CN101721475B - Detection method for Yaotongning capsules - Google Patents

Detection method for Yaotongning capsules Download PDF

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CN101721475B
CN101721475B CN2009102506549A CN200910250654A CN101721475B CN 101721475 B CN101721475 B CN 101721475B CN 2009102506549 A CN2009102506549 A CN 2009102506549A CN 200910250654 A CN200910250654 A CN 200910250654A CN 101721475 B CN101721475 B CN 101721475B
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peaks
volume
mobile phase
parts
strychnine
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CN101721475A (en
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李云霞
鲍建才
李沈明
郭艳玲
刘玺
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Jingfukang Pharmaceutical Group Co., Ltd.
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JINGFUKANG PHARMACEUTICAL GROUP CO Ltd CHENGDE
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Abstract

The invention discloses a quality control method for Yaotongning capsules. The quality control method for the Yaotongning capsules adopts a high performance liquid chromatography to establish a finger print of the Yaotongning capsules, which is applied to the overall process of the Yaotongning capsule production to perform the quality control; and similarity software issued by the pharmacopoeia commission is adopted to set a similarity threshold of the finger print of the Yaotongning capsules to be not lower than 0.87. Experiments prove that the quality control method for the Yaotongning capsules has the advantages of accuracy and reliability, and can embody the characteristic peak of each medicinal material in a formula maximally.

Description

A kind of detection method of Yaotongning capsules
Technical field
The present invention relates to a kind of method of quality control of Chinese medicine, particularly a kind of method of quality control of Yaotongning capsules.
Background technology
Yaotongning capsules records in " the Yaotongning capsules national drug standards (revision) promulgation part " (standard No. WS3-B-3515-2006); On the basis of " the 18 366 pages in the Sanitation Ministry medicine standard Chinese traditional patent formulation preparation " standard, revise quality standard in March, 2006 through the approval of national Bureau of Drugs Supervision; Existing method of quality control has comprised that 5 discriminatings (comprise microscopical identification; Vomiting nut, the discriminating of rhizoma atractylodis, radix cyathulae, Radix Glycyrrhizae); The assay of strychnine and strychnia; Determination of Ephedrine Hydrochloride is measured.
Summary of the invention
The object of the invention is a kind of fingerprint pattern quality control method of Yaotongning capsules.
The present invention seeks to realize through following technical scheme:
The finger-print of Yaotongning capsules is (like Fig. 8):
The finger-print of Yaotongning capsules is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is: with No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138
The finger print measuring method of Yaotongning capsules is a kind of in the following method:
The A method:
These article of getting inclusions, mixing is got 2.0g, and accurate the title, decide; Put in the 50mL tool plug triangular flask, the accurate methyl alcohol 25mL that adds adds concentrated hydrochloric acid 0.63mL, close plug again; Shake up, claim to decide weight, with power 450W, frequency 40kHz carries out sonicated and took out in 45 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 10 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg, and precision is measured above-mentioned reference substance solution 2ml; Put in the 10ml volumetric flask, be diluted to scale, shake up with methyl alcohol; Filter; Get subsequent filtrate, promptly get, contain strychnine 0.1mg among every 1ml;
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
The B method:
These article of getting inclusions, mixing is got 2.0g, and accurate the title, decide, and puts in the 50mL tool plug triangular flask, and the accurate methyl alcohol 25mL that adds adds the 0.63mL concentrated hydrochloric acid again, weighs, and shakes up, and ultrasonic Extraction is taken out, and puts and is chilled to room temperature, weighs, and adds methyl alcohol and supplies weightlessness; Get subsequent filtrate 10ul and inject liquid chromatograph, analyze, chromatographic condition is: according to high effective liquid chromatography for measuring, and HC-C 18Chromatographic column (250mm * 4.6mm, 5 μ m) is a mobile phase A with the acetonitrile; With 0.2% formic acid and 0.2% triethylamine mixed solution is Mobile phase B; Carry out gradient elution, gradient condition is the time: 0～20～50～60 minutes, mobile phase A is: acetonitrile was by 8%～18%～98%～98%; Mobile phase B is: 0.2% formic acid and 0.2% triethylamine mixed solution, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The preparation precision of reference substance solution takes by weighing strychnine reference substance 5mg, puts in the 50ml measuring bottle, adds methenyl choloride 10ml and makes dissolving, adds methyl alcohol to scale, shakes up; Filter with miillpore filter (0.45 μ m), get subsequent filtrate, promptly get (containing strychnine 0.1mg among every 1ml);
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
The C method:
These article of getting inclusions, mixing is got 2.0g, and accurate the title, decide, and puts in the 50mL tool plug triangular flask, and the accurate methyl alcohol 25mL that adds adds the 0.63mL concentrated hydrochloric acid again, weighs, shake up, 450W, 40KH ultrasonic Extraction 45min takes out, and puts and is chilled to room temperature, weighs, and adds methyl alcohol and supplies weightlessness; Get subsequent filtrate 10ul and inject liquid chromatograph, analyze, chromatographic condition is: according to high effective liquid chromatography for measuring, and 4.6 * 250mm, the HC-C of 5 μ 18Chromatographic column is a mobile phase A with the acetonitrile, is Mobile phase B with 0.2% triethylamine mixed solution of 0.2% formic acid; Carry out gradient elution; Gradient condition is the time: 0～20～50～60 minutes, mobile phase A was an acetonitrile, by 8%～18%～98%～98%; Mobile phase B is the mixed solution of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The preparation precision of reference substance solution takes by weighing strychnine reference substance 5mg, puts in the 50ml measuring bottle, adds methenyl choloride 10ml and makes dissolving, adds methyl alcohol to scale, shakes up; Miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, promptly gets the reference substance solution that contains strychnine 0.1mg among every 1ml;
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Yaotongning capsules formula material medicine of the present invention adds conventional auxiliary material, according to common process, can process various preparations such as pharmaceutically acceptable capsule, tablet, pill, granule, oral liquid, powder.
