CN103048408A - Specific chromatogram determination of blood-activating and pain-relieving plaster and quality detection method thereof - Google Patents
Specific chromatogram determination of blood-activating and pain-relieving plaster and quality detection method thereof Download PDFInfo
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Abstract
The invention discloses a specific chromatogram determination method of blood-activating and pain-relieving plaster, comprising the steps: respectively injecting sampling liquid obtained through same extraction operation of the blood-activating and pain-relieving plaster with various standards into a liquid chromatographic instrument, implementing separation experiments under same chromatographic conditions, and introducing obtained chromatograms into a traditional Chinese medicine fingerprint analyzer for detection, thereby obtaining the specific chromatogram of the blood-activating and pain-relieving plaster. The traditional Chinese medicine fingerprint analyzer should be interpreted as equipment which is provided with traditional Chinese medicine fingerprint analyzing software and can run the same. According to the abovementioned method, the specific chromatogram of the blood-activating and pain-relieving plaster can be effectively determined, and the method is convenient for operation and good in repeatability.
Description
Technical field
The present invention relates to the blood-activating analgesic plaster production field, the characteristic spectrum that is specifically related to a kind of blood-activating analgesic plaster is measured and its quality determining method.
Background technology
Blood-activating analgesic plaster is a kind of Chinese patent drug that Anhui Anke Yuliang Medicine Co., Ltd produces, and is used for promoting blood circulation and stopping pain, and channels sootheing and network vessel quickening cures mainly arthralgia and myalgia, muscular paralysis, subcutaneous nodule, joint pain.
At present, mainly be to measure camphor to the quality control of blood-activating analgesic plaster aborning, menthol, borneol, gaultherolin content and to Radix Angelicae Sinensis, dried orange peel, belladonna liquid extract, the component such as rheum officinale and cortex periplocae is carried out thin layer and is differentiated, though said method can be controlled product quality to a certain extent, but can't comprehensively reflect the quality condition of product, for compensating its defect, make its Quality Control Technology more perfect, science also provides a kind of available quality control model to its standardized production from now on, therefore be necessary to set up the standard feature collection of illustrative plates of this product, be used for the quality control of product.
Goal of the invention
Primary and foremost purpose of the present invention provides a kind of characteristic spectrum assay method of blood-activating analgesic plaster, and the method is easy and simple to handle, precision is high, favorable reproducibility.
Its scheme of taking is, a kind of characteristic spectrum assay method of blood-activating analgesic plaster, with each standard blood-activating analgesic plaster sample introduction liquid difference injection liquid chromatography that lixiviate obtains under identical lixiviate operation, under identical chromatographic condition, carry out separation test, then the chromatogram that obtains is imported the traditional Chinese medicine fingerprint analyser and detect the characteristic spectrum that obtains blood-activating analgesic plaster.The traditional Chinese medicine fingerprint analyser is interpreted as the equipment that the traditional Chinese medicine fingerprint analysis software is installed and can moves.
By said method, can effectively determine the characteristic spectrum of the analgesic extract preparation of invigorating blood circulation, and easy to operate, favorable reproducibility.
Another object of the present invention provides a kind of above-mentioned method that characteristic spectrum detects the analgesic extract quality of invigorating blood circulation that records of utilizing.
Its scheme is: a kind of quality determining method of blood-activating analgesic plaster, it is characterized in that: be by data acquisition unit the chromatogram by the blood-activating analgesic plaster product of measuring under the identical experiment condition to be gathered, data acquisition unit inputs to processing unit with the chromatogram that gathers, processing unit is transferred the characteristic spectrum of the blood-activating analgesic plaster that has recorded and the chromatogram of input detects and contrasts, then the output detections consequential signal is to display unit, and display unit shows the quality condition of product.
By said method, can be effectively the product quality of the analgesic extract preparation of invigorating blood circulation be detected, thereby the product quality of the analgesic extract of invigorating blood circulation is control effectively, realize invigorating blood circulation the production of analgesic extract standard preparation.
