CN105717246B - 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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- CN105717246B CN105717246B CN201610059542.5A CN201610059542A CN105717246B CN 105717246 B CN105717246 B CN 105717246B CN 201610059542 A CN201610059542 A CN 201610059542A CN 105717246 B CN105717246 B CN 105717246B
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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 analysiss field, detect loganin, genipin particularly in a kind of JINKUI SHENQI PIAN simultaneously
The method of sweet acid, verbascoside, cinnamic aldehyde and paeonol content.
Background technology
JINKUI SHENQI PIAN system six drugs containing rehmanniae series Chinese patent medicine, prescription is derived from the handss of " holy doctor " Zhang Zhongjing, is recorded in that " gold is deficient will
Slightly ".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 six drugs containing rehmanniae enriching yin and nourishing kidney
Effect, 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, its mirror
Other 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.
Find all there is negative interference between the thin layer chromatography of multiple flavour of a drug by research, there is no the method for efficiently separating to carry out differentiating that it has
Effect composition, leads to existing method of quality control to fail effectively to realize the control of product quality.And existing 2 effective ingredient
Assay, needs to extract test liquid respectively and using different chromatographic system detections, experimental procedure is loaded down with trivial details, and can not be comprehensive
Monitor the quality level of product.
Content of the invention
For above-mentioned technical problem, the present invention provides five kinds of active constituent contents in a kind of JINKUI SHENQI PIAN of detection simultaneously
Method.Effective ingredient described in this method is loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol, described side
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 0.1% phosphate aqueous solution, the body of mobile phase a
Long-pending percentage ratio and mobile phase b percent by volume and remain 100%, carries out linear gradient elution, described linear ladder
Degree eluting, wavelength and flow velocity are as follows:
Gradient 1---0～15min, mobile phase a is (10～20%): (90～80%) with the percent by volume of mobile phase b,
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 be ((10～20%) → (65～
40%)): ((90～80%) → (35～60%)), flow velocity is 1.0ml/min, and Detection wavelength is 230～245nm；
Gradient 3---30～55min, mobile phase a is (40～65%) with the percent by volume of mobile phase b: (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 the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
Described linear gradient elution, wavelength and flow velocity are further:
Gradient 1---0～15min, mobile phase a is 15%: 85% with the percent by volume of mobile phase b, and flow velocity is 1.0ml/
Min, Detection wavelength is 240nm；
Gradient 2---15～30min, mobile phase a is (15% → 55%) with the percent by volume of mobile phase b: (85% →
45%), flow velocity is 1.0ml/min, and Detection wavelength is 240nm；
Gradient 3---30～55min, mobile phase a is 55%: 45% with the percent by volume of mobile phase b, and flow velocity is
1.2ml/min, Detection wavelength is 286nm.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
Described linear gradient elution, wavelength and flow velocity can also be further:
Gradient 1---0～15min, mobile phase a is 10%: 90% with the percent by volume of mobile phase b, and flow velocity is 1.0ml/
Min, Detection wavelength is 235nm；
Gradient 2---15～30min, mobile phase a is (10% → 65%) with the percent by volume of mobile phase b: (90% →
30%), flow velocity is 1.0ml/min, and Detection wavelength is 245nm；
Gradient 3---30～55min, mobile phase a is 65%: 35% with the percent by volume of mobile phase b, and flow velocity is
1.2ml/min, Detection wavelength is 294nm.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
In described linear gradient elution, gradient 3 can also add a transition gradient below, the mobile phase ratio of described transition gradient with
Described gradient 1 is identical, and rinses steady to baseline.So that continuous sample introduction.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
The collocation method of reference substance solution is as follows: precision weighs loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol
Reference substance, is dissolved and is diluted with the methanol solution that percent by volume is 80%, make in every 1ml reference substance solution contain 0.10～
0.14mg loganin, 0.04～0.08mg genipin sweet acid, 0.07～0.11mg verbascoside, 0.15～0.25mg cinnamic aldehyde
With 0.15～0.25mg paeonol.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
The pre-treating method of described JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample to remove coating, finely ground, gained fine powder can pass through
100 mesh sieves, take fine powder 1.8～2.2g, accurately weighed, put in conical flask with cover, and accurate addition percent by volume is 80% first
Alcohol 25ml, weighed weight, soak 1.5 hours at 45 DEG C, put to room temperature after ultrasonic 15min, more weighed weight, use percent by volume
Methanol for 80% supplies the weight of less loss, shakes up, and filters, takes subsequent filtrate as need testing solution.
