CN105021723A - Method for simultaneous determination of content of geniposide, andrographolide and dehydroandrographolide in Zhimai tablets for clearing heat - Google Patents
Method for simultaneous determination of content of geniposide, andrographolide and dehydroandrographolide in Zhimai tablets for clearing heat Download PDFInfo
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Abstract
The present invention discloses a method for simultaneous determination of content of geniposide, andrographolide and dehydroandrographolide in Zhimai tablets for clearing heat by using a binary or higher pump HPLC. The method belongs to the medicine field and comprises the steps of preparation of a reference solution, preparation of a test solution, optimization of chromatographic conditions and selection. The method is as below: using the established HPLC with a C18 reverse phase column, a mobile phase of methanol-water and flow rate of 1.0ml / min; conducting gradient elution; setting program for each measured component wavelength conversion according to the peak time programmed, so as to achieve the simultaneous determination of three active ingredients. The method has the advantages of rapidness, sensitivity, strong specificity, simple operation, low equipment requirements, stability, and good reproducibility, and can be used in quality control of Zhimai tablets for clearing heat and derived products thereof.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the detection method of Gardenoside, andrographolide and Determination of Dehydroandrographoliin in a kind of Simultaneously test Qinghuozhimaipian.
Background technology
Qinghuozhimaipian records in Chinese Pharmacopoeia version in 2010.Be made up of Herba Andrographitis, cape jasmine, the tuber of dwarf lilyturf, there is relieving inflammation or internal heat, detoxify, cool blood, detumescence effect, for abscess of throat, acute tonsillitis, heating, headache, toothache, hot eyes, excessive fire in heart, poor sleep etc.In Chinese Pharmacopoeia 2010 editions with Gardenoside, andrographolide and Dehydro and drographolide for index components carries out quality control to Qinghuozhimaipian, adopt HPLC method, by methanol-water and acetonitrile-water two individual system, andrographolide constituents and Gardenoside are measured respectively, complicated operation.Although there are three kinds of effective constituents in report Simultaneously test Qinghuozhimaipian, the method needs the high performance liquid chromatograph with ternary or the above pumping system of ternary, is unfavorable for the popularization of Basic Enterprise.For solving the problem, inventor proposes the present invention.
The present invention adopts reversed-phased high performace liquid chromatographic, take methanol-water as mobile phase, gradient elution, and each conversion measuring composition wavelength carries out program setting by appearance time order, reaches the object of Simultaneously test 3 kinds of active components.Method rapid sensitive, specificity is strong, easy and simple to handle, stablizes and favorable reproducibility, may be used for the quality control of Qinghuozhimaipian and derivative preparation thereof.
Summary of the invention
For overcoming the deficiencies in the prior art, the present invention is achieved through the following technical solutions Gardenoside, andrographolide and Determination of Dehydroandrographoliin in Simultaneously test Qinghuozhimaipian:
1. the preparation of reference substance solution
1.1 single reference substance solution: getting Gardenoside reference substance methyl alcohol, to be mixed with concentration be 40 ± 5 μ gmL
-1solution, getting andrographolide and Dehydro and drographolide reference substance methyl alcohol, to be mixed with concentration be respectively 80 ± 5 μ gmL
-1solution.
1.2 mixing reference substance solution: get Gardenoside, andrographolide, deoxidation Herba Andrographitis heart lotus lactone reference substance, dissolve with methyl alcohol and are mixed with containing Gardenoside 40 ± 5 μ gmL
-1, andrographolide 80 ± 5 μ gmL
-1, Dehydro and drographolide 80 ± 5 μ gmL
-1mixed solution.
2. for the preparation of test liquid
Get after Qinghuozhimaipian content powder is about 0.5g precise weighing, put in 50mL measuring bottle, add methyl alcohol appropriate, ultrasonic process 30min, lets cool, adds methyl alcohol to scale, shake up, filtration, gets subsequent filtrate after continuous 10ml, to obtain final product.