Further be operating as, also be included in the chromatogram of measuring Physcion under the same experimental conditions, process and obtain the relative retention time of each characteristic peak of blood-activating analgesic plaster characteristic spectrum.By measuring the chromatogram of Physcion, the chromatogram of Physcion and each standard blood-activating analgesic plaster are recorded chromatogram compares, determine common characteristic peak between the two, that is find out the characteristic peak that each standard blood-activating analgesic plaster records the Physcion on the chromatogram, thereby take the relative retention time of Physcion characteristic peak as 1, calculate the relative retention time of other characteristic peaks, be convenient to the detection of the follow-up analgesic extract product of invigorating blood circulation.
Identical condition when the chromatographic condition that wherein, the same experimental conditions of indication refers to lixiviate operation and chromatographic determination among the application all carries out lixiviate operation and chromatographic determination operation with the standard blood-activating analgesic plaster.
Concrete is operating as:
Lixiviate is operating as: get 5～10 standards analgesic extract of invigorating blood circulation and remove lid lining, be cut into fractionlet, the methenyl choloride that adds 50～200ml, soaked 30～60 minutes, stir hypsokinesis and obtain cream liquid one time, then the methyl alcohol that adds 50～100ml, stir, left standstill 5～10 minutes, incline and obtain secondary cream liquid, residue adds the methyl alcohol of 50～100ml again, stir, left standstill 5～10 minutes, incline and obtain cream liquid three times, merge above-mentioned gained cream liquid, water bath method adds methyl alcohol dissolving with the gained lotion, is transferred in the first volumetric flask of 10ml after the filtration and adds methanol constant volume, and then accurate solution 2ml of measuring in the first volumetric flask is transferred in the second volumetric flask of 10ml and adds methanol constant volume, and the solution behind the constant volume is sample introduction liquid.The specification of the analgesic extract of invigorating blood circulation that is operated among the application is 7cm*10cm, when certainly adopting other different big or small standardized products to test, and the variation that the methenyl choloride that adopts and the volume of methyl alcohol can adapt.
Described chromatographic condition is: chromatographic column is take octadecane base key and silica gel as filling agent, mobile phase is A phase and the B phase composition of 35:65～85:15 by volume ratio, A is methyl alcohol or acetonitrile mutually, B is 0.05～0.15% phosphoric acid or 0.1%～1.5% glacial acetic acid for massfraction mutually, gradient elution, the detection wavelength is 254 ± 2nm, flow velocity is 0.5～1.5ml/min, column temperature is 30～40 ℃, the sample size of sample introduction liquid is 10～20 μ l, theoretical cam curve is not less than 6000, and the theoretical cam curve of indication is calculated according to the Physcion peak.
The time of gradient elution and mobile phase ratio are: 0～5min, A phase: 35～40%, B phase: 65～60%; 5～10min, A phase: 40～45%, B phase: 60～55%; 10～25min, A phase: 45～62%, B phase: 55～38%; 25～35min, A phase: 62～83%, B phase: 38～17%; 35～40min, A phase: 83～85%, B phase: 17～15%; 40～65min, A phase: 85～85%, B phase: 15～15%.
At least get 15 batches of standard blood-activating analgesic plasters and carry out respectively lixiviate operation, and with the sample introduction liquid that obtains respectively the injection liquid chromatography test.
Operate by above-mentioned restriction parameter, the separating effect of each component is best in the sample introduction liquid of producing, and the characteristic pattern of the blood-activating analgesic plaster that records is the most accurate.
Among the application, the standard blood-activating analgesic plaster of indication refers to that quality meets the blood-activating analgesic plaster of concerned countries medicine standard fully.The analgesic extract of invigorating blood circulation is to make by following raw material and preparation method.
