CN106324134A - HPLC-DAD fingerprint quality determination method of Periplaneta americana drug material - Google Patents
HPLC-DAD fingerprint quality determination method of Periplaneta americana drug material Download PDFInfo
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- CN106324134A CN106324134A CN201610685828.4A CN201610685828A CN106324134A CN 106324134 A CN106324134 A CN 106324134A CN 201610685828 A CN201610685828 A CN 201610685828A CN 106324134 A CN106324134 A CN 106324134A
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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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- 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 relates to an HPLC-DAD fingerprint quality determination method of a Periplaneta americana drug material. The established chromatographic peak of the HPLC-DAD fingerprint of Periplaneta americana drug material has good separation degree, detection time is reasonable, and repeatability and stability are good. Compared with the HPLC fingerprint technology of Periplaneta americana drug material and kangfuxin liquid formulation in the existing reports, the level of quality control of Periplaneta americana drug material is improved. The used fingerprint technology can more effectively evaluate and control the quality of raw materials of Periplaneta americana drug material. Under the certain condition of preparation technology, it is ensured that the obtained Periplaneta americana extract has stable quality, and the mass of the formulation, which is prepared by the Periplaneta americana extract and contains but not limited to kangfuxin tablet, kangfuxin capsule, kangfuxin liquid, Xinmailong(XML) and Ganlong capsule, can be further effectively controlled.
Description
Technical field
The present invention relates to the measuring method of a kind of Chinese crude drug, particularly to the finger printing quality of periplaneta americana medical material
Assay method.
Background technology
Periplaneta americana medical material records in " Yunnan Province's drug standard " version in 1996.Periplaneta americana medical material mainly contains neuropeptide
The compositions such as class, ucleosides, amino acids, polysaccharide.Though the most existing research report about periplaneta americana medicinal materials fingerprint, but
Sample pre-treatments is the most loaded down with trivial details, will certainly cause the error of sample detection, reduces the degree of accuracy analyzed, and the fingerprint image set up
Spectrum information amount is little, is unfavorable for embodying its " globality " and the distinguishing feature of " ambiguity ", it is difficult to the periplaneta americana to complicated component
Medical material obtains the most comprehensively characterizing.Therefore herein by setting up easy periplaneta americana sample-pretreating method, again can simultaneously
The HPLC finger printing of reflection more information, to the quality offer ginseng for more comprehensively evaluating and control periplaneta americana medical material
Examine.
Summary of the invention
It is an object of the invention to disclose the fingerprint pattern quality determination method of a kind of periplaneta americana medical material, the method has
Good separating effect, highly sensitive, can truly reflect property material contained by periplaneta americana medical material, thus can be as measuring periplaneta americana
The chemical constituent method of alkaloid in medical material, it is possible to as identifying quality control method on Periplaneta americana.
For reaching above-mentioned purpose, technical scheme is as follows:
The fingerprint pattern quality determination method of periplaneta americana of the present invention, the method comprises the steps:
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 1～4 weight portion, puts tool plug conical flask
In, add petroleum ether 50～200 parts by volume, supersound process 1～3h, filter, discard petroleum ether；Medicinal residues add 30%～85% second
Alcohol 25～100 parts by volume, weighed weight, supersound process 1～2h, let cool, more weighed weight；Supply with 30%～85% ethanol and subtract
The weight lost, shakes up, and filters, and precision measures subsequent filtrate 10～50 parts by volume, volatilizes, and residue is dissolved in water, and is transferred to 25～100
In parts by volume measuring bottle, add water constant volume, shakes up, and crosses 0.45 μm microporous filter membrane, takes subsequent filtrate, standby；
(B) preparation of reference substance solution: it is appropriate that precision weighs uracil, hypoxanthine, inosine, adds water and makes every 1mL and contain
Uracil, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3%～4% methanol
Solution (0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
High performance liquid chromatograph condition determination is: filler is octadecylsilane chemically bonded silica；Flowing is 3% methanol mutually
Solution (0.07% acetic acid) (A)-methanol (B)；Employing gradient elution: 0～10min, 100%A；10～20min, 100%A～
80%A；20～35min, 80%A～60%A；35～45min, 60%A～0%A；Detection wavelength is 260nm；Column temperature is 30
℃；Flow velocity is 0.5mL/min；Number of theoretical plate is calculated by hypoxanthine and is not less than 5000.
