CN1760667A - Method for controlling quality of simply single drug - Google Patents
Method for controlling quality of simply single drug Download PDFInfo
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- CN1760667A CN1760667A CN 200410083678 CN200410083678A CN1760667A CN 1760667 A CN1760667 A CN 1760667A CN 200410083678 CN200410083678 CN 200410083678 CN 200410083678 A CN200410083678 A CN 200410083678A CN 1760667 A CN1760667 A CN 1760667A
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Abstract
The invention discloses method for controlling quality of medicinal materials, intermediate of injection, and injection of simply single drug. In quality control of medicinal materials of simply single drug, main steps of method including for checking up fingerprints of medicinal materials, intermediate of injection, and injection are: preparing solutions to be tested, determining, recording spectrums of a graphs, determining referencing peaks and common peaks respectively for medicinal materials, intermediate of injection, and injection. In quality control of injection, main steps of method for measuring content of injection are: in comparison solution, and sampling solution, element of mignonette cannot be less than 0.1mg in 1ml.
Description
Invention field
The present invention relates to the method for quality control of a kind of Tibetan medicine medicinal material, injection intermediate and injection thereof, particularly the method for quality control of the medicinal material of Tibet medicine lamivphlomis root, injection intermediate and lamiophlomis rotata injection.
Background technology
The quality standard of Chinese medicine is very lack of standardization and perfect at present, and the means of quality analysis and evaluation are also very backward, can't reflect the quality condition of existing Chinese crude drug and finished product accurately, becomes Chinese medicine and goes abroad, the obstacle that goes to the world.Chinese crude drug and Chinese patent drug finger-print are the notions of using for reference fingerprint identification in the medical jurisprudence, with medicinal material or the medicine after suitably handling, obtain reflecting the total peak collection of illustrative plates of this medicinal material or medicine feature with certain analysis means.By the analysis of total peak collection of illustrative plates being distinguished, identified the quality of medicinal material or medicine, guarantee the stable and controllable for quality of medicine.Simultaneously, not only can carry out true and false discriminating, also allow the index components of Chinese medicine inherence quantize simultaneously, standardization raw medicinal material in conjunction with the content of the specific indexes composition in medicinal material or the medicine is done quantitative measurement.Be convenient to the quality of Chinese crude drug and Chinese patent drug is monitored.
Summary of the invention:
The object of the invention be open Tibet medicine lamivphlomis root medicinal material, injection intermediate and lamiophlomis rotata injection method of quality control, comprising the fingerprint atlas detection method of medicinal material, injection intermediate and the injection of lamiophlomis rotata and the content assaying method of lamiophlomis rotata injection.
The method of quality control of lamiophlomis rotata medicinal material is as follows, comprising the detection method of lamiophlomis rotata medicinal materials fingerprint is:
The preparation of the need testing solution material powder 4-6g that gets it filled adds ethanol 40-60ml, refluxing extraction 1.5-2.5 hour, filter, reclaim ethanol to there not being the alcohol flavor, residue adds water 15-25ml heating for dissolving, cross macroporous adsorptive resins, wash with water to eluent colourlessly earlier, continue with 8-15% ethanol 80-120ml wash-out, use 60-80% ethanol 80-120ml wash-out again, collect ethanol liquid, water bath method, dissolve with methanol is settled to 15-25m1, shakes up, and uses filtering with microporous membrane, get filtrate, promptly;
Determination method is drawn need testing solution 10 μ l, injects high performance liquid chromatograph, and wherein high performance liquid chromatograph is meant that with octadecyl silane be filling agent; The detection wavelength is 254nm; Column temperature: 35 ℃; Analysis time: 1 hour; Moving phase acetonitrile-0.2% phosphate aqueous solution; Mensuration write down the finger-print of lamiophlomis rotata medicinal material;
The characteristic peak of lamiophlomis rotata medicinal material test sample finger-print is: with No. 5 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as medicinal material.
Being prepared as of need testing solution wherein: get and pulverize the back and cross the medicinal powder 5g that internal diameter is the 5mm sieve, put in the flask, add ethanol 50ml, refluxing extraction 2 hours filters, and reclaims ethanol to there not being the alcohol flavor, residue adds water 20ml heating for dissolving, cross the D101 macroporous adsorptive resins, wash with water to eluent colourlessly earlier, continue with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol liquid, water bath method, dissolve with methanol is settled to 20ml, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly
Wherein moving phase acetonitrile-0.2% phosphate aqueous solution changes over time, adopts gradient elution; Flow velocity: 0.5ml/min; Moving phase situation over time sees Table;
Gradient elution
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Wherein the relative retention time of the characteristic peak of the finger-print of lamiophlomis rotata medicinal material test sample is:
Relative retention time (total peak number): 0.496 ± 0.025 (1), 0.740 ± 0.023 (2), 0.780 ± 0.017 (3), 0.886 ± 0.010 (4), 1 (S) (5), 1.035 ± 0.003 (6), 1.125 ± 0.015 (7), 1.168 ± 0.011 (8), 1.274 ± 0.018 (9), 1.339 ± 0.020 (10), 1.402 ± 0.025 (11), 1.776 ± 0.020 (12)
Wherein the peak area ratio of the characteristic peak of the finger-print of lamiophlomis rotata medicinal material test sample is:
Peak area ratio (total peak number): 0.067 ± 0.028 (1), 0.303 ± 0.176 (2), 0.091 ± 0.072 (3), 0.597 ± 0.185 (4), 1 (S) (5), 1.008 ± 0.419 (6), 0.143 ± 0.061 (7), 0.164 ± 0.062 (8), 0.114 ± 0.018 (9), 0.260 ± 0.048 (10), 0.087 ± 0.033 (11), 0.080 ± 0.051 (12)
See accompanying drawing 1: lamiophlomis rotata medicinal material reference fingerprint.
Lamiophlomis rotata injection intermediates preparation of the present invention is as follows:
Get the lamiophlomis rotata medicinal material, boiling three times, the water logging that adds 15～25 times was for the first time steeped 0.5～1 hour, decocted 0.5～1.5 hour, and respectively added 10～20 times of water gagings for the second time, for the third time, decocted respectively 0.5～1.5 hour, filter, merging filtrate is concentrated into liquor strength and is 1: 1～2 concentrate; Add ethanol and make and contain alcohol amount and reach 60%～80%, in 5～8 ℃ of refrigerations 12～24 hours, filter, filtrate adds ethanol again to be made and contains the alcohol amount and reach 80%～90%, in 5～8 ℃ of refrigerations 12～24 hours, filters; Filtrate is transferred PH to 8～9 with 30%～50% NaOH, in 5～8 ℃ of refrigerations 12～24 hours, filters; Filtrate adds 10%～30% sulfuric acid and transfers PH to 5～6, placed 1～3 hour, filter, reclaim ethanol, add water for injection to dissolving to there not being the alcohol flavor, 5～8 ℃ refrigerate 12～24 hours, filter, filtrate adds activated charcoal 0.05%～0.20%, boils 15～30 minutes, filter, get the lamiophlomis rotata injection intermediate.
Wherein the ratio of volume weight part is ml/g.
The fingerprint atlas detection method that comprises the lamiophlomis rotata injection intermediate in the method for quality control of injection intermediate:
Determination method is drawn need testing solution 10 μ l, injects high performance liquid chromatograph, and wherein high performance liquid chromatograph is meant that with octadecyl silane be filling agent; The detection wavelength is 254nm; Column temperature: 35 ℃; Analysis time: 1 hour; Moving phase acetonitrile-0.2% phosphate aqueous solution; Mensuration write down the finger-print of lamivphlomis root injection liquid intermediate;
The characteristic peak of lamivphlomis root injection liquid intermediate test sample finger-print is: with No. 6 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area value as the parenteral solution intermediate.