Its Raw consists of: 9～18 parts of the roots of Dahurain angelica, 2～4 parts of moutan barks, 4～8 parts of schizonepeta, 9～18 parts of dried orange peels, 16～32 parts of rhizoma zingiberis, 4～8 parts of the roots of Chinese wild ginger, 9～18 parts of Kaempferia galangas, 3～6 parts of the flower buds of lily magnolia, 5～10 parts of unprocessed Arisaema erubescens, 2～4 parts in capsicum, 3～6 parts of Ligusticum wallichiis, 4～8 parts of menthols, 3～6 parts of levisticums, 2～4 parts of myrrhs, 4～8 parts of cortex periplocaes, 8～16 parts of rheum officinales, 5～10 parts of cloves, give birth to 8～16 parts of the tuber of pinellia, 8～16 parts of rhizoma nardostachyos, 4～8 parts of camphors, 5～10 parts of Radix Angelicae Sinensis, 4～8 parts of borneols, 2～4 parts of frankincenses, 6～12 parts of belladonna liquid extracts, 4～8 parts of cassia twig, 5～10 parts in pepper, 2～4 parts of rhizoma atractylodis, 6～12 parts of gaultherolins;
The preparation method is: 28 the flavor raw materials in except menthol, borneol, gaultherolin, belladonna liquid extract, camphor; Remaining 23 flavor is ground into meal, according to the percolation under liquid extract and the extract item, make solvent with 90% ethanol, carry out diacolation, the collection liquid of filtering, the liquid of filtering is condensed into relative density and is about 1.05 clear cream, then adds the residue five tastes raw materials such as above-mentioned menthol, stir evenly, make according to a conventional method again the analgesic extract of invigorating blood circulation;
Or just remaining 23 flavor is ground into that the meal temperature is soaked, CO
2Reflux behind backflow or the supercritical extract, make solvent with 90% ethanol, collect extract, be condensed into relative density and be about 1.05 clear cream, add the residue five tastes such as above-mentioned menthol, stir evenly, make according to a conventional method the analgesic extract of invigorating blood circulation.
According to above-mentioned operation, measured standard feature collection of illustrative plates (as shown in Figure 4) common characteristic peak has 6, No. 5 chromatographic peaks characteristic peak that is Physcion wherein, take the relative retention time of the characteristic peak of Physcion as 1, calculate the relative retention time at other peaks, the standard feature collection of illustrative plates that namely obtains blood-activating analgesic plaster is:
No. 1 peak: relative retention time 0.56～0.62
No. 2 peaks: relative retention time 0.64～0.70
No. 3 peaks: relative retention time 0.78～0.86
No. 4 peaks: relative retention time 0.92～1.01
No. 5 peaks: relative retention time 1.00
No. 6 peaks: relative retention time 1.20～1.33.
When carrying out the product detection, the product of sampling observation is recorded its chromatogram according to same experimental conditions, if processing unit detects the relative retention time error of each characteristic peak in the retention time obtain occurring in the chromatogram 6 identical in the characteristic spectrum each characteristic peak and each characteristic peak and the above-mentioned characteristic spectrum in 5%, this qualified product then, otherwise then defective, thereby fast the analgesic extract preparation of invigorating blood circulation is detected.
Description of drawings
Fig. 1 is the Physcion collection of illustrative plates that records;
Fig. 2 is the characteristic spectrum of the blood-activating analgesic plaster that records of embodiment 2;
Fig. 3 is that the 15 mass products blood analgesic extract chromatograms that record among the embodiment 3 import the sectional drawing of analyzing in the traditional Chinese medicine fingerprint software;
Fig. 4 is the characteristic spectrum that embodiment 3 records blood-activating analgesic plaster;
Embodiment
Embodiment 1 is for measuring the Physcion chromatogram, and embodiment 2,3 is for measuring the characteristic spectrum of the analgesic extract of invigorating blood circulation, and wherein embodiment 1,2,3 used instrument, medicines are as follows:
Instrument and reagent
Instrument: Waters high performance liquid chromatograph, 2489 detecting devices.
Medicine: phosphoric acid, methenyl choloride etc. (it is pure to be analysis); Methyl alcohol (chromatographically pure); Standard blood-activating analgesic plaster (Anhui Anke Yuliang Medicine Co., Ltd's production).