As in figure 2 it is shown, use the present invention to the finger printing of periplaneta americana medical material be characterised by: periplaneta americana medicine
Material has 10 total fingerprint peakses, wherein 3 identical with corresponding object of reference peak retention time respectively；It is that time Huang is fast with reference to peak S
Purine spectral peak, the relative retention time of 10 total fingerprint peakses be respectively as follows: peak, No. 1 peak 0.386 ± 0.039,2 0.640 ±
0.064, No. 3 peak 0.920 ± 0.092, peak, peak 1.139 ± 0.114,5, S peak 1.000 ± 0.000,4 1.327 ±
Peak, peak 1.775 ± 0.178,9, peak 1.662 ± 0.166,8,0.133, No. 6 peak 1.503 ± 0.150,7 2.123 ±
In 0.212,10 total fingerprint peaks, the ratio of 6 peak areas is respectively: No. 1 peak: No. 2 peaks: No. 3 peaks: S peak: No. 4 peaks: 5
Number peak=(0.253～0.469): (0.342～0.636): (0.931～1.397): 1.000: (0.226～0.420):
(0.368～0.612).
The fingerprint pattern quality determination method of periplaneta americana medical material of the present invention, is preferably as follows detection method:
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 2g, puts in tool plug conical flask, adds stone
Oil ether 100ml, supersound process 2h, filter, discard petroleum ether；Medicinal residues add 85% ethanol 50ml, weighed weight, supersound process
1h, lets cool, more weighed weight；Supplying the weight of less loss with 85% ethanol, shake up, filter, precision measures subsequent filtrate 25ml, waves
Dry, residue is dissolved in water, and is transferred in 50ml measuring bottle, and add water constant volume, shakes up, and crosses 0.45 μm microporous filter membrane, takes subsequent filtrate, standby
With；
(B) preparation of reference substance solution: it is appropriate that precision weighs uracil, hypoxanthine, inosine, adds water and makes every 1mL and contain
Uracil, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3% methanol solution
(0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
High performance liquid chromatograph condition determination is: filler is octadecylsilane chemically bonded silica；Flowing is 3% methanol mutually
Solution (0.07% acetic acid) (A)-methanol (B)；Employing gradient elution: 0～10min, 100%A；10～20min, 100%A～
80%A；20～35min, 80%A～60%A；35～45min, 60%A～0%A；Detection wavelength is 260nm；Column temperature is 30
℃；Flow velocity is 0.5mL/min；Number of theoretical plate is calculated by hypoxanthine and is not less than 5000.
As in figure 2 it is shown, use the present invention to the finger printing of periplaneta americana medical material be characterised by: periplaneta americana medicine
Material has 10 total fingerprint peakses, wherein 3 identical with corresponding object of reference peak retention time respectively；It is that time Huang is fast with reference to peak S
Purine spectral peak, the relative retention time of 10 total fingerprint peakses be respectively as follows: peak, No. 1 peak 0.386 ± 0.039,2 0.640 ±
0.064, No. 3 peak 0.920 ± 0.092, peak, peak 1.139 ± 0.114,5, S peak 1.000 ± 0.000,4 1.327 ±
Peak, peak 1.775 ± 0.178,9, peak 1.662 ± 0.166,8,0.133, No. 6 peak 1.503 ± 0.150,7 2.123 ±
In 0.212,10 total fingerprint peaks, the ratio of 6 peak areas is respectively: No. 1 peak: No. 2 peaks: No. 3 peaks: S peak: No. 4 peaks: 5
Number peak=(0.253～0.469): (0.342～0.636): (0.931～1.397): 1.000: (0.226～0.420):
(0.368～0.612).
Foregoing invention weight portion and the relation that relation is g/ml of parts by volume.