The finger-print of drug combination preparation of the present invention is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is: with No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138
The finger print measuring method of drug combination preparation of the present invention is a kind of in the following method:
The A method:
Get drug combination preparation 1-4 weight portion of the present invention, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 15-35 parts by volume that adds; Add concentrated hydrochloric acid 0.5-0.7 parts by volume again, close plug shakes up, and claims to decide weight; With power 250-550W, frequency 30-50kHz carried out sonicated 30-80 minute, took out, and put and was chilled to room temperature; Claim again to decide weight, supply the weight that subtracts mistake, shake up, filter with methyl alcohol; Get subsequent filtrate 0.005-0.020 parts by volume and inject liquid chromatograph, analyze, chromatographic condition is: measure according to high performance liquid chromatography (an appendix VI of pharmacopeia D); With the octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the acetonitrile is mobile phase A, is Mobile phase B with the WS of 0.2% formic acid and 0.2% triethylamine, carries out gradient elution; Gradient condition is the time: 0～20～50～60 minutes; Mobile phase A is: acetonitrile is by 8%～18%～98%～98%, and Mobile phase B is: the WS of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000; It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg; Precision is measured above-mentioned reference substance solution 1-4 parts by volume, puts in the volumetric flask of 5-20ml, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, contains strychnine 0.1mg among every 1ml;
Accurate respectively reference substance solution and each the 0.005-0.020 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets;
The B method:
Get drug combination preparation 1-4 weight portion of the present invention, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 15-35 parts by volume that adds; Add concentrated hydrochloric acid 0.5-0.7 parts by volume again, weigh, shake up ultrasonic Extraction; Take out, put and be chilled to room temperature, weigh, supply weightlessness with methyl alcohol; Get subsequent filtrate 0.005-0.020 parts by volume and inject liquid chromatograph, analyze, chromatographic condition is: according to high effective liquid chromatography for measuring, and HC-C 18Chromatographic column (250mm * 4.6mm, 5 μ m) is a mobile phase A with the acetonitrile; With 0.2% formic acid and 0.2% triethylamine mixed solution is Mobile phase B; Carry out gradient elution, gradient condition is the time: 0～20～50～60 minutes, mobile phase A is: acetonitrile was by 8%～18%～98%～98%; Mobile phase B is: the WS of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The preparation precision of reference substance solution takes by weighing strychnine reference substance 5mg, puts in the 50ml measuring bottle, adds methenyl choloride 10ml and makes dissolving, adds methyl alcohol to scale, shakes up; Filter with miillpore filter (0.45 μ m), get subsequent filtrate, promptly get (containing strychnine 0.1mg among every 1ml);
Accurate respectively reference substance solution and each the 0.005-0.020 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets.
The finger print measuring method of drug combination preparation of the present invention is preferably a kind of in the following method:
The A method:
Get drug combination preparation 2 weight portions of the present invention, the accurate title, decide, and puts in the 50mL tool plug triangular flask, accurate methyl alcohol 25 parts by volume that add; Add concentrated hydrochloric acid 0.63 parts by volume again, close plug shakes up, and claims to decide weight; With power 450W, frequency 40kHz carried out sonicated 45 minutes, took out, and put and was chilled to room temperature; Claim again to decide weight, supply the weight that subtracts mistake, shake up, filter with methyl alcohol; Get subsequent filtrate 0.010 parts by volume and inject liquid chromatograph, analyze, chromatographic condition is: measure according to high performance liquid chromatography (appendix VID of pharmacopeia); With the octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the acetonitrile is mobile phase A, is Mobile phase B with the WS of 0.2% formic acid and 0.2% triethylamine, carries out gradient elution; Gradient condition is the time: 0～20～50～60 minutes; Mobile phase A is: acetonitrile is by 8%～18%～98%～98%, and Mobile phase B is: the WS of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 2 parts by volume, puts in the volumetric flask of 10 parts by volume, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each 0.010 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets;
The B method:
Get drug combination preparation 2.0 weight portions of the present invention, the accurate title, decide, and puts in the 50 parts by volume tool plug triangular flasks, accurate methyl alcohol 25 parts by volume that add; Add 0.63 parts by volume concentrated hydrochloric acid again, weigh, shake up ultrasonic Extraction; Take out, put and be chilled to room temperature, weigh, add methyl alcohol and supply weightlessness; Get subsequent filtrate 0.01 parts by volume and inject liquid chromatograph, analyze, chromatographic condition is: according to high effective liquid chromatography for measuring, and HC-C 18Chromatographic column (250mm * 4.6mm, 5 μ m) is a mobile phase A with the acetonitrile; With 0.2% formic acid and 0.2% triethylamine mixed solution is Mobile phase B; Carry out gradient elution, gradient condition is the time: 0～20～50～60 minutes, mobile phase A is: acetonitrile was by 8%～18%～98%～98%; Mobile phase B is: 0.2% formic acid and 0.2% triethylamine mixed solution, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The preparation precision of reference substance solution takes by weighing strychnine reference substance 0.005 weight portion, puts in the 50 parts by volume measuring bottles, adds methenyl choloride 10 parts by volume and makes dissolving, adds methyl alcohol to scale, shakes up; Filter with miillpore filter (0.45 μ m), get subsequent filtrate, promptly get (containing strychnine 0.1mg among every 1ml).
Accurate respectively reference substance solution and each 0.01 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets.
Yaotongning capsules formula material medicine of the present invention consists of:
Modulation prepared nux vomica 80-160 weight portion ground bettle 15-32 weight portion
Radix cyathulae 15-32 weight portion Chinese ephedra 15-32 weight portion
Frankincense 15-32 weight portion myrrh 15-32 weight portion
Stiff silkworm 15-32 weight portion rhizoma atractylodis 15-32 weight portion
Scorpio 15-32 weight portion Radix Glycyrrhizae 15-32 weight portion.
Yaotongning capsules formula material medicine composition of the present invention is preferably:
Modulation prepared nux vomica 160 weight portion ground bettles 17.5 weight portions
Radix cyathulae 19 weight portion Chinese ephedras 25 weight portions
Frankincense 26 weight portion myrrhs 18 weight portions
Stiff silkworm 20 weight portion rhizoma atractylodis 27 weight portions
Scorpio 28 weight portion Radix Glycyrrhizaes 17 weight portions.
