CN1947740B - Lamiophlomis rotata medicine material, intermediate and its injection liquid finger-print atlas quality testing method - Google Patents
Lamiophlomis rotata medicine material, intermediate and its injection liquid finger-print atlas quality testing method Download PDFInfo
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Abstract
A fingerprint method for testing the quality of common lamiophlomis, its intermediate and its injection includes such steps as preparing the reference solution from quercetin, preparing three solutions of common lamiophlomis, its intermediate and its injection, filling the reference solution and one solution to be tested into liquid-phase chromatograph, and testing the similarity between their fingerprints, which must be greater than 0.90.
Description
Technical field
The present invention relates to the quality determining method of Chinese crude drug, intermediate and parenteral solution thereof, particularly the finger print quality detecting method of lamiophlomis rotata medicinal material, intermediate and parenteral solution thereof.
Background technology
Main chemical constitution mainly contains flavones ingredient and becomes to grade with some fatty acids with the iridoid constituents in the lamiophlomis rotata.Flavones ingredient mainly contains cyanidenon, cyanidenon-7-o-glucoside, Quercetin, Quercetin-3-o-Arabinoside, compositions such as apiolin.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.Traditional Chinese medicine fingerprint is a notion of using for reference fingerprint identification in the medical jurisprudence, through suitably handling, obtains reflecting the total chromatographic peak of Chinese medicine feature with certain analysis means, identifies the true and false of Chinese medicine by having chromatographic peak, guarantees the quality of Chinese medicine.Simultaneously do quantitative measurement, 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 effective in the Chinese medicine.
By relatively screening, because of Quercetin appearance time in finger-print relatively stable, and there is not chromatographic peak to occur at this time period test sample, can be used as " scale " and demarcate each characteristic peak, so the present invention use high performance liquid chromatography with Quercetin as internal standard compound matter, carry out the finger-print research of Chinese crude drug, intermediate, finished product and set up corresponding reference fingerprint, and with this important indicator as control crude drug and end product quality.
Though in No. 200410083678.7 patented claims, also related to the correlation technique content of lamiophlomis rotata finger-print, but this patented claim has been introduced macroporous absorbent resin and carried out pre-treatment in the preparation process of medicinal material, intermediate and injection test sample, thereby may cause the influence of at least two aspects to final result: first macroporous absorbent resin inevitably can be introduced new material in the process of absorption and wash-out; It two is macroporous absorbent resins in the process of handling, and the related substance in the test sample is adsorbed.Can not reflect real fingerprint characteristic.
Summary of the invention
The object of the invention is to provide the finger print quality detecting method of lamiophlomis rotata medicinal material or lamivphlomis root injection liquid intermediate and parenteral solution thereof.
The present invention seeks to be achieved through the following technical solutions
The finger print quality detecting method of lamiophlomis rotata medicinal material of the present invention, this method comprises the steps:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution: preceding 10 minutes, carry out wash-out by moving phase by 10: 90 proportionate relationship of acetonitrile-0.2% phosphate aqueous solution, moving phase becomes acetonitrile-0.2% phosphate aqueous solution 15: 85 by acetonitrile-0.2% phosphate aqueous solution at 10: 90; 10-40 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 30: 70 by acetonitrile-0.2% phosphate aqueous solution at 15: 85; 40-50 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 40: 60 by acetonitrile-0.2% phosphate aqueous solution at 30: 70; 50-60 minute, moving phase remained acetonitrile-0.2% phosphate aqueous solution 40: 60; The preparation of object of reference solution: get the Quercetin reference substance, add the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution; The preparation of need testing solution: get and cross the lamiophlomis rotata medicinal powder 1-10 weight portion that internal diameter is the 5mm sieve after pulverizing, preferred 5 weight portions, put in the flask, add water 50-200 parts by volume, preferred 100 parts by volume, refluxing extraction 1-3 hour, preferred 2 hours, filter water liquid evaporate to dryness, residue adds methyl alcohol and dissolves in right amount, 1000r/min is centrifugal, in the supernatant dislocation 20 parts by volume volumetric flasks, adds object of reference solution 0.4-4 parts by volume, preferred 2 parts by volume, methanol constant volume shakes up to scale, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly;
Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; Test sample finger-print and reference fingerprint similarity are greater than 0.90;
The relative retention time of the total fingerprint peaks of lamiophlomis rotata medicinal material is respectively: No. 1 peak: 0.145 ± 0.002, No. 2 peaks: 0.154 ± 0.002, No. 3 peaks: 0.199 ± 0.001, No. 4 peaks: 0.220 ± 0.001, No. 5 peaks: 0.231 ± 0.001, No. 6 peaks: 0.242 ± 0.001, No. 7 peaks: 0.260 ± 0.002, No. 8 peaks: 0.298 ± 0.003, No. 9 peaks: 0.313 ± 0.002, No. 10 peaks: 0.336 ± 0.003, No. 11 peaks: 0.369 ± 0.003, No. 12 peaks: 0.386 ± 0.004, No. 13 peaks: 0.402 ± 0.001, No. 14 peaks: 0.414 ± 0.002, No. 15 peaks: 0.476 ± 0.006, No. 16 peaks: 0.484 ± 0.004, No. 17 peaks: 0.533 ± 0.003, No. 18 peaks: 0.550 ± 0.001, No. 19 peaks: 0.585 ± 0.002, No. 20 peaks: 0.642 ± 0.005, No. 21 peaks: 0.661 ± 0.003, No. 22 peaks: 0.760 ± 0.001, No. 23 peaks: 0.796 ± 0.007, No. 24 peaks are the S peak: 1, No. 25 peak: 1.077 ± 0.012; Relative peak area ratio is respectively: No. 1 peak: 0.136 ± 0.100, No. 2 peaks: 1.648 ± 0.564, No. 3 peaks: 1.745 ± 0.653, No. 4 peaks: 0.062 ± 0.031, No. 5 peaks: 0.728 ± 0.247, No. 6 peaks: 0.370 ± 0.141, No. 7 peaks: 0.230 ± 0.112, No. 8 peaks: 0.116 ± 0.048, No. 9 peaks: 0.815 ± 0.262, No. 10 peaks: 0.090 ± 0.025, No. 11 peaks: 0.132 ± 0.107, No. 12 peaks: 0.228 ± 0.140, No. 13 peaks: 0.154 ± 0.193, No. 14 peaks: 0.142 ± 0.183, No. 15 peaks: 1.752 ± 0.455, No. 16 peaks: 2.508 ± 1.357, No. 17 peaks: 0.541 ± 0.379, No. 18 peaks: 0.179 ± 0.152, No. 19 peaks: 2.508 ± 2.437, No. 20 peaks: 0.202 ± 0.109, No. 21 peaks: 0.330 ± 0.262, No. 22 peaks: 0.349 ± 0.137, No. 23 peaks: 0.206 ± 0.046, No. 24 peaks are the S peak: 1, No. 25 peak: 0.205 ± 0.077.