Advantage of the present invention is as follows:
1, periplaneta americana finger printing disclosed by the invention, has carried out Efficient Characterization to its property material, it is provided that a kind of
Method periplaneta americana quality of medicinal material being evaluated by overall finger printing information, it is to avoid only measure wherein one or two kind of change
Study point one-sidedness just periplaneta americana medical material weight judged so that periplaneta americana quality of medicinal material controls more accurately visitor
See, it is thus possible to control further effectively by comprising of making of American-cockroach-extract but be not limited only to rehabilitation new film, rehabilitation virgin rubber
The quality of the preparations such as capsule, Kangfuxin Liquid and the new spray of rehabilitation.
2, the fingerprint pattern quality determination method separating degree that the present invention provides is good, and the detection time is reasonable, repeated and stable
Property is good.
Accompanying drawing explanation
Fig. 1 is the HPLC figure of reference substance uracil, hypoxanthine, creatinine content determination；
Fig. 2 is the feature comparison collection of illustrative plates of periplaneta americana medical material；
In figure: peak 1: characteristic peak 1, peak 2: uracil, peak 3: characteristic peak 3, peak (S): hypoxanthine, peak 4: characteristic peak 4, peak
5: characteristic peak 5, peak 6: flesh former times, peak 7: characteristic peak 7, peak 8: characteristic peak 8, peak 9: characteristic peak 9.
Fig. 3 is the chromatogram under periplaneta americana medical material 230nm wavelength condition；
Fig. 4 is the chromatogram under periplaneta americana medical material 254nm wavelength condition；
Fig. 5 is the chromatogram under periplaneta americana medical material 260nm wavelength condition；
Fig. 6 is the chromatogram under periplaneta americana medical material 300nm wavelength condition；
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
Detailed description of the invention
Experimental example 1 periplaneta americana medicinal materials fingerprint has the determination at peak
1. instrument and reagent
High performance liquid chromatograph: U.S.'s Agilent1200 chromatograph system, is equipped with online degasser, quaternary gradient
Pump, diode array (DAD) detector, automatic sampler, column oven；Chromatographic column: Inertsil ODS-3 (4.6mm ×
250mm, 5 μm).
SB-52000 ultrasonic cleaner (Ningbo Xin Yi ultrasonic device company limited)；The excellent general ultra-pure water of Μ LUP-I-10T
Machine (Chengdu Ultra Pure Science & Technology Co., Ltd)；Sartorius BP121s electronic balance (Beijing limited public affairs of Sai Duolisi scientific instrument
Department).
Methanol is chromatographically pure (Fisher), and water is ultra-pure water, and petroleum ether, ethanol, glacial acetic acid etc. are analytical pure (Chengdu
Ke Long chemical reagent factory).
Uracil (lot number: 1000469-201302), hypoxanthine (lot number: 140661-200903), inosine (lot number:
140669-201104) it is purchased from National Institute for Food and Drugs Control.Periplaneta americana medical material is climbed Western medicine industry by the good doctor in Sichuan to be had
Limit responsible company provides.
2, chromatographic condition test
With octadecylsilane chemically bonded silica as filler, with 3% methanol solution (0.07% acetic acid) as mobile phase A, first
Alcohol is Mobile phase B, carries out gradient elution by table 1, and flow velocity is 0.5ml/min, and column temperature is 30 DEG C, and detection wavelength is 260nm.Theoretical
Plate number is calculated by hypoxanthine and is not less than 5000.
Table 1 periplaneta americana test sample analyzes system binary gradient program
| Sequence number | Time (min) | A (%) | B (%) |
| 1 | 0 | 100 | 0 |
| 2 | 10 | 100 | 0 |
| 3 | 20 | 80 | 20 |
| 4 | 35 | 60 | 40 |
| 5 | 45 | 0 | 100 |
3, detection wavelength
Taking periplaneta americana medical material need testing solution of the present invention is to measure object, under above-mentioned chromatographic condition, exists respectively
HPLC mensuration is carried out under 230nm, 254nm, 260nm, 300nm wavelength.Result is shown in Fig. 3-6 in accompanying drawing explanation.Result shows,
There are 7 chromatographic peaks under 230nm wavelength, but seriously drift about at the baseline of 20-30min section；Major part chromatographic peak at 300 nm wavelength
All lack, only at 18min, one bigger chromatographic peak occurs；Under 254nm, 260nm wavelength, the separating degree of each chromatographic peak is good
Good, baseline is steady, and the composition of detection is most, can reflection sample chemical composition information as much as possible.Finally, according to chromatograph
The peak type at peak, symmetry and the difference in height of each chromatographic peak, select 260nm as the detection wavelength of sample HPLC finger printing.