The wherein preparation of need testing solution: get parenteral solution intermediate 20ml by D101 macroporous resin column (Φ 20mm, column length 100mm), water elution is colourless to effluent, with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled to 20ml, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
Wherein moving phase acetonitrile-0.2% phosphate aqueous solution changes over time, adopts gradient elution; Flow velocity: 0.5ml/min; Moving phase situation over time sees Table;
The gradient table
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Wherein the relative retention time of the characteristic peak of the finger-print of lamiophlomis rotata injection intermediate test sample is:
Relative retention time (total peak number): 0.364 ± 0.058 (1), 0.521 ± 0.050 (2), 0.754 ± 0.031 (3), 0.792 ± 0.024 (4), 0.899 ± 0.005 (5), 1 (S) (6), 1.034 ± 0.009 (7), 1.158 ± 0.019 (8), 1.256 ± 0.021 (9), 1.377 ± 0.031 (10), 1.404 ± 0.030 (11), 1.754 ± 0.040 (12)
Wherein the peak area ratio of the characteristic peak of the finger-print of lamiophlomis rotata injection intermediate test sample is:
Peak area ratio (total peak number): 0.066 ± 0.032 (1), 0.106 ± 0.014 (2), 0.576 ± 0.070 (3), 0.042 ± 0.012 (4), 0.143 ± 0.045 (5), 1 (S) (6), 0.021 ± 0.010 (7), 0.133 ± 0.014 (8), 0.132 ± 0.017 (9), 0.081 ± 0.014 (10), 0.053 ± 0.013 (11), 0.125 ± 0.018 (12)
See accompanying drawing 2: lamiophlomis rotata injection intermediate reference fingerprint.
The preparation method of lamiophlomis rotata injection of the present invention is as follows:
Get the lamiophlomis rotata medicinal material, boiling three times, the water logging that adds 15～25 times was for the first time steeped 0.5～1 hour, decocted 0.5～1.5 hour, and respectively added 10～20 times of water gagings for the second time, for the third time, decocted respectively 0.5～1.5 hour, filter, merging filtrate is concentrated into liquor strength and is 1: 1～2 concentrate; Add ethanol and make and contain alcohol amount and reach 60%～80%, in 5～8 ℃ of refrigerations 12～24 hours, filter, filtrate adds ethanol again to be made and contains the alcohol amount and reach 80%～90%, in 5～8 ℃ of refrigerations 12～24 hours, filters; Filtrate is transferred PH to 8～9 with 30%～50% NaOH, in 5～8 ℃ of refrigerations 12～24 hours, filters; Filtrate adds 10%～30% sulfuric acid and transfers PH to 5～6, places 1～3 hour, filters, reclaim ethanol to there not being the alcohol flavor, add water for injection, filter to dissolving 8 ℃ of refrigerations 12～24 hours, filtrate adds activated charcoal 0.05%～0.20%, boils 15～30 minutes, filters, adding water for injection is 1 volume weight part with the medicinal material weight ratio extremely,, filtering with 0.22 μ m miillpore filter, can is in the 5ml ampoule, sterilization, promptly.
Wherein the ratio of volume weight part is ml/g.
The method of quality control of injection, the fingerprint image detection method that wherein contains lamiophlomis rotata injection is as follows:
The preparation precision of need testing solution is measured parenteral solution 15-25ml, cross macroporous adsorptive resins, it is colourless that distilled water is washed till eluent, continues with 8-15% ethanol 80-120ml wash-out, use 60-80% ethanol 80-120ml wash-out again, collect ethanol liquid, water bath method, dissolve with methanol is settled to 15-25ml, shake up, use filtering with microporous membrane, get filtrate, promptly;
Determination method is drawn need testing solution 10 μ l, injects high performance liquid chromatograph, and wherein high performance liquid chromatograph is with being filling agent with octadecyl silane; The detection wavelength is 254nm; Column temperature: 35 ℃; Analysis time: 1 hour; Moving phase is acetonitrile-0.2% phosphate aqueous solution; Mensuration write down the finger-print of lamivphlomis root injection liquid;
The characteristic peak of lamiophlomis rotata injection test sample finger-print is: with No. 6 total peaks, peak of peak area maximum as the reference peak, determine the stable characteristics peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as lamivphlomis root injection liquid finger-print.
Wherein the preparation precision of need testing solution is measured parenteral solution 20ml, by the D101 macroporous resin column, it is colourless that distilled water is eluted to effluent, with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled to 20ml, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
Wherein moving phase acetonitrile-0.2% phosphate aqueous solution changes over time, adopts gradient elution.Flow velocity: 0.5ml/min; Moving phase situation over time is as follows;
Gradient elution
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Wherein the relative retention time of the characteristic peak of the finger-print of lamiophlomis rotata injection test sample is:
Relative retention time (total peak number): 0.329 ± 0.014 (1), 0.482 ± 0.020 (2), 0.728 ± 0.018 (3), 0.754 ± 0.029 (4), 0.894 ± 0.005 (5), 1 (S) (6), 1.039 ± 0.002 (7), 1.175 ± 0.010 (8), 1.276 ± 0.011 (9), 1.404 ± 0.016 (10), 1.428 ± 0.015 (11), 0.937 ± 0.030 (12)
Wherein the peak area ratio of the characteristic peak of the finger-print of lamiophlomis rotata injection test sample is:
Peak area ratio (total peak number): 0.048 ± 0.024 (1), 0.078 ± 0.022 (2), 0.594 ± 0.104 (3), 0.143 ± 0.031 (4), 0.116 ± 0.055 (5), 1 (S) (6), 0.019 ± 0.007 (7), 0.155 ± 0.020 (8), 0.161 ± 0.030 (9), 0.096 ± 0.023 (10), 0.074 ± 0.024 (11), 0.110 ± 0.040 (12).
See accompanying drawing 3: the lamiophlomis rotata injection reference fingerprint.
The method of quality control of injection, wherein contain lamiophlomis rotata injection content assaying method as follows:
It is an amount of that the cyanidenon reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 0.012mg, in contrast product solution.
Parenteral solution 1-3ml is got in the preparation of need testing solution, adds the hydrochloric acid 0.5-1.5ml of 8-12%, water-bath backflow 0.5-1.5 hour, and dissolve with methanol is settled to 15-25ml, shakes up, and uses filtering with microporous membrane, gets filtrate, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, and wherein the liquid chromatograph octadecyl silane is a filling agent; Methyl alcohol-0.2% phosphoric acid solution 50-60: 40-50 is a moving phase; The detection wavelength is 350nm; Number of theoretical plate calculates by the cyanidenon peak should be not less than 2500; Measure wherein and to contain lamiophlomis rotata in the parenteral solution test sample and calculate with cyanidenon, every 1ml must not be less than 0.1mg.
Wherein parenteral solution 2ml is got in the preparation of need testing solution, adds 10% hydrochloric acid 1ml, and water-bath refluxed 1 hour, and dissolve with methanol is settled to 20ml, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly.
Wherein mobile phase methanol-0.2% phosphoric acid solution was the optimal flow phase with 55: 45;
In the experimental study of lamiophlomis rotata medicinal materials fingerprint, stability test is the result show: in 24 hours, similarity does not have obvious variation, and RSD% illustrates that all less than 0.5% need testing solution was stable in 24 hours.Precision test result shows: 5 times sample introduction phase similarity does not have obvious variation mutually, and RSD% shows that all less than 0.3% the precision of instrument is good.Reproducible test results shows that the similarity of measuring behind the 5 duplicate samples sample introductions does not have obvious variation mutually, illustrates that the reappearance of this method is good.The selection test findings that detects wavelength is outstanding in the big feature of response that 330nm and 254nm survey main chromatographic peak in the finger-print, and each chromatographic peak feature is more obvious under 254nm, so definite maximum absorption wavelength is 254nm.The 150mm chromatographic column is finally selected in the selection test of chromatographic column for use.It is 0.125min that gradient determination test retardation time gets gradient retardation time.The result showed after analysis time, sample introduction was investigated 2 hours, and in 60 minutes, all peaks all can be gone out by complete wash-out in the sample, so be defined as 1 hour analysis time.Select for use cyanidenon, Quercetin as object of reference, but these two kinds of compositions all there is not tangible chromatographic peak to occur in the medicinal material chromatogram, therefore fail to adopt; Select for use rutin that absorption is arranged at the 254nm place and Puerarin as internal standard compound, but medicinal material chromatogram complicated component, the degree of separation of internal standard compound and chromatographic peak is poor, does not therefore adopt; The comparison colours spectrogram selects for use No. 5 more stable in the chromatogram total peaks as the reference peak.