The another method that five kinds of active constituent contents in a kind of JINKUI SHENQI PIAN of detection simultaneously are provided of the present invention.Institute in this method
Stating effective ingredient is loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol, and methods described is high-efficient liquid phase color
Spectrometry, 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 0.1% phosphate aqueous solution, the body of mobile phase a
Long-pending percentage ratio and mobile phase b percent by volume and remain 100%, carries out linear gradient elution, described linear ladder
Degree eluting, wavelength and flow velocity are as follows:
Gradient 1---0～35min, the percent by volume of mobile phase a and mobile phase b be ((5～15%) → (65～
45%)): ((95～85%) → (35～55%)), flow velocity is 1.0～1.2ml/min, and Detection wavelength is 230～245nm；
Gradient 2---35～65min, mobile phase a is (40～65%) with the percent by volume of mobile phase b: (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 the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
Described linear gradient elution, wavelength and flow velocity are further:
Gradient 1---0～35min, mobile phase a is (5% → 55%) with the percent by volume of mobile phase b: (95% →
45%), flow velocity is 1.0ml/min, and Detection wavelength is 235nm；
Gradient 2---35～65min, mobile phase a is 55%: 45% with the percent by volume of mobile phase b, and flow velocity is
1.2ml/min, Detection wavelength is 286nm.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
Described linear gradient elution, wavelength and flow velocity can also be further:
Gradient 1---0～35min, mobile phase a is (10% → 60%) with the percent by volume of mobile phase b: (90% →
40%), flow velocity is 1.0ml/min, and Detection wavelength is 240nm；
Gradient 2---35～65min, mobile phase a is 60%: 40% with the percent by volume of mobile phase b, and flow velocity is
1.0ml/min, Detection wavelength is 290nm.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
In described linear gradient elution, gradient 2 can also add a transition gradient below, the mobile phase ratio of described transition gradient with
Described gradient 1 is identical, and rinses steady to baseline.So that continuous sample introduction.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
The collocation method of reference substance solution is as follows: precision weighs loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol
Reference substance, is dissolved and is diluted with the methanol solution that percent by volume is 80%, make in every 1ml reference substance solution contain 0.10～
0.14mg loganin, 0.04～0.08mg genipin sweet acid, 0.07～0.11mg verbascoside, 0.15～0.25mg cinnamic aldehyde
With 0.15～0.25mg paeonol.
As one of embodiment, the present invention detects in the method for five kinds of active constituent contents in JINKUI SHENQI PIAN simultaneously,
The pre-treating method of described JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample to remove coating, finely ground, gained fine powder can pass through
100 mesh sieves, take fine powder 1.8～2.2g, accurately weighed, put in conical flask with cover, and accurate addition percent by volume is 80% first
Alcohol 25ml, close plug, weighed weight, it is heated to reflux 1 hour, put to room temperature, more weighed weight, the first being 80% with percent by volume
The weight of less loss supplied by alcohol, shakes up, and takes supernatant to filter, takes subsequent filtrate, obtain final product.
Compared with existing JINKUI SHENQI PIAN detection meanss, this method application modern efficient liquid chromatography technology, in eluting
During, the gradient elution that gradually change mobile phase a, b concentration proportioning and the invariable isocratic elution of mobile phase a, b proportioning
Combine, form a specific gradient elution program, carry out two gradients or three gradient elutions, simultaneously in gradient elution
Change-detection wavelength.Using this detection method not only can in clean cut separation JINKUI SHENQI PIAN five kinds of effective ingredient detection peak,
Separating degree is good, and specificity is strong, loganin in detection JINKUI SHENQI PIAN while also can be quantitative, genipin sweet acid, verbascoside,
Cinnamic aldehyde and the content of paeonol.This detection method is easy and quick, can effectively improve the quality control level of JINKUI SHENQI PIAN.