3. chromatographic condition: chromatograph: there is binary or binary with the liquid chromatograph of upper pump casing, as VARIAN-LC210, Shimadzu-LC20A, AGILENT-LC160, etc.; The detecting device of high performance liquid chromatograph configuration is UV-detector; Chromatographic column: C
18reverse-phase chromatographic column, as: AgilentC
18(250 × 4.6mm, 5 μm), Welch Ultimate C
18(4.6um × 250mm, 5 μm), Kromasil C
18(250 × 4.6mm, 5 μm), mobile phase is methanol-water, gradient elution, and elution program is: 0 ~ 10min, 35:65,10.01 ~ 25min:53:47; 25.01 ~ 40min:65:35, flow velocity 1.0mLmin
-1; Determined wavelength: Gardenoside: 240nm (0 ~ 10min), andrographolide: 230nm (10.01 ~ 25min), Dehydro and drographolide: 250nm (25.01 ~ 40min); Column temperature: room temperature; Sample size: 10 μ L; Reference substance used provides by National Institute for Food and Drugs Control, and preparation reference substance solution and HPLC chromatography reagent are chromatographically pure rank, and other reagent is for analyzing pure rank.
The present invention has the following advantages:
Gardenoside, andrographolide and Determination of Dehydroandrographoliin in the HPLC assay method energy Simultaneously test Qinghuozhimaipian that the present invention sets up, the method rapid sensitive, specificity is strong, easy and simple to handle, stablize and favorable reproducibility, the quality control for said preparation provides reliable evaluation method.The HPLC assay method of the present invention's foundation in addition, employs comparatively simple methanol-water gradient elution and detects chromatographic technique, decrease (chromatograph containing binary or more pump housing), be beneficial to very much and apply chromatographic requirement.
Accompanying drawing explanation
Fig. 1 Gardenoside, andrographolide and Dehydro and drographolide single contrast liquid chromatography figure
Fig. 2 Gardenoside, andrographolide and Dehydro and drographolide mixing contrast liquid chromatography figure
Fig. 3 Qinghuozhimaipian test liquid chromatogram
The negative sample liquid chromatogram of Fig. 4
Embodiment
Below in conjunction with embodiment, illustrate the present invention further, example only for explaining the present invention, but does not limit scope of the present invention.
The content of Gardenoside, andrographolide and Dehydro and drographolide in HPLC method Simultaneously test Qinghuozhimaipian is utilized to carry out according to following steps:
1. instrument and material
1.1 instruments and reagent
VARIAN 210 type high performance liquid chromatograph (VARIAN Oncology Systems), EL204 ten thousand/gram electronic balance (plum Teller-Tuo benefit instrument Shanghai company limited); KQ-250DE supersonic wave cleaning machine (Kunshan Ultrasonic Instruments Co., Ltd.); DFT-200 Portable high speed Universalpulverizer (Lin great Machinery Co., Ltd. of Wenling city).
Methyl alcohol (chromatographically pure, Xilong Chemical Co., Ltd), water is ultrapure water; Gardenoside reference substance (lot number 110749-200714), andrographolide reference substance (lot number 110797-200307), Dehydro and drographolide reference substance (lot number 110854-201007) are all purchased from National Institute for Food and Drugs Control
1.2 test sample
Qinghuozhimaipian content 10 batches, by medicine resource development centre, Medicinal Plants of Guangxi garden according to " Chinese Pharmacopoeia " 2010 editions fire-clearing zhimai tablet recipes, utilize new technology to manufacture experimently, lot number is respectively 201401,201402,201403,201404,201405,201406,201407,201408,201409,201410.
2. method and result
The preparation of 2.1 reference substance solution
2.1.1 reference substance mixing storing solution: precision takes Gardenoside reference substance 0.0051g, andrographolide reference substance 0.0112g, Dehydro and drographolide reference substance 0.0900g, be placed in same 50ml measuring bottle, dissolve with methyl alcohol and be diluted to scale, shake up, (concentration is respectively: Gardenoside 102 μ gml must to mix reference substance mixing storing solution
-1, andrographolide 224 μ gml
-1, Dehydro and drographolide 180 μ gml
-1).
2.1.2 reference substance mixed solution: precision measures reference substance mixing storing solution 4ml respectively, puts in 10ml measuring bottle, adds methyl alcohol to scale, shake up, obtaining Gardenoside concentration is 40.8 μ gml
-1, andrographolide concentration is 80.8 μ gml
-1, Dehydro and drographolide concentration is 80 μ gml
-1reference substance mixed liquor.
