CN105717246A - Method for simultaneously detecting content of five effective ingredients in Jingui kidney qi tonifying tablets - Google Patents
Method for simultaneously detecting content of five effective ingredients in Jingui kidney qi tonifying tablets Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a method for simultaneously detecting the content of five effective ingredients in Jingui kidney qi tonifying tablets, and relates to the field of pharmaceutical analysis. The effective ingredients comprise loganin, geniposidic acid, verbascoside, cinnamaldehyde and paeonol; high performance liquid chromatography is adopted as the method, and the method comprises the chromatographic conditions that an octadecylsilane chemically bonded silica column is adopted as a reversed phase chromatography column, the sample injection amount is 10 milliliters, acetonitrile is adopted as a first mobile phase, an aqueous phosphoric acid solution with the volume percent of 0.1% is adopted as a second mobile phase, the sum of the volume percent of the first mobile phase and the volume percent of the second mobile phase always keeps 100%, eluting with two or three linear gradients is performed, the flowing velocity is 1.0-1.2 ml/min, and the detection wavelength is 230-294 nm. The method is simple, convenient and rapid, the quality control level of the Jingui kidney qi tonifying tablets is effectively improved, and the method has the technology leading level compared with an existing detection method.
Description
Technical field
The present invention relates to pharmaceutical analysis field, particularly to a kind of method detecting loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol content in JINKUI SHENQI PIAN simultaneously.
Background technology
JINKUI SHENQI PIAN system six drugs containing rehmanniae series Chinese patent medicine, prescription, from the hands of " doctor sage " Zhang Zhongjing, is recorded in " Medical Treasures of the Golden Chamber ".JINKUI SHENQI PIAN is used for edema due to deficiency of the kidney, soreness of the waist and knees, dysuria, aversion to cold and cold limbs.Do not only have the effect of six drugs containing rehmanniae enriching yin and nourishing kidney, and can be supporing yang in nourishing YIN, thus reaching negative and positive with the purpose mended.
JINKUI SHENQI PIAN working standard records in national drug standards WS-10439 (ZD-0439)-2002-2011Z, and its judging standard has recorded 1 microscopical identification, 3 thin layers differentiate and 2 assays, and has 10 taste medicines in pieces of Jin ' gui shenqi tablet recipe.All there is feminine gender interference between the thin layer chromatography finding multiple flavour of a drug by studying, there is no the method for efficiently separating and carry out differentiating its effective ingredient, cause that existing method of quality control fails effectively to realize the control of product quality.And the assay of existing 2 effective ingredient, it is necessary to extracting test liquid respectively and adopt different chromatographic systems to detect, experimental procedure is loaded down with trivial details, and can not comprehensively monitor the quality level of product.
Summary of the invention
For above-mentioned technical problem, the present invention provides a kind of method of five kinds of active constituent contents in JINKUI SHENQI PIAN of detection simultaneously.Effective ingredient described in this method is loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol, and described method is high performance liquid chromatography, and described chromatographic condition is:
Reversed phase chromatographic column: octadecylsilane chemically bonded silica post；Sample size: 10 μ l；
Mobile phase: mobile phase A: acetonitrile, Mobile phase B: percent by volume is the phosphate aqueous solution of 0.1%, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carrying out linear gradient elution, described linear gradient elution, wavelength and flow velocity are as follows:
The percent by volume of gradient 1---0～15min, mobile phase A and Mobile phase B is (10～20%): (90～80%), flow velocity is 1.0ml/min, and detection wavelength is 230～245nm；
Gradient 2---15～30min, the percent by volume of mobile phase A and Mobile phase B is ((10～20%) → (65～40%)): ((90～80%) → (35～60%)), flow velocity is 1.0ml/min, and detection wavelength is 230～245nm；
The percent by volume of gradient 3---30～55min, mobile phase A and Mobile phase B is (40～65%): (60～35%), flow velocity is 1.0～1.2ml/min, and detection wavelength is 284～294nm.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, and described linear gradient elution, wavelength and flow velocity be further:
The percent by volume of gradient 1---0～15min, mobile phase A and Mobile phase B is 15%: 85%, and flow velocity is 1.0ml/min, and detection wavelength is 240nm；
The percent by volume of gradient 2---15～30min, mobile phase A and Mobile phase B is (15% → 55%): (85% → 45%), flow velocity is 1.0ml/min, and detection wavelength is 240nm；