While the present invention provides detection JINKUI SHENQI PIAN in five kinds of active constituent contents method have sensitivity height, favorable reproducibility,
The feature of good stability, can accurate qualitative and quantitative detection JINKUI SHENQI PIAN five kinds of effective ingredient, time-consuming, save examination
Agent.The quality of product can more effectively be controlled it is ensured that the curative effect of medicine using the method for the present invention.
In order to more fully understand and implement, describe the present invention below in conjunction with the accompanying drawings in detail.
Brief description
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.
Specific embodiment
Instrument: high performance liquid chromatograph agilent1260 infinity, g1315d and g1314d.Chromatographic column: gl
Sciences, 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), (science popularization ultrasonic electronic technology in Guangzhou has Ultrasound Instrument
Limit company, model: kp-q600).
Quality standard: test sample every contains wine-prepared fructus corni with loganin (c17h26o10) meter, 0.25mg must not be less than；Every contains
Semen Plantaginiss are with genipin sweet acid (c16h22o10) and verbascoside (c29h36o15) meter, distinguish must not less than 0.23mg with
0.20mg；Every contains Ramulus Cinnamomi with cinnamic aldehyde (c9h8O) count, 0.45mg must not be less than；Every contains Cortex Moutan with paeonol (c9h10o3)
Meter, must not be less than 0.70mg.
Reference substance: loganin (lot number: 111640-201005, Nat'l Pharmaceutical ＆ Biological Products Control Institute provides), genipin are sweet
Acid (lot number: 111828-201102, Nat'l Pharmaceutical ＆ Biological Products Control Institute provides), verbascoside (lot number: 111530-
201310, Nat'l Pharmaceutical ＆ Biological Products Control Institute provide), paeonol (lot number: 110708-200506, Chinese pharmaceutical biological product
Calibrating is provided) and cinnamic aldehyde (lot number: 110710-201016, Nat'l Pharmaceutical ＆ Biological Products Control Institute provides).
Embodiment 1
Laboratory sample: (Guangdong Taicheng Pharmaceutical Co., Ltd produces JINKUI SHENQI PIAN, lot number: 20150101).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for flowing
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 volume of mobile phase b
Percentage ratio and remain 100%, carry out gradient elution by table 1.Number of theoretical plate is calculated by loganin peak and is not less than 5000,
Each detection target peak should be greater than 1.5 with the separating degree of its respective adjacent peak.
＜ table 1 ＞
The preparation of reference substance solution: precision weighs loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol
Appropriate reference substance, plus the methanol solution that percent by volume is 80% dissolve and dilute make every 1ml contain 0.12mg loganin,
0.06mg genipin sweet acid, the mixed solution of 0.09mg verbascoside, 0.20mg cinnamic aldehyde and 0.20mg paeonol, obtain final product.
The preparation of need testing solution: take test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, takes fine powder
2.0g, accurately weighed, put in conical flask with cover, accurate addition percent by volume is 80% methanol solution 25ml, close plug is weighed
Weight, is heated to reflux 1 hour, puts to room temperature, more weighed weight, supplies less loss with the methanol solution that percent by volume is 80%
Weight, shakes up, and takes supernatant to filter, takes subsequent filtrate, obtain final product.
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, records chromatograph
Scheme and by external standard method with calculated by peak area, result is referring to accompanying drawing 1～2, every 0.43mg containing loganin, genipin sweet acid
0.37mg, verbascoside 0.38mg, cinnamic aldehyde 0.66mg, paeonol 0.97mg.