Embodiment 1 measures the Physcion collection of illustrative plates
The preparation of sample introduction liquid: it is an amount of to get the Physcion reference substance, and precise weighing adds the sample introduction liquor that methyl alcohol is made 40 μ g/ml.
Chromatographic determination operation: get 20 μ l sample introductions and carry out sample introduction to the liquid high performance liquid chromatograph, concrete operations are by " regulation of " high performance liquid chromatography test " operates in Chinese pharmacopoeia (2010 editions) the First appendix, obtain retention time and the chromatogram of Physcion characteristic peak, as shown in Figure 1.
Chromatographic condition wherein: chromatographic column is take octadecane base key and silica gel as filling agent, and take methyl alcohol as the A phase, 0.08～0.10% phosphoric acid is B phase composition mobile phase; Adopt the gradient elution mode, its elution time and mobile phase ratio are: 0～5min, A phase: 35～40%, B phase: 65～60%; 5～10min, A phase: 40～45%, B phase: 60～55%; 10～25min, A phase: 45～62%, B phase: 55～38%; 25～35min, A phase: 62～83%, B phase: 38～17%; 35～40min, A phase: 83～85%, B phase: 17～15%; 40～65min, A phase: 85～85%, B phase: 15～15%; The detection wavelength is 254 ± 2nm, flow velocity 0.5～1.5ml/min, and 30～40 ℃ of column temperatures, theoretical cam curve is not less than 6000.
The preparation of sample introduction liquid: get 15 batches of standards analgesic extract of invigorating blood circulation, prepare 15 parts of sample introduction liquid, label A1～A15 according to following lixiviate operation respectively.
Lixiviate operation: get 5～10 standards analgesic extract of invigorating blood circulation and remove the lid lining, be cut into fractionlet, the methenyl choloride that adds 50～200ml, soaked 30～60 minutes, stir hypsokinesis and obtain cream liquid one time, then the methyl alcohol that adds 50～100ml, stir, left standstill 5～10 minutes, incline and obtain secondary cream liquid, residue adds the methyl alcohol of 50～100ml again, stir, left standstill 5～10 minutes, incline and obtain cream liquid three times, merge above-mentioned gained cream liquid, water bath method adds methyl alcohol dissolving with the gained lotion, is transferred in the first volumetric flask of 10ml after the filtration and adds methanol constant volume, and then accurate solution 2ml of measuring in the first volumetric flask is transferred in the second volumetric flask of 10ml and adds methanol constant volume, and the solution behind the constant volume is sample introduction liquid.
Chromatographic determination operation: 15 batches of sample introduction liquid are got respectively 10 μ l carry out sample introduction to the liquid high performance liquid chromatograph, concrete operations are by " regulation of " high performance liquid chromatography test " operates in Chinese pharmacopoeia (2010 editions) the First appendix, obtains the invigorate blood circulation chromatogram of analgesic extract of 15 standards.
Chromatographic condition wherein: chromatographic column is take octadecane base key and silica gel as filling agent, and take acetonitrile as the A phase, 0.1%～1.5% glacial acetic acid is B phase composition mobile phase; Adopt the gradient elution mode, its elution time and mobile phase ratio are: 0～5min, A phase: 20～30%, B phase: 80～40%; 5～10min, A phase: 30～40%, B phase: 70～60%; 10～25min, A phase: 40～57%, B phase: 60～43%; 25～35min, A phase: 57～62%, B phase: 43～38%; 35～45min, A phase: 62～67%, B phase: 38～33%; 45～65min, A phase: 67～85%, B phase: 33～15%; The detection wavelength is 254 ± 2nm, flow velocity 0.5～1.5ml/min, 30～40 ℃ of column temperatures, sample size 20 μ l; Theoretical cam curve should be not less than 6000 by the Physcion peak.
Characteristic spectrum: compare comparison by the 15 batches of standard blood-activating analgesic plasters recording and the chromatogram of Physcion, determine that the blood-activating analgesic plaster chromatogram has 6 total peaks, label 1～6, No. 5 chromatographic peaks characteristic peak that is Physcion wherein, the relative retention time of setting No. 5 peaks is 1, calculates the relative retention time at other peaks.The chromatogram of 15 analgesic extracts of invigorating blood circulation obtaining is imported in the traditional Chinese medicine fingerprint software, obtain the characteristic spectrum (as shown in Figure 3) of blood-activating analgesic plaster behind the corresponding operating.