The preparation of 2.2 confession test liquids and negative sample liquid
2.2.1 the preparation of need testing solution: get Qinghuozhimaipian content powder and be about in the rearmounted 50mL measuring bottle of 0.5g precise weighing, add methyl alcohol appropriate, ultrasonic process 30min, lets cool, adds methyl alcohol to scale, shake up, and filters, gets the subsequent filtrate after 10ml, to obtain final product.
2.2.2 the preparation of negative sample liquid: in the ratio of fire-clearing zhimai tablet recipe, preparation does not contain the negative sample of cape jasmine and Herba Andrographitis, porphyrize, takes appropriate, by method operation under " 2.2.1 " item, to obtain final product.
2.3 chromatographic condition
Liquid chromatograph: VARIAN-LC210, detecting device: UV-detector, chromatographic column: Agilent C
18(250 × 4.6mm, 5 μm), mobile phase is methanol-water, gradient elution, and elution program is: 0 ~ 10min, 35:65,10.01 ~ 25min:53:47; 25.01 ~ 40min:65:35, flow velocity 1.0mLmin
-1; Determined wavelength: Gardenoside: 240nm (0 ~ 10min), andrographolide: 230nm (10.01 ~ 25min), Dehydro and drographolide: 250nm (25.01 ~ 40min); Column temperature: room temperature; Sample size: 10 μ L.
The investigation of 2.4 linear relationships
Respectively precision measure reference substance mixing storing solution 1,2,3,4,5,6,7,8ml, put in 10ml measuring bottle, add methyl alcohol to scale.Shake up rear respectively sample introduction 10 μ L, record peak area, with sample size X for horizontal ordinate to peak area Y for ordinate returns, investigate linear relationship.As shown in Table 1, Gardenoside is at 10.2 ~ 81.6 μ gml
-1, andrographolide is at 22.4 ~ 179.2 μ gml
-1, Dehydro and drographolide is at 18.0 ~ 144.0 μ gml
-1concentration range in, reference substance solution concentration and absorbance log are good linear relationship.
The typical curve of table 1 Gardenoside, andrographolide and Dehydro and drographolide, related coefficient, range of linearity measurement result
2.5 precision, repeatability and stability test
Get Qinghuozhimaipian content dry powder and be about 0.5g, need testing solution is made by the compound method of need testing solution, sample introduction 10 μ L under condition determination, repeat sample introduction 6 times, record peak area, the RSD value recording Gardenoside, andrographolide and Dehydro and drographolide is respectively: 1.06%, 1.19%, 1.06%, result shows that the precision of instrument is good.
Get Qinghuozhimaipian content dry powder and be about 0.5g, need testing solution is made by the compound method of need testing solution, ambient temperatare is put, in 0,2,4,6,8,10,12 hour sample introduction 10 μ L, record peak area, the RSD value of Gardenoside, andrographolide and Dehydro and drographolide is respectively 1.22%, 1.84%, 1.10%, result shows that need testing solution is good at 12 hours internal stabilities.
Get 6 parts, Qinghuozhimaipian content dry powder, every part of about 0.5g, makes need testing solution by the compound method of need testing solution, every part of sample introduction 10 μ L, record peak area, and the average relative medicinal material content recording Gardenoside is 23.755mg/g, RSD is 1.45%; Andrographolide average relative medicinal material content is 4.888mg/g, RSD is 1.92%; Dehydro and drographolide average relative medicinal material content is 4.405mg/g, RSD is 1.18%, and result shows that this method repeatability is good.
2.6 average recovery tests
Get Qinghuozhimaipian extraction dry extract test sample and be about 0.25g, accurately weighed, totally 9 parts, 3 parts respectively precision add reference substance stock solution 3mL, 3 parts respectively precision add reference substance stock solution 5mL, 3 parts respectively precision add reference substance stock solution 7mL, prepare by the compound method of need testing solution, obtain application of sample reclaim test liquid, sample introduction 10 μ L respectively, measure peak area, calculate the recovery, result is as table 2-4.Result confirms that the method set up has higher accuracy for Gardenoside, andrographolide and Determination of Dehydroandrographoliin in Simultaneously test Qinghuozhimaipian.