The percent by volume of gradient 3---30～55min, mobile phase A and Mobile phase B is 55%: 45%, and flow velocity is 1.2ml/min, and detection wavelength is 286nm.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, and described linear gradient elution, wavelength and flow velocity can also be further:
The percent by volume of gradient 1---0～15min, mobile phase A and Mobile phase B is 10%: 90%, and flow velocity is 1.0ml/min, and detection wavelength is 235nm；
The percent by volume of gradient 2---15～30min, mobile phase A and Mobile phase B is (10% → 65%): (90% → 30%), flow velocity is 1.0ml/min, and detection wavelength is 245nm；
The percent by volume of gradient 3---30～55min, mobile phase A and Mobile phase B is 65%: 35%, and flow velocity is 1.2ml/min, and detection wavelength is 294nm.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, in described linear gradient elution, gradient 3 can also add a transition gradient below, and the mobile phase ratio of described transition gradient is identical with described gradient 1, and rinses steady to baseline.So as continuous sample introduction.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, the collocation method of reference substance solution is as follows: precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol reference substance, dissolve with the methanol solution that percent by volume is 80% and dilute, making in every 1ml reference substance solution containing 0.10～0.14mg loganin, the sweet acid of 0.04～0.08mg genipin, 0.07～0.11mg verbascoside, 0.15～0.25mg cinnamic aldehyde and 0.15～0.25mg paeonol.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, the pre-treating method of described JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample and removes coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder 1.8～2.2g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol 25ml of 80%, weighed weight, soak 1.5 hours at 45 DEG C, put after ultrasonic 15min to room temperature, weighed weight again, the weight of less loss is supplied with the methanol that percent by volume is 80%, shake up, filter, take subsequent filtrate as need testing solution.
The present invention separately provides a kind of method of five kinds of active constituent contents in JINKUI SHENQI PIAN of detection simultaneously.Effective ingredient described in this method is loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol, and described method is high performance liquid chromatography, and described chromatographic condition is:
Reversed phase chromatographic column: octadecylsilane chemically bonded silica post；Sample size: 10 μ l；
Mobile phase: mobile phase A: acetonitrile, Mobile phase B: percent by volume is the phosphate aqueous solution of 0.1%, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carrying out linear gradient elution, described linear gradient elution, wavelength and flow velocity are as follows:
Gradient 1---0～35min, the percent by volume of mobile phase A and Mobile phase B is ((5～15%) → (65～45%)): ((95～85%) → (35～55%)), flow velocity is 1.0～1.2ml/min, and detection wavelength is 230～245nm；
The percent by volume of gradient 2---35～65min, mobile phase A and Mobile phase B is (40～65%): (60～35%), flow velocity is 1.0～1.2ml/min, and detection wavelength is 284～294nm.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, and described linear gradient elution, wavelength and flow velocity be further:
The percent by volume of gradient 1---0～35min, mobile phase A and Mobile phase B is (5% → 55%): (95% → 45%), flow velocity is 1.0ml/min, and detection wavelength is 235nm；
The percent by volume of gradient 2---35～65min, mobile phase A and Mobile phase B is 55%: 45%, and flow velocity is 1.2ml/min, and detection wavelength is 286nm.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, and described linear gradient elution, wavelength and flow velocity can also be further:
The percent by volume of gradient 1---0～35min, mobile phase A and Mobile phase B is (10% → 60%): (90% → 40%), flow velocity is 1.0ml/min, and detection wavelength is 240nm；
The percent by volume of gradient 2---35～65min, mobile phase A and Mobile phase B is 60%: 40%, and flow velocity is 1.0ml/min, and detection wavelength is 290nm.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, in described linear gradient elution, gradient 2 can also add a transition gradient below, and the mobile phase ratio of described transition gradient is identical with described gradient 1, and rinses steady to baseline.So as continuous sample introduction.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, the collocation method of reference substance solution is as follows: precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol reference substance, dissolve with the methanol solution that percent by volume is 80% and dilute, making in every 1ml reference substance solution containing 0.10～0.14mg loganin, the sweet acid of 0.04～0.08mg genipin, 0.07～0.11mg verbascoside, 0.15～0.25mg cinnamic aldehyde and 0.15～0.25mg paeonol.
