CN101101280A - Use of pulse beat restoring capsule fingerprint pattern detect technology - Google Patents
Use of pulse beat restoring capsule fingerprint pattern detect technology Download PDFInfo
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Abstract
The invention relates to the fingerprint detection technology of pumice stone compound pharmaceutics Shengmai capsules and application of its quality control of Shengmai capsules in the production. It includes selection and preparation test solution of sample from the Shengmai capsule sample by solvent, under the definite chromatogram condition, testing liquid chromatogram fingerprint of solution, calculating semblance of Shengmai capsule test solution fingerprint by auxiliary similarity evaluation software of computer, according to the semblance of fingerprint should be more than 0.09, and get purpose to control the quality of the Shengmai capsule. It has been found that the invention has the function of good quality control for the production of Shengmai capsules; it's a key technology of evaluation the production quality of pumice stone compound pharmaceutics.
Description
Technical field
The present invention relates to a kind of finger-print detection technique and the application in the control of the SHENGMAI JIAONANG quality of production thereof of compound Chinese medicinal preparation SHENGMAI JIAONANG.Specifically, promptly utilize the drug quality of fingerprint pattern technology control SHENGMAI JIAONANG.
Background technology
Shengmai San is the famous ancient prescription of China's tradition, it is generally acknowledged and comes from " interior wound is debated puzzled opinion " (1213).In recent years through relevant expert textual criticism, think that Shengmai San comes from 1186 " medicine origin " the earliest, 821 years so far.Shengmai San comprises genseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine three flavor medicinal materials.Function with nourishing generate fluid, astringing YIN to stop sweating.Cure mainly the deficiency of both qi and yin of thermal burn vigour, cloudy liquid loss, card sees that sweat is many, thirsty, dry throat, breathe heavily that urgency is desired to take off, body tired lazy, feeble pulse is unable, the tongue extra dry red wine does not have rule, or prolonged illness cardiopulmonary two void, deficiency of both vital energy and Yin, to cough few phlegm of choking, the spontaneous perspiration of losing heart, mouth parched and tongue scorched and pulse condition void soft.We still widely use so far through the long-term clinical practice determined curative effect.Since the beginning of the seventies so far the world of medicine this side has been carried out big quantity research, clinical application range constantly enlarges, and has been used for the treatment of anti-shock, anti-heart failure in the cardiovascular disease, the aspect illness such as resist myocardial ischemia; The also existing over one hundred piece of report of pharmacological research.Domestic priority has been developed novel formulation such as Shengmai San oral liquid, granule, capsule, injection since the eighties.
SHENGMAI JIAONANG ties up on the basis of traditional ancient prescription " Shengmai San ", with a kind of natural Chinese traditional medicines preparation that modern science advanced technologies refining refinement forms, is widely used in treatment coronary heart disease, angina pectoris, heart and brain thrombus, improves organism adaptation ability etc.SHENGMAI JIAONANG comprises genseng, the tuber of dwarf lilyturf, and the fruit of Chinese magnoliavine three flavor medicinal materials, genseng is aided with the nourishing Yin and promoting production of body fluid tuber of dwarf lilyturf based on invigorating the lung and benefiting vital QI in the side, the fruit of Chinese magnoliavine hidroschesis of astringing the lung, three medicines share, and one mends one clear one holds back, and can play Yiqi and vein recovery altogether, the effect of nourishing Yin and promoting production of body fluid.Pharmacological research proves that we have the increase coronary flow, strengthens myocardial contractive power, improves the tolerance of cardiac muscle to hypoxic-ischemic, reduces the effect of oxygen utilization.
Clinical research shows that (clinical research official written reply application numbers: protect the Shen in 2002 Zhejiang and face word No. 5) SHENGMAI JIAONANG treatment Syndrome in TCM belongs to the obstruction of qi in the chest (coronary disease and angina pectoris) due to the deficiency of both qi and yin, palpitaition (vital myocarditis) all has the good clinical curative effect, and the no obvious toxic-side effects of safety.
Belong in the randomized controlled trial of the obstruction of qi in the chest due to the deficiency of both qi and yin (coronary disease and angina pectoris) in the treatment Syndrome in TCM, test group 46 examples are finished final test, and overall assessment (curative effect of disease) obvious effective rate is 13.0%, and total effective rate is 69.6%; Tcm syndrome clinical trial group total effective rate 93.5%; Curative effect to treat angina pectoris test group obvious effective rate is 34.8%, total effective rate 80.4%; ECG curative effect test group total effective rate 63.0%.Open trial has 87 examples to finish final test, and overall assessment (curative effect of disease) obvious effective rate is 14.9%, and total effective rate is 65.5%; Tcm syndrome curative effect total effective rate is 89.7%; The curative effect to treat angina pectoris total effective rate is 73.6%; The ECG curative effect total effective rate is 67.8%, and the prompting SHENGMAI JIAONANG has certain curative effect to above-mentioned each index.The clinical testing the long and confirms: the clinical efficacy of the obstruction of qi in the chest (coronary disease and angina pectoris) is definite due to the SHENGMAI JIAONANG treatment deficiency of both qi and yin.
Belong in the treatment Syndrome in TCM that palpitaition (vital myocarditis) randomized controlled trial shows that test group 41 examples are finished final test due to the deficiency of both qi and yin, overall assessment (curative effect of disease) obvious effective rate is 22.0%, and total effective rate is 73.2%; Tcm syndrome clinical trial group total effective rate 85.4%; Premature beat clinical trial group total effective rate 65.9%.Belong in the treatment Syndrome in TCM that palpitaition (vital myocarditis) open trial has 80 examples to finish final test due to the deficiency of both qi and yin, overall assessment (curative effect of disease) obvious effective rate is 45.0%, and total effective rate is 91.3%; Premature beat curative effect total effective rate is 83.8%; Tcm syndrome curative effect total effective rate is 95.0%; Clinical test results confirms: the clinical efficacy of palpitaition (vital myocarditis) is remarkable due to the SHENGMAI JIAONANG treatment deficiency of both qi and yin.
But regrettably, the SHENGMAI JIAONANG practical clinical of determined curative effect often shows as the curative effect instability, has greatly hindered the clinical practice of this product.Discover in a large number through us, the main cause that causes this phenomenon is a unstable product quality, in the existing SHENGMAI JIAONANG quality standard (WS3-B-1312-93) only among the other side constituent of ginseng carry out thin-layer chromatography and differentiate, and the composition thin-layer chromatography was differentiated and was not all had the fruit of Chinese magnoliavine, the tuber of dwarf lilyturf.Obvious above-mentioned standard can not objectively respond SHENGMAI JIAONANG total quality situation, is difficult to effectively control SHENGMAI JIAONANG quality.
Often adopt finger-print that autonomic drug is carried out quality control in the world, effect is remarkable, as Korea S, foreign country-Japan, foreign country (Germany) etc.They relatively pay attention to the development and utilization to autonomic drug, and foreign country (Germany) is with the ginkgo agent of fingerprint pattern technology control of quality, in the whole world-have very strong competitive power in the world.After the entry into the World Trade Organization, the big country that produces autonomic drug also all will enter China and be at war with, and the traditional Chinese medicine enterprise of China is faced with stern challenge.
Traditional Chinese medicine fingerprint has two characteristics: the one, by the characteristic of finger-print, can effectively differentiate the true and false or the place of production of sample; The 2nd, by the area at finger-print principal character peak or the formulation of ratio, can effectively control the quality of product, guarantee the consistent relatively of product quality.At present, finger-print has become the most effective means of the whole world-internationally recognized control Chinese medicine or natural drug quality.
