Peak situation is gone out under the conditions of 360nm；But the index components ephedrine hydrochloride of the Herba Ephedrae of medicated wine Chinese crude drug large usage quantity and hydrochloric acid puppet fiber crops
Yellow alkali goes out peak at 210nm.Therefore, present invention determine that 210 and 300nm as detection wavelength.
For the selection of chromatographic column, the present invention compares the chromatographic column of 3 kinds of different brands models, from chromatographic peak peak shape, divides
Compare from aspects such as degree, preferably Ecosil C18 post (4.6 × 250mm, 5 μm) or Kromasil C18 post (4.6 × 250mm,
5 μm) for testing chromatographic column.
The optimum results of flowing phase shows, respectively with methanol-0.1% formic acid, methanol-0.2% acetic acid, acetonitrile-0.1% phosphorus
Acid or methanol-0.1% phosphoric acid are as flowing phase, and result show that chromatographic peak is the most undesirable；And select methanol-(0.1% acetic acid:
0.1% phosphoric acid=1:1) as flow phase system time, eluting power is strong, and chromatogram contains much information, and chromatographic peak profile is good, separating effect
Good, baseline is steady；In flowing mutually, phosphoric acid increases the detection of ultraviolet absorption value, beneficially ephedrine；Other peaks are had relatively by acetic acid
Good separating effect.Therefore, the present invention selects methanol-(0.1% acetic acid: 0.1% phosphoric acid=1:1) as flow phase system.
Precision test shows, according to the method for building up of finger printing of the present invention, takes FENGLIAOXING FENGSHI DIEDA YAOJIU parallel
Result importing " similarity evaluation " is also carried out chromatographic peak coupling by sample introduction 6 times, and 15 are total to as a result
There are the equal ＜ of relative retention time RSD 1% at peak, the equal ＜ of relative peak area RSD 5%, show that precision is good.Stability test table
Bright, take FENGLIAOXING FENGSHI DIEDA YAOJIU respectively at 0-24h sample introduction, the equal ＜ of relative retention time RSD at 15 total peaks as a result
1%, the equal ＜ of relative peak area RSD 5%, show that the inventive method has good stability.Replica test shows, the property rheumatism by Feng
Medical liquor for treating injury need testing solution 6 parts sample introduction, the as a result equal ＜ of relative retention time RSD at 15 total peaks 1%, face, relative peak respectively
The long-pending equal ＜ of RSD 5%, shows that the inventive method repeatability is good.
The present invention further discloses the FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing that described method is set up, it is preferred that institute
State FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing as shown in Figure of description Fig. 3.Feng that the present invention is set up property rheumatic bone-setting
Medicated wine finger printing can be applied to quality control or the quality evaluation of FENGLIAOXING FENGSHI DIEDA YAOJIU.The similarity of finger printing
Evaluation result shows, the similarity of 10 batches of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing is substantially more than 0.9, and precision is good.
Technical solution of the present invention compared with prior art, has the advantages that
The present invention uses HPLC-UV atlas analysis to establish the finger printing of FENGLIAOXING FENGSHI DIEDA YAOJIU, the method letter
Easy row, precision is good, reproducible, reliable and stable.The FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing that the present invention is set up
Highly sensitive, good stability, be a kind of effective FENGLIAOXING FENGSHI DIEDA YAOJIU quality evaluating method, property rheumatism will fall for Feng
The production of laxative wine and quality control provide scientific basis.
Accompanying drawing explanation
Fig. 1 is the finger printing of FENGLIAOXING FENGSHI DIEDA YAOJIU and reference substance solution；Wherein, the unit of abscissa is min；
Fig. 2 is the HPLC finger printing 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 printing of FENGLIAOXING FENGSHI DIEDA YAOJIU；Wherein, 1-15 is characterized fingerprint peaks；The unit of abscissa
For min；
What Fig. 4 was 300nm as detection wavelength goes out peak situation；
What Fig. 5 was 246nm as detection wavelength goes out peak situation；
What Fig. 6 was 274nm as detection wavelength goes out peak situation；
What Fig. 7 was 340nm as detection wavelength goes out peak situation；
What Fig. 8 was 360nm as detection wavelength goes out peak situation；
What Fig. 9 was 210nm as detection wavelength goes out peak situation；
Figure 10 be methanol-0.1% formic acid be flowing phase chromatographic peak；
Figure 11 be methanol-0.2% acetic acid be flowing phase chromatographic peak；
Figure 12 be acetonitrile-0.1% phosphoric acid be flowing phase chromatographic peak；
Figure 13 be methanol-0.1% phosphoric acid be flowing phase chromatographic peak；
Figure 14 be methanol-(0.1% acetic acid: 0.1% phosphoric acid=1:1) be flowing phase chromatographic peak.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
1, instrument and reagent
1.1 instrument
Agilent high performance liquid chromatograph (Agilent company of the U.S., model 1260 series), electronic balance (prunus mume (sieb.) sieb.et zucc. Teller-torr
Benefit, XS105DU).