Yaotongning capsules formula material medicine composition of the present invention is preferably:
Modulation prepared nux vomica 80 weight portion ground bettles 23 weight portions
Radix cyathulae 24.5 weight portion Chinese ephedras 17.5 weight portions
Frankincense 15 weight portion myrrhs 31 weight portions
Stiff silkworm 29 weight portion rhizoma atractylodis 19 weight portions
Scorpio 18 weight portion Radix Glycyrrhizaes 26 weight portions.
Yaotongning capsules formula material medicine composition of the present invention is preferably:
Modulation prepared nux vomica 120 weight portion ground bettles 21 weight portions
Radix cyathulae 21 weight portion Chinese ephedras 21 weight portions
Frankincense 21 weight portion myrrhs 21 weight portions
Stiff silkworm 21 weight portion rhizoma atractylodis 21 weight portions
Scorpio 21 weight portion Radix Glycyrrhizaes 21 weight portions.
Press practice of pharmacy; Can the above-mentioned raw materials medicine be prepared into various clinical or pharmaceutically acceptable formulations, include but not limited to a kind of in the middle of the following formulation as: capsule, tablet, sustained release tablets, controlled release tablet, oral solution, oral suspensions, Orally taken emulsion, mucilage, oral liquid, emulsion, colloidal solution, mixture, tincture, drops, suspendible drops, pill, dripping pill, granule, enteric coated granule, powder, Babu plaster or rubber ointment etc.
Wherein, modulation prepared nux vomica of the present invention is according to the conventional method in the pharmacopeia, or following method preparation: whenever get Semen Strychni (processed) 100 weight portions; Be ground into fine powder; After measuring strychnine content according to method under the assay item of Pharmacopoeia of People's Republic of China vomiting nut, add starch 5-100 weight portion, make strychnine C21H22N2O2 content count 1.09%～1.15% by dry product after; Mixing is promptly processed the modulation prepared nux vomica.
The ratio of weight portion of the present invention and parts by volume is a grams per milliliter.
Description of drawings
Fig. 1: the total peak of methodological study
Fig. 2: finger-print precision
Fig. 3: finger-print repeatability
Fig. 4: stability
Fig. 5: at Agilent 1100, Agilent 1200, the collection of illustrative plates that obtains on Tianjin, island-10A instrument
Fig. 6: the chromatogram that on the Waters high performance liquid chromatograph, obtains
Fig. 7: confirm that fingerprint peaks ownership uses collection of illustrative plates
Fig. 8: the reference fingerprint that " chromatographic fingerprints of Chinese materia medica similarity evaluation system " generates
Fig. 9: representational 15 batches of Yaotongning capsules finger-prints
The present invention adopts efficient liquid-phase chromatography method to set up the finger-print of Yaotongning capsules, and the overall process that is applied to Yaotongning capsules production is carried out quality control; The similarity software that adopts pharmacopeia to issue must not be decided to be Yaotongning capsules finger-print similarity threshold and is lower than 0.87.Accurate through this method of experiment proof, reliably, can embody the characteristic peak of each prescription medicinal material to greatest extent.
Experimental example 1 is on wavelength is selected; Through studying the wavelength coverage of tentatively having confirmed high performance liquid chromatogram, between 210-260nm, use HPLC/DAD that the Yaotongning capsules analytic sample is carried out full wavelength scanner then; Scheme and the long 2D stacking diagram of all-wave at 190～400nm full wavelength scanner figure and 3D thereof according to the DAD detecting device; Full wavelength scanner 254nm place chromatographic peak most number, the solvent background is low, disturbs little; Most component color spectral strength maximums and separation case are better, therefore select the detection wavelength of 254nm as the Yaotongning capsules finger-print.
The optimization of experimental example 2 extraction conditions, extraction time, extracting mode
Introduce the thought and the method for uniform Design, comparative studies six kinds extract solvent compositions: 100% ethanol, 70% ethanol; 50% ethanol, 30% ethanol, 100% water; 100% methyl alcohol and methyl alcohol add 2.5% concentrated hydrochloric acid, and the sample introduction analysis is compared under identical HPLC condition, and methyl alcohol adds 2.5% concentrated hydrochloric acid extraction chromatography peak number order and total peak area is maximum; Help the Yaotongning capsules characteristic information and express, effect is superior to the extraction of methanol solvate, therefore selects for use methyl alcohol to add 2.5% concentrated hydrochloric acid and is the basic solvent that extracts; Multiple different method for distilling such as reflux extraction, ultrasonic extraction, water extract-alcohol precipitation, diafiltration and mixed and extracted mode have been compared; Select for use methyl alcohol and hydrochloric acid as extraction conditions; Sample introduction analysis under identical HPLC condition; Adopting power is that 450W ultrasonic Extraction mode chromatogram peak number is close with the finger-print that refluxing extraction obtains with total peak area, because ultrasonic Extraction is simple to operate, the time short, good reproducibility, therefore selects for use ultrasonic as extracting mode; Also extraction time (30,45,60,80,90,120min) has been carried out optimization process simultaneously; Select ultrasonic Extraction 45min (450W for use; 40KH); When adopting power to be the 450W ultrasonic Extraction, the sample stripping reaches stable state behind the extraction time 45min, and the peak number and the total peak area of 45min and 60min finger-print are basic identical.Confirm that at last adopting extraction time is 45min.
The optimization of experimental example 3 chromatographic separation conditions considers that the vomiting nut medicinal material is a monarch drug in a prescription in the Yaotongning capsules prescription, in prescription, occupies leading position, and vomiting nut has toxic action simultaneously, is the emphasis of quality control.So the optimization aim of finger-print chromatographic condition is set at strychnia, these 2 compositions of strychnine must reach baseline separation; Need take into account other composition chromatographic peaks simultaneously; Embody maximum chromatographic peak information as much as possible; Guaranteeing that the chromatogram gradient condition is simple as far as possible under the necessary separating effect situation, helping repeatability that finger-print obtains and stable.In the research process; Select acetonitrile (A)-water (B) and methyl alcohol (A)-two kinds of basic moving phase systems of water (B) respectively; Comparative result shows; Chromatographic peak number and total peak area are maximum in the finger-print that acetonitrile-water moving phase system obtains, and help the Yaotongning capsules characteristic information and express, and effect is superior to methyl alcohol-pure aquatic system.Investigated in the research in moving phase and to have added formic acid and the effect of triethylamine to chromatographic resolution, preliminary test prompting interpolation formic acid and triethylamine in moving phase improve significantly to the chromatographic resolution tool really.Moving phase: acetonitrile is a mobile phase A, and the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B;
Carry out gradient elution, gradient condition is the time: 0～20～50～60 minutes, mobile phase A (acetonitrile) was by 8%～18%～98%～98%, and Mobile phase B (0.2% triethylamine mixed solution of 0.2% formic acid) is by 92%～82%～2%～2%.