Domesticly be engaged in traditional Chinese medicine fingerprint research and can track the sixties the earliest, formulate the finger-print etc. of Chinese crude drug as adopting thin-layer chromatography, ultraviolet spectrum, infrared spectrum.But people paid attention to inadequately this at that time, and there is a certain distance in the relative developed country with achievement of research speed.In recent years, along with the modernization of Chinese medicine, the whole world-international deeply development, government, enterprise and researchist recognize more and more clear-headedly, the quality of control Chinese medicine is one of our gordian technique of needing to be resolved hurrily, and traditional Chinese medicine fingerprint then is the feasible of control crude drug, semi-manufacture, final products authenticity, consistance and stability and effective means.Because the singularity of traditional Chinese medicine, at present domestic a large amount of finger-prints research concentrates on traditional Chinese medicine aspect, the less finger-print research work of carrying out oral preparation of Chinese traditional medicinal substantially.
The existing at present report that relates to traditional Chinese medicine injection and the research of Chinese crude drug finger-print in a large number, people such as Gaoyang have delivered the research of ginseng crude drug's fingerprint analysis method, and (No.4 p473), indicates 18 total peaks in ginseng crude drug's collection of illustrative plates altogether for Chinese patent drug, Vol.28; (He'nan University's journal (medical science version), Vol.22, No.3 such as Chen Hongxuan, p17), (Chinese patent drug such as Feng Wei, Vol.28, No.4, p477), (chemistry and bioengineering such as Feng Xuesong, Vol.23, No.5, p60) and (Northeast China Normal University's journal natural science edition, Vol.36 such as Fan Zhuowen, No.2 p50) studies the medicinal materials fingerprint analytical approach of the fruit of Chinese magnoliavine; Because the finger-print of compound preparation is not the simple superposition of several medicinal materials fingerprints, can't be directly used in the finger-print detection of compound preparation basically to the finger-print detection technique of genseng or the single medicinal material of the fruit of Chinese magnoliavine.
People such as Xia Jing have delivered the research of shengmai injection HPLC finger-print, standard finger-print by 20 characteristic peaks form (Chinese patent drug, Vol.28, No.8, p1093).The method of quality control that Chinese patent CN 1836717A discloses shengmai injection on September 27th, 2006, this method comprises finger-print test, discriminating and assay; Formulated based on red ginseng and the tuber of dwarf lilyturf composition characteristics finger-print and control the quality of injection based on the finger-print of fruit of Chinese magnoliavine composition characteristics comprehensively, its method that adopts is that each medicinal material collection of illustrative plates is compared one by one, step is many, quality control inconvenience during the big production of actual medicine is unfavorable for that the suitability for industrialized production quality control uses.
Though SHENGMAI JIAONANG and shengmai injection all are to develop from the basis of traditional ancient prescription " Shengmai San " to form, both have very big difference, see following table 1 for details: SHENGMAI JIAONANG and shengmai injection comparison sheet.
We are easy to reach a conclusion from table 1, no matter SHENGMAI JIAONANG and shengmai injection still are method for making from daily crude drug amount, all have than big-difference, single from the genseng angle, SHENGMAI JIAONANG contains a large amount of genseng crude drug crude drug powder, then have only the ethanol extract of genseng not contain genseng crude drug crude drug powder in the shengmai injection, thereby the material base of SHENGMAI JIAONANG and shengmai injection obviously is different, the fingerprint test method of shengmai injection can not directly apply to SHENGMAI JIAONANG.
For a long time, the technology that obtains a kind of effective control SHENGMAI JIAONANG quality is the target of pursuing in the industry always.We once attempted revising and enlarging the thin-layer chromatography discriminating of the fruit of Chinese magnoliavine, the tuber of dwarf lilyturf and the liquid chromatography assay of monarch drug in a prescription genseng is controlled product quality, but effect still can not be satisfactory.Through great efforts, we are in conjunction with the trend of liquid-phase chromatograph finger print atlas technical development, found suitable SHENGMAI JIAONANG finger-print detection technique, comprise the test liquid that the SHENGMAI JIAONANG sample is prepared sample with solvent extraction, test test liquid liquid-phase chromatograph finger print atlas under certain chromatographic condition, utilize the similarity of area of computer aided similarity evaluation computed in software SHENGMAI JIAONANG test liquid finger-print, obtain finger-print similarity numerical value, thereby reach the purpose of control SHENGMAI JIAONANG drug quality.Experiment showed, that finger-print detection technique of the present invention can play good quality control effect to the production of SHENGMAI JIAONANG, is a kind of gordian technique of estimating the compound Chinese medicinal preparation quality of production.
Table 1: SHENGMAI JIAONANG and shengmai injection comparison sheet
| Prescription | Usage and dosage | Amount to every day the crude drug consumption | Method for making | |
| SHENGMAI JIAONANG | Genseng (330g) tuber of dwarf lilyturf (660g) fruit of Chinese magnoliavine (330g) | Oral, one |
Genseng 2.97g, the tuber of dwarf lilyturf 5.94g, fruit of Chinese magnoliavine 2.97g | More than three the flavor, get genseng 200g, be ground into fine powder, standby; All the other ginseng pulverates are broken into meal, according to the percolation under liquid extract and the extract item (17 pages of appendix), make solvent with 75% ethanol, flood and carry out diacolation after 24 hours, collect the liquid of filtering.Fructus Schisandrae Chinensis powder is broken into meal, distillation is extracted, residue and the boiling tuber of |
| Shengmai injection | Genseng (100g) tuber of dwarf lilyturf (312g) fruit of Chinese magnoliavine (156g) | Intramuscular injection: a 2～4ml, 1～2 time on the one.Drip-feed: a 20～60ml, use with 5% glucose injection, 250～500ml dilution back, or follow the doctor's advice. | Intramuscular injection: ginseng 0.2-0.8g 0.624-2.496g tuber of dwarf lilyturf fruit of Chinese magnoliavine 0.312-1.248g drip-feed: ginseng 2-6g 6.24-18.72g tuber of dwarf lilyturf fruit of Chinese magnoliavine 3.12-9.36g | More than three the flavor, red ginseng powder is broken into particulate, with |
Summary of the invention
SHENGMAI JIAONANG comprises genseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine three flavor medicinal materials.The genseng principal ingredient is the genseng saponins in the side, mainly contains flavonoids, saponins etc. in the tuber of dwarf lilyturf, and the fruit of Chinese magnoliavine mainly contains lignanoids compositions such as schizandrin, schizandrin A, more than the principal ingredients of three flavor medicinal materials have all that ultraviolet or ultraviolet are terminal to be absorbed.
SHENGMAI JIAONANG finger-print detection technique of the present invention, comprise the test liquid that the SHENGMAI JIAONANG sample is prepared sample with solvent extraction, test test liquid finger-print under certain chromatographic condition, test sample finger-print and reference fingerprint be (Chinese Pharmacopoeia Commission's hosting of the auxiliary similarity evaluation software of machine as calculated, the traditional Chinese medicine fingerprint similarity software for calculation of units such as Zhejiang University's pharmaceutical engineering exploitation) calculated fingerprint collection of illustrative plates similarity obtains finger-print similarity numerical value.According to similarity numerical value, the finger-print detection technique is used for the quality control of SHENGMAI JIAONANG.
The preparation method of test liquid, comprise following steps: get the about 1g of SHENGMAI JIAONANG content, the accurate title, decide, add the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby for test.
Employed certain chromatographic condition comprises following factor: chromatographic column; Moving phase; The linear gradient elution program; Column temperature: 30 ℃; Flow velocity is 0.8ml/min; UV-detector detects; The detection wavelength is 203nm; Sample size: 10 μ L; Operation 90min.
The acquisition methods of reference fingerprint is for getting 10 batches of SHENGMAI JIAONANG samples, preparation method by above-mentioned test liquid prepares need testing solution, obtaining 10 batches of SHENGMAI JIAONANG HPLC finger-prints by above-mentioned chromatographic condition, is reference fingerprint with these 10 batches of average finger-prints of SHENGMAI JIAONANG.