Through the finger-print experimental study of lamiophlomis rotata injection intermediate, Precision test result shows: similarity changes not remarkable, and RSD% illustrates that all less than 0.40% precision is good.Stability test is the result show: in 24 hours, similarity is mutually all greater than 0.98, and RSD%J illustrates that all less than 0.50% need testing solution is basicly stable in 24 hours.Reproducible test results shows that the finger-print similarity of measuring behind the 5 duplicate samples sample introductions is all greater than 0.98, and similarity changes not remarkable, illustrates that the reappearance of this method is good.
Lamivphlomis root injection liquid fingerprint image methodological study: Precision test result shows: similarity changes not remarkable, and RSD% illustrates that all less than 0.40% precision is good.Stability test is the result show: in 24 hours, similarity is mutually all greater than 0.98, changes not obviously, illustrates that test sample was stable in 24 hours.Reproducible test results shows that the finger-print similarity of measuring behind the 5 duplicate samples sample introductions is all greater than 0.98, and RSD% illustrates that all less than 0.60% the reappearance of this method is good.
Correlativity by parenteral solution and intermediate and medicinal materials fingerprint compares, and each chromatographic peak in the medicinal materials fingerprint substantially all has embodiment in intermediate and preparation finger, show that medicinal material and intermediate and preparation also have good correlativity; The collection of illustrative plates of intermediate and preparation is similar substantially, and the main chromatogram of intermediate is divided all embodiment in the chromatogram in preparation, show that intermediate and preparation have good correlativity.
The experimental study of the assay of lamiophlomis rotata injection, the mensuration of maximum absorption wavelength, according to spectrophotometry, the maximum absorption wavelength of cyanidenon is 350nm.Reference substance purity is investigated test findings and is shown that the purity of this reference substance is 100%.The test findings of interference test data sheet chromatogram shows that no chromatographic peak occurs on the corresponding position of cyanidenon reference substance chromatographic peak, shows blank noiseless.The investigation test of linear relationship promptly obtains the result by regression equation and shows, has favorable linearity in the scope of 0.076～0.380 μ g.Stability test is the result show, need testing solution was through 12 hours study on the stability, and the peak area integrated value changes less, and RSD% is 0.47%, illustrates that need testing solution was stable in 12 hours.Precision test result shows that the RSD% of 5 sample introductions is 0.46%, shows that precision is good.Replica test is the result show, the content mean value that 5 tests record cyanidenon in this batch sample is 0.127mg/ml, and RSD1.17% shows that repeatability is good.The average recovery rate of 5 average recovery tests is 99.74%, and RSD is 2.00%, shows that the recovery is good.The assay test determination result of sample shows that minimum content is 0.113mg/ml, floats downward 10%, is defined as the every ml of this injection and contains lamiophlomis rotata with cyanidenon (C
15O
6H
10) meter, must not be less than 0.1mg.
Following experimental example is used to further specify but is not limited to the present invention.
Experimental example 1: the selection of moving phase test in the measuring method:
By being that moving phase contrast is tested with methyl alcohol-0.2% phosphate aqueous solution and acetonitrile-0.2% phosphate aqueous solution, the result shows that when acetonitrile-0.2% phosphate aqueous solution was moving phase, the peak shape of chromatographic peak was good, each peak separates more complete, so select for use acetonitrile-0.2% phosphate aqueous solution as moving phase.Compare investigation with the moving phase of constant current and different gradient as the chromatographic condition of measuring respectively simultaneously, through test of many times, the content assaying method of injection: methyl alcohol-0.2% phosphoric acid solution 50-60: 40-50 is a moving phase; Wherein the optimal proportion of methyl alcohol-0.2% phosphoric acid solution is 55: 45; The moving phase of fingerprint map analyzing is all separated preferably with each component in the following gradient operation sample, and baseline is steady, so select for use as moving phase.
The gradient table
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Flow velocity is comparatively suitable with 0.5ml/min, and with this understanding, each peak separates better.
Experimental example 2: preparation method's optimization test of medicinal material need testing solution
Through overtesting: use high performance liquid chromatography, select simultaneously to test as detecting index, set up the finger-print of medicinal material with flavones ingredient in the lamiophlomis rotata.The test sample of medicinal material derives from fixedly Gansu Province's Wudu County of the place of production, makes medicinal material have enough representativenesses.
Need testing solution mainly is to extract and the rich flavones ingredient that amasss in the medicinal material, because the D101 macroreticular resin has the long-pending effect of good richness to flavones ingredient, and can remove desaccharification, the pigment impurity component plays the effect of purification of samples.By D101 macroporous adsorptive resins and the chromatogram comparison test of not passing through this post, better by each peak degree of separation in the chromatogram of macroporous adsorptive resins, characteristic is strong, and baseline is steady.Mistake D101 macroporous resin column was tested after therefore adding selected medicinal material extract for use.
Experimental example 3: its major parameter of lamiophlomis rotata medicinal material collection of illustrative plates characteristic peak is determined
The lamiophlomis rotata crude drug source
| Sample number into spectrum | The place of | Collecting time | |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 | Gande, Qinghai, Changdu, autonomous county of southern Mongols Tibet is scolded in Shiqu Qinghai, Ganzi, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu, Maqu, Gansu | 2002.9 2002.10 2002.9 2002.9 2002.9 2002.10 2001.10 2001.9 2001.8 2002.9 2002.8 2002.9 2002.8 2002.9 |
The lamiophlomis rotata medicinal materials fingerprint is analyzed, relatively, determined the characteristic fingerprint chromatographic peak of 12 total peaks as medicinal material.Because the used medicinal material of preparation is the medicinal material that produce the Maqu, Gansu, place of production relative fixed, therefore select fixedly 1～No. 10 test sample fingerprint map analyzing in the place of production, determine total fingerprint chromatogram peak, as the reference peak, calculate relative retention time (total peak number), peak area ratio (total peak number), average relative retention time and average peak area ratio with No. 5 total peaks.
Fixedly the ten batches of lamiophlomis rotata medicinal materials fingerprints in place of production test figure imports traditional Chinese medicine fingerprint similarity software, as calculated, draw the common pattern of lamiophlomis rotata medicinal materials fingerprint, the finger-print of each batch sample shows with similarity result: same place of production medicinal materials fingerprint is similar, and similarity result is all more than 0.9; The medicinal material in the different places of production is because the difference of natural climate condition, corresponding major component peak has appearred, but the ratio at each peak is not quite similar, and similarity differs greatly, and the medicinal material in the commercially available same place of production is owing to reasons such as collecting season and storages, similarity is starkly lower than other medicinal material, the medicinal materials fingerprint that this method is formulated has certain specificity, can obviously distinguish different produce medicinal material difference, for the discriminating of medicinal material provides reliable foundation, guaranteed the quality of the pharmaceutical preparations simultaneously.
Experimental example 4: the preparation method of intermediate need testing solution selects test
1. accurately measure midbody solution 2ml and put in the volumetric flask of 20ml, add methanol constant volume, shake up,, get filtrate, promptly with 0.45 μ m filtering with microporous membrane to scale.