4, finger printing
According to the relevant parameter given by 20 batches of periplaneta americana medical material HPLC collection of illustrative plates, aforementioned preparation side pressed by periplaneta americana medical material
Method is measured the main chromatographic peak of gained to be occurred in 43 minutes；The need testing solution chromatogram of 1 hour show 43 minutes with
After chromatographic peak does not occurs.Relatively the chromatogram of each batch sample be found to have 10 peaks be each batch total, thus Criterion fingerprint
Collection of illustrative plates.
5, the demarcation of total fingerprint peaks
According to the result of calculation of 20 batches of periplaneta americana medical material test sample finger printing related datas, total peak average relative is protected
The time (peak number) is stayed to be followed successively by peak 0.920, peak 0.640,3, No. 1 peak 0.386,2, peak, S peak 1.000,4 1.139,5
Peak 1.327, peak 2.123, peak 1.775,9, peak 1.662,8, No. 6 peaks 1.503,7, demarcate in this, as total fingerprint peaks
Foundation, it is allowed to relative deviation is ± 10%.
6, total fingerprint peaks relative retention time
In 20 batches of periplaneta americana medical material test sample finger printing, the relative retention time of total fingerprint peaks is shown in Table 2.Table 2 is beautiful
Continent big Lian medical material has fingerprint peaks relative retention time (n=20)
The RSD of the relative retention time of 20 batches of test samples is respectively less than 2.0%, shows that the concordance of each batch sample is preferable.
7, total fingerprint peaks relative peak area
In 20 batches of periplaneta americana medical material test sample finger printing, the relative peak area of total fingerprint peaks is shown in Table 3.Table 3 America
Big Lian medical material has fingerprint peaks relative peak area (n=20)
With reference to the related request of " technology of Chinese medicine finger printing research requires (provisional) ", each total fingerprint peaks
Area ratio must be relatively fixed.Ratio and each total peak area in finger printing to total peak area each in test sample collection of illustrative plates
Odds ratio relatively, retention time was less than or equal to the total peak of 30 minutes: unimodal area accounts for total peak area more than or equal to 20%
Total peak, its difference cannot be greater than ± 20%；Unimodal area accounts for total peak area more than or equal to 10%, and being total to less than 20%
Having peak, its difference cannot be greater than ± 25%；Unimodal area accounts for total peak area and is more than or equal to 5%, and is less than the total peak of 10%,
Its difference cannot be greater than ± 30%；Unimodal area accounts for the total peak area total peak less than 5%, the rule that peak area ratio is not required
Fixed.The retention time total peak more than 30 minutes: unimodal area accounts for the total peak area total peak less than 10%, and peak area ratio is not
It is required, but relative retention time must be demarcated.Total peak 1 in peak, total peak 2, total peak 4 etc. are had at periplaneta americana medical material
Three total peak-to-peak areas account for the 5%～10% of total peak area, and average relative peak area is respectively 0.361,0.489 and 0.323,
RSD is 1.21%, 1.71% and 0.79%；The peak area at total peak 3 accounts for total peak area and is more than 20%, and average relative peak area divides
Not being 1.164, RSD is 1.11%；Total peak 5 peak area accounts for the 10%～20% of total peak area, and average relative peak area is
0.490, RSD is 2.07%.So that it is determined that the relative peak area at six total peaks more than: No. 1 peak: No. 2 peaks: No. 3 peaks: No. S
Peak: No. 4 peaks: No. 5 peak=(0.253～0.469): (0.342～0.636): (0.931～1.397): 1.000: (0.226～
0.420): (0.368～0.612).