What chromatographic column was selected for use is the C18 post, comprises wherein a kind of of Agilent Zorbax SB-C18, XDB-C18 or InertsilODS-3.Moving phase refers to acetonitrile (B phase)-water (A phase) system or acetonitrile (B phase)-0.03% phosphoric acid water (A phase) system.The linear gradient elution program, actual conditions is as follows: moving phase: water (A)-acetonitrile (B), 0min 2%B, 35min 35%B, 45min 40%B, 60min 55%B, 75min 100%B.
Through research, adopted the preparation method of following need testing solution: get the about 1g of SHENGMAI JIAONANG content, the accurate title, decide, add the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
Through research, adopted following chromatographic condition: chromatographic column: Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m); Moving phase: water (A)-acetonitrile (B), linear gradient elution program: 0min 2%B, 35min 35%B, 45min 40%B, 60min 55%B, 75min 100B%, operation 90min; Flow velocity: 0.8ml/min; Detect wavelength: 203nm; Column temperature: 30 ℃; Sample size: 10 μ L.The SHENGMAI JIAONANG reference fingerprint that obtains under this chromatographic condition is seen Fig. 1.
By our studies show that: the SHENGMAI JIAONANG finger-print has 32 characteristic peaks, the collection of illustrative plates total length is 90min, significantly be different from the shengmai injection (research of the shengmai injection HPLC finger-print that people such as Xia Jing delivers, standard finger-print is made up of 20 characteristic peaks, collection of illustrative plates total length 100min (Chinese patent drug, Vol.28, No.8, p1093)).Studies show that, wherein the chromatographic peak retention time at peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22, peak 23, peak 25, peak 27, peak 29, peak 31, peak 32 and spectrogram are consistent with the chromatographic peak in the genseng finger-print, and these 23 chromatographic peaks belong to the ginseng crude drug; The chromatographic peak retention time at peak 1, peak 4, peak 24 and spectrogram are consistent with the chromatographic peak in the fruit of Chinese magnoliavine finger-print, and these 3 chromatographic peaks belong to schisandra chinensis medicinal material; To be the chromatographic peak retention time of 28.0min and spectrogram consistent with the chromatographic peak in the tuber of dwarf lilyturf finger-print for retention time in the finger-print, and this chromatographic peak derives from the medicinal material tuber of dwarf lilyturf.
Use the present invention, the finger-print similarity that we press specification product should be the check quality control standard greater than 0.90, SHENGMAI JIAONANG finger-print detection technique is successfully applied to the production testing quality control of SHENGMAI JIAONANG, the products obtained therefrom steady quality is reliable, the inconsistency of curative effect is expected to basic solution, really reach safe and effective, reliable in quality.
It is as follows that foundation of SHENGMAI JIAONANG fingerprint analysis method and quality stability are estimated concrete condition:
1.1 instrument and reagent
Instrument: Agilent 1100 type high performance liquid chromatographs are joined degasser, quaternary gradient pump, automatic sampler, column oven, diode array detector, UV-detector (Agilent company); Alltech ELSD 2000 evaporative light-scattering detector (Alltech company).
Reagent: acetonitrile, methyl alcohol are chromatographically pure (MERCK company), and it is pure that all the other reagent are analysis, and water is the Milli-Q pure water.
SHENGMAI JIAONANG (Zhengda Qingchunbao Pharmaceutical Co., Ltd, lot number: 0505001,0506001,0507001,0509001,0509002,0510001,0511002,0512002,0512003,0602001).
1.2 chromatographic condition optimization
1.2.1 chromatographic column is selected
Under same chromatographic condition, compared phenomenex (250mm * 4.6mm i.d. respectively, 4 μ m), Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m), Agilent Zorbax XDB-C18 (250mm * 4.6mm i.d., 5 μ m), the Inertsil ODS-3 of enlightening horse (250mm * 4.0mm i.d., 5 μ m) four root chromatogram column chromatographic resolution situations the results are shown in Figure 2.Phenomenex post separating effect is not good, and there is conditions of streaking at the peak, and Agilent Zorbax SB-C18, XDB-C18 and Inertsil ODS-3 chromatographic column all present separating effect preferably, the peak shape symmetry, but better with Zorbax SB-C18 separating effect.It is many to detect peak number, the arteries and veins capsule fingerprint pattern analysis chromatographic column so selection Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m) makes a living.
1.2.2 detecting wavelength selects
Consider that principal ingredient is the genseng saponins in the SHENGMAI JIAONANG, mainly contain flavonoids in the tuber of dwarf lilyturf, saponins, saccharide compound, the fruit of Chinese magnoliavine mainly contains schizandrin, schizandrin A, the second element, first-class lignanoids composition of schisantherin and organic acid composition, the selection that detects wavelength should take into account all kinds of chemical compositions, the bibliographical information saponin component all has only terminal the absorption, the flavones ingredient maximum absorption wavelength is 230,280nm, and the maximum absorption wavelength of lignan component is 254nm in the fruit of Chinese magnoliavine, investigated 203 simultaneously, 230,250, SHENGMAI JIAONANG sample chromatogram figure under the 280nm wavelength, result such as Fig. 3 are maximum with the chromatogram chromatogram peak number under the 203nm wavelength.230,250, many peaks disappearance among the SHENGMAI JIAONANG sample chromatogram figure under the 280nm wavelength.Select 203nm to use and detect wavelength for the SHENGMAI JIAONANG analysis.
1.2.3 the moving phase system is selected
Compare methanol-water, acetonitrile-water, acetonitrile-0.03% phosphoric acid water system, the gained chromatogram is seen Fig. 4.The methanol-water system is under the 203nm wavelength as a result, chromatogram baseline fluctuation and drift are bigger, the separating effect at peak is relatively poor, and the chromatogram peak under the acetonitrile-water system, acetonitrile-0.03% phosphoric acid water system separates better, the two equal baseline is steady, and separation case shows slightly difference in the chromatogram different time sections, and quality is respectively arranged, consider that experiment condition is good to be reduced to, selecting the acetonitrile-water system is SHENGMAI JIAONANG used in chromatograph moving phase system.
1.2.4 column temperature is selected
Analyze SHENGMAI JIAONANG under 20,25,30,35,40 ℃ of column temperatures, the gained chromatogram is seen Fig. 5, the result: column temperature raises, and the post drops is moved after the chromatographic peak retention time in 40～70min zone is most, and the chromatographic peak degree of separation has improvement slightly, but does not have significant difference.Column temperature raises, and the post drops help the protection of chromatographic column, but column temperature is too high, and filler in the chromatographic column is had adverse effect, also easily causes the variation of labile element in the sample, takes all factors into consideration the each side factor, and the stratographic analysis column temperature is selected 30 ℃.
1.2.5 the selection of flow velocity
Investigated 0.6,0.8, the influence of flow velocity of 1.0ml/min to separating, result such as Fig. 6, with the flow velocity separating effect of 0.8ml/min preferable.
1.2.6 eluent gradient is selected
Adopt Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m) chromatographic column; Flow velocity: 0.8ml/min; Detect wavelength: 203nm; Column temperature: 30 ℃; Sample size: 10 μ L; With water is the A phase, and acetonitrile is the B phase, has investigated multiple condition of gradient elution, and four linear gradient conditions wherein are:
(1) 0min 5%B, 10min 10%B, 30min 35%B, 50min 55%B, 70min 100B%, operation 90min;
(2) 0min 5%B, 10min 8%B, 50min 55%B, 70min 100B%, operation 90min;
(3) 0min 10%B, 10min 10%B, 60min 55%B, 80min 100B%, operation 90min;
(4) 0min 2%B, 35min 35%B, 45min 40%B, 60min 55%B, 75min 100B%, operation 90min;
Corresponding chromatogram is seen Fig. 7; The chromatogram that obtains with gradient (4) separate better and also the spectrum peak more, can provide bigger quantity of information, so select with the chromatographic condition of gradient (4) as the SHENGMAI JIAONANG fingerprint map analyzing.