As one of embodiment, the present invention detects in JINKUI SHENQI PIAN in the method for five kinds of active constituent contents simultaneously, the pre-treating method of described JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample and removes coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder 1.8～2.2g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol 25ml of 80%, close plug, weighed weight, it is heated to reflux 1 hour, put to room temperature, weighed weight again, the weight of less loss is supplied with the methanol that percent by volume is 80%, shake up, take supernatant to filter, take subsequent filtrate, obtain.
Compared with existing JINKUI SHENQI PIAN detection means, this method application modern efficient liquid chromatography technology, in elution process, the invariable isocratic elution of the gradient elution gradually change mobile phase A, B concentration proportioning and mobile phase A, B proportioning combines, form a specific gradient elution program, carry out two gradients or three gradient elutions, simultaneously the change-detection wavelength when gradient elution.Adopt this detection method not only can the detection peak of five kinds of effective ingredient in clean cut separation JINKUI SHENQI PIAN, separating degree is good, specificity is strong, also can quantitative while the content of loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol in detection JINKUI SHENQI PIAN.This detection method is easy and quick, can be effectively improved the quality control level of JINKUI SHENQI PIAN.In the JINKUI SHENQI PIAN of detection simultaneously provided by the invention, the method for five kinds of active constituent contents has feature highly sensitive, favorable reproducibility, good stability, can five kinds of effective ingredient of qualitative and quantitative detection JINKUI SHENQI PIAN accurately, save the time, save reagent.The method adopting the present invention can the quality of more effective control product, it is ensured that the curative effect of medicine.
In order to be more fully understood that and implement, describe the present invention in detail below in conjunction with accompanying drawing.
Accompanying drawing explanation
Fig. 1 is the reference substance HPLC collection of illustrative plates that embodiment 1 measures gained；
Fig. 2 is the test sample HPLC collection of illustrative plates that embodiment 1 measures gained；
Fig. 3 is the test sample HPLC collection of illustrative plates that embodiment 3 measures gained；
Fig. 4 is the reference substance HPLC collection of illustrative plates that embodiment 3 measures gained；
Fig. 5 is the test sample HPLC collection of illustrative plates that embodiment 4 measures gained；
Fig. 6 is the reference substance HPLC collection of illustrative plates that embodiment 4 measures gained.
Detailed description of the invention
Instrument: high performance liquid chromatograph Agilent1260Infinity, G1315D and G1314D.Chromatographic column: GLSciences, C18 (4.6 × 250mm, 5 μm, post number: 1A7134917)；Shiseido C18 (4.6 × 250mm, 5 μm, post number: AKAD08072), 100,000/one-level electronic balance (model: CPA225D), Ultrasound Instrument (Guangzhou Kepu Ultrasonic Electronic Technological Ltd., model: KP-Q600).
Quality standard: test sample every containing wine Fructus Corni with loganin (C17H26O10) meter, must not less than 0.25mg；Every containing Semen Plantaginis with the sweet acid (C of genipin16H22O10) and verbascoside (C29H36O15) meter, must not distinguish less than 0.23mg and 0.20mg；Every containing Ramulus Cinnamomi with cinnamic aldehyde (C9H8O) meter, must not less than 0.45mg；Every containing Cortex Moutan with paeonol (C9H10O3) meter, must not less than 0.70mg.
Reference substance: loganin (lot number: 111640-201005, Nat'l Pharmaceutical & Biological Products Control Institute provide), genipin sweet acid (lot number: 111828-201102, Nat'l Pharmaceutical & Biological Products Control Institute provide), verbascoside (lot number: 111530-201310, Nat'l Pharmaceutical & Biological Products Control Institute provide), paeonol (lot number: 110708-200506, Nat'l Pharmaceutical & Biological Products Control Institute provides) and cinnamic aldehyde (lot number: 110710-201016, Nat'l Pharmaceutical & Biological Products Control Institute provides).
Embodiment 1
Laboratory sample: JINKUI SHENQI PIAN (Guangdong Taicheng Pharmaceutical Co., Ltd produces, lot number: 20150101).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for mobile phase A, with percent by volume be 0.1% phosphoric acid solution for Mobile phase B, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carry out gradient elution by table 1.Number of theoretical plate calculates by loganin peak and is not less than 5000, and each separating degree detecting target peak adjacent peak respective with it should be greater than 1.5.