8, non-shared peak area
Removing the peak retained without post, the non-shared peak gross area calculating 20 batches of test samples accounts for the ratio of total peak area,
The results are shown in Table 4.
The non-shared peak gross area of table 4 periplaneta americana medical material accounts for the percentage ratio (n=20) of total peak area
The ratio that each batch of periplaneta americana medical material non-shared peak gross area accounts for the gross area is respectively less than 10%, meets " Chinese medicine injection
Agent finger printing research technology require (provisional) " regulation.
9, periplaneta americana medicinal materials fingerprint similarity evaluation
By 20 batches of periplaneta americana medical materials, detect as stated above, obtain the finger printing of 20 batches of medical materials, use China
The area of computer aided similarity evaluation software (establishment of Central South University's modernization of cmm center) that committee of pharmacopeia is recommended calculates,
Similarity is shown in Table 5.
Table 5 periplaneta americana medicinal materials fingerprint similarity
| Lot number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Similarity | 0.991 | 0.998 | 0.977 | 0.981 | 0.999 | 0.994 | 0.979 | 0.992 | 0.996 | 0.998 |
| Lot number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Similarity | 0.994 | 0.998 | 0.993 | 0.997 | 0.993 | 0.986 | 0.962 | 0.992 | 0.979 | 0.991 |
Experimental example 2 periplaneta americana medical material need testing solution precision test
Take periplaneta americana medical material need testing solution of the present invention for measure as, under chromatographic condition of the present invention, even
Continuous mensuration 6 times, records relative retention time and the relative peak area of each total chromatographic peak, the results are shown in Table 6～7.Result shows, respectively
The relative retention time of chromatographic peak and the RSD of peak area are respectively less than 3%.Prove that the precision of instrument is good, meet HPLC fingerprint
Graphical spectrum technology requirement.
Table 6 periplaneta americana precision test relative retention time
Table 7 periplaneta americana precision test relative peak area
Experimental example 3 periplaneta americana medical material need testing solution stability test
Taking periplaneta americana medical material need testing solution of the present invention is to measure object, under chromatographic condition of the present invention, point
Not 0,2,4,6,8,12h detect, record related data.With the hypoxanthic retention time of object of reference as reference, conversion
Go out relative retention time and the ratio of peak area at each total peak, the results are shown in Table 8～9.Result shows, the relative guarantor of each chromatographic peak
The RSD staying time and relative peak area is respectively less than 3%, it was demonstrated that sample is stable in 12h, meets HPLC fingerprint pattern technology and wants
Ask.
Table 8 periplaneta americana stability test relative retention time
Table 9 periplaneta americana stability test relative peak area
Experimental example 4 periplaneta americana medical material need testing solution repeatability is tested
Taking periplaneta americana medical material test sample of the present invention 6 parts, be analyzed under chromatographic condition of the present invention, record is relevant
Data.With the hypoxanthic retention time of object of reference as reference, converse the relative retention time at each total peak and peak area
Ratio, the results are shown in Table 10～11.Result shows, the relative retention time of each chromatographic peak and the RSD of relative peak area are respectively less than
3%, show that the repeatability of the method is good, meet fingerprint pattern technology requirement.
Table 10 periplaneta americana stability test relative retention time
Table 11 periplaneta americana stability test relative peak area
Experimental example 5 periplaneta americana medical material need testing solution precision test
Take periplaneta americana medical material need testing solution of the present invention for measure as, under chromatographic condition of the present invention, even
Continuous mensuration 6 times, records relative retention time and the relative peak area of each total chromatographic peak, the results are shown in Table 12～13.Result shows,
The relative retention time of each chromatographic peak and the RSD of peak area are respectively less than 3%.Prove that the precision of instrument is good, meet HPLC and refer to
Stricture of vagina graphical spectrum technology requirement.