Embodiment 2
Laboratory sample: (Guangdong Taicheng Pharmaceutical Co., Ltd produces JINKUI SHENQI PIAN, lot number: 20150201).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for flowing
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 volume of mobile phase b
Percentage ratio and remain 100%, carry out gradient elution by table 2.Number of theoretical plate is calculated by loganin peak and is not less than 5000,
Each detection target peak should be greater than 1.5 with the separating degree of its respective adjacent peak.
＜ table 2 ＞
The preparation of reference substance solution: precision weighs loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol
Appropriate reference substance, plus the dissolving of methanol solution that percent by volume is 80% makes every 1ml and contains 0.10mg loganin, 0.04mg capital
The flat sweet acid of Buddhist nun, the mixed solution of 0.07mg verbascoside, 0.15mg cinnamic aldehyde and 0.15mg paeonol, obtain final product.
The preparation of need testing solution: take test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, takes fine powder
2.0g, accurately weighed, put in conical flask with cover, accurate addition percent by volume is 80% methanol solution 25ml, weighed weight,
Warm macerating (45 DEG C) 1.5 hours, puts to room temperature after ultrasonic 15min, more weighed weight, the methanol solution being 80% with percent by volume
Supply the weight of less loss, shake up, 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, records chromatograph
Scheme and by external standard method with calculated by peak area.Result: every 0.45mg containing loganin, genipin sweet acid 0.36mg, verbascoside
0.44mg, cinnamic aldehyde 0.69mg, paeonol 0.92mg.
Embodiment 3
Laboratory sample: (Guangdong Taicheng Pharmaceutical Co., Ltd produces JINKUI SHENQI PIAN, lot number: 20150202).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for flowing
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 volume of mobile phase b
Percentage ratio and remain 100%, carry out gradient elution by table 3.Number of theoretical plate is calculated by loganin peak and is not less than 5000,
Each detection target peak should be greater than 1.5 with the separating degree of its respective adjacent peak.
＜ table 3 ＞
The preparation of reference substance solution: precision weighs loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol
Appropriate reference substance, plus the dissolving of methanol solution that percent by volume is 80% makes every 1ml and contains 0.12mg loganin, 0.06mg capital
The flat sweet acid of Buddhist nun, the mixed solution of 0.09mg verbascoside, 0.20mg cinnamic aldehyde and 0.20mg paeonol, obtain final product.
The preparation of need testing solution: take test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, takes fine powder
1.8g, accurately weighed, put in conical flask with cover, accurate addition percent by volume is 80% methanol solution 25ml, close plug is weighed
Weight, is heated to reflux 1 hour, puts to room temperature, more weighed weight, supplies less loss with the methanol solution that percent by volume is 80%
Weight, shakes up, and takes supernatant to filter, takes subsequent filtrate, obtain final product.
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, records chromatograph
Scheme and by external standard method with calculated by peak area, result is referring to accompanying drawing 3～4, every 0.41mg containing loganin, genipin sweet acid
0.38mg, verbascoside 0.38mg, cinnamic aldehyde 0.65mg, paeonol 0.96mg.
Embodiment 4
Laboratory sample: (Guangdong Taicheng Pharmaceutical Co., Ltd produces JINKUI SHENQI PIAN, lot number: 20150101).
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With acetonitrile for flowing
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 volume of mobile phase b
Percentage ratio and remain 100%, carry out gradient elution by table 4.Number of theoretical plate is calculated by loganin peak and is not less than 5000,
Each detection target peak should be greater than 1.5 with the separating degree of its respective adjacent peak.
＜ table 4 ＞
The preparation of reference substance solution: precision weighs loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol
Appropriate reference substance, plus the dissolving of methanol solution that percent by volume is 80% makes every 1ml and contains 0.12mg loganin, 0.06mg capital
The flat sweet acid of Buddhist nun, the mixed solution of 0.09mg verbascoside, 0.20mg cinnamic aldehyde and 0.20mg paeonol, obtain final product.