< table 1 >
The preparation of reference substance solution: it is appropriate that precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol reference substance, add the methanol solution that percent by volume is 80% to dissolve and dilute and make every 1ml and contain the mixed solution of 0.12mg loganin, the sweet acid of 0.06mg genipin, 0.09mg verbascoside, 0.20mg cinnamic aldehyde and 0.20mg paeonol, to obtain final product.
The preparation of need testing solution: taking test sample and remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder 2.0g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol solution 25ml of 80%, close plug, weighed weight, it is heated to reflux 1 hour, puts to room temperature, more weighed weight, supply the weight of less loss with the methanol solution that percent by volume is 80%, shake up, take supernatant and filter, take subsequent filtrate, to obtain final product.
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, record chromatogram also presses external standard method with calculated by peak area, result is referring to accompanying drawing 1～2, and every containing loganin 0.43mg, genipin sweet acid 0.37mg, verbascoside 0.38mg, cinnamic aldehyde 0.66mg, paeonol 0.97mg.
Embodiment 2
Laboratory sample: JINKUI SHENQI PIAN (Guangdong Taicheng Pharmaceutical Co., Ltd produces, lot number: 20150201).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for mobile phase A, with percent by volume be 0.1% phosphoric acid solution for Mobile phase B, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carry out gradient elution by table 2.Number of theoretical plate calculates by loganin peak and is not less than 5000, and each separating degree detecting target peak adjacent peak respective with it should be greater than 1.5.
< table 2 >
The preparation of reference substance solution: it is appropriate that precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol reference substance, add methanol solution that percent by volume is 80% to dissolve and make every 1ml and contain the mixed solution of 0.10mg loganin, the sweet acid of 0.04mg genipin, 0.07mg verbascoside, 0.15mg cinnamic aldehyde and 0.15mg paeonol, to obtain final product.
The preparation of need testing solution: taking test sample and remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder 2.0g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol solution 25ml of 80%, weighed weight, warm macerating (45 DEG C) 1.5 hours, put after ultrasonic 15min to room temperature, weighed weight again, supplies the weight of less loss, shakes up with the methanol solution that percent by volume is 80%, filter, take subsequent filtrate as need testing solution；
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, and record chromatogram also presses external standard method with calculated by peak area.Result: every containing loganin 0.45mg, genipin sweet acid 0.36mg, verbascoside 0.44mg, cinnamic aldehyde 0.69mg, paeonol 0.92mg.
Embodiment 3
Laboratory sample: JINKUI SHENQI PIAN (Guangdong Taicheng Pharmaceutical Co., Ltd produces, lot number: 20150202).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for mobile phase A, with percent by volume be 0.1% phosphoric acid solution for Mobile phase B, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carry out gradient elution by table 3.Number of theoretical plate calculates by loganin peak and is not less than 5000, and each separating degree detecting target peak adjacent peak respective with it should be greater than 1.5.
< table 3 >
The preparation of reference substance solution: it is appropriate that precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol reference substance, add methanol solution that percent by volume is 80% to dissolve and make every 1ml and contain the mixed solution of 0.12mg loganin, the sweet acid of 0.06mg genipin, 0.09mg verbascoside, 0.20mg cinnamic aldehyde and 0.20mg paeonol, to obtain final product.
The preparation of need testing solution: taking test sample and remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder 1.8g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol solution 25ml of 80%, close plug, weighed weight, it is heated to reflux 1 hour, puts to room temperature, more weighed weight, supply the weight of less loss with the methanol solution that percent by volume is 80%, shake up, take supernatant and filter, take subsequent filtrate, to obtain final product.
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, record chromatogram also presses external standard method with calculated by peak area, result is referring to accompanying drawing 3～4, and every containing loganin 0.41mg, genipin sweet acid 0.38mg, verbascoside 0.38mg, cinnamic aldehyde 0.65mg, paeonol 0.96mg.