2. get midbody solution 2ml, by D101 macroporous resin column (Φ 20mm, column length 100mm), water elution is colourless to effluent, with 10% ethanol 100ml wash-out, use the 100ml70% ethanol elution again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled in the 20ml measuring bottle, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
Draw above-mentioned two kinds of solution 10ul respectively, sample introduction is measured under above-mentioned chromatographic condition, the record chromatogram.The result shows: the test sample chromatogram that 1. method prepares, and the baseline injustice, the degree of separation at each peak is poor, though the test sample chromatogram that 2. the chromatographic peak ratio method prepares is many, characteristic is less, so selecting method 2 preparation need testing solutions.
Experimental example 5: the finger-print experimental study of lamiophlomis rotata injection intermediate
Do not have tangible composition in the finger-print as object of reference, do not have suitable material yet, therefore select metastable total peak as the reference peak as interior mark.Ten batches of lamiophlomis rotata intermediate finger-prints are analyzed, relatively determined total fingerprint chromatogram peak.With No. 6 total peaks as the reference peak, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio, the result: lamiophlomis rotata intermediate finger-print is isolated more than 20 chromatographic peak, through more therefrom having selected the stable characteristics peak of 12 total peaks as lamiophlomis rotata injection intermediate finger-print.Ten batches of lamiophlomis rotata intermediate test figures are imported computing machine, through traditional Chinese medicine fingerprint similarity computed in software, draw intermediate finger-print common pattern, each sample all compares with common pattern, composes similarity entirely and calculates.The result shows: similarity is not significant to be changed, and shows relative the stablizing of technology, can be used for the suitability for industrialized production of preparation.Show that simultaneously the intermediate finger-print that this method is formulated has certain specificity, can be used for the monitoring of technology, guarantee the quality of finished product.
Experimental example 6: the experimental study of lamiophlomis rotata injection finger-print
Ten batches of lamivphlomis root injection liquid finger-prints are analyzed, relatively determined total fingerprint chromatogram peak.With No. 6 total peaks as the reference peak, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio, the result: lamivphlomis root injection liquid finger-print is isolated more than 20 chromatographic peak, through more therefrom having selected the stable characteristics peak of individual 12 total peaks as lamivphlomis root injection liquid finger-print.Ten batches of lamivphlomis root injection liquid finger-print test figures are imported traditional Chinese medicine fingerprint similarity software, through traditional Chinese medicine fingerprint similarity computed in software, draw the common pattern of lamivphlomis root injection liquid finger-print, each sample all compares with common pattern, composes similarity entirely and calculates.
The result shows that the lamivphlomis root injection liquid finger-print that this method is formulated has certain specificity, can be used for the control of end product quality, guarantees the quality of finished product.
Experimental example 7: the experimental study of the assay of lamiophlomis rotata injection
With octadecyl silane is filling agent; Methyl alcohol-0.2% phosphoric acid solution (55: 45) is a moving phase; The detection wavelength is 350nm; Flow velocity is 1.0ml/min, and column temperature is 30 ℃, and number of theoretical plate is calculated as 3598 by the cyanidenon peak, should be lower than 3000 so number of theoretical plate calculates by the cyanidenon peak.
Content assaying method mainly is a spectrophotometric method, and the HPLC method is reference substance mensuration content with the cyanidenon, but mainly exists with the form of glycosides because of cyanidenon in the sample, and therefore free cyanidenon is seldom tested to designing and will be measured after the sample hydrolysis.
Determining of I acid addition
The acid addition is investigated
| 10% hydrochloric acid addition | 0.5ml | 1ml | 2ml |
| Content (mg/ml) | 0.117 | 0.131 | 0.129 |
Determining of II hydrolysis time
The investigation of hydrolysis time
| Hydrolysis time | 0.5 | 1 | 2 hours |
| Content (mg/ml) | 0.117 | 0.133 | 0.132 |
Therefore determine that precision is measured each 2ml of sample, add 10% hydrochloric acid 1ml, hydrolysis, 1.0 hours, methanol constant volume shakes up to putting in the 20ml measuring bottle, with 0.45 μ m filtering with microporous membrane, promptly.
Further specify the present invention below in conjunction with drawings and Examples
Description of drawings:
Accompanying drawing 1: lamiophlomis rotata medicinal material reference fingerprint
Accompanying drawing 2: lamiophlomis rotata injection intermediate reference fingerprint
Accompanying drawing 3: lamiophlomis rotata injection reference fingerprint
Embodiment 1: the method for quality control of lamiophlomis rotata medicinal material
(1) instrument and reagent: SeriesIII type high performance liquid chromatograph; SPD-6A type UV-detector; The N2010 chromatographic work station, Jiangsu Chinese nation chromatographic column (C18,150mm * 4.6mm, 5 μ m) BS110S type analysis balance, Sartorius AG produces; Computer aided similarity evaluation system software (1.290), Central South University modernization of Chinese medicine center, The Hong Kong Polytechnic University; Acetonitrile, chromatographically pure, import, MERCK company; Water is ultrapure water; It is pure that all the other reagent are analysis.
(2) collection of test sample: ten batches of medicinal materials getting different batches prepare test sample.
After getting and pulverize, the preparation of need testing solution crosses the medicinal powder 5g that internal diameter is the 5mm sieve, put in the flask, add ethanol 50ml, refluxing extraction 2 hours filters, reclaim ethanol to there not being the alcohol flavor, residue adds water 20ml heating for dissolving, crosses D101 macroporous adsorptive resins (Φ 20mm, column length 10cm), wash with water to eluent colourless earlier, continue with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol liquid, water bath method, dissolve with methanol is settled in the 20ml volumetric flask, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly
[finger-print] is according to the high performance liquid chromatography test.
Chromatographic condition and system suitability test are filling agent with octadecyl silane; The detection wavelength is 254nm.Column temperature: 35 ℃.Flow velocity: 0.5ml/min; Analysis time: 1 hour.
Gradient elution
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Determination method is drawn need testing solution 10 μ l, injects liquid chromatograph, measures.The record chromatogram.Test sample finger-print and reference fingerprint be machine similarity computed in software as calculated, and similarity meets the requirements greater than 0.90.
The characteristic peak of the test sample finger-print of lamiophlomis rotata medicinal material is: with No. 5 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as medicinal material.
Relative retention time (total peak number): 0.496 ± 0.025 (1), 0.740 ± 0.023 (2), 0.780 ± 0.017 (3), 0.886 ± 0.010 (4), 1 (S) (5), 1.035 ± 0.003 (6), 1.125 ± 0.015 (7), 1.168 ± 0.011 (8), 1.274 ± 0.018 (9), 1.339 ± 0.020 (10), 1.402 ± 0.025 (11), 1.776 ± 0.020 (12)
Peak area ratio (total peak number): 0.067 ± 0.028 (1), 0.303 ± 0.176 (2), 0.091 ± 0.072 (3), 0.597 ± 0.185 (4), 1 (S) (5), 1.008 ± 0.419 (6), 0.143 ± 0.061 (7), 0.164 ± 0.062 (8), 0.114 ± 0.018 (9), 0.260 ± 0.048 (10), 0.087 ± 0.033 (11), 0.080 ± 0.051 (12).
Embodiment 2: the preparation of lamiophlomis rotata injection intermediate and method of quality control
The method for making of lamiophlomis rotata injection intermediate: get the lamiophlomis rotata medicinal material, clean, be cut into 1～2cm segment, take by weighing 1000g, boiling three times adds 20 times water for the first time, soaked 0.5 hour, and decocted 1 hour, for the second time, respectively add 15 times of water gagings for the third time, decocted respectively 1 hour, and filtered merging filtrate, be concentrated into about 500ml, add ethanol and make and contain alcohol amount and reach 70%, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate adds ethanol again made content reach 85%, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate was transferred PH to 8～9 with 40% NaOH, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate adds 20% sulfuric acid and transfers PH5～6, places 1 hour, filter, reclaim ethanol, add water for injection to 800ml to there not being the alcohol flavor, 5～8 ℃ refrigerate 12 hours, filter, filtrate adds activated charcoal 0.05%, boils 15 minutes, filter, promptly get intermediate.