Table 1 linear gradient elution time-program(me)
Through analysis-by-synthesis, confirm that finally the finger-print condition is: get these article inclusions, mixing is got 2.0g; The accurate title, decide, and puts in the 50mL tool plug triangular flask, and the accurate methyl alcohol 25mL that adds adds the 0.63mL concentrated hydrochloric acid again; Weigh, shake up, (450W 40KH) takes out ultrasonic Extraction 45min; Put and be chilled to room temperature, weigh, add methyl alcohol and supply weightlessness.Get subsequent filtrate 10ul and inject liquid chromatograph, analyze, chromatographic condition is: measure HC-C according to high performance liquid chromatography (appendix VI D) 18Chromatographic column (4.6 * 250mm, 5 μ), gradient elution is a mobile phase A with the acetonitrile, is Mobile phase B with 0.2% triethylamine solution of 0.2% formic acid, gradient condition such as table 1; Detect wavelength 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min.Number of theoretical plate calculates by the strychnine peak should be not less than 6000.See accompanying drawing 1: the total peak of methodological study.
Experimental example 4 methodological study aspects:
The precision of traditional Chinese medicine fingerprint analytical approach, repeatability and stable method for building up are measured similar with conventional analysis.But in the application of traditional Chinese medicine fingerprint, this three's identification is divided into part and whole two aspects, and the former comprises the relative retention time at index components or total peak and the investigation of relative integral area; The latter is for measuring the global similarity property investigation between the collection of illustrative plates.In research process, respectively part and whole two aspects are studied.
At first, the relative integral area at total peak is investigated, is made a concrete analysis of as follows:
The Yaotongning capsules finished product of producing with the multiple health Pharma Inc. of Chengde neck is an analytic target, and more than 10 fingerprint peakses among the figure are object, have investigated the precision of analytical approach, repeatability, and stability of sample.
1. precision is investigated: the precision of mainly investigating instrument.Get Yaotongning capsules (lot number: 070121) test sample, prepare test sample according to the method for distilling of Yaotongning capsules finger-print, assay method is with the Yaotongning capsules fingerprint spectrum method.Continuous sample introduction 6; Write down the retention time and the integral area of each total chromatographic peak; (No. 3 peaks with strychnine; S) retention time and integral area are reference, converse the relative retention time and relative integral 4 areas at each total peak, with 9 relative retention times of investigating chromatogram 8 peaks, the consistance of peak area ratio.The result shows that the HPLC instrument and the total system precision of method employing is good, and analysis result is stable, credible.Calculate the RSD%=0.08% of the similarity of 6 sample introductions according to similarity.See accompanying drawing 2: finger-print precision
Table 2 Yaotongning capsules fingerprint spectrum method precision is investigated
Figure G2009102506549D00111
2. repeatability is investigated: according to preceding method, repeat to prepare 6 parts of need testing solutions (080402), the sample introduction analysis is an index with the peak area, the repeatability of investigation method, and the result sees table 6.Calculate the RSD%=0.12% of the similarity of 6 sample introductions according to similarity.See accompanying drawing 3: finger-print repeatability
Table 3 Yaotongning capsules fingerprint spectrum method repeatability is investigated
Figure G2009102506549D00112
3. study on the stability: the stability of mainly investigating test sample.Get the Yaotongning capsules test sample of same lot number (070402); The different time continuous sample introduction detects within 48 hours respectively; Write down the retention time and the integral area of each total chromatographic peak; Retention time and integral area with the strychnine peak are reference, converse the relative retention time and the relative integral area at each total peak, investigate the relative retention time of chromatographic peak, the consistance of peak area ratio.Presentation of results better can guarantee that in the 48h time science of experimental result is reliable through sample stability under the normal temperature experiment condition of preparation, sees table 7 and Figure 32.Calculate the RSD%=0.10% of the similarity of 6 sample introductions according to similarity, see accompanying drawing 4: stability.
Table 4 Yaotongning capsules fingerprint spectrum method study on the stability
Figure G2009102506549D00113
Figure G2009102506549D00121
Secondly, be object with 10 fingerprint peakses, adopt global similarity property method that precision, repeatability and the stability of Yaotongning capsules fingerprint analysis method are investigated, it is as shown in the table for the result.
Table 5 Yaotongning capsules fingerprint spectrum method is learned and is investigated similarity result of calculation
Figure G2009102506549D00122
The above results shows, no matter is index, and overall similarity all has data presentation preferably.The Yaotongning capsules finger print measuring method has believable precision and repeatability, and sample is stable in 48 hours, can obtain reliable finger-print.
4. on the applicability of instrument, compare same lot sample article (070124), adopted same root chromatogram column on the high performance liquid chromatograph of different model, to detect, adopted finger-print similarity software to compare similarity.The high performance liquid chromatograph that adopts comprises: Agilent 1100, and Agilent 1200, island Tianjin-10A, waters.The result shows that at Agilent 1100, Agilent 1200, and the chromatogram that obtains on island Tianjin-10A and the waters high performance liquid chromatograph is consistent basically, and similarity reaches more than 0.95, explains that this method can be applicable to different liquid chromatographs.See accompanying drawing 5: at Agilent 1100, Agilent 1200, collection of illustrative plates that obtains on Tianjin, island-10A instrument and accompanying drawing 6: the chromatogram that on the Waters high performance liquid chromatograph, obtains.