The average recovery test findings of Gardenoside in table 2 Qinghuozhimaipian
The average recovery test findings of andrographolide in table 3 Qinghuozhimaipian
The average recovery test findings of Dehydro and drographolide in table 4 Qinghuozhimaipian
2.7 sample determination
Precision takes the sample 0.5g of 10 different batches, prepares by the compound method of need testing solution, obtains ten batches of test sample sample liquids.Measure under above-mentioned chromatographic condition, measurement result is in table 5.
In table 50 batches of different batches Qinghuozhimaipians, Gardenoside, andrographolide and Dehydro and drographolide are containing surveying result
3. conclusion
What in Chinese Pharmacopoeia 2010 editions Qinghuozhimaipians, the assay of Gardenoside, andrographolide and Dehydro and drographolide adopted is separate determination method.The HPLC detection method that this experiment is set up, adopt water-methanol elution system, the content of Gardenoside, andrographolide and Dehydro and drographolide three kinds of materials in energy Simultaneously test Qinghuozhimaipian, the method rapid sensitive, lower to the requirement of equipment, easy and simple to handle, overcome the shortcoming that former method is consuming time and loaded down with trivial details, for the quality control of Qinghuozhimaipian and derived product thereof provides reliably and the method for quality control of economy.
Claims (2)
1. the detection method of Gardenoside, andrographolide and Determination of Dehydroandrographoliin in Simultaneously test Qinghuozhimaipian, is characterized in that following steps:
The preparation of 1.1 reference substance solution
Get Gardenoside, andrographolide, Dehydro and drographolide reference substance methyl alcohol is mixed with the independent reference substance solution of each reference substance and the mixing reference substance solution of 3 kinds of reference substances;
The concentration of 1.2 each reference substance solution
Independent reference substance solution: Gardenoside: 40 ± 5 μ gmL
-1, andrographolide: 80 ± 5 μ gmL
-1, Dehydro and drographolide: 80 ± 5 μ gmL
-1; Mixing reference substance solution: containing Gardenoside: 40 ± 5 μ gmL
-1, andrographolide: 80 ± 5 μ gmL
-1, Dehydro and drographolide: 80 ± 5 μ gmL
-1;
The preparation of 1.3 confession test liquids
Get Qinghuozhimaipian content powder and be about 0.5g, in accurately weighed rearmounted 50mL measuring bottle, add methyl alcohol appropriate, ultrasonic process 30min, lets cool, adds methyl alcohol to scale, shake up, and filters, and collects the subsequent filtrate after 10ml, to obtain final product;
1.4 chromatographic conditions: chromatographic column: C
18reverse-phase chromatographic column, as: Agilent C
18(250 × 4.6mm, 5 μm), Welch Ultimate C
18(4.6um × 250mm, 5 μm), Kromasil C
18(250 × 4.6mm, 5 μm) etc., mobile phase is methanol-water, gradient elution, and elution program is: 0 ~ 10min:35:65,10.01 ~ 25min:53:47,25.01 ~ 40min:65:35, flow velocity 1.0mLmin
-1, determined wavelength: Gardenoside: 240nm (0 ~ 10min), andrographolide: 230nm (10.01 ~ 25min), Dehydro and drographolide: 250nm (25.01 ~ 40min), column temperature: room temperature, sample size: 10 μ L.
2. according to claim 1, the high performance liquid chromatograph that in Simultaneously test Qinghuozhimaipian, Gardenoside, andrographolide and Determination of Dehydroandrographoliin method adopt must configure the pump housing of more than binary or binary, as VARIAN-LC210, Shimadzu-LC20A, AGILENT-LC160 etc., the detecting device of high performance liquid chromatograph configuration is UV-detector, reference substance used provides by National Institute for Food and Drugs Control or internationally recognized mechanism, reference substance solution and HPLC chromatography reagent are chromatographically pure rank, and other reagent is for analyzing pure rank.
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| CN105929066A (en) * | 2016-04-25 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for determining andrographolide and dehydroandrographoline in andrographis tablet by using HPLC |
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Application publication date: 20151104 |