The intermediate finger-print:
(1) instrument and reagent: with the instrument and the reagent of the determining fingerprint pattern of lamiophlomis rotata medicinal material.
(2) collection of test sample: ten batches of intermediate preparation test samples getting different batches.
The preparation of need testing solution: get the intermediate 20ml of parenteral solution, by D101 macroporous resin column (Φ 20mm, column length 100mm), water elution is colourless to effluent, with 10% ethanol 100ml wash-out, use the 100ml70% ethanol elution again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled in the 20ml measuring bottle, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
[finger-print] is according to the high performance liquid chromatography test.
Gradient elution is adopted in chromatographic condition and system suitability test, and moving phase situation over time sees the following form.
The gradient table
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Flow velocity: 0.5ml/min; Detect wavelength: 254nm.
Column temperature: 35 ℃; Analysis time: 1 hour.
Determination method is drawn need testing solution 10 μ l, injects liquid chromatograph, measures.The record chromatogram.Test sample finger-print and reference fingerprint be machine similarity computed in software as calculated, and similarity meets the requirements greater than 0.90.
The characteristic peak of lamivphlomis root injection liquid intermediate test sample finger-print is: with No. 6 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as medicinal material.
Lamiophlomis rotata intermediate finger-print characteristic peak is:
Relative retention time (total peak number): 0.364 ± 0.058 (1), 0.521 ± 0.050 (2), 0.754 ± 0.031 (3), 0.792 ± 0.024 (4), 0.899 ± 0.005 (5), 1 (S) (6), 1.034 ± 0.009 (7), 1.158 ± 0.019 (8), 1.256 ± 0.021 (9), 1.377 ± 0.031 (10), 1.404 ± 0.030 (11), 1.754 ± 0.040 (12)
Peak area ratio (total peak number): 0.066 ± 0.032 (1), 0.106 ± 0.014 (2), 0.576 ± 0.070 (3), 0.042 ± 0.012 (4), 0.143 ± 0.045 (5), 1 (S) (6), 0.021 ± 0.010 (7), 0.133 ± 0.014 (8), 0.132 ± 0.017 (9), 0.081 ± 0.014 (10), 0.053 ± 0.013 (11), 0.125 ± 0.018 (12)
Embodiment 3: the preparation of lamiophlomis rotata injection and method of quality control
Lamiophlomis rotata injection method for making: get the lamiophlomis rotata medicinal material, clean, be cut into 1～2cm segment, take by weighing 1000g, boiling three times adds 20 times water for the first time, soaked 0.5 hour, and decocted 1 hour, for the second time, respectively add 15 times of water gagings for the third time, decocted respectively 1 hour, and filtered merging filtrate, be concentrated into about 500ml, add ethanol and make and contain alcohol amount and reach 70%, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate adds ethanol again made content reach 85%, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate was transferred PH to 8～9 with 40% NaOH, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate adds 20% sulfuric acid and transfers PH5～6, places 1 hour, filter, reclaim ethanol, add water for injection to 800ml to there not being the alcohol flavor, 5～8 ℃ refrigerate 12 hours, filter, filtrate adds activated charcoal 0.05%, boils 15 minutes, filter, add water for injection to 1000ml, filter with 0.22 μ m miillpore filter, can is in the 5ml ampoule, sterilization, promptly.
(1) instrument and reagent: with the instrument and the reagent of the determining fingerprint pattern of lamiophlomis rotata medicinal material.
(2) collection of test sample: ten batches of injections getting different batches prepare test sample.
Parenteral solution 20ml is got in the preparation of need testing solution, by the D101 macroporous resin column (Φ 20mm, 10cm), it is colourless that distilled water is eluted to effluent, with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled in the 20ml volumetric flask, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
[finger-print] is according to the high performance liquid chromatography test.
Chromatographic condition and system suitability test are filling agent with octadecyl silane; The detection wavelength is 254nm.Column temperature: 35 ℃.Flow velocity: 0.5ml/min; Analysis time: 1 hour.
Gradient elution
| Time (min) | Acetonitrile (%) | 0.2% phosphate aqueous solution (%) |
| 0 10 40 50 60 | 15 15 30 40 40 | 85 85 70 60 60 |
Determination method is drawn need testing solution 10 μ l, injects liquid chromatograph, measures.Peak with peak area maximum in the chromatogram is with reference to the peak.
Test sample finger-print and reference fingerprint be machine similarity computed in software as calculated, and similarity meets the requirements greater than 0.90.
The characteristic peak of lamivphlomis root injection liquid test sample finger-print is: with No. 6 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as medicinal material.
Relative retention time (total peak number): 0.329 ± 0.014 (1), 0.482 ± 0.020 (2), 0.728 ± 0.018 (3), 0.754 ± 0.029 (4), 0.894 ± 0.005 (5), 1 (S) (6), 1.039 ± 0.002 (7), 1.175 ± 0.010 (8), 1.276 ± 0.011 (9), 1.404 ± 0.016 (10), 1.428 ± 0.015 (11), 0.937 ± 0.030 (12)
Peak area ratio (total peak number): 0.048 ± 0.024 (1), 0.078 ± 0.022 (2), 0.594 ± 0.104 (3), 0.143 ± 0.031 (4), 0.116 ± 0.055 (5), 1 (S) (6), 0.019 ± 0.007 (7), 0.155 ± 0.020 (8), 0.161 ± 0.030 (9), 0.096 ± 0.023 (10), 0.074 ± 0.024 (11), 0.110 ± 0.040 (12)
Embodiment 4: the method for quality control assay of lamiophlomis rotata injection
Test according to high performance liquid chromatography.
Chromatographic condition and system suitability test are filling agent with octadecyl silane; Methyl alcohol-0.2% phosphoric acid solution (55: 45) is a moving phase; The detection wavelength is 350nm; Number of theoretical plate calculates by the cyanidenon peak should be not less than 2500.
It is an amount of that the cyanidenon reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 0.012mg, in contrast product solution.
This product 2ml is got in the preparation of need testing solution, adds 10% hydrochloric acid 1ml, and water-bath refluxed 1 hour, and dissolve with methanol is settled in the 20ml measuring bottle, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, and calculate by external standard method.
This product contains lamiophlomis rotata to be calculated with cyanidenon, and every 1ml must not be less than 0.1mg.
Claims (26)
1, a kind of method of quality control of lamiophlomis rotata medicinal material is characterized in that the assay method that comprises finger-print in this method is:
The preparation of the need testing solution material powder 4-6g that gets it filled adds ethanol 40-60ml, refluxing extraction 1.5-2.5 hour, filter, reclaim ethanol to there not being the alcohol flavor, residue adds water 15-25ml heating for dissolving, cross macroporous adsorptive resins, wash with water to eluent colourlessly earlier, continue with 8-15% ethanol 80-120ml wash-out, use 60-80% ethanol 80-120ml wash-out again, collect ethanol liquid, water bath method, dissolve with methanol is settled to 15-25ml, shakes up, with 0.25～0.65 μ m filtering with microporous membrane, get filtrate, promptly;
Determination method is drawn need testing solution 10 μ l, injects high performance liquid chromatograph, and wherein the high performance liquid chromatograph octadecyl silane is a filling agent; The detection wavelength is 254nm; Column temperature: 35 ℃; Analysis time: 1 hour; Moving phase is acetonitrile-0.2% phosphate aqueous solution; Mensuration write down the finger-print of lamiophlomis rotata medicinal material;
The characteristic peak of the finger-print of lamiophlomis rotata medicinal material is: with No. 5 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as medicinal material.