Since differences such as the pump discharge precision of different manufacturers instrument, gradient hybrid mode, detection sensitivity, dead volume size, and it is different to cause the chromatographic resolution situation to have, and same chromatographic peak retention time on different instruments has certain difference.Though this method has than extensive applicability the high performance liquid chromatograph utensil; But when selecting surveying instrument, still advise paying the utmost attention to consistent with standard finger-print; Particularly chromatographic gradient retardation time, and the high pressure mixing of the pump mode of mixing with low pressure is paid attention to need select instrument the time.
5. on the applicability of chromatographic column; The sample extraction method that adopts the Yaotongning capsules finger-print to establish; Adopt same high performance liquid chromatograph (Agilent 1100) that same sample (070124) is analyzed; In analytic process, change different chromatographic columns, model is: and A:Apollo (4.6 * 250mm, 5um); B:Agilent HC-C18 (4.6 * 250mm, 5um); C:Agilent TC-C18 (4.6 * 250mm, 5um); D:Interisl (4.6 * 250mm, 5um).Adopt finger-print similarity software to handle to the collection of illustrative plates of gathering, the result shows: when adopting same high performance liquid chromatograph to measure same lot sample article, and basically identical as a result, wherein, (4.6 * 250mm, 5um) effect is best with B:Agilent HC-C18.The above results shows; Agilent HC-C18 (4.6 * 250mm; 5um) the C18 chromatographic column of chromatographic column and same size, other brand that character is close all might be applicable to the mensuration of Yaotongning capsules finger-print; But, recommend to adopt Agilent HC-C18 (4.6 * 250mm, 5um) chromatographic column in order to guarantee that the peaceful finger-print of pain in the back has better stability.
6. the ownership of fingerprint peaks in the peaceful finger-print of pain in the back is studied; The selection at the total peak of the characteristic of the standard finger-print of Yaotongning capsules is the basis with 21 chromatographic peaks (like figure below) of analyzing through the chromatographic peak belongingness; In conjunction with mensuration to 40 batches of Yaotongning capsules finished product finger-prints;, repeatability good and stability according to total peak that in all batches finger-print, all has and separation case better, certain principles such as medicinal material representativeness are arranged; Definite selection 10 chromatographic peaks wherein are as the characteristic peak of reference fingerprint; No. 1, No. 3, No. 5, No. 6, No. 7, No. 10, No. 11, No. 15, No. 16, No. 21 of 21 chromatographic peaks of correspondence respectively, medicinal materials such as vomiting nut, radix cyathulae, Radix Glycyrrhizae, rhizoma atractylodis have been represented.Wherein original No. 2 chromatographic peaks corresponding to the Chinese ephedra medicinal material; Possibly be the mixing peak of ephedrine and two kinds of isomeride of pseudoephedrine; In addition since the characteristic absorption of Herba Ephedrae alkaloid in the terminal uptake zone (203nm) of ultraviolet; A little less than the detection wavelength 254nm place response that finger-print is set, repeatability is undesirable, does not therefore bring in 10 common characteristic peaks of final finger-print.Correspondingly, the method for quantitatively determining of setting up ephedrine and pseudoephedrine separately can complement one another with the finger-print Quality Control as the quantitative quality control index of multi-target ingredient.See accompanying drawing 7: confirm that fingerprint peaks ownership uses collection of illustrative plates.
7. 15 batches of Yaotongning capsules products producing with our company have generated the standard finger-print of Yaotongning capsules.Confirm that through test this finger-print has good representativeness.
The generation of typical fingerprint collection of illustrative plates is the finger-print research through a series of samples; Therefrom select one to have typical meaning or representational finger-print as the contrast spectrum; Or the relative retention value at the total peak of finger-print as contrast, the reference fingerprint that can also adopt the method simulation of the comprehensive all samples information of data (mean vector or median vector) to generate.
The reference fingerprint of Yaotongning capsules adopts multisample median vector comprehensively as the common pattern vector, has avoided the harmful effect of extraordinary sample in the multisample, makes experimental result more steadily and surely feasible.
Utilize the Yaotongning capsules finger print measuring method of confirming; 15 batches of Yaotongning capsules products with our company's production; With pharmacopeia committee finger-print software is that appraisal tool adopts the principle match of median to generate reference fingerprint, as the standard finger-print of Yaotongning capsules.The generation standard diagram is following.See accompanying drawing 8: the reference fingerprint that " chromatographic fingerprints of Chinese materia medica similarity evaluation system " generates.
In order to confirm the zone of reasonableness of Yaotongning capsules finger-print similarity threshold, 48 batches of Yaotongning capsules products producing later in 06 year are analyzed, explain that finger-print that this standard generates can reflect the quality of product preferably.The finger-print similarity result of calculation of 48 batches of specification product is 0.867 ± 0.044 (mean value ± SD).Take all factors into consideration, the similarity numerical standard of Yaotongning capsules finger-print qualified samples is decided to be is not less than 0.87.See accompanying drawing 9: representational 15 batches of Yaotongning capsules finger-prints
48 batches of Yaotongning capsules products of table 6 similarity is measured the result
Figure G2009102506549D00141
Embodiment
Embodiment 1: the determining fingerprint pattern of Yaotongning capsules
Modulation prepared nux vomica 120g ground bettle 21g
Radix cyathulae 21g Chinese ephedra 21g
Frankincense 21g myrrh 21g
Stiff silkworm 21g rhizoma atractylodis 21g
Scorpio 21g Radix Glycyrrhizae 21g;
Get bulk drug, add conventional auxiliary material, process capsule by conventional method;
According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), require to measure in conjunction with finger-print.
The finger print measuring method of Yaotongning capsules is: get these article inclusions, mixing is got 2.0g, and accurate the title decides; Put in the 50mL tool plug triangular flask, the accurate methyl alcohol 25mL that adds adds concentrated hydrochloric acid 0.63mL, close plug again; Shake up, claim to decide weight, with power 450W, 40kHz carries out sonicated and took out in 45 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 10 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 2ml, puts in the 10ml volumetric flask, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Finger-print and each item technical parameter test sample chromatogram should with the standard finger-print basically identical, corresponding 10 characteristic peaks are arranged, the characteristic peak appearance time should be within 60 minutes.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare with standard finger-print, calculate similarity, similarity should be not less than 0.87;
The finger-print of Yaotongning capsules is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is:
With No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138.