1.2.7 different detecting devices are investigated
In order to investigate the influence of different detecting devices to finger-print, searching can provide the detection method than large information capacity, under same chromatographic condition, adopts UV-detector, evaporative light-scattering detector to detect respectively.UV-detector parameter: detect wavelength 203nm; The ELSD parameter: atomization gas is a nitrogen, and atomization gas flow 2.0L/min, drift tube temperature are 105 ℃.The gained chromatogram is seen Fig. 8.Can find that from figure the chromatographic peak number that evaporative light-scattering detects gained SHENGMAI JIAONANG finger-print obviously is less than the ultraviolet detection result, the information that provides is less, therefore, selects UV-detector to detect.
1.3 the SHENGMAI JIAONANG sample preparation methods is optimized
1.3.1 extracting method is selected
Because in SHENGMAI JIAONANG technology, genseng adopts 75% ethanol percolation, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine adopt water extraction and alcohol precipitation method to extract, in the end also sneak into part genseng crude drug powder in the preparation process and make capsule, analyze in view of the above, the component that is contained in SHENGMAI JIAONANG is based on pure soluble components, still contain the strong polar component that all can dissolve in low amounts of water and the alcohol, and because of in capsule, having mixed part genseng crude drug powder, should consider in the specimen preparation process extraction to ginsenoside constituents in the genseng crude drug is considered in the stripping of three flavor medicinal substances extracts again.Take all factors into consideration above technological factor, at first selecting methyl alcohol is to extract solvent, investigates the difference between backflow and two kinds of methods of ultrasonic Extraction respectively.Concrete comparative approach is as follows: get the same batch of about 1g of SHENGMAI JIAONANG content, the accurate title, decide, the accurate 50.0ml methyl alcohol that adds adopts respectively and refluxes and ultrasonic extracting method extraction 2h, filters, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, add dissolve with methanol and be settled to 2.0ml, the centrifugal 10min of 10000rpm gets supernatant, carry out stratographic analysis, the results are shown in Figure 9.The refluxing extraction method is extracted more complete, selects the refluxing extraction mode to carry out the SHENGMAI JIAONANG specimen preparation.
1.3.2 extracting solvent investigates
Get the same batch of about 1g of SHENGMAI JIAONANG content, the accurate title, decide, accurate respectively 50.0ml methanol/ethanol/60% methyl alcohol/60% ethanol that adds, refluxing extraction 2h filters, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, add dissolve with methanol and be settled to 2.0ml, the centrifugal 10min of 10000rpm gets supernatant, carry out stratographic analysis, the results are shown in Figure 10.The peak number amount that with methyl alcohol is solvent extraction is more, and peak area is the highest, and the strong polar impurity that extracts is few.Select methyl alcohol for extracting solvent.
1.3.3 extracting quantity of solvent investigates
Get the same batch of about 1g of SHENGMAI JIAONANG content, precision claims fixed, accurate respectively adding 25.0,50.0,75.0,100.0ml methyl alcohol, refluxing extraction 2h, filter, precision is measured and is extracted half subsequent filtrate of total amount, is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, get supernatant, carry out stratographic analysis, the results are shown in Figure 11.When extracting quantity of solvent 〉=50ml, the no significant difference of each peak-to-peak area size is considered from reducing consumption of organic solvent and cost angle in the extract chromatogram, extracts quantity of solvent selection 50ml.
1.3.4 extraction time is investigated:
Get the same batch of about 1g of SHENGMAI JIAONANG content, the accurate title, decide, the accurate 50.0ml methyl alcohol that adds, refluxing extraction 1.5,2h, 2.5h filter respectively, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, add dissolve with methanol and be settled to 2.0ml, the centrifugal 10min of 10000rpm gets supernatant, carry out stratographic analysis, the results are shown in Figure 12.The extraction time 〉=during 2.0h, more composition can be proposed, extract more complete.Extraction time is selected 2h.
1.4 SHENGMAI JIAONANG fingerprint spectrum method demonstration
1.4.1 instrument precision
Get same SHENGMAI JIAONANG need testing solution, continuous sample introduction 6 times the results are shown in Table 1, Figure 13.Each peak retention time RSD is less than 0.06% as a result, unimodal area greater than the peak-to-peak area RSD of total peak area 1% less than 2.32%.For the first time to analyze the gained finger-print is contrast, analyzes gained chromatogram and reference fingerprint comparison other 5 times, and similarity result of calculation is: 1.000,1.000,1.000,1.000,1.000, and instrument precision is good.
Table 1-1 precision experimental result (peak area)
| Peak number | A-1 | A-2 | A-3 | A-4 | A-5 | A-6 | Mean value | RSD(％) |
| 1 2 3 4 5 6 8 10 12 17 18 21 22 23 25 29 32 | 10805 4141 5635 10541 13130 1592 1583 724 870 776 1573 1330 1504 2525 852 2331 1899 | 10997 4079 5615 10547 13206 1612 1599 722 876 776 1580 1334 1508 2524 859 2335 1892 | 11011 4091 5761 10520 13308 1611 1659 732 863 775 1590 1335 1493 2530 869 2355 1916 | 11294 4067 5861 10586 13377 1613 1655 738 866 782 1595 1342 1498 2535 878 2366 1917 | 11085 4115 5706 10574 13416 1623 1638 728 873 783 1598 1351 1503 2536 878 2377 1926 | 10992 4088 5702 10472 13452 1628 1603 732 880 785 1604 1354 1500 2543 888 2386 1917 | 11031 4097 5713 10540 13315 1613 1623 729 871 780 1590 1341 1501 2532 871 2358 1911 | 1.44 0.65 1.57 0.39 0.94 0.77 1.97 0.78 0.73 0.56 0.72 0.73 0.35 0.28 1.54 0.94 0.68 |
Table 1-2 precision experimental result (retention time)
| Peak number | t R-1 | t R-2 | t R-3 | t R-4 | t R-5 | t R-6 | Mean value | RSD(％) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 | 5.04 9.59 11.29 11.88 14.81 31.22 38.94 39.73 40.22 40.75 41.53 41.91 42.22 43.16 44.91 54.75 55.98 57.14 58.83 59.41 61.20 66.80 67.49 69.97 70.31 70.83 71.60 72.71 76.81 77.40 77.80 79.42 | 5.04 9.60 11.30 11.89 14.81 31.22 38.94 39.74 40.23 40.76 41.54 41.91 42.23 43.17 44.91 54.76 56.00 57.15 58.84 59.42 61.20 66.80 67.49 69.97 70.30 70.83 71.59 72.70 76.80 77.40 77.79 79.42 | 5.04 9.60 11.30 11.89 14.81 31.23 38.95 39.74 40.23 40.75 41.54 41.91 42.23 43.18 44.93 54.78 56.02 57.16 58.85 59.42 61.21 66.80 67.50 69.97 70.31 70.83 71.60 72.71 76.81 77.40 77.80 79.42 | 5.04 9.60 11.30 11.89 14.81 31.26 38.96 39.75 40.23 40.75 41.54 41.90 42.22 43.16 44.93 54.75 55.99 57.14 58.83 59.41 61.19 66.80 67.49 69.96 70.30 70.83 71.59 72.70 76.80 77.40 77.79 79.42 | 5.04 9.60 11.29 11.88 14.80 31.24 38.94 39.74 40.22 40.74 41.53 41.90 42.22 43.17 44.91 54.76 56.00 57.14 58.84 59.41 61.18 66.80 67.49 69.95 70.29 70.83 71.58 72.70 76.79 77.39 77.79 79.41 | 5.04 9.60 11.30 11.89 14.82 31.26 38.97 39.75 40.24 40.76 41.55 41.93 42.24 43.21 44.97 54.80 56.04 57.19 58.87 59.45 61.23 66.83 67.51 69.97 70.31 70.84 71.60 72.71 76.82 77.42 77.81 79.43 | 5.04 9.60 11.30 11.89 14.81 31.24 38.95 39.74 40.23 40.75 41.54 41.91 42.23 43.18 44.93 54.77 56.01 57.15 58.84 59.42 61.20 66.81 67.50 69.97 70.30 70.83 71.59 72.70 76.81 77.40 77.80 79.42 | 0.03 0.04 0.05 0.03 0.03 0.06 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.04 0.05 0.04 0.04 0.04 0.03 0.03 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 |
1.4.2 method reappearance
Get 6 parts of same batch of SHENGMAI JIAONANG contents, make need testing solution by the test sample preparation method respectively, the sample introduction analysis the results are shown in Table 2, Figure 14.Each peak retention time RSD is less than 0.30% as a result, unimodal area greater than the peak-to-peak area RSD of total peak area 1% less than 4.21%.With a chromatogram wherein is standard finger-print, other chromatograms and this finger-print relatively, similarity is respectively 0.999,0.999,0.998,0.999,0.998, all greater than 0.998, shows this method tool good reproduction.