Table 12 periplaneta americana precision test relative retention time
Table 13 periplaneta americana precision test relative peak area
Experimental example 6 periplaneta americana medical material need testing solution stability test
Taking periplaneta americana medical material need testing solution of the present invention is to measure object, under chromatographic condition of the present invention, point
Not 0,2,4,6,8,12h detect, record related data.With the hypoxanthic retention time of object of reference as reference, conversion
Go out relative retention time and the ratio of peak area at each total peak, the results are shown in Table 14～15.Result shows, each chromatographic peak relative
The RSD of retention time and relative peak area is respectively less than 3%, it was demonstrated that sample is stable in 12h, meets HPLC fingerprint pattern technology and wants
Ask.
Table 14 periplaneta americana stability test relative retention time
Table 15 periplaneta americana stability test relative peak area
Experimental example 7 periplaneta americana medical material need testing solution repeatability is tested
Taking periplaneta americana medical material test sample of the present invention 6 parts, be analyzed under chromatographic condition of the present invention, record is relevant
Data.With the hypoxanthic retention time of object of reference as reference, converse the relative retention time at each total peak and peak area
Ratio, the results are shown in Table 16～17.Result shows, the relative retention time of each chromatographic peak and the RSD of relative peak area are respectively less than
3%, show that the repeatability of the method is good, meet fingerprint pattern technology requirement.
Table 16 periplaneta americana stability test relative retention time
Table 17 periplaneta americana stability test relative peak area
Embodiment 1: the fingerprint pattern quality determination method of periplaneta americana medical material
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 1g, puts in tool plug conical flask, adds stone
Oil ether 50ml, supersound process 1h, filter, discard petroleum ether；Medicinal residues add 65% ethanol 25ml, weighed weight, supersound process (merit
Rate 160W, frequency 45kHz) 1h, let cool, more weighed weight；Supply the weight of less loss with 65% ethanol, shake up, filter, accurate amount
Taking subsequent filtrate 10ml, volatilize, residue is dissolved in water, and is transferred in 50ml measuring bottle, and add water constant volume, shakes up, and crosses 0.45 μm micropore filter
Film, takes subsequent filtrate, standby；
(B) preparation of reference substance solution: it is appropriate that precision weighs uracil, hypoxanthine, inosine, adds water and makes every 1mL and contain
Uracil, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3% methanol solution
(0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
High performance liquid chromatograph condition determination is: filler is octadecylsilane chemically bonded silica；Flowing is 3% methanol mutually
Solution (0.07% acetic acid) (A)-methanol (B)；Employing gradient elution: 0～10min, 100%A；10～20min, 100%A～
80%A；20～35min, 80%A～60%A；35～45min, 60%A～0%A；Detection wavelength is 260nm；Column temperature is 30
℃；Flow velocity is 0.5mL/min；Number of theoretical plate is calculated by hypoxanthine and is not less than 5000.
Use the present invention to periplaneta americana medical material have 10 total fingerprint peakses, wherein join with corresponding respectively for 3
Identical according to thing peak retention time；Being hypoxanthine spectral peak with reference to peak S, the relative retention time of 10 total fingerprint peakses is respectively as follows:
Peak 0.925, peak 0.647,3, No. 1 peak 0.382,2, peak 1.510, peak 1.335,6, peak 1.143,5, S peak 1.000,4,
In the total fingerprint peaks in 2.121,10, peak, peak 1.772,9, No. 7 peaks 1.668,8, the ratio of 6 peak areas is respectively: No. 1 peak
: No. 2 peaks: No. 3 peaks: S peak: No. 4 peaks: No. 5 peak=0.358: 0.477: 1.170: 1.000: 0.330: 0.292.