Experimental example 3 is measured the blood-activating analgesic plaster characteristic spectrum
The preparation of sample introduction liquid: get 15 batches of standards analgesic extract of invigorating blood circulation, prepare 15 parts of sample introduction liquid, label S1～S15 according to following lixiviate operation respectively.
Lixiviate operation: get 5～10 standards analgesic extract of invigorating blood circulation and remove the lid lining, be cut into fractionlet, the methenyl choloride that adds 50～200ml, soaked 30～60 minutes, stir hypsokinesis and obtain cream liquid one time, then the methyl alcohol that adds 50～100ml, stir, left standstill 5～10 minutes, incline and obtain secondary cream liquid, residue adds the methyl alcohol of 50～100ml again, stir, left standstill 5～10 minutes, incline and obtain cream liquid three times, merge above-mentioned gained cream liquid, water bath method adds methyl alcohol dissolving with the gained lotion, is transferred in the first volumetric flask of 10ml after the filtration and adds methanol constant volume, and then accurate solution 2ml of measuring in the first volumetric flask is transferred in the second volumetric flask of 10ml and adds methanol constant volume, and the solution behind the constant volume is sample introduction liquid.
Chromatographic determination operation: 15 batches of sample introduction liquid are got respectively 20 μ l carry out sample introduction to the liquid high performance liquid chromatograph, concrete operations are by " regulation of " high performance liquid chromatography test " operates in Chinese pharmacopoeia (2010 editions) the First appendix, obtains the invigorate blood circulation chromatogram of analgesic extract of 15 standards.
Chromatographic condition wherein: chromatographic column is take octadecane base key and silica gel as filling agent, and take methyl alcohol as the A phase, 0.08～0.10% phosphoric acid is B phase composition mobile phase; Adopt the gradient elution mode, its elution time and mobile phase ratio are: 0～5min, A phase: 35～40%, B phase: 65～60%; 5～10min, A phase: 40～45%, B phase: 60～55%; 10～25min, A phase: 45～62%, B phase: 55～38%; 25～35min, A phase: 62～83%, B phase: 38～17%; 35～40min, A phase: 83～85%, B phase: 17～15%; 40～65min, A phase: 85～85%, B phase: 15～15%; The detection wavelength is 254 ± 2nm, flow velocity 0.5～1.5ml/min, 30～40 ℃ of column temperatures, sample size 20 μ l; Theoretical cam curve should be not less than 6000 by the Physcion peak.
Characteristic spectrum: the 15 mass products blood and relieving pain cream that will record and the chromatogram of Physcion compare, the chromatogram of determining blood-activating analgesic plaster has 6 total peaks, label 1～6, No. 5 chromatographic peaks characteristic peak that is Physcion wherein, the relative retention time of setting No. 5 peaks is 1, calculate the relative retention time at other peaks, concrete outcome sees table 1 mutually for details.15 collection of illustrative plates that obtain are imported in the traditional Chinese medicine fingerprint software (as shown in Figure 2), obtain the characteristic spectrum (as shown in Figure 3) of blood-activating analgesic plaster behind the corresponding operating.
The invigorate blood circulation relative retention time statistics of characteristic peak of analgesic extract collection of illustrative plates of 15 batches of standards among table 1 embodiment 3
Claims (7)
1. the characteristic spectrum assay method of a blood-activating analgesic plaster, with each standard blood-activating analgesic plaster sample introduction liquid difference injection liquid chromatography that lixiviate obtains under identical lixiviate operation, under identical chromatographic condition, carry out separation test, then the chromatogram that obtains is imported the traditional Chinese medicine fingerprint analyser and detect the characteristic spectrum that obtains blood-activating analgesic plaster.