Table 2-1 reappearance experimental result (peak area)
| Peak number | A-1 | A-2 | A-3 | A-4 | A-5 | A-6 | Mean value | RSD(％) |
| 1 2 3 4 5 6 8 10 12 17 18 21 22 23 25 29 32 | 10858 4471 5049 11361 11940 1489 1608 705 765 781 1467 1277 1500 2301 844 2232 1669 | 10790 4601 4976 11885 12355 1519 1626 689 739 790 1488 1306 1521 2300 813 2284 1679 | 10770 4326 4704 11867 11385 1479 1566 691 745 743 1394 1203 1405 2311 766 2045 1538 | 11539 4387 4991 11571 11304 1509 1611 706 734 783 1446 1237 1475 2488 771 2193 1584 | 11531 4660 4573 11915 12189 1578 1528 713 787 798 1494 1276 1435 2384 760 2263 1695 | 11138 4638 4937 11021 12080 1566 1453 711 796 819 1485 1274 1487 2502 804 2265 1685 | 11104 4514 4872 11603 11876 1523 1565 702 761 786 1462 1262 1470 2381 793 2214 1642 | 3.23 3.10 3.87 3.10 3.65 2.67 4.21 1.45 3.45 3.20 2.59 2.86 2.93 3.93 4.14 4.01 3.94 |
Table 2-2 reappearance experimental result (retention time)
| Peak number | t R-1 | t R-2 | t R-3 | t R-4 | t R-5 | t R-6 | Mean value | RSD(％) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 | 5.02 9.55 11.24 11.82 14.77 31.36 39.10 39.94 40.43 40.98 41.74 42.15 42.44 43.47 45.22 55.03 56.26 57.41 59.10 59.67 61.29 | 5.03 9.54 11.23 11.81 14.76 31.39 39.14 39.97 40.46 41.03 41.78 42.22 42.51 43.55 45.32 55.12 56.36 57.51 59.20 59.77 61.30 | 4.99 9.56 11.25 11.84 14.78 31.33 39.07 39.89 40.39 40.93 41.70 42.11 42.41 43.43 45.17 54.99 56.23 57.38 59.07 59.65 61.30 | 5.03 9.59 11.30 11.87 14.82 31.34 39.06 39.85 40.36 40.88 41.66 42.05 42.37 43.35 45.08 54.90 56.14 57.28 58.96 59.53 61.26 | 5.04 9.58 11.28 11.87 14.80 31.21 38.94 39.74 40.24 40.75 41.53 41.90 42.22 43.18 44.93 54.78 56.01 57.16 58.85 59.42 61.19 | 5.03 9.58 11.27 11.86 14.79 31.24 38.94 39.74 40.25 40.76 41.53 41.91 42.22 43.18 44.94 54.78 56.01 57.16 58.86 59.43 61.19 | 5.03 9.57 11.26 11.85 14.79 31.31 39.04 39.86 40.36 40.89 41.66 42.06 42.36 43.36 45.11 54.93 56.17 57.32 59.01 59.58 61.26 | 0.33 0.21 0.21 0.24 0.17 0.23 0.21 0.25 0.23 0.28 0.25 0.31 0.28 0.36 0.35 0.25 0.25 0.24 0.24 0.23 0.08 |
| 22 23 24 25 26 27 28 29 30 31 32 | 66.99 67.67 69.99 70.32 70.96 71.62 72.70 76.78 77.37 77.76 79.35 | 67.04 67.72 69.98 70.31 70.98 71.62 72.69 76.76 77.34 77.73 79.32 | 66.97 67.65 70.00 70.33 70.94 71.62 72.72 76.80 77.39 77.78 79.39 | 66.88 67.56 69.98 70.32 70.88 71.60 72.71 76.80 77.40 77.79 79.41 | 66.82 67.50 69.96 70.30 70.84 71.59 72.70 76.81 77.41 77.80 79.43 | 66.81 67.50 69.96 70.30 70.84 71.59 72.70 76.81 77.41 77.80 79.42 | 66.92 67.60 69.98 70.31 70.91 71.61 72.70 76.80 77.39 77.78 79.39 | 0.14 0.13 0.02 0.02 0.09 0.02 0.01 0.03 0.03 0.04 0.06 |
1.4.3 sample stability
Get same SHENGMAI JIAONANG need testing solution, place 0,2,4,8,12,18 in room temperature respectively, sample introduction analysis behind the 24h, the results are shown in Table 3, Figure 15.Each peak retention time RSD is less than 0.34% as a result, unimodal area greater than the peak-to-peak area RSD of total peak area 1% less than 2.93%.Analyzing the gained finger-print with 0 o'clock is contrast, other times analyze the gained chromatogram and this finger-print compares, similarity is respectively 1.000,1.000,1.000,0.999,0.999, all greater than 0.999, shows that SHENGMAI JIAONANG need testing solution sample stability in room temperature 24h is good.