Embodiment 2: the fingerprint pattern quality determination method of periplaneta americana medical material
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 2g, puts in tool plug conical flask, adds stone
Oil ether 100ml, supersound process 2h, filter, discard petroleum ether；Medicinal residues add 85% ethanol 50ml, weighed weight, supersound process
(power 160W, frequency 45kHz) 1h, lets cool, more weighed weight；Supply the weight of less loss with 85% ethanol, shake up, filter, essence
Close measuring subsequent filtrate 25ml, volatilize, residue is dissolved in water, and is transferred in 50ml measuring bottle, and add water constant volume, shakes up, and crosses 0.45 μm micro-
Hole filter membrane, takes subsequent filtrate, standby；
(B) preparation of reference substance solution: it is appropriate that precision weighs uracil, hypoxanthine, inosine, adds water and makes every 1mL and contain
Uracil, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3% methanol solution
(0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
High performance liquid chromatograph condition determination is: filler is octadecylsilane chemically bonded silica；Flowing is 3% methanol mutually
Solution (0.07% acetic acid) (A)-methanol (B)；Employing gradient elution: 0～10min, 100%A；10～20min, 100%A～
80%A；20～35min, 80%A～60%A；35～45min, 60%A～0%A；Detection wavelength is 260nm；Column temperature is 30
℃；Flow velocity is 0.5mL/min；Number of theoretical plate is calculated by hypoxanthine and is not less than 5000.
Use the present invention to periplaneta americana medical material have 10 total fingerprint peakses, wherein join with corresponding respectively for 3
Identical according to thing peak retention time；Being hypoxanthine spectral peak with reference to peak S, the relative retention time of 10 total fingerprint peakses is respectively as follows: 1
Number peak 0.928, peak 0.651,3, peak 0.389,2, peak 1.504,7, peak 1.333,6, peak 1.139,5, S peak 1.000,4
In number total fingerprint peaks in 2.125,10, peak, peak 1.781,9, peak 1.661,8, the ratio of 6 peak areas is respectively: No. 1 peak:
No. 2 peaks: No. 3 peaks: S peak: No. 4 peaks: No. 5 peak=0.358: 0.487: 1.170: 1.000: 0.328: 0.286.
The fingerprint pattern quality determination method of embodiment 3 periplaneta americana medical material
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 4g, puts in tool plug conical flask, adds stone
Oil ether 200ml, supersound process 3h, filter, discard petroleum ether；Medicinal residues add 65% ethanol 100ml, weighed weight, supersound process
(power 160W, frequency 45kHz) 2h, lets cool, more weighed weight；Supply the weight of less loss with 85% ethanol, shake up, filter, essence
Close measuring subsequent filtrate 50ml, volatilize, residue is dissolved in water, and is transferred in 100ml measuring bottle, and add water constant volume, shakes up, and crosses 0.45 μm micro-
Hole filter membrane, takes subsequent filtrate, standby；
(B) preparation of reference substance solution: it is appropriate that precision weighs uracil, hypoxanthine, inosine, adds water and makes every 1mL and contain
Uracil, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3% methanol solution
(0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
High performance liquid chromatograph condition determination is: filler is octadecylsilane chemically bonded silica；Flowing is 3% methanol mutually
Solution (0.07% acetic acid) (A)-methanol (B)；Employing gradient elution: 0～10min, 100%A；10～20min, 100%A～
80%A；20～35min, 80%A～60%A；35～45min, 60%A～0%A；Detection wavelength is 260nm；Column temperature is 30
℃；Flow velocity is 0.5mL/min；Number of theoretical plate is calculated by hypoxanthine and is not less than 5000.
Use the present invention to periplaneta americana medical material have 10 total fingerprint peakses, wherein join with corresponding respectively for 3
Identical according to thing peak retention time；Being hypoxanthine spectral peak with reference to peak S, the relative retention time of 10 total fingerprint peakses is respectively as follows: 1
Number peak 0.925, peak 0.647,3, peak 0.389,2, peak 1.506,7, peak 1.331,6, peak 1.144,5, S peak 1.000,4
In number total fingerprint peaks in 2.125,10, peak, peak 1.772,9, peak 1.661,8, the ratio of 6 peak areas is respectively: No. 1 peak:
No. 2 peaks: No. 3 peaks: S peak: No. 4 peaks: No. 5 peak=0.363: 0.487: 1.167: 1.000: 0.328: 0.287.