2, the method for quality control of lamiophlomis rotata medicinal material as claimed in claim 1 is characterized in that wherein the preparation method of need testing solution is:
Get and cross the medicinal powder 5g that internal diameter is the 5mm sieve after pulverizing, put in the flask, add ethanol 50ml, refluxing extraction 2 hours filters, and reclaims ethanol to there not being the alcohol flavor, residue adds water 20ml heating for dissolving, crosses the D101 macroporous adsorptive resins, washes with water to eluent colourless earlier, continue with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol liquid, water bath method, dissolve with methanol is settled to 20ml, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
3, the method for quality control of lamiophlomis rotata medicinal material as claimed in claim 1 or 2 is characterized in that wherein moving phase acetonitrile-0.2% phosphate aqueous solution changes over time, adopts gradient elution; Flow velocity: 0.5ml/min; Moving phase situation over time is as follows:
The proportionate relationship that adds 15% acetonitrile solution by 85% 0.2% phosphate aqueous solution in the time of 0 to 10 minute is carried out wash-out, and from 10 to 40 minutes, reduce to 70% by 0.2% phosphate aqueous solution by 85%, acetonitrile is raised to 30% proportionate relationship by 15% and carries out wash-out; From 40 to 50 minutes, reduce to 60% by 0.2% phosphate aqueous solution by 70%, acetonitrile is raised to 40% proportionate relationship by 30% and carries out wash-out; The proportionate relationship that adds 40% acetonitrile solution by 60% 0.2% phosphate aqueous solution in the time of 50 to 60 minutes is carried out wash-out.
4, the method for quality control of lamiophlomis rotata medicinal material as claimed in claim 1 or 2 is characterized in that the relative retention time of characteristic peak of the finger-print of lamiophlomis rotata medicinal material is:
No. 1 peak: 0.496 ± 0.025, No. 2 peaks: 0.740 ± 0.023, No. 3 peaks: 0.780 ± 0.017, No. 4 peaks: 0.886 ± 0.010, No. 5 peaks: 1 (S), No. 6 peaks: 1.035 ± 0.003, No. 7 peaks: 1.125 ± 0.015, No. 8 peaks: 1.168 ± 0.011, No. 9 peaks: 1.274 ± 0.018, No. 10 peaks: 1.339 ± 0.020, No. 11 peaks: 1.402 ± 0.025, No. 12 peaks: 1.776 ± 0.020.
5, the method for quality control of lamiophlomis rotata medicinal material as claimed in claim 1 or 2 is characterized in that the peak area ratio of characteristic peak of the finger-print of lamiophlomis rotata medicinal material is:
No. 1 peak: 0.067 ± 0.028, No. 2 peaks: 0.303 ± 0.176, No. 3 peaks: 0.091 ± 0.072, No. 4 peaks: 0.597 ± 0.185,5S peak: 1, No. 6 peaks: 1.008 ± 0.419, No. 7 peaks: 0.143 ± 0.061, No. 8 peaks: 0.164 ± 0.062, No. 9 peaks: 0.114 ± 0.018, No. 10 peaks: 0.260 ± 0.048, No. 11 peaks: 0.087 ± 0.033, No. 12 peaks: 0.080 ± 0.051.
6, the finger-print of a kind of lamiophlomis rotata medicinal material as claimed in claim 3 is characterized in that the relative retention time of characteristic peak of the finger-print of lamiophlomis rotata medicinal material is:
No. 1 peak: 0.496 ± 0.025, No. 2 peaks: 0.740 ± 0.023, No. 3 peaks: 0.780 ± 0.017, No. 4 peaks: 0.886 ± 0.010,5S peak: 1, No. 6 peaks: 1.035 ± 0.003, No. 7 peaks: 1.125 ± 0.015, No. 8 peaks: 1.168 ± 0.011, No. 9 peaks: 1.274 ± 0.018, No. 10 peaks: 1.339 ± 0.020, No. 11 peaks: 1.402 ± 0.025, No. 12 peaks: 1.776 ± 0.020.
7, the finger-print of a kind of lamiophlomis rotata medicinal material as claimed in claim 3, it is characterized in that the lamiophlomis rotata medicinal material finger-print characteristic peak peak area ratio be:
No. 1 peak: 0.067 ± 0.028 (1), No. 2 peaks: 0.303 ± 0.176 (2), No. 3 peaks: 0.091 ± 0.072, No. 4 peaks: 0.597 ± 0.185, No. 5 peaks: 1 (S), No. 6 peaks: 1.008 ± 0.419, No. 7 peaks: 0.143 ± 0.061, No. 8 peaks: 0.164 ± 0.062, No. 9 peaks: 0.114 ± 0.018, No. 10 peaks: 0.260 ± 0.048, No. 11 peaks: 0.087 ± 0.033, No. 12 peaks: 0.080 ± 0.051.
8, a kind of intermediates preparation of lamiophlomis rotata injection is characterized in that this method comprises the steps:
Get the lamiophlomis rotata medicinal material, boiling three times, the water logging that adds 15～25 times was for the first time steeped 0.5～1 hour, decocted 0.5～1.5 hour, and respectively added 10～20 times of water gagings for the second time, for the third time, decocted respectively 0.5～1.5 hour, filter, merging filtrate is concentrated into liquor strength and is 1: 1～2 concentrate; Add ethanol and make and contain alcohol amount and reach 60%～80%, in 5～8 ℃ of refrigerations 12～24 hours, filter, filtrate adds ethanol again makes content reach 80%～90%, in 5～8 ℃ of refrigerations 12～24 hours, filters; Filtrate is transferred PH to 8～9 with 30%～50% NaOH, in 5～8 ℃ of refrigerations 12～24 hours, filters; Filtrate adds 10%～30% sulfuric acid and transfers PH to 5～6, placed 1～3 hour, filter, reclaim ethanol, add water for injection to dissolving to there not being the alcohol flavor, 5～8 ℃ refrigerate 12～24 hours, filter, filtrate adds activated charcoal 0.05%～0.20%, boils 15～30 minutes, filter, get the intermediate of lamiophlomis rotata injection.
9, the intermediates preparation of lamiophlomis rotata injection as claimed in claim 8 is characterized in that this method comprises the steps:
Get the lamiophlomis rotata medicinal material, boiling three times, the water logging that adds 20 times was for the first time steeped 0.5 hour, decocted 1 hour, for the second time, respectively add 15 times of water gagings for the third time and soaked 0.5 hour, decocted respectively 1 hour, filter, merging filtrate is concentrated into liquor strength and is 1: 2 concentrate, add ethanol and make and contain alcohol amount and reach 70%,, filter in 5～8 ℃ of refrigerations 12 hours, filtrate adds ethanol again makes content reach 85%, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate is transferred PH to 8～9 with 40% NaOH, in 5～8 ℃ of refrigerations 12 hours, filter, filtrate adds 20% sulfuric acid and transfers PH5～6, places 1 hour, filter, reclaim ethanol to there not being the alcohol flavor, add water for injection, filter to dissolving 8 ℃ of refrigerations 12 hours, filtrate adds activated charcoal 0.05%, boiled 15 minutes, and filtered, get the intermediate of lamiophlomis rotata injection.
10, a kind of method of quality control of intermediate of lamiophlomis rotata injection is characterized in that the assay method that comprises finger-print in this method is:
The preparation precision of need testing solution is measured parenteral solution midbody solution 15～25ml and is crossed macroporous adsorptive resins, it is colourless that distilled water is washed till eluent, continue with 8-15% ethanol 80-120ml wash-out, use 60-80% ethanol 80-120ml wash-out again, collect ethanol liquid, water bath method, dissolve with methanol is settled to 15-25ml, shakes up, and uses filtering with microporous membrane, get filtrate, promptly;
Determination method is drawn need testing solution 10 μ l, injects high performance liquid chromatograph, and wherein high performance liquid chromatograph is meant that with octadecyl silane be filling agent; The detection wavelength is 254nm; Column temperature: 35 ℃; Analysis time: 1 hour; Moving phase acetonitrile-0.2% phosphate aqueous solution; Mensuration write down the finger-print of lamiophlomis rotata injection intermediate;
The characteristic peak of lamivphlomis root injection liquid intermediate test sample finger-print is: with No. 6 total peaks, peak of peak area maximum as the reference peak, determine the characteristic fingerprint chromatographic peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as the injection intermediate.