The preparation of need testing solution: take test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, takes fine powder
2.2g, accurately weighed, put in conical flask with cover, accurate addition percent by volume is 80% methanol solution 25ml, close plug is weighed
Weight, is heated to reflux 1 hour, puts to room temperature, more weighed weight, supplies less loss with the methanol solution that percent by volume is 80%
Weight, shakes up, and takes supernatant to filter, takes subsequent filtrate, obtain final product.
Measure: precision measures reference substance solution and each 10 μ l injection chromatograph of liquid of need testing solution respectively, records chromatograph
Scheme and by external standard method with calculated by peak area, result is referring to accompanying drawing 5～6, every 0.44mg containing loganin, genipin sweet acid
0.37mg, verbascoside 0.65mg, cinnamic aldehyde 0.64mg, paeonol 0.99mg.
Method validation embodiment
By " Chinese Pharmacopoeia " version in 2010 one " annex xviii quality standards in Chinese drugs analysis method verification guide principle "
Requirement carries out Method validation test.
Laboratory sample: JINKUI SHENQI PIAN (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；JINKUI SHENQI PIAN wine-prepared fructus corni is cloudy
Property preparation (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；JINKUI SHENQI PIAN Semen Plantaginiss feminine gender preparation (Guangdong TV station city pharmacy stock
Part company limited produces)；JINKUI SHENQI PIAN Ramulus Cinnamomi feminine gender preparation (Guangdong Taicheng Pharmaceutical Co., Ltd's production)；Pieces of Jin ' gui shenqi
Piece 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 respectively
9.25mg, verbascoside reference substance 13.98mg, cinnamic aldehyde reference substance 33.45mg and paeonol reference substance 32.31mg, by it
Put in same 25ml volumetric flask, dissolved and be diluted to scale with the methanol solution that percent by volume is 80%, shake up, as right
According to product stock solution.
1. specificity test
Method according to embodiment 1 prepares reference substance solution, need testing solution, with reference to need testing solution in embodiment 1
Compound method is prepared JINKUI SHENQI PIAN and is lacked wine-prepared fructus corni, Semen Plantaginiss, Ramulus Cinnamomi, the negative sample solution of Cortex Moutan each feminine gender preparation, essence
Close measure each 10 μ l of above-mentioned solution, be injected separately into chromatograph of liquid, by embodiment 1-4 assay condition measure.
Result shows, each negative sample solution chromatogram divides no dry on corresponding position in reference substance solution chromatogram each group
Disturb.In need testing solution chromatogram, number of theoretical plate is calculated by loganin peak and is not less than 5000, each detection target peak and its respective phase
The separating degree at adjacent peak is all higher than 1.5, meets " Chinese Pharmacopoeia " version assay related request in 2015.Table 5 is illustrated that employing
The specificity experimental result of the assay method gained of embodiment 1.Tested using the specificity that the assay method of embodiment 2-4 records
Result is similar.
Table 5 specificity result of the test
2. linear test
Precision measures reference substance stock solution 1ml, 1ml, 1ml, 2ml, 3ml, 5ml, be respectively placed in 50ml, 25ml, 10ml,
In 10ml, 10ml, 10ml volumetric flask and plus percent by volume be that 80% methanol solution is diluted to scale, shake up, obtain final product for
The linear each concentrations control product solution investigated；
Precision measures each 10 μ l of above-mentioned solution, is injected separately into chromatograph of liquid, according to the condition determination record color of embodiment 1
Spectrogram.As shown in table 6, loganin is in good with its peak area in the range of concentration 0.0159mg/ml～0.3968mg/ml to result
Good linear relationship, genipin sweet acid is in good with its peak area in the range of concentration 0.0074mg/ml 1～0.1850mg/ml
Good linear relationship, verbascoside is in good with its peak area in the range of concentration 0.0112mg/ml～0.2796mg/ml
Linear relationship, cinnamic aldehyde is in good linear with its peak area in the range of concentration 0.0268mg/ml～0.6690mg/ml
Relation, paeonol is in good linear relationship with its peak area in the range of concentration 0.025mg/ml～0.6462mg/ml.Adopt
Result with the condition determination gained of embodiment 2-4 is substantially similar to using embodiment 1 condition determination acquired results, and here is no longer
Repeat.