Embodiment 4
Laboratory sample: JINKUI SHENQI PIAN (Guangdong Taicheng Pharmaceutical Co., Ltd produces, lot number: 20150101).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for mobile phase A, with percent by volume be 0.1% phosphoric acid solution for Mobile phase B, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carry out gradient elution by table 4.Number of theoretical plate calculates by loganin peak and is not less than 5000, and each separating degree detecting target peak adjacent peak respective with it should be greater than 1.5.
< table 4 >
The preparation of reference substance solution: it is appropriate that precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol reference substance, add methanol solution that percent by volume is 80% to dissolve and make every 1ml and contain the mixed solution of 0.12mg loganin, the sweet acid of 0.06mg genipin, 0.09mg verbascoside, 0.20mg cinnamic aldehyde and 0.20mg paeonol, to obtain final product.
The preparation of need testing solution: taking test sample and remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder 2.2g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol solution 25ml of 80%, close plug, weighed weight, it is heated to reflux 1 hour, puts to room temperature, more weighed weight, supply the weight of less loss with the methanol solution that percent by volume is 80%, shake up, take supernatant and filter, take subsequent filtrate, to obtain final product.
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, record chromatogram also presses external standard method with calculated by peak area, result is referring to accompanying drawing 5～6, and every containing loganin 0.44mg, genipin sweet acid 0.37mg, verbascoside 0.65mg, cinnamic aldehyde 0.64mg, paeonol 0.99mg.
Method validation embodiment
Method validation test is carried out by the requirement of " Chinese Pharmacopoeia " version in 2010 " annex xVIII quality standards in Chinese drugs analyzes method validation guideline ".
Laboratory sample: JINKUI SHENQI PIAN (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；JINKUI SHENQI PIAN wine Fructus Corni feminine gender preparation (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；JINKUI SHENQI PIAN Semen Plantaginis feminine gender preparation (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；JINKUI SHENQI PIAN Ramulus Cinnamomi feminine gender preparation (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；JINKUI SHENQI PIAN Cortex Moutan feminine gender preparation (Guangdong Taicheng Pharmaceutical Co., Ltd's production).
The preparation of reference substance stock solution: precision claims to obtain loganin reference substance 19.84mg, genipin sweet acid reference substance 9.25mg, verbascoside reference substance 13.98mg, cinnamic aldehyde reference substance 33.45mg and paeonol reference substance 32.31mg respectively, they are put in same 25ml volumetric flask, dissolve with the methanol solution that percent by volume is 80% and be diluted to scale, shake up, as reference substance stock solution.
1. specificity test
Reference substance solution, need testing solution is prepared according to the method for embodiment 1, the negative sample solution of wine Fructus Corni, Semen Plantaginis, Ramulus Cinnamomi, each negative preparation of Cortex Moutan is lacked with reference to the compound method preparation JINKUI SHENQI PIAN of need testing solution in embodiment 1, precision measures each 10 μ l of above-mentioned solution, it is injected separately into chromatograph of liquid, measures by the assay condition of embodiment 1-4.
Result shows, each negative sample solution chromatogram is noiseless on the corresponding position of each component of reference substance solution chromatogram.In need testing solution chromatogram, number of theoretical plate calculates by loganin peak and is not less than 5000, and each separating degree detecting target peak adjacent peak respective with it is all higher than 1.5, meets " Chinese Pharmacopoeia " version assay related request in 2015.Table 5 is illustrated that the specificity experimental result of the assay method gained adopting embodiment 1.Adopt the specificity experimental result that the assay method of embodiment 2-4 records similarly.
Table 5 specificity result of the test
2. linear test
Precision measures reference substance stock solution 1ml, 1ml, 1ml, 2ml, 3ml, 5ml, it is respectively placed in 50ml, 25ml, 10ml, 10ml, 10ml, 10ml volumetric flask and adds the methanol solution that percent by volume is 80% and be diluted to scale, shake up, obtain each concentrations control product solution for linearly investigating；
Precision measures each 10 μ l of above-mentioned solution, is injected separately into chromatograph of liquid, according to the condition determination record chromatogram of embodiment 1.Result is as shown in table 6, loganin in the scope of concentration 0.0159mg/ml～0.3968mg/ml with the linear relationship that its peak area is good, the sweet acid of genipin in the scope of concentration 0.0074mg/ml1～0.1850mg/ml with the linear relationship that its peak area is good, verbascoside in the scope of concentration 0.0112mg/ml～0.2796mg/ml with the linear relationship that its peak area is good, cinnamic aldehyde in the scope of concentration 0.0268mg/ml～0.6690mg/ml with the linear relationship that its peak area is good, paeonol in the scope of concentration 0.025mg/ml～0.6462mg/ml with the linear relationship that its peak area is good.The result adopting the condition determination gained of embodiment 2-4 is basic similar to adopting embodiment 1 condition determination acquired results, does not repeat them here.