11, the method for quality control of the intermediate of lamiophlomis rotata injection as claimed in claim 10 is characterized in that wherein the preparation method of need testing solution is:
Precision is measured parenteral solution midbody solution 20ml, by the D101 macroporous resin column, water elution is colourless to effluent, with 10% ethanol 100ml wash-out, use the 100ml70% ethanol elution again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled to 20ml, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
12, as the method for quality control of the intermediate of claim 10 or 11 described lamiophlomis rotata injections, it is characterized in that wherein moving phase acetonitrile-0.2% phosphate aqueous solution changes over time, adopt gradient elution; Flow velocity: 0.5ml/min; Moving phase situation over time is as follows:
The proportionate relationship that adds 15% acetonitrile solution by 85% 0.2% phosphate aqueous solution in the time of 0 to 10 minute is carried out wash-out, and from 10 to 40 minutes, reduce to 70% by 0.2% phosphate aqueous solution by 85%, acetonitrile is raised to 30% proportionate relationship by 15% and carries out wash-out; From 40 to 50 minutes, reduce to 60% by 0.2% phosphate aqueous solution by 70%, acetonitrile is raised to 40% proportionate relationship by 30% and carries out wash-out; The proportionate relationship that adds 40% acetonitrile solution by 60% 0.2% phosphate aqueous solution in the time of 50 to 60 minutes is carried out wash-out.
13,, it is characterized in that the relative retention time of characteristic peak of finger-print of the intermediate of lamiophlomis rotata injection is as the method for quality control of the intermediate of claim 10 or 11 described lamiophlomis rotata injections:
No. 1 peak: 0.364 ± 0.058, No. 2 peaks: 0.521 ± 0.050, No. 3 peaks: 0.754 ± 0.031, No. 4 peaks: 0.792 ± 0.024, No. 5 peaks: 0.899 ± 0.005, No. 6 peaks: 1 (S), No. 7 peaks: 1.034 ± 0.009, No. 8 peaks: 1.158 ± 0.019, No. 9 peaks: 1.256 ± 0.021, No. 10 peaks: 1.377 ± 0.031, No. 11 peaks: 1.404 ± 0.030, No. 12 peaks: 1.754 ± 0.040.
14,, it is characterized in that the peak area ratio of characteristic peak of finger-print of the intermediate of lamiophlomis rotata injection is as the method for quality control of the intermediate of claim 10 or 11 described lamiophlomis rotata injections:
No. 1 peak: 0.066 ± 0.032, No. 2 peaks: 0.106 ± 0.014, No. 3 peaks: 0.576 ± 0.070, No. 4 peaks: 0.042 ± 0.012, No. 5 peaks: 0.143 ± 0.045, No. 6 peaks: 1 (S), No. 7 peaks: 0.021 ± 0.010, No. 8 peaks: 0.133 ± 0.014, No. 9 peaks: 0.132 ± 0.017, No. 10 peaks: 0.081 ± 0.014, No. 11 peaks: 0.053 ± 0.013, No. 12 peaks: 0.125 ± 0.018.
15, the finger-print of the intermediate of a kind of lamiophlomis rotata injection as claimed in claim 12 is characterized in that the relative retention time of characteristic peak of finger-print of the intermediate of lamiophlomis rotata injection is:
No. 1 peak: 0.364 ± 0.058, No. 2 peaks: 0.521 ± 0.050, No. 3 peaks: 0.754 ± 0.031, No. 4 peaks: 0.792 ± 0.024, No. 5 peaks: 0.899 ± 0.005, No. 6 peaks: 1 (S), No. 7 peaks: 1.034 ± 0.009, No. 8 peaks: 1.158 ± 0.019, No. 9 peaks: 1.256 ± 0.021, No. 10 peaks: 1.377 ± 0.031, No. 11 peaks: 1.404 ± 0.030, No. 12 peaks: 1.754 ± 0.040.
16, the finger-print of the intermediate of a kind of lamiophlomis rotata injection as claimed in claim 12, it is characterized in that lamiophlomis rotata injection intermediate finger-print characteristic peak peak area ratio be:
No. 1 peak: 0.066 ± 0.032, No. 2 peaks: 0.106 ± 0.014, No. 3 peaks: 0.576 ± 0.070, No. 4 peaks: 0.042 ± 0.012, No. 5 peaks: 0.143 ± 0.045, No. 6 peaks: 1 (S), No. 7 peaks: 0.021 ± 0.010, No. 8 peaks: 0.133 ± 0.014, No. 9 peaks: 0.132 ± 0.017, No. 10 peaks: 0.081 ± 0.014, No. 11 peaks: 0.053 ± 0.013, No. 12 peaks: 0.125 ± 0.018.
17, a kind of method of quality control of lamiophlomis rotata injection is characterized in that comprising the detection method of lamiophlomis rotata injection finger-print being:
The preparation precision of need testing solution is measured parenteral solution solution 15～25ml, cross macroporous adsorptive resins, it is colourless that distilled water is washed till eluent, continues with 8-15% ethanol 80-120ml wash-out, use 60-80% ethanol 80-120ml wash-out again, collect ethanol liquid, water bath method, dissolve with methanol is settled to 15-25ml, shake up, with 0.25～0.65 μ m filtering with microporous membrane, get filtrate, promptly;
Determination method is drawn need testing solution 10 μ l, injects high performance liquid chromatograph, and wherein the high performance liquid chromatograph octadecyl silane is a filling agent; The detection wavelength is 254nm; Column temperature: 35 ℃; Analysis time: 1 hour; Moving phase is acetonitrile-0.2% phosphate aqueous solution; Mensuration write down the finger-print of lamiophlomis rotata medicinal material;
The characteristic peak of lamiophlomis rotata injection finger-print is: with No. 6 total peaks, peak of peak area maximum as the reference peak, determine the stable characteristics peak of 12 total peaks, calculate relative retention time, peak area ratio, average relative retention time and average peak area ratio as lamivphlomis root injection liquid finger-print.
18, the method for quality control of injection as claimed in claim 17, its feature preparation method of need testing solution therein are:
Precision is measured parenteral solution solution 20ml, by the D101 macroporous resin column, it is colourless that distilled water is eluted to effluent, with 10% ethanol 100ml wash-out, use 70% ethanol 100ml wash-out again, collect 70% ethanol eluate, water bath method, dissolve with methanol is settled to 20ml, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
19, as the method for quality control of claim 17 or 18 described lamiophlomis rotata injections, it is characterized in that wherein moving phase acetonitrile-0.2% phosphate aqueous solution changes over time, adopt gradient elution; Flow velocity: 0.5ml/min; Moving phase situation over time is as follows:
The proportionate relationship that adds 15% acetonitrile solution by 85% 0.2% phosphate aqueous solution in the time of 0 to 10 minute is carried out wash-out, and from 10 to 40 minutes, reduce to 70% by 0.2% phosphate aqueous solution by 85%, acetonitrile is raised to 30% proportionate relationship by 15% and carries out wash-out; From 40 to 50 minutes, reduce to 60% by 0.2% phosphate aqueous solution by 70%, acetonitrile is raised to 40% proportionate relationship by 30% and carries out wash-out; The proportionate relationship that adds 40% acetonitrile solution by 60% 0.2% phosphate aqueous solution in the time of 50 to 60 minutes is carried out wash-out.