1.2 reagent
Reference substance scopoletin (110768-200504), cinnamic aldehyde (110710-201418), chlorogenic acid (110753-
201314), neohesperidin (11857-201102), ephedrine hydrochloride (171241-201508), pseudoephedrine hydrochloride (171237-
201208), naringin (110722-201312) is provided by Nat'l Pharmaceutical & Biological Products Control Institute；Scopolin (purity >=
98%, lot number Y24S6Y17687) provided by Yuan Ye bio tech ltd, Shanghai.
FENGLIAOXING FENGSHI DIEDA YAOJIU (traditional Chinese medicines group Feng property (Foshan) pharmaceutcal corporation, Ltd produces, 500mL/ bottle).First
Alcohol is chromatographically pure, and water is ultra-pure water, and other reagent are analytical pure.
The foundation of embodiment 1 FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing
1, experimental technique
The foundation of 1.1 FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing
1.1.1 chromatographic condition
Kromasil C18 post (4.6 × 250mm, 5 μm)；Flowing is (meter by volume) methanol (A)-0.1% acetic acid mutually:
The aqueous solution (B) of 0.1% phosphoric acid=1:1；(0～20min, A are 12% to gradient elution (meter by volume)；20～30min, A by
12% rises progressively 25%；30～50min, A are risen progressively 38% by 25%；50～60min, A are 38%；60～75min, A by
38% rises progressively 60%；75～95min, A are risen progressively 90% by 60%；90～100min, A are 90%)；Detection wavelength (0-
10min be 300nm, 10-20min be 210nm, 20-100min be 300nm)；Flow velocity 1ml/min；Column temperature 30 DEG C；Sample size 5 μ
l。
1.1.2 the preparation of need testing solution
Precision measures FENGLIAOXING FENGSHI DIEDA YAOJIU 10ml, puts in 20ml measuring bottle, adds methanol dilution to scale, shakes up, mistake
0.45 μm microporous filter membrane, takes subsequent filtrate, to obtain final product.
1.1.3 the preparation of reference substance solution
Precision weighs scopoletin, cinnamic aldehyde, chlorogenic acid, neohesperidin, ephedrine hydrochloride, pseudoephedrine hydrochloride, Fructus Citri grandis
Skin glycosides, scopolin reference substance are appropriate, add 80% methanol aqueous solution dissolving and make scopoletin (40 μ g/ml), cinnamic aldehyde (10
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), naringin (80 μ g/ml), scopolin (212 μ g/ml) solution, to obtain final product.
1.2 methodological study
1.2.1 precision test
Take FENGLIAOXING FENGSHI DIEDA YAOJIU, prepare need testing solution according to the method for 1.1.2, according to the chromatostrip of 1.1.1
Part, parallel sample introduction 6 times, and result is imported " similarity evaluation " 2004A version, carry out chromatographic peak
Coupling.
1.2.2 stability test
Take FENGLIAOXING FENGSHI DIEDA YAOJIU, prepare need testing solution according to the method for 1.1.2, according to the chromatostrip of 1.1.1
Part, respectively at 0,2,4,8,12,24h sample introductions, investigate the stability of the inventive method.
1.2.3 replica test
Take FENGLIAOXING FENGSHI DIEDA YAOJIU, prepare need testing solution 6 parts, according to the color of 1.1.1 according to 1.1.2 method is parallel
Spectral condition, respectively sample introduction, investigate the repeatability of the inventive method.
2, experimental result
The foundation of 2.1 finger printing and analysis
2.1.1 the foundation of finger printing
The finger printing of FENGLIAOXING FENGSHI DIEDA YAOJIU test sample of the present invention and reference substance solution is shown in Fig. 1.
The present invention is measured by the chromatographic condition of above-mentioned 1.1.1, obtains FENGLIAOXING FENGSHI DIEDA YAOJIU S1-S10 for examination
The HPLC finger printing (Fig. 2) of product, according to the relative retention time of chromatographic peak each in chromatogram, determines total peak, and chooses 15
Individual total peak is characterized fingerprint peaks (Fig. 3).Because collection of illustrative plates Content of Chlorogenic Acid separating degree is good, appearance time is moderate, and peak area is relatively big and steady
Fixed, therefore as chromatograph with reference to peak S.