The pass of above-mentioned parts by volume and weight portion is milliliter and the relation that restrains.
The finger print quality detecting method of lamivphlomis root injection liquid intermediate of the present invention, this method comprises the steps:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution: preceding 10 minutes, carry out wash-out by moving phase by 10: 90 proportionate relationship of acetonitrile-0.2% phosphate aqueous solution, moving phase becomes acetonitrile-0.2% phosphate aqueous solution 15: 85 by acetonitrile-0.2% phosphate aqueous solution at 10: 90; 10-40 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 30: 70 by acetonitrile-0.2% phosphate aqueous solution at 15: 85; 40-50 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 40: 60 by acetonitrile-0.2% phosphate aqueous solution at 30: 70; 50-60 minute, moving phase remained acetonitrile-0.2% phosphate aqueous solution 40: 60; The preparation of object of reference solution: get the Quercetin reference substance, add the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution; The preparation of need testing solution: get lamivphlomis root injection liquid intermediate 0.5-2 parts by volume, preferred 1 parts by volume, put in the volumetric flask of 10 parts by volume, add object of reference solution 0.5-2 parts by volume, preferred 1 parts by volume, methyl alcohol is diluted to 10 parts by volume, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly;
Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; Test sample finger-print and reference fingerprint similarity are greater than 0.90;
The relative retention time of the total fingerprint peaks of lamivphlomis root injection liquid intermediate is respectively: No. 1 peak: 0.143 ± 0.001, No. 2 peaks: 0.153 ± 0.001, No. 3 peaks: 0.165 ± 0.001, No. 4 peaks: 0.198 ± 0.001, No. 5 peaks: 0.217 ± 0.001, No. 6 peaks: 0.230 ± 0.001, No. 7 peaks: 0.242 ± 0.002, No. 8 peaks: 0.260 ± 0.001, No. 9 peaks: 0.312 ± 0.001, No. 10 peaks: 0.391 ± 0.008, No. 11 peaks: 0.475 ± 0.002, No. 12 peaks: 0.484 ± 0.002, No. 13 peaks: 0.532 ± 0.001, No. 14 peaks: 0.554 ± 0.004, No. 15 peaks: 0.586 ± 0.001, No. 16 peaks: 0.640 ± 0.003, No. 17 peaks: 0.660 ± 0.002, No. 18 peaks: 0.717 ± 0.001, No. 19 the peak is the S peak: 1, No. 20 peak: 1.070 ± 0.007; The relative peak area ratio of total fingerprint peaks is respectively: No. 1 peak: 0.029 ± 0.019, No. 2 peaks: 0.384 ± 0.138, No. 3 peaks: 0.028 ± 0.013, No. 4 peaks: 0.260 ± 0.013, No. 5 peaks: 0.094 ± 0.005, No. 6 peaks: 0.121 ± 0.004, No. 7 peaks: 0.058 ± 0.007, No. 8 peaks: 0.086 ± 0.010, No. 9 peaks: 0.127 ± 0.003, No. 10 peaks: 0.037 ± 0.001, No. 11 peaks: 0.352 ± 0.014, No. 12 peaks: 0.619 ± 0.026, No. 13 peaks: 0.119 ± 0.012, No. 14 peaks: 0.032 ± 0.006, No. 15 peaks: 1.402 ± 0.054, No. 16 peaks: 0.051 ± 0.006, No. 17 peaks: 0.183 ± 0.010, No. 18 peaks: 0.066 ± 0.042, No. 19 the peak is the S peak: 1, No. 20 peak: 0.036 ± 0.005.
Above-mentioned lamivphlomis root injection liquid intermediates preparation is as follows, but is not limited to this method:
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, and filtered, reclaim ethanol to there not being the alcohol flavor, adding water for injection is diluted in right amount, 5～8 ℃ refrigerate 12～24 hours, filter, and filtrate adds activated charcoal 0.05%～0.25%, boiled 15～30 minutes, filter, it is 20 volume weight parts with the medicinal material weight ratio extremely that filtrate adds water for injection, gets the lamiophlomis rotata injection intermediate.