Table 3-1 stability experiment result (peak area)
| Peak number | A-0h | A-2h | A-4h | A-8h | A-12h | A-18h | A-24h | Mean value | RSD(％) |
| 1 2 3 4 5 6 8 10 12 17 18 21 22 23 25 29 32 | 10756 4282 4649 11973 11271 1468 1620 694 773 741 1378 1193 1366 2309 806 2041 1494 | 10665 4301 4670 11918 11239 1469 1592 672 742 732 1381 1190 1400 2284 760 2037 1491 | 10770 4326 4704 11867 11385 1479 1628 691 745 743 1394 1203 1405 2311 766 2045 1538 | 10607 4246 4689 11791 11373 1465 1644 664 721 738 1396 1216 1432 2243 788 2061 1501 | 10588 4303 4844 11791 11575 1476 1611 678 737 744 1404 1234 1442 2173 788 2068 1473 | 10911 4480 4944 11900 11786 1519 1705 700 761 797 1454 1248 1437 2298 793 2127 1561 | 11130 4457 4959 11958 11605 1436 1578 719 762 742 1468 1256 1415 2330 815 2131 1595 | 10775 4342 4780 11885 11462 1473 1625 688 749 748 1411 1220 1414 2278 788 2073 1522 | 1.78 2.07 2.78 0.62 1.74 1.68 2.55 2.74 2.37 2.93 2.54 2.16 1.88 2.37 2.51 1.92 2.90 |
Table 3-2 stability experiment result (retention time)
| Peak number | t R-0h | t R-2h | t R-4h | t R-8h | t R-12h | t R-18h | t R-24h | Mean value | RSD(％) |
| 1 2 3 | 5.00 9.57 11.26 | 5.00 9.58 11.27 | 4.99 9.56 11.25 | 4.99 9.53 11.22 | 5.00 9.55 11.24 | 5.01 9.60 11.28 | 5.02 9.62 11.31 | 5.00 9.57 11.26 | 0.18 0.31 0.25 |
| 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 | 11.84 14.78 31.36 39.10 39.93 40.43 40.98 41.73 42.15 42.44 43.47 45.23 55.03 56.27 57.41 59.10 59.68 61.31 66.99 67.67 70.01 70.34 70.96 71.63 72.72 76.80 77.40 77.79 79.40 | 11.86 14.80 31.36 39.10 39.92 40.42 40.97 41.73 42.14 42.44 43.46 45.21 55.02 56.26 57.40 59.09 59.66 61.32 66.97 67.65 70.01 70.34 70.95 71.63 72.73 76.82 77.41 77.80 79.41 | 11.84 14.78 31.33 39.07 39.89 40.39 40.93 41.70 42.11 42.41 43.43 45.17 54.99 56.23 57.38 59.07 59.65 61.30 66.97 67.65 70.00 70.33 70.94 71.62 72.72 76.80 77.39 77.78 79.39 | 11.80 14.75 31.39 39.14 39.96 40.45 41.02 41.77 42.20 42.49 43.52 45.30 55.09 56.33 57.47 59.18 59.76 61.31 67.04 67.72 69.99 70.33 70.99 71.63 72.70 76.77 77.35 77.74 79.34 | 11.81 14.77 31.43 39.19 40.03 40.52 41.11 41.85 42.31 42.58 43.66 45.42 55.21 56.45 57.59 59.29 59.86 61.37 67.12 67.79 70.03 70.36 71.04 71.67 72.72 76.77 77.35 77.74 79.34 | 11. 88 14.81 31.23 38.95 39.73 40.24 40.74 41.53 41.90 42.22 43.17 44.90 54.79 56.02 57.18 58.86 59.44 61.19 66.83 67.51 69.95 70.29 70.84 71.58 72.70 76.80 77.40 77.79 79.41 | 11.91 14.83 31.24 38.97 39.77 40.27 40.78 41.56 41.94 42.26 43.22 44.97 54.81 56.04 57.19 58.88 59.45 61.24 66.84 67.52 69.98 70.32 70.85 71.61 72.72 76.83 77.33 77.81 79.44 | 11.85 14.79 31.34 39.07 39.89 40.39 40.93 41.69 42.11 42.40 43.42 45.17 54.99 56.23 57.38 59.07 59.64 61.29 66.97 67.64 70.00 70.33 70.94 71.63 72.72 76.80 77.38 77.78 79.39 | 0.32 0.18 0.24 0.23 0.27 0.25 0.32 0.27 0.34 0.30 0.39 0.40 0.27 0.27 0.26 0.26 0.26 0.10 0.16 0.15 0.03 0.03 0.10 0.04 0.02 0.03 0.04 0.04 0.05 |
1.4.4 method durability
1.4.4.1 the chromatogram reappearance relatively between same type chromatographic column
Analyze same batch of SHENGMAI JIAONANG sample with three same type Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m) chromatographic column (SN number be respectively USCL013112, USCL013107, USCL012253), the results are shown in Figure 16.The finger-print of measuring with a root chromatogram column (SN:USCL012253) wherein is reference, calculate the similarity of two root chromatogram columns (SN:USCL013112, USCL013107) gained finger-print in addition, the result is 0.999,0.971, shows that to adopt the finger-print reappearance of same type chromatogram column analysis good.
1.4.4.2 SHENGMAI JIAONANG finger-print reappearance relatively between different instruments
The same a collection of SHENGMAI JIAONANG finger-prints of 3 Agilent 1100 liquid chromatographs records relatively the results are shown in Table 4, Figure 17.Can find that the finger-print of different Instrument measurings is similar on the whole, but compose peak retention time difference, deviation is arranged slightly.With 1 Agilent 1100 chromatograph wherein is reference, calculates the similarity with a collection of SHENGMAI JIAONANG finger-print of surveying on other 2 Agilent 1100 instruments, and the result is: 0.945,0.990.This is caused by flow velocity precision difference, gradient not equal factor retardation time between different instruments.But this method applies to finger-print that different instrument obtains, though spectrum peak retention time has drift slightly, the peak-to-peak degree of separation influence of each chromatogram is little, shows that the SHENGMAI JIAONANG chromatographic fingerprinting method of being set up has good durability.
SHENGMAI JIAONANG finger-print retention time relatively between the different instruments of table 4
| Peak number | Retention time (min) | ||
| Agilent 1100 (DE03010408) | Agilent 1100 (DE14916574) | Agilent 1100 (DE14919166) | |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 | 5.16 9.72 11.41 12.02 14.97 31.36 39.07 39.86 40.37 40.88 41.66 42.05 42.36 43.35 45.06 54.87 56.11 57.26 58.94 59.51 61.39 66.90 67.58 70.10 70.43 70.93 71.71 72.82 76.91 77.47 77.93 79.59 | 5.21 9.73 11.40 11.98 14.91 31.39 39.08 39.86 40.39 40.90 41.69 42.06 42.38 43.35 45.07 54.86 56.07 57.21 58.87 59.45 61.38 66.80 67.49 70.07 70.41 70.85 71.52 72.80 76.90 77.57 77.90 79.59 | 5.11 9.77 11.58 12.11 15.06 31.81 39.62 40.60 41.01 41.74 42.39 43.01 43.99 44.41 46.22 55.77 56.96 58.09 59.87 60.44 61.89 67.51 68.16 70.25 70.57 71.29 71.85 72.90 76.98 77.65 77.99 79.65 |
Through above-mentioned research, we have adopted the preparation method of following need testing solution: get the about 1g of SHENGMAI JIAONANG content, the accurate title, decide, add the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
Through above-mentioned research, the present invention has adopted following chromatographic condition: chromatographic column: Agilent ZorbaxSB-C18 (250mm * 4.6mm i.d., 5 μ m); Moving phase: water (A)-acetonitrile (B), linear gradient elution program: 0min 2%B, 35min 35%B, 45min 40%B, 60min 55%B, 75min 100B%, operation 90min; Flow velocity: 0.8ml/min; Detect wavelength: 203nm; Column temperature: 30 ℃; Sample size: 10 μ L.
Description of drawings
Fig. 1 SHENGMAI JIAONANG reference fingerprint
The different chromatographic columns of Fig. 2 are to the influence of SHENGMAI JIAONANG chromatographic resolution
Fig. 3 detects wavelength and selects
Fig. 4 moving phase system relatively
Fig. 5 column temperature is to the influence of SHENGMAI JIAONANG chromatographic resolution
Fig. 6 flow velocity is to the influence of SHENGMAI JIAONANG chromatographic resolution
Gained SHENGMAI JIAONANG chromatogram under the different gradient condition of Fig. 7
The finger-print that Fig. 8 UV and ELSD detect relatively
The selection refluxing extraction chromatogram of Fig. 9 extracting method
Figure 10 extracts choice of Solvent
Figure 11 extracts the selection of quantity of solvent
The selection of Figure 12 extraction time
Figure 13 instrument precision experimental result
Figure 14 method reappearance experimental result
Figure 15 sample stability experimental result
Figure 16 is different, and the lot number chromatographic column influences the SHENGMAI JIAONANG finger-print
The different instruments of Figure 17 are to the influence of SHENGMAI JIAONANG finger-print
Ten batches of SHENGMAI JIAONANG HPLC of Figure 18 finger-print
Figure 19 SHENGMAI JIAONANG and ginseng crude drug's finger-print contrast figure
Figure 20 shortage of staff joins SHENGMAI JIAONANG and SHENGMAI JIAONANG finger-print
Figure 21 SHENGMAI JIAONANG and medicinal materials fingerprint contrast tuber of dwarf lilyturf figure
Figure 22 SHENGMAI JIAONANG and fruit of Chinese magnoliavine finger-print contrast figure
The continuous 5 batches of SHENGMAI JIAONANG finger-print control experiment results of Figure 23
Embodiment
Below in conjunction with embodiment the present invention is further specified.