2. the characteristic spectrum assay method of blood-activating analgesic plaster as claimed in claim 1 is characterized in that: also be included in the chromatogram of measuring Physcion under the same experimental conditions, process and obtain the relative retention time of each characteristic peak of blood-activating analgesic plaster characteristic spectrum.。
3. such as the characteristic spectrum assay method of claim 1 or 3 described blood-activating analgesic plasters, it is characterized in that, described chromatographic condition is: chromatographic column is take octadecane base key and silica gel as filling agent, mobile phase is A phase and the B phase composition of 35:65～85:15 by volume ratio, A is methyl alcohol or acetonitrile mutually, B is 0.05～0.15% phosphoric acid or 0.1%～1.5% glacial acetic acid for massfraction mutually, gradient elution, the detection wavelength is 254 ± 2nm, flow velocity is 0.5～1.5ml/min, column temperature is 30～40 ℃, and the sample size of sample introduction liquid is 10～20 μ l.
4. the characteristic spectrum assay method of blood-activating analgesic plaster as claimed in claim 2, it is characterized in that: the time of gradient elution and mobile phase ratio are: 0～5min, A phase: 35～40%, B phase: 65～60%; 5～10min, A phase: 40～45%, B phase: 60～55%; 10～25min, A phase: 45～62%, B phase: 55～38%; 25～35min, A phase: 62～83%, B phase: 38～17%; 35～40min, A phase: 83～85%, B phase: 17～15%; 40～65min, A phase: 85～85%, B phase: 15～15%.
5. the characteristic spectrum assay method of blood-activating analgesic plaster as claimed in claim 1 or 2 is characterized in that: get at least 15 batches of standard blood-activating analgesic plasters and carry out respectively the lixiviate operation, and the sample introduction liquid that obtains is tested in the injection liquid chromatography respectively.
6. the characteristic spectrum assay method of blood-activating analgesic plaster as claimed in claim 1 or 2, it is characterized in that, lixiviate is operating as: get 5～10 standards analgesic extract of invigorating blood circulation and remove lid lining, be cut into fractionlet, add the methenyl choloride of 50～200ml, soaked 30～60 minutes, stir hypsokinesis and obtain cream liquid one time, then the methyl alcohol that adds 50～100ml, stir, left standstill 5～10 minutes, incline and obtain secondary cream liquid, residue adds the methyl alcohol of 50～100ml again, stir, left standstill 5～10 minutes, incline and obtain cream liquid three times, merge above-mentioned gained cream liquid, water bath method adds methyl alcohol dissolving with the gained lotion, is transferred in the first volumetric flask of 10ml after the filtration and adds methanol constant volume, and then accurate solution 2ml of measuring in the first volumetric flask is transferred in the second volumetric flask of 10ml and adds methanol constant volume, and the solution behind the constant volume is sample introduction liquid.
7. the quality determining method of a blood-activating analgesic plaster, it is characterized in that: be by data acquisition unit the chromatogram by the blood-activating analgesic plaster product of measuring under the identical experiment condition to be gathered, data acquisition unit inputs to processing unit with the chromatogram that gathers, processing unit is transferred the characteristic spectrum of the blood-activating analgesic plaster that has recorded and the chromatogram of input detects and contrasts, then the output detections consequential signal is to display unit, and display unit shows the quality condition of product.
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| CN111487351A (en) * | 2020-05-25 | 2020-08-04 | 江苏弘典中药产业研究院有限公司 | Method for detecting fingerprint of blood-activating pain-relieving capsule |
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| CN106770850A (en) * | 2016-11-30 | 2017-05-31 | 安徽安科余良卿药业有限公司 | The structure and its quality determining method of the standard feature collection of illustrative plates of analgesic rheumatoid plaster |
| CN107976494A (en) * | 2017-11-10 | 2018-05-01 | 安徽安科余良卿药业有限公司 | The structure and its quality determining method of health skin tincture standard feature collection of illustrative plates |
| CN111487351A (en) * | 2020-05-25 | 2020-08-04 | 江苏弘典中药产业研究院有限公司 | Method for detecting fingerprint of blood-activating pain-relieving capsule |
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