The finger print quality detecting method of lamivphlomis root injection liquid of the present invention, this method comprises the steps:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution: preceding 10 minutes, carry out wash-out by moving phase by 10: 90 proportionate relationship of acetonitrile-0.2% phosphate aqueous solution, moving phase becomes acetonitrile-0.2% phosphate aqueous solution 15: 85 by acetonitrile-0.2% phosphate aqueous solution at 10: 90; 10-40 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 30: 70 by acetonitrile-0.2% phosphate aqueous solution at 15: 85; 40-50 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 40: 60 by acetonitrile-0.2% phosphate aqueous solution at 30: 70; 50-60 minute, moving phase remained acetonitrile-0.2% phosphate aqueous solution 40: 60; The preparation of object of reference solution: get the Quercetin reference substance, add the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution; The preparation of need testing solution: get lamivphlomis root injection liquid 0.5-2 parts by volume, preferred 1 parts by volume is put in the volumetric flask of 10 parts by volume, adds object of reference solution 0.5-2 parts by volume, preferred 1 parts by volume, methyl alcohol is diluted to 10 parts by volume, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly;
Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; Test sample finger-print and reference fingerprint; Similarity is greater than 0.90;
The relative retention time of the total fingerprint peaks of lamivphlomis root injection liquid is respectively: No. 1 peak: 0.141 ± 0.005, No. 2 peaks: 0.153 ± 0.001, No. 3 peaks: 0.199 ± 0.001, No. 4 peaks: 0.218 ± 0.002, No. 5 peaks: 0.233 ± 0.002, No. 6 peaks: 0.242 ± 0.001, No. 7 peaks: 0.260 ± 0.001, No. 8 peaks: 0.312 ± 0.001, No. 9 peaks: 0.475 ± 0.001, No. 10 peaks: 0.484 ± 0.001, No. 11 peaks: 0.530 ± 0.001, No. 12 peaks: 0.550 ± 0.001, No. 13 peak: 0.0.585 ± 0.001, No. 14 peaks: 0.659 ± 0.001, S peak: 1; The relative peak area ratio of total fingerprint peaks is respectively: No. 1 peak: 0.023 ± 0.028, No. 2 peaks: 0.168 ± 0.140, No. 3 peaks: 0.163 ± 0.078, No. 4 peaks: 0.054 ± 0.021, No. 5 peaks: 0.067 ± 0.029, No. 6 peaks: 0.031 ± 0.022, No. 7 peaks: 0.047 ± 0.048, No. 8 peaks: 0.057 ± 0.049, No. 9 peaks: 0.164 ± 0.110, No. 10 peaks: 0.325 ± 0.174, No. 11 peaks: 0.066 ± 0.066, No. 12 peaks: 0.048 ± 0.050, No. 13 peaks: 0.591 ± 0.423, No. 14 peaks: 0.091 ± 0.071, S peak: 1.
Above-mentioned lamivphlomis root injection liquid and preparation method thereof can be following method, but is not limited to this method:
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, and filtered, reclaim ethanol to there not being the alcohol flavor, adding water for injection is diluted in right amount, 5～8 ℃ refrigerate 12～24 hours, filter, and filtrate adds activated charcoal 0.05%～0.25%, boiled 15～30 minutes, filter, it is 10 volume weight parts with the medicinal material weight ratio extremely that filtrate adds water for injection, stirs evenly, add sodium chloride then, add 0.05%～0.25% activated charcoal, be heated to 80 ℃, boiled 15～30 minutes, filter, filtrate adds injection and is diluted with water in right amount, and with dilute sulfuric acid adjust pH 5.5～6.0, adding water for injection is 20 volume weight parts with the medicinal material weight ratio extremely, filter with 0.22 μ m miillpore filter, can is sterilized, promptly in the 5ml ampoule.The ratio of above-mentioned volume weight part is ml/g.
Lamiophlomis rotata medicinal material in the present invention, the characteristic peak quantity of lamivphlomis root injection liquid intermediate and three kinds of test samples of lamivphlomis root injection liquid is successively decreased and has been reflected accurately that also the lamiophlomis rotata medicinal material is in being prepared into lamivphlomis root injection liquid process, the phenomenon that unnecessary material is constantly removed with preparation process, reflect more accurately and showed lamiophlomis rotata and in preparation process, constantly make with extra care, remove and the irrelevant relevant composition of treatment, be beneficial in process of production and accurately product quality controlled and monitored.
The application does not introduce macroporous absorbent resin and carries out pre-treatment in the preparation process of medicinal material, intermediate and injection test sample, guaranteed that effective constituent embodies in finger-print to greatest extent, can reflect real fingerprint characteristic.
Experimental study shows: the need testing solution of lamiophlomis rotata medicinal material of the present invention, intermediate and parenteral solution finger print quality detecting method thereof is stable in 24 hours; The degree of separation at each peak is better, and baseline is more steady, medicinal materials fingerprint and preparation finger better correlativity arranged; Precision good; The reappearance of method is good.Show by the result, medicinal materials fingerprint of the present invention, has certain specificity, can obviously distinguish the medicinal material difference in the different places of production, for the discriminating of medicinal material provides foundation, difference medicinal material of poor quality provides precursor and guarantee for producing qualified preparation, what technology was relative stablizes, and can be used for the suitability for industrialized production of preparation; The intermediate finger-print that the present invention formulates has certain specificity, can be used for the monitoring of technology, guarantees the quality of finished product; Lamivphlomis root injection liquid finger-print of the present invention has certain specificity, can be used for the control of end product quality, guarantees the quality of finished product.
Description of drawings:
Fig. 1 is a lamiophlomis rotata medicinal material standard finger-print;
Fig. 2 is a lamiophlomis rotata intermediate standard finger-print;
Fig. 3 is a lamivphlomis root injection liquid standard finger-print.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
The experiment of experimental example 1 lamiophlomis rotata medicinal materials fingerprint
(1) source of medicinal material
Crude drug source is mainly Gansu Province's Wudu County, and the place of production is more fixing, and the physical environment of medicinal material growth is identical, therefore collects the medicinal material of different local medicinal materials in this area or different collecting seasons and commercially available medicinal material as test sample; The medicinal material of collecting the different places of production simultaneously as test sample by comparison makes medicinal material have enough representativenesses.See Table 1.