Table 6 linear test result
3. precision test
3.1 it is repeated
Take 20150101 batches of JINKUI SHENQI PIAN appropriate, finely ground after removing coating, gained fine powder can pass through 100 mesh sieves, accurate
Weigh totally 6 parts of fine powder 1.8～2.2g, make need testing solution according to the method for embodiment 1 respectively；Separately precision weigh loganin,
Genipin sweet acid, verbascoside, cinnamic aldehyde and each reference substance of paeonol are appropriate, and the method according to embodiment 1 makes reference substance
Solution, is measured by the condition determination of embodiment 1-4.It was found that the condition determination acquired results phase using embodiment 1-4
Seemingly, and measurement result is respectively provided with good repeatability, rsd value is respectively less than 2%.Table 7 below shows the mensure bar using embodiment 1
The replica test result that part records.
The preparation of need testing solution: take test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh sieves, takes fine powder about
2.0g, accurately weighed, put in conical flask with cover, accurate addition percent by volume is 80% methanol solution 25ml, close plug is weighed
Weight, is heated to reflux 1 hour, puts to room temperature, more weighed weight, supplies less loss with the methanol solution that percent by volume is 80%
Weight, shakes up, and takes supernatant to filter, takes subsequent filtrate, 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 press essence per capita
The method under " 3.1 repeatability " item in density test respectively prepares 3 parts of need testing solutions, by the assay bar of embodiment 1-4
Part is measured, and table 8 below shows the Intermediate precision result of the test recording using the content assaying method of embodiment 1.(adopt
The Intermediate precision measurement result of the content assaying method of embodiment 2-4 is similar to embodiment 1, will not be described here)
Table 8 Intermediate precision result of the test
From Intermediate precision result of the test, the content assaying method of the present invention has the characteristics that favorable reproducibility.
4. stability test
Method according to embodiment 1 prepares need testing solution and each 1 part of reference substance solution, places 0,4,8,12,18,24 little
Constantly, precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, according to embodiment 1-4
Assay condition is measured, it is found that need testing solution and reference substance solution are basicly stable in 24 hours, below will
The stability test result being recorded using the assay method of embodiment 1 is shown in table 9 below.
Table 9 stability test result
From stability test result, the assay method of the present invention measured value rsd of peak area in 24 hours is less than
2.0, good stability.
4. average recovery
The preparation of recovery test need testing solution: take the JINKUI SHENQI PIAN of 20150101 batches of known content to remove bag
Finely ground after clothing, gained fine powder can pass through 100 mesh sieves, and precision weighs totally 6 parts of fine powder about 1.0g, is respectively placed in 6 conical flask with covers
In, each accurate addition recovery test reference substance stock solution 5ml and methanol solution 20ml that percent by volume is 80%, close plug,
Weighed weight, is heated to reflux 1 hour, puts to room temperature, more weighed weight, is supplied with the methanol solution that percent by volume is 80% and subtracts
The weight lost, shakes up, and takes supernatant to filter, takes subsequent filtrate as recovery test need testing solution, according to the mensure of embodiment 1-4
Condition is measured, and gained response rate meansigma methodss all reach more than 98%, rsd and are respectively less than 2.0%.Below by using embodiment 1
The recovery test result that assay method records is illustrated in table 10 as representative:
Table 10 recovery test result
Knowable to the result of the test of the response rate, good accuracy is had using the detection method that the present invention provides.
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
As a result, after carrying out comprehensive analysis, determine and investigate accurately with 50% and 250% of quality standard described in five kinds of effective ingredient for scope
Degree.