Claims (4)
1. the HPLC-DAD fingerprint pattern quality determination method of a periplaneta americana medical material, it is characterised in that the method includes as follows
Step:
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 1～4 weight portion, puts in tool plug conical flask, adds
Enter petroleum ether 50～200 parts by volume, supersound process 1～3h, filter, discard petroleum ether；Medicinal residues add 30%～85% ethanol 25
～100 parts by volume, weighed weight, supersound process (power 160W, frequency 45kHz) 1～2h, let cool, more weighed weight；With 30%
～85% ethanol supply the weight of less loss, shake up, filter, precision measures subsequent filtrate 10～50 parts by volume, volatilizes, and residue adds water-soluble
Solving, be transferred in 25～100 parts by volume measuring bottles, add water constant volume, shakes up, and crosses 0.45 μm microporous filter membrane, takes subsequent filtrate, standby；
(B) preparation of reference substance solution: precision weighs uracil, hypoxanthine, inosine in right amount, adding water, it is phonetic containing urine to make every 1mL
Pyridine, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3%～4% methanol solution
(0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
The HPLC-DAD fingerprint pattern quality determination method of periplaneta americana medical material the most according to claim 1, its feature exists
In high performance liquid chromatograph condition determination it is: filler is octadecylsilane chemically bonded silica；Flowing is 3% methanol solution mutually
(0.07% acetic acid) (A)-methanol (B)；Employing gradient elution: 0～10min, 100%A；10～20min, 100%A～80%A；
20～35min, 80%A～60%A；35～45min, 60%A～0%A；Detection wavelength is 260nm；Column temperature is 30 DEG C；Flow velocity
For 0.5mL/min；Number of theoretical plate is calculated by hypoxanthine and is not less than 5000.
3. the finger obtained according to the HPLC-DAD fingerprint pattern quality determination method of the periplaneta americana medical material described in claim 1,2
Stricture of vagina collection of illustrative plates, it is characterised in that: periplaneta americana medical material has 10 total fingerprint peakses, wherein 3 respectively with corresponding object of reference peak
Retention time is identical；Being hypoxanthine spectral peak with reference to peak S, the relative retention time of 10 total fingerprint peakses is respectively as follows: No. 1 peak
Peak 0.920 ± 0.092,0.386 ± 0.039, No. 2 peaks 0.640 ± 0.064,3, peak 1.139, S peak 1.000 ± 0.000,4
Peak, peak 1.662 ± 0.166,8, peak 1.503 ± 0.150,7, ± 0.114, No. 5 peaks 1.327 ± 0.133,6 1.775 ±
In 0.178, No. 9 total fingerprint peaks in 2.123 ± 0.212,10, peak, the ratio of 6 peak areas is respectively: No. 1 peak: No. 2 peaks: 3
Number peak: S peak: No. 4 peaks: No. 5 peak=(0.253～0.469): (0.342～0.636): (0.931～1.397): 1.000:
(0.226～0.420): (0.368～0.612).
4. the HPLC-DAD fingerprint pattern quality determination method of periplaneta americana medical material described in 1 is required according to profit, it is characterised in that
The method comprises the steps:
(A) preparation of need testing solution: accurately weighed periplaneta americana medicinal powder 2g, puts in tool plug conical flask, adds petroleum ether
100ml, supersound process 2h, filter, discard petroleum ether；Medicinal residues add 85% ethanol 50ml, weighed weight, supersound process (power
160W, frequency 45kHz) 1h, let cool, more weighed weight；Supplying the weight of less loss with 85% ethanol, shake up, filter, precision measures
Subsequent filtrate 25ml, volatilizes, and residue is dissolved in water, and is transferred in 50ml measuring bottle, and add water constant volume, shakes up, and crosses 0.45 μm microporous filter membrane,
Take subsequent filtrate, standby；
(B) preparation of reference substance solution: precision weighs uracil, hypoxanthine, inosine in right amount, adding water, it is phonetic containing urine to make every 1mL
Pyridine, hypoxanthine and inosine are respectively 15 μ g, 30 μ g and the mixed solution of 45 μ g, standby；
(C) measure: draw test sample 10 μ ls each with reference substance solution and inject high performance liquid chromatograph, with 3% methanol solution
(0.07% acetic acid) (A)-methanol (B) is flowing phase, uses gradient elution, obtains the finger printing of periplaneta americana medical material.
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