Table 1 lamiophlomis rotata crude drug source
Main chemical constitution mainly contains flavones ingredient and becomes to grade with some fatty acids with the iridoid constituents in the lamiophlomis rotata.Flavones ingredient mainly contains cyanidenon, cyanidenon-7-o-glucoside, Quercetin, Quercetin-3-o-Arabinoside, compositions such as apiolin.The experimental applications high performance liquid chromatography experimentizes as detecting index with flavones ingredient in the lamiophlomis rotata, sets up the finger-print of medicinal material.
(2) instrument and reagent
Agilent 1100 high performance liquid chromatographs, Chemistation chromatographic work station, Thermo ODS-2HYPERSIL (C
18, 250mm * 4.6mm, 5 μ m) and chromatographic column.
Sartorius type analysis balance (sensibility reciprocal: 0.1mg, 0.01mg), Sartorius AG produces.Acetonitrile, chromatographically pure, import, MERCK company.Water is ultrapure water; It is pure that all the other reagent are analysis.
(3) selection of chromatographic condition
1. the selection of moving phase
Selection is that moving phase experimentizes 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.
Because complicated component in the sample, under constant current conditions, each component separating degree in the sample is poor, the difficult overall permanence that embodies finger-print; Moving phase is moved with following gradient, and each component all has separation preferably in the sample, and baseline is steady, so select for use as moving phase.
Table 2 gradient table:
Flow velocity is comparatively suitable with 1ml/min, and with this understanding, each peak separates better.
2. detect the selection of wavelength
Use the DAD detecting device test sample has been carried out the long mensuration of all-wave relatively, the result is bigger in the response that 254nm surveys main chromatographic peak in the finger-print, and each chromatographic peak feature is outstanding, so determine that maximum absorption wavelength is 254nm.
3. the selection of chromatographic column
Because Main Ingredients and Appearance is the flavonoids composition in the sample, therefore adopt general octadecyl silane to separate for the filling agent chromatographic column.Thermo ODS-2 HYPERSIL (C
18, 250mm * 4.6mm, 5 μ m) and chromatographic column.
Sample introduction is investigated under above-mentioned chromatographic condition, and results sample is fine in this filler chromatographic column degree of separation, determines to select for use 5u C
18(2) 100A, 4.6mm * 250mm, chromatographic column.
(4) gradient is measured retardation time
Replacing chromatographic column with a zero volume connector, is solvent orange 2 A with methyl alcohol, and the methyl alcohol that contains 0.1% acetone is solvent B, and the detection wavelength is 260nm, and the gradient operation sees the following form:
The operation of table 3 gradient
The result: gradient retardation time is 0min.
(5) preparation of need testing solution
Need testing solution mainly is to extract and the rich flavones ingredient that amasss in the medicinal material, for the ease of the correlativity of contrast medicinal material and preparation, and with reference to the preparation technology of preparation, the following extracting method of experimental design:
1. get and cross the medicinal powder 5g that internal diameter is the 5mm sieve after pulverizing, put in the flask, add water 100ml, refluxing extraction 2 hours filters, and is concentrated into 20ml, adding ethanol to determining alcohol is 75%, and refrigerator was placed 12 hours, filters, reclaim ethanol, residue adds 95% dissolve with ethanol, and constant volume shakes up in the volumetric flask of 20ml, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly; 2. get and cross the medicinal powder 5g that internal diameter is the 5mm sieve after pulverizing, put in the flask, add 95% ethanol 100ml, refluxing extraction 2 hours, filter, evaporate to dryness, residue add 95% dissolve with ethanol constant volume and shake up in the volumetric flask of 20ml, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly; 3. get and cross the medicinal powder 5g that internal diameter is the 5mm sieve after pulverizing, put in the flask, add water 100ml, refluxing extraction 2 hours, filter, evaporate to dryness, residue add an amount of dissolve with methanol, centrifugal (1000r/min), the supernatant constant volume shakes up in the volumetric flask of 20ml, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
Drawing the 10ul sample introduction respectively measures.The result shows: each peak degree of separation is better in the need testing solution chromatogram that 1. method prepares, and stronger characteristic is arranged, but the method complexity, the time is longer, therefore can not adopt; Though the method 2. degree of separation at each peak is better, baseline is more steady, owing to differ bigger with preparation process, the correlativity that records medicinal materials fingerprint and preparation finger is relatively poor, therefore also not employing; The method 3. degree of separation at each peak is better, and baseline is more steady, records the better correlativity of having of medicinal materials fingerprint and preparation finger, therefore determines to adopt this method to prepare the medicinal material need testing solution.
(6) analysis time
Following sample introduction at above-mentioned chromatographic condition is investigated 2 hours, and the result shows that 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.
(7) methodological study
1. stability experiment
Get same need testing solution,, investigate finger-print similarity situation of change, the results are shown in Table 4 respectively in the different time sample detection.
Table 4. stability experiment similarity evaluation result
Experimental result shows: in 24 hours, similarity does not have obvious variation, and RSD% illustrates that less than 0.3% need testing solution was stable in 24 hours.
2. precision experiment
Get same need testing solution continuous sample introduction 5 times, investigate finger-print similarity situation of change, the results are shown in Table 5.
Table 5 precision experiment similarity evaluation result
Experimental result shows: the similarity of 5 sample introductions does not have obvious variation mutually, and RSD% shows that less than 0.3% the precision of instrument is good.
3. reappearance experiment
Get 5 parts of same lot number medicinal materials, the preparation method of shining need testing solution is equipped with need testing solution with legal system, detects under these conditions, to investigate test sample preparation method's reappearance, the results are shown in Table 6.