Embodiment 1Obtaining and the quality stability evaluation of reference fingerprint;
Get 10 batches of (lot numbers: 0505001,0506001,0507001,0509001,0509002,0510001,0511002,0512002,0512003,0602001) SHENGMAI JIAONANG sample, make the SHENGMAI JIAONANG need testing solution by need testing solution preparation method under " A " item, by the chromatographic condition analysis down of " B " item, obtain 10 batches of SHENGMAI JIAONANG HPLC finger-prints (Figure 18), with these 10 batches of average finger-prints of SHENGMAI JIAONANG is reference fingerprint (Fig. 1), calculate each batch SHENGMAI JIAONANG finger-print and reference fingerprint similarity relatively respectively, as a result, each batch SHENGMAI JIAONANG finger-print similarity is respectively 0.995,0.996,0.996,0.989,0.999,0.989,0.987,0.991,0.989,0.981.Similarity is all greater than 0.98, and the result shows: quality homogeneous, stable between each batch of SHENGMAI JIAONANG that Zhengda Qingchunbao Pharmaceutical Co., Ltd produces.
(A) preparation method of need testing solution: get the about 1g of SHENGMAI JIAONANG content, the accurate title, decide, and adds the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
(B) chromatographic condition: chromatographic column: Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m); Moving phase: water (A)-acetonitrile (B), linear gradient elution program: 0min 2%B, 35min 35%B, 45min 40%B, 60min 55%B, 75min 100B%, operation 90min; Flow velocity: 0.8ml/min; Detect wavelength: 203nm; Column temperature: 30 ℃; Sample size: 10 μ L.
Embodiment 2SHENGMAI JIAONANG finger-print and ginseng crude drug's finger-print correlativity
1.1 genseng need testing solution preparation: precision takes by weighing genseng meal 0.4g, according to SHENGMAI JIAONANG preparation technology, with 75% alcohol dipping 24 hours, diacolation is collected percolate, and decompression recycling ethanol is done near, 60 ℃ of drying under reduced pressure become extract powder, add 0.6g genseng meal mixing, add the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, add dissolve with methanol and be settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
1.2 the genseng finger-print and with SHENGMAI JIAONANG finger-print correlativity: press embodiment 1 " B " chromatographic condition down, precision is measured genseng need testing solution 10 μ L, inject liquid chromatograph, the record chromatogram, compare with each chromatographic peak retention time and spectrogram in the SHENGMAI JIAONANG finger-print that obtains under the same chromatographic condition, the peak 6 in the SHENGMAI JIAONANG finger-print, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22, peak 23, peak 25, peak 27, peak 29, peak 31, the chromatographic peak retention time at peak 32 and spectrogram are consistent with the chromatographic peak in the genseng finger-print, infer that these 23 chromatographic peaks derive from ginseng crude drug (Figure 19) in the SHENGMAI JIAONANG finger-print.
1.3 the shortage of staff joins obtaining of SHENGMAI JIAONANG finger-print
1.3.1 the shortage of staff joins SHENGMAI JIAONANG need testing solution preparation: take by weighing the 10g tuber of dwarf lilyturf, fruit of Chinese magnoliavine 5g is ground into meal respectively, the distillation of fruit of Chinese magnoliavine meal is extracted, residue and boiling tuber of dwarf lilyturf secondary filter merging filtrate, be concentrated into an amount of, add ethanol and make and contain alcohol amount and reach 60%, left standstill 24 hours, filter, decompression recycling ethanol is done near, and 60 ℃ of drying under reduced pressure become extract powder standby.Get the above-mentioned extract powder that is equivalent to 1g SHENGMAI JIAONANG content, the accurate title, decide, and adds the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
1.3.2 press embodiment 1 " B " chromatographic condition down, accurate respectively absorption SHENGMAI JIAONANG need testing solution, shortage of staff join SHENGMAI JIAONANG need testing solution 10 μ L, inject liquid chromatograph, the record chromatogram, SHENGMAI JIAONANG and shortage of staff join SHENGMAI JIAONANG need testing solution finger-print and see Figure 20, as seen from the figure, the shortage of staff joins retention time in the SHENGMAI JIAONANG finger-print in the peak of 30～50min disappearance, show in the SHENGMAI JIAONANG retention time at the peak of 30～50min majority from genseng.
Embodiment 3The tuber of dwarf lilyturf medicinal materials fingerprint and SHENGMAI JIAONANG finger-print correlativity
1.1 the need testing solution preparation tuber of dwarf lilyturf: take by weighing tuber of dwarf lilyturf 2g and be ground into meal, press the SHENGMAI JIAONANG extraction process, the boiling secondary, filter, merging filtrate is concentrated into an amount of, add ethanol and make and contain alcohol amount and reach 60%, left standstill 24 hours, filter, decompression recycling ethanol is done near, adds the 50.0ml methanol eddy and extracts 2h, filters, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, add dissolve with methanol and be settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
1.2 the tuber of dwarf lilyturf finger-print and with SHENGMAI JIAONANG finger-print correlativity: press embodiment 1 " B " chromatographic condition down, precision is measured the need testing solution 10 μ L tuber of dwarf lilyturf, inject liquid chromatograph, the record chromatogram, compare with each chromatographic peak retention time and spectrogram in the SHENGMAI JIAONANG finger-print that obtains under the same chromatographic condition, to be the chromatographic peak retention time of 28.0min and spectrogram consistent with the chromatographic peak in the tuber of dwarf lilyturf finger-print for retention time in the SHENGMAI JIAONANG finger-print, infers that this chromatographic peak derives from the medicinal material tuber of dwarf lilyturf (Figure 21) in the SHENGMAI JIAONANG finger-print.
Embodiment 4SHENGMAI JIAONANG finger-print and schisandra chinensis medicinal material finger-print correlativity
1.1 the preparation of fruit of Chinese magnoliavine need testing solution: precision takes by weighing fruit of Chinese magnoliavine meal 1g, boiling three times filters, merging filtrate is concentrated in right amount, adds ethanol and makes and contain the alcohol amount and reach 60%, left standstill 24 hours, and filtered, decompression recycling ethanol is done near, add the 50.0ml methanol eddy and extract 2h, filter, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, and adds dissolve with methanol and is settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby.
1.2 fruit of Chinese magnoliavine finger-print and with SHENGMAI JIAONANG finger-print correlativity: press embodiment 1 " B " chromatographic condition down, precision is measured fruit of Chinese magnoliavine need testing solution 10 μ L, inject liquid chromatograph, the record chromatogram, compare with each chromatographic peak retention time and spectrogram in the SHENGMAI JIAONANG finger-print that obtains under the same chromatographic condition, the peak 1 in the SHENGMAI JIAONANG finger-print, peak 4, peak 24, chromatographic peak retention time and spectrogram consistent with the chromatographic peak in the fruit of Chinese magnoliavine finger-print, infer that these 3 chromatographic peaks derive from schisandra chinensis medicinal material (Figure 22) in the SHENGMAI JIAONANG finger-print.