Prepared by limit 50% need testing solution: take 20150101 batches of JINKUI SHENQI PIAN appropriate, remove finely ground, gained after coating
Fine powder can pass through 100 mesh sieves, takes 6 parts of fine powder about 0.5g, accurately weighed, and every part all adds each 1ml of reference substance stock solution, afterwards
By the preparation method preparation of the need testing solution of embodiment 1, the condition determination according to embodiment 1-4 measures each component content, meter
Calculate the response rate.Table 11 below shows the result recording according to the condition determination of embodiment 1, according to the condition determination of embodiment 2-4
Measured result is substantially identical with embodiment 1.
Limit 250% test sample: take 20150101 batches of JINKUI SHENQI PIAN appropriate, remove finely ground, gained fine powder energy after coating
By 100 mesh sieves, take 6 parts of fine powder about 2.5g, accurately weighed, every part all adds each 5ml of reference substance stock solution, by embodiment 1
The preparation method preparation of need testing solution, the condition determination according to embodiment 1-4 measures each component content, calculates the response rate.Under
Table 12 shows the result recording according to the condition determination of embodiment 1, according to the result measured by the condition determination of embodiment 2-4
Basic identical with embodiment 1.
Table 11 low strength range (by limit 50%)
Table 12 high concentration range (by limit 250%)
From the result of the accuracy test of measurement range, each composition limit in JINKUI SHENQI PIAN 50%～
In the range of 250%, measurement result is reliable and stable, and accuracy is high.
By the result of above method checking, using the detection method of the present invention, gold not only can be detected simultaneously
The content of five kinds of effective ingredient in deficient kidney qi piece, and this detection method accuracy, repeatability and precision are all good, are measuring
In the range of linear relationship good, in 24 hours, need testing solution and reference substance are all basicly stable.Method validation result of the test shows
Show that this law can be used as the survey of loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol content in JINKUI SHENQI PIAN
Determine method.Measure again can be qualitative while content for quantitation, can effectively to wine-prepared fructus corni in pieces of Jin ' gui shenqi tablet recipe, Semen Plantaginiss,
Ramulus Cinnamomi and 4 flavour of a drug of Cortex Moutan are differentiated.
The invention is not limited in above-mentioned embodiment, if the various changes to the present invention or deformation are without departing from the present invention
Spirit and scope, if these are changed and within the scope of deformation belongs to claim and the equivalent technologies of the present invention, then this
Bright it is also intended to comprise these and changes and deform.
Claims (6)
1. a kind of simultaneously detection JINKUI SHENQI PIAN in five kinds of active constituent contents method it is characterised in that: described effective ingredient
For loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol, methods described is high performance liquid chromatography, described color
Spectral 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 0.1% phosphate aqueous solution, the volume hundred of mobile phase a
Point than with the percent by volume of mobile phase b and remain 100%, carry out linear gradient elution；
Described linear gradient elution, wavelength and flow velocity are:
Gradient 1---0～15min, mobile phase a is 15%:85% with the percent by volume of mobile phase b, and flow velocity is 1.0ml/min,
Detection wavelength is 240nm；
Gradient 2---15～30min, mobile phase a is (15% → 55%) with the percent by volume of mobile phase b: (85% →
45%), flow velocity is 1.0ml/min, and Detection wavelength is 240nm；
Gradient 3---30～55min, mobile phase a is 55%:45% with the percent by volume of mobile phase b, and flow velocity is 1.2ml/
Min, Detection wavelength is 286nm；
Or, described linear gradient elution, wavelength and flow velocity are:
Gradient 1---0～15min, mobile phase a is 10%:90% with the percent by volume of mobile phase b, and flow velocity is 1.0ml/min,
Detection wavelength is 235nm；
Gradient 2---15～30min, mobile phase a is (10% → 65%) with the percent by volume of mobile phase b: (90% →
35%), flow velocity is 1.0ml/min, and Detection wavelength is 245nm；
Gradient 3---30～55min, mobile phase a is 65%:35% with the percent by volume of mobile phase b, and flow velocity is 1.0ml/
Min, Detection wavelength is 294nm.