Table 6 reappearance experiment similarity evaluation result
The result shows that the similarity of measuring behind the 5 duplicate samples sample introductions does not have obvious variation, and RSD% illustrates that less than 0.3% the reappearance of this method is good.
(8) similarity evaluation
Ten batches of lamiophlomis rotata medicinal materials of table 7 similarity evaluation
Experimental example 2 lamiophlomis rotata intermediates and injection finger-print chromatographic condition are selected
Adopt gradient elution, moving phase situation over time sees Table 8.
Table 8 gradient table
Flow velocity: 1ml/min; Detect wavelength: 254nm.
Column temperature: 30 ℃; Analysis time: 1 hour.
The result shows, measures under this chromatographic condition, and a chromatographic peak feature is outstanding in the sample, and degree of separation is good, and baseline is steady.
Experimental example 3 lamiophlomis rotata intermediate finger-print stability experiments
Get lamiophlomis rotata intermediate 1ml, put in the dissolve measuring bottle of 10ml, it is an amount of that the Quercetin reference substance is got in adding, add methyl alcohol and make the solution 1ml of 0.4mg/ml, methyl alcohol is diluted to scale, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, get need testing solution, detect at different time sample introduction 10 μ l respectively, the record chromatogram, investigate finger-print similarity situation of change, the results are shown in Table 9.
Table 9 stability experiment similarity evaluation result
Experimental result shows: in 24 hours, similarity is all greater than 0.98, and RSD is 0.05%, illustrates that need testing solution is basicly stable in 24 hours.
The experiment of experimental example 4 lamiophlomis rotata intermediate finger-print reappearances
Table 10 reappearance experiment similarity evaluation result
The result shows that the finger-print similarity of measuring behind the 5 duplicate samples sample introductions is all greater than 0.98, and RSD is 0.09%, and similarity changes not remarkable, illustrates that the reappearance of this method is good.
Experimental example 5 lamiophlomis rotata intermediate finger-print similarity evaluations
Ten batches of lamiophlomis rotata intermediates of table 11 finger-print similarity evaluation
The result shows: each batch similarity is not significant to be changed, and RSD% is 0.26%, less than 3.0%, shows that the intermediate finger-print that this method is formulated has certain specificity, can be used for the monitoring of technology, guarantees the quality of finished product.
Experimental example 6 lamiophlomis rotata intermediates and parenteral solution finger-print need testing solution preparation method select experiment
1. get lamiophlomis rotata intermediate or parenteral solution stoste as need testing solution.
2. get lamiophlomis rotata intermediate or parenteral solution 1ml, put in the dissolve measuring bottle of 10ml, add methyl alcohol and be diluted to scale, as need testing solution.
Getting above-mentioned need testing solution 10 μ l sample introductions respectively and measure, the record chromatogram. the result shows: sample stoste sample introduction, the degree of separation of each chromatographic peak is relatively poor, and baseline is not steady; Behind the sample methyl alcohol dilution sample introduction, the degree of separation of each chromatographic peak is better, baseline is steady. and chromatogram and each chromatographic peak ratio of stoste sample introduction chromatogram do not have marked difference, and 2. therefore global feature that can objective comprehensive response preparation determine to adopt method as the preparation method of need testing solution.
Experimental example 7 lamivphlomis root injection liquid finger-print stability experiments
Get same need testing solution, respectively in different time 10 μ l sample detection, the record chromatogram is investigated finger-print similarity situation of change, the results are shown in Table 12.
Table 12 stability experiment similarity evaluation result
Experimental result shows: in 24 hours, similarity changes not obvious, and RSD% illustrates that less than 3.0% test sample was stable in 24 hours.
Experimental example 8 lamivphlomis root injection liquid finger-print test sample preparation methods' reappearance experiment
Get 5 parts in same lot number sample, the preparation method of shining need testing solution is equipped with need testing solution with legal system, and sample introduction is measured under above-mentioned chromatographic condition, calculates similarity.The results are shown in Table 13:
Table 13 reappearance experiment similarity evaluation result
The result shows that the finger-print similarity of measuring behind the 5 duplicate samples sample introductions is all greater than 0.98, and RSD is 0.79%, and RSD% illustrates that less than 3.0% the reappearance of this method is good.
Experimental example 9 lamivphlomis root injection liquid finger-print similarity evaluations
Ten batches of lamivphlomis root injection liquid of table 14 finger-print similarity evaluation
The result shows: each batch sample similarity is not significant to be changed, and RSD% is 2.65%, less than 3.0%, shows relative the stablizing of technology, can be used for the suitability for industrialized production of preparation.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.
Following embodiment all can realize the described effect of above-mentioned experimental example.