Described 15 the total peaks relative retention time with chlorogenic acid as reference is shown in Table 1.
The relative retention time at 15 total peaks of table 1 S1-S10 test sample HPLC finger printing and relative peak area
The present invention is compared by retention time, uv-spectrogram and reference substance comparison, identifies 8 chromatographs from total peak
Peak, respectively ephedrine hydrochloride (2, i.e. have peak numbering, lower with), pseudoephedrine hydrochloride (3), scopolin (4), chlorogenic acid
(5), scopoletin (7), naringin (11), neohesperidin (12) and cinnamic aldehyde (14).
2.1.2 the similarity evaluation of finger printing
Use " chromatographic fingerprints of Chinese materia medica similarity evaluation software " 2004A version (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 YAOJIU 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 |
Result shows, the similarity of 10 batches of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing is substantially more than 0.9, and precision is good
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 " chromatographic fingerprints of Chinese materia medica phase
Like degree evaluation system, " 2004A version carries out chromatographic peak coupling, the results are shown in Table 3.
Result shows (table 3, S1-S6 is sample introduction 6 secondary data, for peak area data) 15 total peaks (numbered 1-15)
The equal ＜ of relative retention time RSD 1%, the equal ＜ of relative peak area RSD 5%, show instrument stabilizer, and precision is good.
Table 3 Precision test result
| 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, and 2,4,8,12,24h sample introductions (S1-S6), result (table 4,
S1-S6 is sample introduction 6 secondary data, for peak area data) the equal ＜ of relative retention time RSD at 15 total peaks (numbered 1-15)
1%, the equal ＜ of relative peak area RSD 5%, show that need testing solution is stable in 24h, having good stability of the inventive method.
Table 4 stability test result
| 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 prepares need testing solution 6 parts sample introduction, result (table 5, S1-respectively
S6 is the peak area data of sample introduction 6 times) the equal ＜ of relative retention time RSD 1% at 15 total peaks (numbered 1-15), relative peak
The equal ＜ of area RSD 5%, shows that the inventive method repeatability is good.
Table 5 replica test result
| 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 | 17*** | 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 experimental example 1 FENGLIAOXING FENGSHI DIEDA YAOJIU fingerprint
1, the selection of wavelength is detected
Need testing solution (preparing need testing solution according to 1.1.2 in embodiment 1) is entered by present invention ultraviolet DAD detector
Row detection, it is the abundantest that result (Fig. 4) display goes out peak under the conditions of 300nm, hence it is evident that is better than at 246nm, 274nm, 340nm, 360nm
Under the conditions of go out peak situation (Fig. 5-Fig. 8)；But the index components ephedrine hydrochloride of the Herba Ephedrae of medicated wine Chinese crude drug large usage quantity and hydrochloric acid
Pseudoephedrine to go out peak (Fig. 9) at 210nm.
Therefore, present invention determine that 210 and 300nm as detection wavelength.
2, the selection of chromatographic column
The present invention compares the chromatographic column of 3 different brands models, compares in terms of chromatographic peak peak shape, separating degree etc., excellent
Select Ecosil C18 post (4.6 × 250mm, 5 μm), Kromasil C18 post (4.6 × 250mm, 5 μm) for test chromatographic column.
The selection of the phase that 3, flows
The present invention, under conditions of the preparation of need testing solution and chromatographic condition are with embodiment 1, have selected (volume hundred respectively
Proportion by subtraction) methanol-0.1% formic acid, methanol-0.2% acetic acid, acetonitrile-0.1% phosphoric acid, methanol-0.1% phosphoric acid as flowing enter mutually
Row test, result draws chromatographic peak the most undesirable (Figure 10-Figure 13)；And select methanol-(0.1% acetic acid: 0.1% phosphoric acid=1:
1), time 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 flowing mutually, phosphoric acid increases the detection of ultraviolet absorption value, beneficially ephedrine；Other peaks are had preferably by acetic acid
Separating effect.
Therefore, the present invention selects methanol-(0.1% acetic acid: 0.1% phosphoric acid=1:1) as flow phase system.
Table 6 methanol-(0.1% acetic acid: 0.1% phosphoric acid=1:1) is as the chromatograph testing result of flowing phase
Claims (10)
1. the method for building up of a FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing, it is characterised in that comprise the following steps: (1) will
FENGLIAOXING FENGSHI DIEDA YAOJIU dilutes, and filters, and collects filtrate as need testing solution；(2) need testing solution is carried out high-efficient liquid
Analysis of hplc, obtains HPLC finger printing；(3) determine total according to the relative retention time of chromatographic peak in HPLC finger printing
Peak；(4) from total peak, select characteristic fingerprint peak, obtain FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing.