Embodiment 2: the preparation of pharmaceutical composition tablet of the present invention
Modulation prepared nux vomica 120g ground bettle 21g radix cyathulae 21g
Chinese ephedra 21g frankincense 21g myrrh 21g
Stiff silkworm 21g rhizoma atractylodis 21g scorpio 21g
Radix Glycyrrhizae 21g;
Get bulk drug, add conventional auxiliary material, process tablet by conventional method.
Embodiment 3: the preparation of drug composition oral liquid preparation of the present invention
Get the stiff silkworm 29g of modulation prepared nux vomica 80g ground bettle 23g radix cyathulae 24.5g Chinese ephedra 17.5g frankincense 15g myrrh 31g rhizoma atractylodis 19g scorpio 18g Radix Glycyrrhizae 26g; Add auxiliary materials such as solvent, flavouring, emulsifying agent; According to common process, process oral administration solution.
Embodiment 4: the preparation of pharmaceutical composition sustained-release tablet of the present invention
Get the stiff silkworm 20g of modulation prepared nux vomica 160g ground bettle 17.5g radix cyathulae 19g Chinese ephedra 25g frankincense 26g myrrh 18g rhizoma atractylodis 27g scorpio 28g Radix Glycyrrhizae 17g, add conventional auxiliary material, process sustained release tablets by conventional method.
Embodiment 5: the preparation of medicament composition granule agent of the present invention
Get the stiff silkworm 18g of modulation prepared nux vomica 154g ground bettle 20g radix cyathulae 17.5g Chinese ephedra 23g frankincense 24.5g myrrh 19g rhizoma atractylodis 26g scorpio 31g Radix Glycyrrhizae 15g, add conventional auxiliary material, process granule by conventional method.。
Embodiment 6: the preparation of medicinal composition powders of the present invention
Get the stiff silkworm 23g of modulation prepared nux vomica 108g ground bettle 24g radix cyathulae 32g Chinese ephedra 16g frankincense 17.5g myrrh 28g rhizoma atractylodis 15g scorpio 20g Radix Glycyrrhizae 30g, process powder according to common process.
Embodiment 7: the preparation of medicament composition dropping pills of the present invention
Get the stiff silkworm 16g of modulation prepared nux vomica 136g ground bettle 19g radix cyathulae 15g Chinese ephedra 32g frankincense 28g myrrh 17.5g rhizoma atractylodis 30g scorpio 23g Radix Glycyrrhizae 18g, add conventional auxiliary material, process dripping pill by conventional method.
Embodiment 8: the determining fingerprint pattern of pharmaceutical composition tablet of the present invention
According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), require to measure in conjunction with finger-print.
The finger print measuring method of pharmaceutical composition tablet of the present invention is:
Get the tablet 2.5g of embodiment 2 preparations, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 28mL that adds; Add concentrated hydrochloric acid 0.68mL again, close plug shakes up; Claim to decide weight, with power 480W, 45kHz carries out sonicated and took out in 60 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 12 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 3.5ml, puts in the 15ml volumetric flask, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 12 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Finger-print and each item technical parameter test sample chromatogram should with the standard finger-print basically identical, corresponding 10 characteristic peaks are arranged, the characteristic peak appearance time should be within 60 minutes.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare with standard finger-print, calculate similarity, similarity should be not less than 0.87;
The finger-print of pharmaceutical composition tablet of the present invention is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is:
With No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138.
Embodiment 9: the determining fingerprint pattern of medicament composition dropping pills of the present invention
According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), require to measure in conjunction with finger-print.
The finger print measuring method of medicament composition dropping pills of the present invention is:
Get the dripping pill 3g of embodiment 7 preparations, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 32mL that adds; Add concentrated hydrochloric acid 0.7mL again, close plug shakes up; Claim to decide weight, with power 420W, 38kHz carries out sonicated and took out in 55 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 15 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 3ml, puts in the 15ml volumetric flask, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 14 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Finger-print and each item technical parameter test sample chromatogram should with the standard finger-print basically identical, corresponding 10 characteristic peaks are arranged, the characteristic peak appearance time should be within 60 minutes.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare with standard finger-print, calculate similarity, similarity should be not less than 0.87;
The finger-print of medicament composition dropping pills agent of the present invention is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is:
With No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138.
Embodiment 10: the determining fingerprint pattern of drug composition oral liquid preparation of the present invention
According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), require to measure in conjunction with finger-print.
The finger print measuring method of drug composition oral liquid preparation of the present invention is:
Get the oral liquid 1.8ml of embodiment 3 preparations, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 20mL that adds; Add concentrated hydrochloric acid 0.58mL again, close plug shakes up; Claim to decide weight, with power 360W, 40kHz carries out sonicated and took out in 60 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 9 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 2.5ml, puts in the 5ml volumetric flask, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 8 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Finger-print and each item technical parameter test sample chromatogram should with the standard finger-print basically identical, corresponding 10 characteristic peaks are arranged, the characteristic peak appearance time should be within 60 minutes.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare with standard finger-print, calculate similarity, similarity should be not less than 0.87;
The finger-print of drug composition oral liquid preparation of the present invention is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is:
With No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138.
Embodiment 11: the determining fingerprint pattern of medicament composition granule agent of the present invention
According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), require to measure in conjunction with finger-print.
The finger print measuring method of medicament composition granule agent of the present invention is:
Get the granule 4g of embodiment 5 preparations, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 32mL that adds; Add concentrated hydrochloric acid 0.6mL again, close plug shakes up; Claim to decide weight, with power 320W, 40kHz carries out sonicated and took out in 60 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 13 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 4ml, puts in the 20ml volumetric flask, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 13 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Finger-print and each item technical parameter test sample chromatogram should with the standard finger-print basically identical, corresponding 10 characteristic peaks are arranged, the characteristic peak appearance time should be within 60 minutes.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare with standard finger-print, calculate similarity, similarity should be not less than 0.87;
The finger-print of medicament composition granule agent of the present invention is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is:
With No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138.
Embodiment 12: the determining fingerprint pattern of medicinal composition powders of the present invention
According to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), require to measure in conjunction with finger-print.