Embodiment
Embodiment 1: the finger print quality detecting method of lamiophlomis rotata medicinal material
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution:
Gradient elution
The preparation of object of reference solution: it is an amount of to get the Quercetin reference substance, adds the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution;
The preparation of need testing solution: get and cross the medicinal powder 5g that internal diameter is the 5mm sieve after pulverizing, put in the flask, add water 100ml, refluxing extraction 2 hours filters water liquid evaporate to dryness, residue adds methyl alcohol and dissolves in right amount, and 1000r/min is centrifugal, in the supernatant dislocation 20ml volumetric flask, the accurate object of reference solution 2ml that adds, methanol constant volume shakes up to scale, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly;
Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; Test sample finger-print and reference fingerprint similarity are greater than 0.90;
The preferred relative retention time of the total fingerprint peaks of lamiophlomis rotata medicinal material is respectively: 0.145 ± 0.002 (1), 0.154 ± 0.002 (2), 0.199 ± 0.001 (3), 0.220 ± 0.001 (4), 0.231 ± 0.001 (5), 0.242 ± 0.001 (6), 0.260 ± 0.002 (7), 0.298 ± 0.003 (8), 0.313 ± 0.002 (9), 0.336 ± 0.003 (10), 0.369 ± 0.003 (11), 0.386 ± 0.004 (12), 0.402 ± 0.001 (13), 0.414 ± 0.002 (14), 0.476 ± 0.006 (15), 0.484 ± 0.004 (16), 0.533 ± 0.003 (17), 0.550 ± 0.001 (18), 0.585 ± 0.002 (19), 0.642 ± 0.005 (20), 0.661 ± 0.003 (21), 0.760 ± 0.001 (22), 0.796 ± 0.007 (23), 1 (S) (24), 1.077 ± 0.012 (25); Preferred relative peak area ratio is respectively: 0.136 ± 0.100 (1), 1.648 ± 0.564 (2), 1.745 ± 0.653 (3), 0.062 ± 0.031 (4), 0.728 ± 0.247 (5), 0.370 ± 0.141 (6), 0.230 ± 0.112 (7), 0.116 ± 0.048 (8), 0.815 ± 0.262 (9), 0.090 ± 0.025 (10), 0.132 ± 0.107 (11), 0.228 ± 0.140 (12), 0.154 ± 0.193 (13), 0.142 ± 0.183 (14), 1.752 ± 0.455 (15), 2.508 ± 1.357 (16), 0.541 ± 0.379 (17), 0.179 ± 0.152 (18), 2.508 ± 2.437 (19), 0.202 ± 0.109 (20), 0.330 ± 0.262 (21), 0.349 ± 0.137 (22), 0.206 ± 0.046 (23), 1 (S) (24), 0.205 ± 0.077 (25).
Embodiment 2: the finger print quality detecting method of lamiophlomis rotata intermediate
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution:
Gradient elution
The preparation of object of reference solution: it is an amount of to get the Quercetin reference substance, adds the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution;
The preparation of need testing solution: get lamivphlomis root injection liquid intermediate 1ml, put in the dissolve measuring bottle of 10ml, the accurate object of reference solution 1ml that adds, methyl alcohol is diluted to scale, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly;
Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; Test sample finger-print and reference fingerprint similarity are greater than 0.90;
The preferred relative retention time of the total fingerprint peaks of lamiophlomis rotata intermediate is respectively: 0.143 ± 0.001 (1), 0.153 ± 0.001 (2), 0.165 ± 0.001 (3), 0.198 ± 0.001 (4), 0.217 ± 0.001 (5), 0.230 ± 0.001 (6), 0.242 ± 0.002 (7), 0.260 ± 0.001 (8), 0.312 ± 0.001 (9), 0.391 ± 0.008 (10), 0.475 ± 0.002 (11), 0.484 ± 0.002 (12), 0.532 ± 0.001 (13), 0.554 ± 0.004 (14), 0.586 ± 0.001 (15), 0.640 ± 0.003 (16), 0.660 ± 0.002 (17), 0.717 ± 0.001 (18), 1 (19) (S), and 1.070 ± 0.007 (20); The preferred relative peak area ratio of total fingerprint peaks is respectively: 0.029 ± 0.019 (1), 0.384 ± 0.138 (2), 0.028 ± 0.013 (3), 0.260 ± 0.013 (4), 0.094 ± 0.005 (5), 0.121 ± 0.004 (6), 0.058 ± 0.007 (7), 0.086 ± 0.010 (8), 0.127 ± 0.003 (9), 0.037 ± 0.001 (10), 0.352 ± 0.014 (11), 0.619 ± 0.026 (12), 0.119 ± 0.012 (13), 0.032 ± 0.006 (14), 1.402 ± 0.054 (15), 0.051 ± 0.006 (16), 0.183 ± 0.010 (17), 0.066 ± 0.042 (18), 1 (19) (S), 0.036 ± 0.005.
Embodiment 3: the finger print quality detecting method of lamivphlomis root injection liquid
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution:
Gradient elution
The preparation of object of reference solution: it is an amount of to get the Quercetin reference substance, adds the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution;
The preparation of need testing solution: get lamivphlomis root injection liquid 1ml, put in the volumetric flask of 10ml, add object of reference solution 1ml, methyl alcohol is diluted to scale, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly;
Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; Test sample finger-print and reference fingerprint; Similarity is greater than 0.90;
The preferred relative retention time of the total fingerprint peaks of lamivphlomis root injection liquid is respectively: 0.141 ± 0.005 (1), 0.153 ± 0.001 (2), 0.199 ± 0.001 (3), 0.218 ± 0.002 (4), 0.233 ± 0.002 (5), 0.242 ± 0.001 (6), 0.260 ± 0.001 (7), 0.312 ± 0.001 (8), 0.475 ± 0.001 (9), 0.484 ± 0.001 (10), 0.530 ± 0.001 (11), 0.550 ± 0.001 (12), 0.0.585 ± 0.001 (13), ± 0.001 (14), 1 0.659 (S) (15); The preferred relative peak area ratio of total fingerprint peaks is respectively: 0.023 ± 0.028 (1), 0.168 ± 0.140 (2), 0.163 ± 0.078 (3), 0.054 ± 0.021 (4), 0.067 ± 0.029 (5), 0.031 ± 0.022 (6), 0.047 ± 0.048 (7), 0.057 ± 0.049 (8), 0.164 ± 0.110 (9), 0.325 ± 0.174 (1 (10), 0.066 ± 0.066 (11), 0.048 ± 0.050 (12), 0.591 ± 0.423 (13), ± 0.071 (14), 1 0.091 (S) (15).