Table 6 linear test result
3. precision test
3.1 repeatability
Taking 20150101 batches of JINKUI SHENQI PIAN appropriate, finely ground after removing coating, gained fine powder can pass through 100 mesh sieves, and precision weighs fine powder 1.8～2.2g totally 6 parts, makes need testing solution according to the method for embodiment 1 respectively；It is appropriate that another precision weighs loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and each reference substance of paeonol, makes reference substance solution according to the method for embodiment 1, is measured by the condition determination of embodiment 1-4.It was found that the condition determination acquired results of employing embodiment 1-4 is similar, and measurement result is respectively provided with good repeatability, and RSD value is respectively less than 2%.Table 7 below illustrates the replica test result adopting the condition determination of embodiment 1 to record.
The preparation of need testing solution: taking test sample and remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder and be about 2.0g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol solution 25ml of 80%, close plug, weighed weight, it is heated to reflux 1 hour, puts to room temperature, more weighed weight, supply the weight of less loss with the methanol solution that percent by volume is 80%, shake up, take supernatant and filter, take subsequent filtrate, to obtain final product.
Table 7 replica test result
3.2 Intermediate precision
Take 20150101 batches of JINKUI SHENQI PIAN, by A, B, C tri-people respectively at not same date, distinct device, often prepare 3 parts of need testing solutions by the method under " 3.1 repeatability " item in precision test per capita, being measured by the assay condition of embodiment 1-4, table 8 below illustrates the Intermediate precision result of the test adopting the content assaying method of embodiment 1 to record.(the Intermediate precision measurement result adopting the content assaying method of embodiment 2-4 is similar to embodiment 1, does not repeat them here)
Table 8 Intermediate precision result of the test
By Intermediate precision result of the test it can be seen that the content assaying method of the present invention has the feature of favorable reproducibility.
4. stability test
Need testing solution and each 1 part of reference substance solution is prepared according to the method for embodiment 1, placement 0,4,8,12,18,24 is little constantly, precision draws reference substance solution and each 10 μ l of need testing solution respectively, inject chromatograph of liquid, it is measured according to the assay condition of embodiment 1-4, found that need testing solution and reference substance solution are basicly stable in 24 hours, below the stability test result that the assay method adopting embodiment 1 records is shown below 9.
Table 9 stability test result
By stability test result it can be seen that the assay method of the present invention in 24 hours the measured value RSD of peak area less than 2.0, good stability.
4. average recovery
The preparation of recovery test need testing solution: take after the JINKUI SHENQI PIAN of 20150101 batches of known content removes coating finely ground, gained fine powder can pass through 100 mesh sieves, precision weighs fine powder and is about 1.0g totally 6 parts, it is respectively placed in 6 tool plug conical flasks, each accurate addition recovery test reference substance stock solution 5ml and the methanol solution 20ml that percent by volume is 80%, close plug, weighed weight, it is heated to reflux 1 hour, put to room temperature, weighed weight again, the weight of less loss is supplied with the methanol solution that percent by volume is 80%, shake up, take supernatant to filter, take subsequent filtrate as recovery test need testing solution, it is measured according to the condition determination of embodiment 1-4, gained response rate meansigma methods all reaches more than 98%, RSD is respectively less than 2.0%.Below the recovery test result that the assay method adopting embodiment 1 records representatively is illustrated in table 10:
Table 10 recovery test result
From the result of the test of the response rate it can be seen that adopt detection method provided by the invention to have good accuracy.
5. the accuracy test of measurement range
The extraction rate of transform according to each qualified medical material content limit and each medical material effective ingredient and this product study on the stability result, after carrying out comprehensively analyzing, it is determined that with described in five kinds of effective ingredient 50% and the 250% of quality standard for scope investigation accuracy.