The finger print measuring method of medicinal composition powders of the present invention is:
Get the powder 1.5g of embodiment 6 preparations, the accurate title, decide, and puts in the 50mL tool plug triangular flask, the accurate methyl alcohol 18mL that adds; Add concentrated hydrochloric acid 0.56mL again, close plug shakes up; Claim to decide weight, with power 250W, 50kHz carries out sonicated and took out in 30 minutes; Put and be chilled to room temperature, claim again to decide weight, supply the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate 8 μ l and inject liquid chromatograph; Analyze, chromatographic condition is: measuring according to high performance liquid chromatography (an appendix VI of pharmacopeia D), is the chromatographic column of filling agent with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A; With the WS that contains 0.2% formic acid and 0.2% triethylamine is Mobile phase B, carries out gradient elution, and gradient condition is the time: 0～20～50～60 minutes; Mobile phase A (acetonitrile) is by 8%～18%～98%～98%, and Mobile phase B (WS that contains 0.2% formic acid and 0.2% triethylamine) is by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg.Precision is measured above-mentioned reference substance solution 1.5ml, puts in the 5ml volumetric flask, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 8 μ l of need testing solution of drawing of determination method inject liquid chromatograph, write down 60 minutes chromatogram, promptly get.
Finger-print and each item technical parameter test sample chromatogram should with the standard finger-print basically identical, corresponding 10 characteristic peaks are arranged, the characteristic peak appearance time should be within 60 minutes.The test sample chromatogram is imported chromatographic fingerprints of Chinese materia medica similarity evaluation system (2004, the 1.0B version), compare with standard finger-print, calculate similarity, similarity should be not less than 0.87;
The finger-print of medicinal composition powders of the present invention is: the test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is:
With No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1;
No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813;
No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439;
No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1;
No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074;
No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036;
No. 10 peaks: 0.305 ± 0.138.

Claims (11)

1. the detection method of a drug combination preparation is characterized in that a kind of in the following method of being determined as of finger-print in this method:
The A method:
Chromatographic condition is the chromatographic column of filling agent according to the high effective liquid chromatography for measuring of an appendix VID of pharmacopeia with the octadecylsilane chemically bonded silica, is mobile phase A with the acetonitrile; The WS with 0.2% formic acid and 0.2% triethylamine is Mobile phase B; Carry out gradient elution, gradient condition is the time: 0～20～50～60 minutes, mobile phase A is: acetonitrile was by 8%～18%～98%～98%; Mobile phase B is: the WS of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The configuration of the need testing solution compositions preparation 1-4 weight portion of getting it filled is accurately claimed surely, puts in the 50mL tool plug triangular flask the accurate methyl alcohol 15-35 parts by volume that adds; Add concentrated hydrochloric acid 0.5-0.7 parts by volume again, close plug shakes up, and claims to decide weight; With power 250-550W, frequency 30-50kHz carried out sonicated 30-80 minute, took out, and put and was chilled to room temperature; Claim again to decide weight, supply the weight that subtracts mistake, shake up, filter with methyl alcohol; Get subsequent filtrate 0.005-0.020 parts by volume and inject liquid chromatograph, analyze;
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg; Precision is measured above-mentioned solution 1-4 parts by volume, puts in the volumetric flask of 5-20ml, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, contains strychnine 0.1mg among every 1ml;
Accurate respectively reference substance solution and each the 0.005-0.020 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets;
The B method:
Chromatographic condition is the HC-C of 250mm * 4.6mm granularity 5 μ m according to high effective liquid chromatography for measuring with the specification 18Chromatographic column; With the acetonitrile is mobile phase A, is Mobile phase B with 0.2% formic acid and 0.2% triethylamine mixed solution, carries out gradient elution; Gradient condition is the time: 0～20～50～60 minutes; Mobile phase A is: acetonitrile is by 8%～18%～98%～98%, and Mobile phase B is: the WS of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The configuration of the need testing solution compositions preparation 1-4 weight portion of getting it filled is accurately claimed surely, puts in the 50mL tool plug triangular flask the accurate methyl alcohol 15-35 parts by volume that adds; Add concentrated hydrochloric acid 0.5-0.7 parts by volume again, weigh, shake up ultrasonic Extraction; Take out, put and be chilled to room temperature, weigh, supply weightlessness with methyl alcohol; Get subsequent filtrate 0.005-0.020 parts by volume and inject liquid chromatograph, analyze;
The preparation precision of reference substance solution takes by weighing strychnine reference substance 5mg, puts in the 50ml measuring bottle, adds methenyl choloride 10ml and makes dissolving, adds methyl alcohol to scale, shakes up; Miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each the 0.005-0.020 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets;
The bulk drug of wherein said drug combination preparation of the present invention consists of:
Figure FSB00000695249600021
2. the detection method of a drug combination preparation is characterized in that a kind of in the following method of being determined as of finger-print in this method:
The A method:
Chromatographic condition is the chromatographic column of filling agent according to the high effective liquid chromatography for measuring of an appendix VID of pharmacopeia with the octadecylsilane chemically bonded silica, is mobile phase A with the acetonitrile; The WS with 0.2% formic acid and 0.2% triethylamine is Mobile phase B; Carry out gradient elution, gradient condition is the time: 0～20～50～60 minutes, mobile phase A is: acetonitrile was by 8%～18%～98%～98%; Mobile phase B is: the WS of 0.2% formic acid and 0.2% triethylamine, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The configuration of need testing solution compositions preparation 2 weight portions of getting it filled are accurately claimed surely, put in the 50mL tool plug triangular flask accurate methyl alcohol 25 parts by volume that add; Add concentrated hydrochloric acid 0.63 parts by volume again, close plug shakes up, and claims to decide weight; With power 450W, frequency 40kHz carried out sonicated 45 minutes, took out, and put and was chilled to room temperature; Claim again to decide weight, supply the weight that subtracts mistake, shake up, filter with methyl alcohol; Get subsequent filtrate 0.010 parts by volume and inject liquid chromatograph, analyze;