Claims (6)
1. the detection method of a lamiophlomis rotata medicinal material is characterized in that the detection step of finger-print in this method is:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution: preceding 10 minutes, carry out wash-out by moving phase by 10: 90 proportionate relationship of acetonitrile-0.2% phosphate aqueous solution, moving phase becomes acetonitrile-0.2% phosphate aqueous solution 15: 85 by acetonitrile-0.2% phosphate aqueous solution at 10: 90; 10-40 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 30: 70 by acetonitrile-0.2% phosphate aqueous solution at 15: 85; 40-50 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 40: 60 by acetonitrile-0.2% phosphate aqueous solution at 30: 70; 50-60 minute, moving phase remained acetonitrile-0.2% phosphate aqueous solution 40: 60; The preparation of object of reference solution: get the Quercetin reference substance, add the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution; The preparation of need testing solution: get and cross the lamiophlomis rotata medicinal powder 1-10 weight portion that internal diameter is the 5mm sieve after pulverizing, put in the flask, add water 50-200 parts by volume, refluxing extraction 1-3 hour, filter water liquid evaporate to dryness, residue adds methyl alcohol and dissolves in right amount, and 1000r/min is centrifugal, in the supernatant dislocation 20 parts by volume volumetric flasks, add object of reference solution 0.4-4 parts by volume, methanol constant volume shakes up to scale, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly; Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; The relative retention time of total fingerprint peaks is respectively: No. 1 peak: 0.145 ± 0.002, No. 2 peaks: 0.154 ± 0.002, No. 3 peaks: 0.199 ± 0.001, No. 4 peaks: 0.220 ± 0.001, No. 5 peaks: 0.231 ± 0.001, No. 6 peaks: 0.242 ± 0.001, No. 7 peaks: 0.260 ± 0.002, No. 8 peaks: 0.298 ± 0.003, No. 9 peaks: 0.313 ± 0.002, No. 10 peaks: 0.336 ± 0.003, No. 11 peaks: 0.369 ± 0.003, No. 12 peaks: 0.386 ± 0.004, No. 13 peaks: 0.402 ± 0.001, No. 14 peaks: 0.414 ± 0.002, No. 15 peaks: 0.476 ± 0.006, No. 16 peaks: 0.484 ± 0.004, No. 17 peaks: 0.533 ± 0.003, No. 18 peaks: 0.550 ± 0.001, No. 19 peaks: 0.585 ± 0.002, No. 20 peaks: 0.642 ± 0.005, No. 21 peaks: 0.661 ± 0.003, No. 22 peaks: 0.760 ± 0.001, No. 23 peaks: 0.796 ± 0.007, No. 24 peaks are the S peak: 1, No. 25 peak: 1.077 ± 0.012; The relative peak area ratio of total fingerprint peaks is respectively: No. 1 peak: 0.136 ± 0.100, No. 2 peaks: 1.648 ± 0.564, No. 3 peaks: 1.745 ± 0.653, No. 4 peaks: 0.062 ± 0.031, No. 5 peaks: 0.728 ± 0.247, No. 6 peaks: 0.370 ± 0.141, No. 7 peaks: 0.230 ± 0.112, No. 8 peaks: 0.116 ± 0.048, No. 9 peaks: 0.815 ± 0.262, No. 10 peaks: 0.090 ± 0.025, No. 11 peaks: 0.132 ± 0.107, No. 12 peaks: 0.228 ± 0.140, No. 13 peaks: 0.154 ± 0.193, No. 14 peaks: 0.142 ± 0.183, No. 15 peaks: 1.752 ± 0.455, No. 16 peaks: 2.508 ± 1.357, No. 17 peaks: 0.541 ± 0.379, No. 18 peaks: 0.179 ± 0.152, No. 19 peaks: 2.508 ± 2.437, No. 20 peaks: 0.202 ± 0.109, No. 21 peaks: 0.330 ± 0.262, No. 22 peaks: 0.349 ± 0.137, No. 23 peaks: 0.206 ± 0.046, No. 24 peaks are the S peak: 1, No. 25 peak: 0.205 ± 0.077.
2. the detection method of lamiophlomis rotata medicinal material according to claim 1, the preparation process that it is characterized in that need testing solution in this method is: get and cross lamiophlomis rotata medicinal powder 5 weight portions that internal diameter is the 5mm sieve after pulverizing, put in the flask, add water 100 parts by volume, refluxing extraction 2 hours, filter, water liquid evaporate to dryness, residue adds methyl alcohol and dissolves in right amount, 1000r/min is centrifugal, in the supernatant dislocation 20 parts by volume volumetric flasks, add object of reference solution 2 parts by volume, methanol constant volume is to scale, shake up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
3. the detection method of a lamiophlomis rotata injection intermediate is characterized in that the detection step of finger-print in this method is:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution: preceding 10 minutes, carry out wash-out by moving phase by 10: 90 proportionate relationship of acetonitrile-0.2% phosphate aqueous solution, moving phase becomes acetonitrile-0.2% phosphate aqueous solution 15: 85 by acetonitrile-0.2% phosphate aqueous solution at 10: 90; 10-40 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 30: 70 by acetonitrile-0.2% phosphate aqueous solution at 15: 85; 40-50 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 40: 60 by acetonitrile-0.2% phosphate aqueous solution at 30: 70; 50-60 minute, moving phase remained acetonitrile-0.2% phosphate aqueous solution 40: 60; The preparation of object of reference solution: get the Quercetin reference substance, add the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution; The preparation of need testing solution: get lamivphlomis root injection liquid intermediate 0.5-2 parts by volume, put in the volumetric flask of 10 