20,, it is characterized in that the relative retention time of characteristic peak of the finger-print of lamiophlomis rotata injection is as the method for quality control of claim 17 or 18 described lamiophlomis rotata injections:
No. 1 peak: 0.329 ± 0.014, No. 2 peaks: 0.482 ± 0.020, No. 3 peaks: 0.576 ± 0.070, No. 4 peaks: 0.754 ± 0.029, No. 5 peaks: 0.894 ± 0.005, No. 6 peaks: 1 (S), No. 7 peaks: 1.039 ± 0.002, No. 8 peaks: 1.175 ± 0.010, No. 9 peaks: 1.276 ± 0.011, No. 10 peaks: 1.404 ± 0.016, No. 11 peaks: 1.428 ± 0.015, No. 12 peaks: 0.937 ± 0.030.
21,, it is characterized in that the peak area ratio of characteristic peak of the finger-print of lamiophlomis rotata injection is as the method for quality control of claim 17 or 18 described lamiophlomis rotata injections:
No. 1 peak: 0.048 ± 0.024, No. 2 peaks: 0.078 ± 0.022, No. 3 peaks: 0.594 ± 0.104, No. 4 peaks: 0.143 ± 0.031, No. 5 peaks: 0.116 ± 0.055, No. 6 peaks: 1 (S), No. 7 peaks: 0.019 ± 0.007, No. 8 peaks: 0.155 ± 0.020, No. 9 peaks: 0.161 ± 0.030, No. 10 peaks: 0.096 ± 0.023, No. 11 peaks: 0.074 ± 0.024, No. 12 peaks: 0.110 ± 0.040.
22, the method for quality control of lamiophlomis rotata injection as claimed in claim 19 is characterized in that the relative retention time of characteristic peak of the finger-print of lamiophlomis rotata injection is:
No. 1 peak: 0.329 ± 0.014, No. 2 peaks: 0.482 ± 0.020, No. 3 peaks: 0.576 ± 0.070, No. 4 peaks: 0.754 ± 0.029, No. 5 peaks: 0.894 ± 0.005, No. 6 peaks: 1 (S), No. 7 peaks: 1.039 ± 0.002, No. 8 peaks: 1.175 ± 0.010, No. 9 peaks: 1.276 ± 0.011, No. 10 peaks: 1.404 ± 0.016, No. 11 peaks: 1.428 ± 0.015, No. 12 peaks: 0.937 ± 0.030.
23, the method for quality control of lamiophlomis rotata injection as claimed in claim 19 is characterized in that the peak area ratio of characteristic peak of the finger-print of lamiophlomis rotata injection is:
No. 1 peak: 0.048 ± 0.024, No. 2 peaks: 0.078 ± 0.022, No. 3 peaks: 0.594 ± 0.104, No. 4 peaks: 0.143 ± 0.031, No. 5 peaks: 0.116 ± 0.055, No. 6 peaks: 1 (S), No. 7 peaks: 0.019 ± 0.007, No. 8 peaks: 0.155 ± 0.020, No. 9 peaks: 0.161 ± 0.030, No. 10 peaks: 0.096 ± 0.023, No. 11 peaks: 0.074 ± 0.024, No. 12 peaks: 0.110 ± 0.040.
24, a kind of method of quality control of lamiophlomis rotata injection is characterized in that comprising the lamiophlomis rotata injection content assaying method being:
It is an amount of that the cyanidenon reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 0.012mg, in contrast product solution;
Parenteral solution 1-3ml is got in the preparation of need testing solution, adds the hydrochloric acid 0.5-1.5ml of 8-12%, water-bath backflow 0.5-1.5 hour, and dissolve with methanol is settled to 15-25ml, shakes up, and with 0.25～0.65 μ m filtering with microporous membrane, gets filtrate, promptly;
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, and wherein the liquid chromatograph octadecyl silane is a filling agent; Methyl alcohol-0.2% phosphoric acid solution 50-60:40-50 is a moving phase; The detection wavelength is 350nm; Number of theoretical plate calculates by the cyanidenon peak should be not less than 2500; Contain lamiophlomis rotata in the mensuration parenteral solution test sample and calculate with cyanidenon, every 1ml must not be less than 0.1mg.
25, the method for quality control of lamiophlomis rotata injection as claimed in claim 24 is characterized in that wherein the preparation method of need testing solution is:
Get parenteral solution 2ml, add 10% hydrochloric acid 1ml, water-bath refluxed 1 hour, and dissolve with methanol is settled to 20ml, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly.
26,, it is characterized in that wherein with 55: 45 methyl alcohol-0.2% phosphoric acid solution as moving phase as the method for quality control of claim 24 or 25 described lamiophlomis rotata injections.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1947740B (en) * | 2006-11-09 | 2010-05-12 | 成都优他制药有限责任公司 | Lamiophlomis rotata medicine material, intermediate and its injection liquid finger-print atlas quality testing method |
| CN101078713B (en) * | 2007-06-28 | 2010-08-11 | 上海现代中医药技术发展有限公司 | Fingerprint detection method of gynostemma pentaphylla medicine added with internal standard |
| CN102397324A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院兰州化学物理研究所 | Method for determining phenylpropanoid glycoside substances in Tibetan medicine Lamiophlomis rotata |
| CN104237441A (en) * | 2014-10-09 | 2014-12-24 | 成都中医药大学 | Method for simultaneous detection of iridoid glycoside, phenylethanoid glycoside, flavone and dicaffeoyl ingredients in lamiophlomis rotata |
| CN104237440A (en) * | 2014-10-09 | 2014-12-24 | 成都中医药大学 | Lamiophlomis rotata kudo HPLC (High Performance Liquid Chromatography) detection method and finger-print detection technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP4886933B2 (en) * | 2001-01-12 | 2012-02-29 | カウンセル オブ サイエンティフィック アンド インダストリアル リサーチ | A novel method for standardization of chromatographic fingerprints and single medicines and formulations |
| CN1141575C (en) * | 2001-06-29 | 2004-03-10 | 天津市金士力药物研究开发有限公司 | Red sage medicine fingerprint establishing method and standard fingerprint atlas |
| US20040018260A1 (en) * | 2002-06-19 | 2004-01-29 | Novemed Group Limited | Novel botanical extract of Tripterygium Wilfordii Hook F. |
| CN1333248C (en) * | 2003-07-14 | 2007-08-22 | 北京中医药大学 | Chinese medicine quality control method |
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| CN1947740B (en) * | 2006-11-09 | 2010-05-12 | 成都优他制药有限责任公司 | Lamiophlomis rotata medicine material, intermediate and its injection liquid finger-print atlas quality testing method |
| CN101078713B (en) * | 2007-06-28 | 2010-08-11 | 上海现代中医药技术发展有限公司 | Fingerprint detection method of gynostemma pentaphylla medicine added with internal standard |
| CN102397324A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院兰州化学物理研究所 | Method for determining phenylpropanoid glycoside substances in Tibetan medicine Lamiophlomis rotata |
| CN102397324B (en) * | 2010-09-10 | 2013-11-27 | 中国科学院兰州化学物理研究所 | Method for determining phenylpropanoid glycoside substances in Tibetmedicine Lamiophlomis rotata |
| CN104237441A (en) * | 2014-10-09 | 2014-12-24 | 成都中医药大学 | Method for simultaneous detection of iridoid glycoside, phenylethanoid glycoside, flavone and dicaffeoyl ingredients in lamiophlomis rotata |
| CN104237440A (en) * | 2014-10-09 | 2014-12-24 | 成都中医药大学 | Lamiophlomis rotata kudo HPLC (High Performance Liquid Chromatography) detection method and finger-print detection technology |
| CN104237441B (en) * | 2014-10-09 | 2015-07-22 | 成都中医药大学 | Method for simultaneous detection of iridoid glycoside, phenylethanoid glycoside, flavone and dicaffeoyl ingredients in lamiophlomis rotata |
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