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:
Chromatographic column is Kromasil C18 post, and specification is 4.6 × 250mm, 5 μm, or, Ecosil C18 post, specification is 4.6 ×
250mm, 5 μm；Flowing is selected from mutually: count by volume, methanol (A)-0.1% acetic acid: the aqueous solution (B) of 0.1% phosphoric acid=1:1,
Methanol (A)-0.1% formic acid (B), methanol (A)-0.2% acetic acid (B), acetonitrile (A)-0.1% phosphoric acid (B) or methanol (A)-0.1%
Any one in phosphoric acid (B)；Gradient elution；Detection wavelength is 210-360nm；Flow velocity 1ml/min；Column temperature 30 DEG C；Sample size 5
μl。
3. according to the method for building up described in claim 2, it is characterised in that described flowing is mutually: count by volume, methanol (A)-
0.1% acetic acid: the aqueous solution (B) of 0.1% phosphoric acid=1:1.
4. according to the method for building up described in claim 2, it is characterised in that the step of described gradient elution includes: according to volume
Mark meter, 0-20min, A are 12%；20-30min, A are risen progressively 25% by 12%；30-50min, A are risen progressively 38% by 25%；
50-60min, A are 38%；60-75min, A are risen progressively 60% by 38%；75-95min, A are risen progressively 90% by 60%；90-
100min, A are 90%.
5. according to the method for building up described in claim 2, it is characterised in that described detection wavelength is 210nm and 300nm；Preferably
, described detection wavelength is: 0-10min be 300nm, 10-20min be 210nm, 20-100min be 300nm.
6. according to the method for building up described in claim 1, it is characterised in that step (4) selects 15 total peaks to be characterized fingerprint
Peak, described 15 the total peaks relative retention time with chlorogenic acid as reference is respectively as follows:
No. 1 peak average relative retention time is 0.265, RSD ＜ 1%, it is preferred that RSD is 0.37%；
No. 2 peak average relative retention times are 0.494, RSD ＜ 1%, it is preferred that RSD is 0.74%；
No. 3 peak average relative retention times are 0.525, RSD ＜ 1%, it is preferred that RSD is 0.61%；
No. 4 peak average relative retention times are 0.960, RSD ＜ 1%, it is preferred that RSD is 0.23%；
No. 5 peak average relative retention times are 1.000, RSD ＜ 1%, it is preferred that RSD is 0.2%；
No. 6 peak average relative retention times are 1.078, RSD ＜ 1%, it is preferred that RSD is 0.17%；
No. 7 peak average relative retention times are 1.368, RSD ＜ 1%, it is preferred that RSD is 0.15%；
No. 8 peak average relative retention times are 1.621, RSD ＜ 1%, it is preferred that RSD is 0.15%；
No. 9 peak average relative retention times are 1.691, RSD ＜ 1%, it is preferred that RSD is 0.16%；
No. 10 peak average relative retention times are 1.713, RSD ＜ 1%, it is preferred that RSD is 0.17%；
No. 11 peak average relative retention times are 1.745, RSD ＜ 1%, it is preferred that RSD is 0.18%；
No. 12 peak average relative retention times are 1.824, RSD ＜ 1%, it is preferred that RSD is 0.2%；
No. 13 peak average relative retention times are 2.000, RSD ＜ 1%, it is preferred that RSD is 0.28%；
No. 14 peak average relative retention times are 2.047, RSD ＜ 1%, it is preferred that RSD is 0.09%；
No. 15 peak average relative retention times are 2.081, RSD ＜ 1%, it is preferred that RSD is 0.14%.
7. according to the method for building up described in claim 1, it is characterised in that step is diluted to described in (1): with methanol by Feng Liaoxing
Rheumatism-traumatic medicinal liquor dilutes 1-3 times, is preferably dilution 1 times；
0.45 μm microporous filter membrane it was filtered into described in step (1).
8. the FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing that method for building up described in claim 1 to 7 any one is set up.
9. according to the FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing described in claim 8, it is characterised in that: described Feng property rheumatism
Medical liquor for treating injury finger printing is as shown in Figure 3.
10. the FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing described in claim 8 or 9 is in the quality of FENGLIAOXING FENGSHI DIEDA YAOJIU
Application in control.
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