2. method according to claim 1 it is characterised in that: in described linear gradient elution, gradient 3 can also add below
Plus a transition gradient, the mobile phase ratio of described transition gradient is identical with described gradient 1, and rinses steady to baseline.
3. method according to claim 1 is it is characterised in that also including the pre-treating method of described JINKUI SHENQI PIAN, described
The pre-treating method of JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh
Sieve, takes described fine powder 1.8～2.2g, accurately weighed, puts in conical flask with cover, and accurate addition percent by volume is 80% methanol
25ml, weighed weight, soak 1.5 hours at 45 DEG C, put to room temperature after ultrasonic 15min, more weighed weight, with percent by volume it is
80% methanol supplies the weight of less loss, shakes up, and filters, takes subsequent filtrate as need testing solution.
4. a kind of simultaneously detection JINKUI SHENQI PIAN in five kinds of active constituent contents method it is characterised in that: described effective ingredient
For loganin, genipin sweet acid, verbascoside, cinnamic aldehyde and paeonol, methods described is high performance liquid chromatography, described color
Spectral 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 0.1% phosphate aqueous solution, the volume hundred of mobile phase a
Point than with the percent by volume of mobile phase b and remain 100%, carry out linear gradient elution；
Described linear gradient elution, wavelength and flow velocity are:
Gradient 1---0～35min, mobile phase a is (5% → 55%): (95% → 45%) with the percent by volume of mobile phase b,
Flow velocity is 1.0ml/min, and Detection wavelength is 235nm；
Gradient 2---35～65min, mobile phase a is 55%:45% with the percent by volume of mobile phase b, and flow velocity is 1.2ml/
Min, Detection wavelength is 286nm；
Or, described linear gradient elution, wavelength and flow velocity are:
Gradient 1---0～35min, mobile phase a is (10% → 60%): (90% → 40%) with the percent by volume of mobile phase b,
Flow velocity is 1.0ml/min, and Detection wavelength is 240nm；
Gradient 2---35～65min, mobile phase a is 60%:40% with the percent by volume of mobile phase b, and flow velocity is 1.0ml/
Min, Detection wavelength is 290nm.
5. method according to claim 4 it is characterised in that: in described linear gradient elution, gradient 2 can also add below
Plus a transition gradient, the mobile phase ratio of described transition gradient is identical with described gradient 1, and rinses steady to baseline.
6. method according to claim 4 is it is characterised in that also including the pre-treating method of described JINKUI SHENQI PIAN, described
The pre-treating method of JINKUI SHENQI PIAN is: takes JINKUI SHENQI PIAN test sample to remove coating, finely ground, gained fine powder can pass through 100 mesh
Sieve, takes described fine powder 1.8～2.2g, accurately weighed, puts in conical flask with cover, and accurate addition percent by volume is 80% methanol
25ml, close plug, weighed weight, it is heated to reflux 1 hour, put to room temperature, more weighed weight, the methanol being 80% with percent by volume
Supply the weight of less loss, shake up, take supernatant to filter, take subsequent filtrate, obtain final product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102872295A (en) * | 2011-07-14 | 2013-01-16 | 天津丹溪国药研究所 | New process for preparing cinnamon, Radix aconiti lateralis preparata and rehmannia root preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102872295A (en) * | 2011-07-14 | 2013-01-16 | 天津丹溪国药研究所 | New process for preparing cinnamon, Radix aconiti lateralis preparata and rehmannia root preparation |
Non-Patent Citations (4)
| Title |
|---|
| Screening and analysis of metabolic components in rat urine and feces after oral administration of total glycosides of Jinkuishenqi pills by HPLC-ESI/MSn;LI Wen-Lan等;《Chinese Journal of Natural Medicines》;20031130;第56-62页 * |
| 李文兰等.金匮肾气丸总苷类成分HPLC-DAD-ELSD指纹图谱的建立.《中国药学杂志》.2008, * |
| 林林等.HPLC 法测定金匮肾气丸中马钱苷、芍药苷、毛蕊花糖苷和丹皮酚.《中成药》.2012, * |
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