Prepared by limit 50% need testing solution: take 20150101 batches of JINKUI SHENQI PIAN appropriate, remove after coating finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder and be about 0.5g6 part, accurately weighed, every part all adds each 1ml of reference substance stock solution, prepares by the preparation method of the need testing solution of embodiment 1 afterwards, measure each component content according to the condition determination of embodiment 1-4, calculate the response rate.Table 11 below illustrates that the result measured by the condition determination of embodiment 2-4 is basic identical with embodiment 1 according to the result that the condition determination of embodiment 1 records.
Limit 250% test sample: take 20150101 batches of JINKUI SHENQI PIAN appropriate, remove after coating finely ground, gained fine powder can pass through 100 mesh sieves, take fine powder and be about 2.5g6 part, accurately weighed, every part all adds each 5ml of reference substance stock solution, prepares by the preparation method of the need testing solution of embodiment 1, measure each component content according to the condition determination of embodiment 1-4, calculate the response rate.Table 12 below illustrates that the result measured by the condition determination of embodiment 2-4 is basic identical with embodiment 1 according to the result that the condition determination of embodiment 1 records.
Table 11 low strength range (by limit 50%)
Table 12 high concentration range (by limit 250%)
By the result of the accuracy test of measurement range it can be seen that in the scope of 50%～250% of each composition limit in JINKUI SHENQI PIAN, measurement result is reliable and stable, accuracy is high.
Result by above method checking, adopt the detection method of the present invention, it is possible not only to detect the content of five kinds of effective ingredient in JINKUI SHENQI PIAN simultaneously, and this detection method accuracy, repeatability and precision are all good, good in measurement range internal linear relation, in 24 hours, need testing solution and reference substance are all basicly stable.Method validation result of the test shows that this law can as the assay method of loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol content in JINKUI SHENQI PIAN.Again can be qualitative while quantitatively measuring content, can effectively wine Fructus Corni, Semen Plantaginis, Ramulus Cinnamomi and 4 flavour of a drug of Cortex Moutan in pieces of Jin ' gui shenqi tablet recipe be differentiated.
The invention is not limited in above-mentioned embodiment, if to the various changes of the present invention or deformation without departing from the spirit and scope of the present invention, if these are changed and deform within the claim and the equivalent technologies scope that belong to the present invention, then the present invention is also intended to comprise these changes and deformation.
Claims (10)
1. one kind is detected the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously, it is characterized in that: described effective ingredient is loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol, described method is high performance liquid chromatography, and described chromatographic condition is:
Reversed phase chromatographic column: octadecylsilane chemically bonded silica post；Sample size: 10 μ l；
Mobile phase: mobile phase A: acetonitrile, Mobile phase B: percent by volume is the phosphate aqueous solution of 0.1%, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carrying out linear gradient elution, described linear gradient elution, wavelength and flow velocity are as follows:
The percent by volume of gradient 1---0～15min, mobile phase A and Mobile phase B is (10～20%): (90～80%), flow velocity is 1.0ml/min, and detection wavelength is 230～245nm；
Gradient 2---15～30min, the percent by volume of mobile phase A and Mobile phase B is ((10～20%) → (65～40%)): ((90～80%) → (35～60%)), flow velocity is 1.0ml/min, and detection wavelength is 230～245nm；
The percent by volume of gradient 3---30～55min, mobile phase A and Mobile phase B is (40～65%): (60～35%), flow velocity is 1.0～1.2ml/min, and detection wavelength is 284～294nm.
2. method according to claim 1, it is characterised in that: described linear gradient elution, wavelength and flow velocity are as follows:
The percent by volume of gradient 1---0～15min, mobile phase A and Mobile phase B is 15%: 85%, and flow velocity is 1.0ml/min, and detection wavelength is 240nm；
The percent by volume of gradient 2---15～30min, mobile phase A and Mobile phase B is (15% → 55%): (85% → 45%), flow velocity is 1.0ml/min, and detection wavelength is 240nm；
The percent by volume of gradient 3---30～55min, mobile phase A and Mobile phase B is 55%∶45%, flow velocity is 1.2ml/min, and detection wavelength is 286nm.