parts by volume, add object of reference solution 0.5-2 parts by volume, methyl alcohol is diluted to 10 parts by volume, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly; Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; The relative retention time of total fingerprint peaks is respectively: No. 1 peak: 0.143 ± 0.001, No. 2 peaks: 0.153 ± 0.001, No. 3 peaks: 0.165 ± 0.001, No. 4 peaks: 0.198 ± 0.001, No. 5 peaks: 0.217 ± 0.001, No. 6 peaks: 0.230 ± 0.001, No. 7 peaks: 0.242 ± 0.002, No. 8 peaks: 0.260 ± 0.001, No. 9 peaks: 0.312 ± 0.001, No. 10 peaks: 0.391 ± 0.008, No. 11 peaks: 0.475 ± 0.002, No. 12 peaks: 0.484 ± 0.002, No. 13 peaks: 0.532 ± 0.001, No. 14 peaks: 0.554 ± 0.004, No. 15 peaks: 0.586 ± 0.001, No. 16 peaks: 0.640 ± 0.003, No. 17 peaks: 0.660 ± 0.002, No. 18 peaks: 0.717 ± 0.001, No. 19 the peak is the S peak: 1, No. 20 peak: 1.070 ± 0.007; The relative peak area ratio of total fingerprint peaks is respectively: No. 1 peak: 0.029 ± 0.019, No. 2 peaks: 0.384 ± 0.138, No. 3 peaks: 0.028 ± 0.013, No. 4 peaks: 0.260 ± 0.013, No. 5 peaks: 0.094 ± 0.005, No. 6 peaks: 0.121 ± 0.004, No. 7 peaks: 0.058 ± 0.007, No. 8 peaks: 0.086 ± 0.010, No. 9 peaks: 0.127 ± 0.003, No. 10 peaks: 0.037 ± 0.001, No. 11 peaks: 0.352 ± 0.014, No. 12 peaks: 0.619 ± 0.026, No. 13 peaks: 0.119 ± 0.012, No. 14 peaks: 0.032 ± 0.006, No. 15 peaks: 1.402 ± 0.054, No. 16 peaks: 0.051 ± 0.006, No. 17 peaks: 0.183 ± 0.010, No. 18 peaks: 0.066 ± 0.042, No. 19 the peak is the S peak: 1, No. 20 peak: 0.036 ± 0.005.
4. the detection method of lamiophlomis rotata injection intermediate as claimed in claim 3, the preparation process that it is characterized in that need testing solution in this method is: get lamivphlomis root injection liquid intermediate 1 parts by volume, put in the volumetric flask of 10 parts by volume, add object of reference solution 1 parts by volume, methyl alcohol is diluted to 10 parts by volume, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
5. the detection method of a lamivphlomis root injection liquid is characterized in that the detection step of finger-print in this method is:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; Flow velocity: 1ml/min; The detection wavelength is 254nm; Column temperature: 30 ℃; Analysis time: 1 hour; Carry out gradient elution: preceding 10 minutes, carry out wash-out by moving phase by 10: 90 proportionate relationship of acetonitrile-0.2% phosphate aqueous solution, moving phase becomes acetonitrile-0.2% phosphate aqueous solution 15: 85 by acetonitrile-0.2% phosphate aqueous solution at 10: 90; 10-40 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 30: 70 by acetonitrile-0.2% phosphate aqueous solution at 15: 85; 40-50 minute, moving phase became acetonitrile-0.2% phosphate aqueous solution 40: 60 by acetonitrile-0.2% phosphate aqueous solution at 30: 70; 50-60 minute, moving phase remained acetonitrile-0.2% phosphate aqueous solution 40: 60; The preparation of object of reference solution: get the Quercetin reference substance, add the solution that methyl alcohol is made 40 μ g/ml, as object of reference solution; The preparation of need testing solution: get lamivphlomis root injection liquid 0.5-2 parts by volume, put in the volumetric flask of 10 parts by volume, add object of reference solution 0.5-2 parts by volume, methyl alcohol is diluted to 10 parts by volume, shakes up, and with 0.45 μ m filtering with microporous membrane, gets filtrate, promptly; Determination method: draw each 10 μ l of object of reference solution and need testing solution, inject liquid chromatograph, measure; The relative retention time of total fingerprint peaks is respectively: No. 1 peak: 0.141 ± 0.005, No. 2 peaks: 0.153 ± 0.001, No. 3 peaks: 0.199 ± 0.001, No. 4 peaks: 0.218 ± 0.002, No. 5 peaks: 0.233 ± 0.002, No. 6 peaks: 0.242 ± 0.001, No. 7 peaks: 0.260 ± 0.001, No. 8 peaks: 0.312 ± 0.001, No. 9 peaks: 0.475 ± 0.001, No. 10 peaks: 0.484 ± 0.001, No. 11 peaks: 0.530 ± 0.001, No. 12 peaks: 0.550 ± 0.001, No. 13 peak: 0.0.585 ± 0.001, No. 14 peaks: 0.659 ± 0.001, S peak: 1; Total fingerprint peaks relative peak area ratio is respectively: No. 1 peak: 0.023 ± 0.028, No. 2 peaks: 0.168 ± 0.140, No. 3 peaks: 0.163 ± 0.078, No. 4 peaks: 0.054 ± 0.021, No. 5 peaks: 0.067 ± 0.029, No. 6 peaks: 0.031 ± 0.022, No. 7 peaks: 0.047 ± 0.048, No. 8 peaks: 0.057 ± 0.049, No. 9 peaks: 0.164 ± 0.110, No. 10 peaks: 0.325 ± 0.174, No. 11 peaks: 0.066 ± 0.066, No. 12 peaks: 0.048 ± 0.050, No. 13 peaks: 0.591 ± 0.423, No. 14 peaks: 0.091 ± 0.071, S peak: 1.
6. the detection method of lamivphlomis root injection liquid as claimed in claim 5, the preparation process that it is characterized in that need testing solution in this method is: get lamivphlomis root injection liquid 1 parts by volume, put in the volumetric flask of 10 parts by volume, add object of reference solution 1 parts by volume, methyl alcohol is diluted to 10 parts by volume, shakes up, with 0.45 μ m filtering with microporous membrane, get filtrate, promptly.
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