Embodiment 5The SHENGMAI JIAONANG finger-print control experiment that different times is 5 batches
(Zhengda Qingchunbao Pharmaceutical Co., Ltd produces to get 5 batches of quantity-produced SHENGMAI JIAONANGs, lot number: 0602003,0602004,0603001,0603002,0603003), make need testing solution by test sample preparation method under embodiment 1 " A " item respectively, press embodiment 1 " B " chromatographic condition down, sample introduction is analyzed, and the results are shown in Figure 23.This figure center line 1 is a reference fingerprint, and line 2-6 is followed successively by lot number 0602003,0602004,0603001,0603002,0603003.Each batch chromatogram and reference fingerprint are relatively, similarity is respectively 0.999,0.998,0.999,0.999,0.998 as calculated, all greater than 0.99, show quantity-produced SHENGMAI JIAONANG quality homogeneous, stable, the method for quality control that this SHENGMAI JIAONANG is described is easily and effectively.
The present invention's concrete technical scheme required for protection is not limited to the concrete combination of the expressed technical scheme of the foregoing description.
Claims (15)
1, a kind of application of SHENGMAI JIAONANG finger-print detection technique.Comprise the test liquid that the SHENGMAI JIAONANG sample is prepared sample with solvent extraction, test test liquid finger-print under certain chromatographic condition, test liquid finger-print and reference fingerprint be the similarity of the auxiliary similarity evaluation software calculated fingerprint collection of illustrative plates of machine as calculated, obtain similarity numerical value, should control the quality of SHENGMAI JIAONANG as quality control standard greater than 0.90 according to the finger-print similarity.
2, the application of the described SHENGMAI JIAONANG finger-print of claim 1 detection technique, the preparation method of described test liquid, comprise following steps: get the about 1g of SHENGMAI JIAONANG content, the accurate title, decide, and adds the 50.0ml methanol eddy and extract 2h, filters, precision is measured subsequent filtrate 25.0ml and is evaporated near doing, add dissolve with methanol and be settled to 2.0ml, the centrifugal 10min of 10000rpm, supernatant is standby for test.
3, the application of the described SHENGMAI JIAONANG finger-print of claim 1 detection technique, described certain chromatographic condition comprises following factor: chromatographic column; Moving phase; The linear gradient elution program; Column temperature: 30 ℃; Flow velocity is 0.8ml/min; UV-detector detects; The detection wavelength is 203nm; Sample size: 10 μ L; Operation 90min.
4, the application of the described SHENGMAI JIAONANG finger-print of claim 3 detection technique, what the chromatographic column in described certain chromatographic condition was selected for use is the C18 post.
5, the application of the described SHENGMAI JIAONANG finger-print of claim 4 detection technique, the chromatographic column in described certain chromatographic condition refers to wherein a kind of of Agilent Zorbax SB-C18, XDB-C18 or Inertsil ODS-3.
6, the application of the described SHENGMAI JIAONANG finger-print of claim 5 detection technique, the chromatographic column in described certain chromatographic condition refers to Agilent Zorbax SB-C18.
7, the application of the described SHENGMAI JIAONANG finger-print of claim 3 detection technique, the moving phase in described certain chromatographic condition refers to acetonitrile (B phase)-water (A phase) system or acetonitrile (B phase)-0.03% phosphoric acid water (A phase) system.
8, the application of the described SHENGMAI JIAONANG finger-print of claim 7 detection technique, the moving phase in described certain chromatographic condition refers to acetonitrile (B phase)-water (A phase) system.
9, the application of the described SHENGMAI JIAONANG finger-print of claim 3 detection technique, linear gradient elution program in described certain chromatographic condition, actual conditions is as follows: moving phase: water (A)-acetonitrile (B), 0min 2%B, 35min 35%B, 45min 40%B, 60min 55%B, 75min 100%B.
10, the application of claim 1 or 3 described SHENGMAI JIAONANG finger-print detection techniques, described certain chromatographic condition is as follows: chromatographic column: Agilent Zorbax SB-C18 (250mm * 4.6mm i.d., 5 μ m); Moving phase: water (A)-acetonitrile (B), linear gradient elution program: 0min 2%B, 35min 35%B, 45min40%B, 60min 55%B, 75min 100%B, operation 90min; Flow velocity: 0.8ml/min; Detect wavelength: 203nm; Column temperature: 30 ℃; Sample size: 10 μ L.
11, the application of the described SHENGMAI JIAONANG finger-print of claim 10 detection technique, the acquisition methods of described reference fingerprint is: get 10 batches of SHENGMAI JIAONANG samples, prepare need testing solution by the described method of claim 2, by certain chromatographic condition analysis, obtaining 10 batches of SHENGMAI JIAONANG HPLC finger-prints, is reference fingerprint with these 10 batches of average finger-prints of SHENGMAI JIAONANG.
12, the application of the described SHENGMAI JIAONANG finger-print of claim 11 detection technique, described reference fingerprint has 32 characteristic peaks, and the collection of illustrative plates total length is 90min.
13, the application of the described SHENGMAI JIAONANG finger-print of claim 12 detection technique, described reference fingerprint has 32 characteristic peaks, and wherein peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22, peak 23, peak 25, peak 27, peak 29, peak 31, peak 32 these 23 chromatographic peaks belong to the ginseng crude drug.
14, the application of the described SHENGMAI JIAONANG finger-print of claim 12 detection technique, described reference fingerprint has 32 characteristic peaks, and wherein peak 1, peak 4, peak 24 these 3 chromatographic peaks belong to schisandra chinensis medicinal material.
15, the application of the described SHENGMAI JIAONANG finger-print of claim 12 detection technique, retention time is that the chromatographic peak of 28.0min derives from the medicinal material tuber of dwarf lilyturf in the described reference fingerprint.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101879270A (en) * | 2009-05-07 | 2010-11-10 | 天津天士力之骄药业有限公司 | Chinese medicinal injectable powder and quality control method thereof |
| CN108195988A (en) * | 2018-02-01 | 2018-06-22 | 华润三九（雅安）药业有限公司 | Using the schisandra chinensis medicinal material of the construction method of HPLC finger-prints and its pulse invigorating injection liquid formulation |
| CN109212121A (en) * | 2018-09-10 | 2019-01-15 | 华润三九（雅安）药业有限公司 | Red ginseng medicinal materials fingerprint and its construction method with formulation ingredients relevance |
| CN111380964A (en) * | 2018-12-27 | 2020-07-07 | 云南生物谷药业股份有限公司 | Method for establishing fingerprint of schisandra chinensis medicinal material and erigeron breviscapus pulse-activating capsule |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1621836A (en) * | 2004-12-13 | 2005-06-01 | 贵阳云岩西创药物科技开发有限公司 | Quality controlling method for pulse restoring injection |
| CN100529753C (en) * | 2004-12-13 | 2009-08-19 | 贵阳云岩西创药物科技开发有限公司 | Quality controlling method for pulse restoring injection |
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2007
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101879270A (en) * | 2009-05-07 | 2010-11-10 | 天津天士力之骄药业有限公司 | Chinese medicinal injectable powder and quality control method thereof |
| CN101879270B (en) * | 2009-05-07 | 2014-02-12 | 天津天士力之骄药业有限公司 | Traditional Chinese medicine injectable powder and quality control method thereof |
| CN108195988A (en) * | 2018-02-01 | 2018-06-22 | 华润三九（雅安）药业有限公司 | Using the schisandra chinensis medicinal material of the construction method of HPLC finger-prints and its pulse invigorating injection liquid formulation |
| CN109212121A (en) * | 2018-09-10 | 2019-01-15 | 华润三九（雅安）药业有限公司 | Red ginseng medicinal materials fingerprint and its construction method with formulation ingredients relevance |
| CN111380964A (en) * | 2018-12-27 | 2020-07-07 | 云南生物谷药业股份有限公司 | Method for establishing fingerprint of schisandra chinensis medicinal material and erigeron breviscapus pulse-activating capsule |
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