3. method according to claim 1, it is characterised in that: described linear gradient elution, wavelength and flow velocity are as follows:
The percent by volume of gradient 1---0～15min, mobile phase A and Mobile phase B is 10%: 90%, and flow velocity is 1.0ml/min, and detection wavelength is 235nm；
The percent by volume of gradient 2---15～30min, mobile phase A and Mobile phase B is (10% → 65%): (90% → 30%), flow velocity is 1.0ml/min, and detection wavelength is 245nm；
The percent by volume of gradient 3---30～55min, mobile phase A and Mobile phase B is 65%: 35%, and flow velocity is 1.2ml/min, and detection wavelength is 294nm.
4. according to the method in claim 2 or 3, it is characterised in that: in described linear gradient elution, gradient 3 can also add a transition gradient below, and the mobile phase ratio of described transition gradient is identical with described gradient 1, and rinses steady to baseline.
5. according to the method in claim 2 or 3, characterized by further comprising the pre-treating method of described JINKUI SHENQI PIAN, the pre-treating method of described JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample and removes coating, finely ground, gained fine powder can pass through 100 mesh sieves, take described fine powder 1.8～2.2g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol 25ml of 80%, weighed weight, soak 1.5 hours at 45 DEG C, put after ultrasonic 15min to room temperature, weighed weight again, the weight of less loss is supplied with the methanol that percent by volume is 80%, shake up, filter, take subsequent filtrate as need testing solution.
6. one kind is detected the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously, it is characterized in that: described effective ingredient is loganin, the sweet acid of genipin, verbascoside, cinnamic aldehyde and paeonol, described method is high performance liquid chromatography, and described chromatographic condition is:
Reversed phase chromatographic column: octadecylsilane chemically bonded silica post；Sample size: 10 μ l；
Mobile phase: mobile phase A: acetonitrile, Mobile phase B: percent by volume is the phosphate aqueous solution of 0.1%, the percent by volume of mobile phase A and the percent by volume of Mobile phase B and remain 100%, carrying out linear gradient elution, described linear gradient elution, wavelength and flow velocity are as follows:
Gradient 1---0～35min, the percent by volume of mobile phase A and Mobile phase B is ((5～15%) → (65～45%)): ((95～85%) → (35～55%)), flow velocity is 1.0～1.2ml/mm, and detection wavelength is 230～245nm；
The percent by volume of gradient 2---35～65min, mobile phase A and Mobile phase B is (40～65%): (60～35%), flow velocity is 1.0～1.2ml/min, and detection wavelength is 284～294nm.
7. method according to claim 6, it is characterised in that: described linear gradient elution, wavelength and flow velocity are as follows:
The percent by volume of gradient 1---0～35min, mobile phase A and Mobile phase B is (5% → 55%): (95% → 45%), flow velocity is 1.0ml/min, and detection wavelength is 235nm；
The percent by volume of gradient 2---35～65min, mobile phase A and Mobile phase B is 55%: 45%, and flow velocity is 1.2ml/mm, and detection wavelength is 286nm.
8. method according to claim 6, it is characterised in that: described linear gradient elution, wavelength and flow velocity are as follows:
The percent by volume of gradient 1---0～35min, mobile phase A and Mobile phase B is (10% → 60%): (90% → 40%), flow velocity is 1.0ml/min, and detection wavelength is 240nm；
The percent by volume of gradient 2---35～65min, mobile phase A and Mobile phase B is 60%: 40%, and flow velocity is 1.0ml/mm, and detection wavelength is 290nm.
9. the method according to claim 7 or 8, it is characterised in that: in described linear gradient elution, gradient 2 can also add a transition gradient below, and the mobile phase ratio of described transition gradient is identical with described gradient 1, and rinses steady to baseline.
10. the method according to claim 7 or 8, characterized by further comprising the pre-treating method of described JINKUI SHENQI PIAN, the pre-treating method of described JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample and removes coating, finely ground, gained fine powder can pass through 100 mesh sieves, take described fine powder 1.8～2.2g, accurately weighed, put in tool plug conical flask, accurate addition percent by volume is the methanol 25ml of 80%, close plug, weighed weight, it is heated to reflux 1 hour, put to room temperature, weighed weight again, the weight of less loss is supplied with the methanol that percent by volume is 80%, shake up, take supernatant to filter, take subsequent filtrate, obtain.
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| CN115327019A (en) * | 2022-08-04 | 2022-11-11 | 国药集团德众（佛山）药业有限公司 | Quality control method of mulberry twig |
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