It is an amount of that the preparation precision of reference substance solution takes by weighing the strychnine reference substance, adds methenyl choloride and process the solution that every 1ml contains strychnine 0.5mg; Precision is measured above-mentioned solution 2 parts by volume, puts in the volumetric flask of 10 parts by volume, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets, and contains strychnine 0.1mg among wherein every 1ml;
Accurate respectively reference substance solution and each 0.010 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets;
The B method:
Chromatographic condition is according to high effective liquid chromatography for measuring, and adopting specification is the HC-C of 250mm * 4.6mm granularity 5 μ m 18Chromatographic column; With the acetonitrile is mobile phase A, is Mobile phase B with 0.2% formic acid and 0.2% triethylamine mixed solution, carries out gradient elution; Gradient condition is the time: 0～20～50～60 minutes; Mobile phase A is: acetonitrile is by 8%～18%～98%～98%, and Mobile phase B is: 0.2% formic acid and 0.2% triethylamine mixed solution, by 92%～82%～2%～2%; The detection wavelength is 254nm; 25 ℃ of column temperatures; Flow velocity is 1ml/min; Number of theoretical plate calculates by the strychnine peak should be not less than 6000;
The configuration of need testing solution compositions preparation 2.0 weight portions of getting it filled are accurately claimed surely, put in the 50 parts by volume tool plug triangular flasks accurate methyl alcohol 25 parts by volume that add; Add 0.63 parts by volume concentrated hydrochloric acid again, weigh, shake up ultrasonic Extraction; Take out, put and be chilled to room temperature, weigh, add methyl alcohol and supply weightlessness; Get subsequent filtrate 0.01 parts by volume and inject liquid chromatograph, analyze;
The preparation precision of reference substance solution takes by weighing strychnine reference substance 0.005 weight portion, puts in the 50 parts by volume measuring bottles, adds methenyl choloride 10 parts by volume and makes dissolving, adds methyl alcohol to scale, shakes up; Miillpore filter with 0.45 μ m filters, and gets subsequent filtrate, promptly gets the reference substance solution that every 1ml contains strychnine 0.1mg;
Accurate respectively reference substance solution and each 0.01 parts by volume of need testing solution drawn of determination method injected liquid chromatograph, writes down 60 minutes chromatogram, promptly gets;
The bulk drug of wherein said drug combination preparation of the present invention consists of:
Figure FSB00000695249600031
3. according to claim 1 or claim 2 a kind of detection method of drug combination preparation is characterized in that the finger-print of this method is:
The test sample chromatogram should with the standard finger-print basically identical; Corresponding 10 characteristic peaks are arranged; The characteristic peak appearance time should import chromatographic fingerprints of Chinese materia medica similarity evaluation system with the test sample chromatogram within 60 minutes, compare with standard finger-print; Calculate similarity, similarity should be not less than 0.87; Standard finger-print is: with No. 3 peaks is with reference to the peak, and the relative retention time of 10 characteristic peaks is:
No. 1 peak: 0.389 ± 0.162; No. 2: 0.575 ± 0.253; No. 3 peaks: 1; No. 4 peaks: 1.059 ± 0.131; No. 5 peaks: 1.151 ± 0.268; No. 6 peaks: 1.315 ± 0.813; No. 7 peaks: 1.405 ± 0.392; No. 8 peaks: 1.987 ± 0.248; No. 9 peaks: 2.204 ± 0.439; No. 10 peaks: 2.922 ± 0.137;
With No. 3 peaks is with reference to the peak, and the relative peak area of 10 characteristic peaks is:
No. 1: 0.063 ± 0.047; No. 2: 0.366 ± 0.183; No. 3 peaks: 1; No. 4 peaks: 0.416 ± 0.101; No. 5 peaks: 0.063 ± 0.057; No. 6 peaks: 0.153 ± 0.074; No. 7 peaks: 0.131 ± 0.086; No. 8 peaks: 0.191 ± 0.065; No. 9 peaks: 0.090 ± 0.036; No. 10 peaks: 0.305 ± 0.138.
4. according to claim 1 or claim 2 a kind of detection method of drug combination preparation is characterized in that the drug combination preparation in this method is processed by following method:
Press practice of pharmacy, bulk drug is prepared into various capsule, tablet, oral solution, oral suspensions, mucilage, emulsion, pill, granule, powder, Babu plaster or rubber ointments clinical or that pharmaceutically accept.
5. according to claim 1 or claim 2 a kind of detection method of drug combination preparation is characterized in that the drug combination preparation in this method is processed by following method:
Press practice of pharmacy, bulk drug is prepared into various sustained release tablets, controlled release tablet, Orally taken emulsion, oral liquid, colloidal solution, mixture, tincture, drops, dripping pill, enteric coated granules clinical or that pharmaceutically accept.
6. the detection method of a kind of drug combination preparation as claimed in claim 3 is characterized in that the drug combination preparation in this method is processed by following method:
Press practice of pharmacy, bulk drug is prepared into various capsule, tablet, oral solution, oral suspensions, mucilage, emulsion, pill, granule, powder, Babu plaster or rubber ointments clinical or that pharmaceutically accept.
7. the detection method of a kind of drug combination preparation as claimed in claim 3 is characterized in that the drug combination preparation in this method is processed by following method:
Press practice of pharmacy, bulk drug is prepared into various sustained release tablets, controlled release tablet, Orally taken emulsion, oral liquid, colloidal solution, mixture, tincture, drops, dripping pill, enteric coated granules clinical or that pharmaceutically accept.
8. like the detection method of claim 1,2,6 or 7 described a kind of drug combination preparations, it is characterized in that the bulk drug of the pharmaceutical composition in this method consists of:
Figure FSB00000695249600051
Figure FSB00000695249600052
Figure FSB00000695249600053
9. the detection method of a kind of drug combination preparation as claimed in claim 3 is characterized in that the bulk drug of the pharmaceutical composition in this method consists of:
Figure FSB00000695249600054
Figure FSB00000695249600055
Figure FSB00000695249600062
10. the detection method of a kind of drug combination preparation as claimed in claim 4 is characterized in that the bulk drug of the pharmaceutical composition in this method consists of:
Figure FSB00000695249600063
Figure FSB00000695249600064
Figure FSB00000695249600065
11. the detection method of a kind of drug combination preparation as claimed in claim 5 is characterized in that the bulk drug of the pharmaceutical composition in this method consists of:
Figure FSB00000695249600066
Figure FSB00000695249600071
Figure FSB00000695249600072
Figure FSB00000695249600073
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