CN104634911B - A kind of 4 kinds of flavonoids effective constituent detection methods of CHUANKEZHI ZHUSHEYE - Google Patents
A kind of 4 kinds of flavonoids effective constituent detection methods of CHUANKEZHI ZHUSHEYE Download PDFInfo
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Abstract
The present invention relates to a kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method, by liquid chromatography mass coupling, can concurrently separate and measure Epimedin A, Epimedin B, epimedin and four kinds of effective ingredient of icariin, the method is reliable and stable and can truly reflect the quality of product, is suitable for the daily production testing of industrialization.
Description
Technical field
The present invention relates to field of medicaments, especially relate to the detection method of a kind of CHUANKEZHI ZHUSHEYE effective ingredient.
Background technology
CHUANKEZHI ZHUSHEYE is national two class new Chinese medicines, system is made up of Herba Epimedii and Radix Morindae Officinalis medical material, there is gentle the kidney invigorating, antiasthmatic-antitussive, there is antiallergic, strengthen the humoral immunization function with cellular immunization, and toxicity is little, medical mechanism is clear and definite, wide in the potential applicability in clinical practice of immunologic function disorder disease such as asthma, chronic bronchitis.
Containing being mainly composed of flavonoid glycoside in CHUANKEZHI ZHUSHEYE, the content of flavonoid glycoside respectively Epimedin A, Epimedin B, epimedin and icariin, these flavonoid glycosides are from epimedium herb. Epimedium herb source is many, and complicated component poor quality controls. Epimedium sagittatum medical material used by current CHUANKEZHI ZHUSHEYE has no the quantitative analysis to Epimedin A, Epimedin B, existing CHUANKEZHI ZHUSHEYE content assaying method only need to investigate the amount of epimedin and icariin, but without the content analysis to other flavones ingredient.
Liquid chromatography mass spectrometric multiple techniques because analyzing quickly, sample treatment is succinct, highly sensitive and is more and more frequently used in quality analysis of Chinese medicine. Therefore this research adopt high performance liquid chromatography-tandem mass method quickly, the content of Epimedin A, Epimedin B, epimedin and these 4 kinds of flavonoid glycosides of icariin in Accurate Determining CHUANKEZHI ZHUSHEYE, provide more complete reliable quality control method for CHUANKEZHI ZHUSHEYE.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of 4 kinds of flavonoids effective constituent detection methods of CHUANKEZHI ZHUSHEYE, the method is reliable and stable, it is possible to really reflect the inherent quality of product, simple to operate, is suitable for the routine check work of industrialization.
A kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method of the present invention, comprises the steps:
(1) preparation of reference substance solution: take reference substance Epimedin A, Epimedin B, epimedin and icariin, dissolves with methanol or mobile phase and get final product;
(2) preparation of inner mark solution: take naringin reference substance, dissolves with methanol or mobile phase and get final product;
(3) preparation of need testing solution: take CHUANKEZHI ZHUSHEYE methanol or mobile phase dilutes and get final product;
(4) detection method: combined gas chromatography mass spectrometry, described chromatographic condition, chromatographic column C18, mobile phase: acetonitrile-aqueous acid; Described Mass Spectrometry Conditions, auxiliary gasification electric spray ion source ESI, many reactive ions monitoring MRM pattern anionic textiles.
It is preferred that the reference substance Epimedin A described in step (1), Epimedin B, epimedin and icariin are each configured to the reference substance stock solution of 1mg/ml; Precision measures reference substance storing solution and is placed in same measuring bottle respectively again, adds methanol dilution and is configured to Epimedin A, Epimedin B, epimedin and icariin and is the mixing reference substance storing solution of 1000ng/ml.
Further each about 5mg of reference substance Epimedin A described in preferred steps (1), Epimedin B, epimedin and icariin, accurately weighed, is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, is configured to 4 kinds of reference substance storing solutions; Precision measures 4 kinds of reference substance storing solutions and is placed in same 10ml measuring bottle respectively again, adds methanol dilution to scale and is configured to Epimedin A, Epimedin B, epimedin and icariin and is the mixing reference substance storing solution of 1000ng/ml.
The preparation of inner mark solution in above-mentioned steps (2), further, the naringin reference substance described in step (2) is configured to the interior mark stock solution of 1mg/ml, is configured to 2 �� g/ml inner mark solutions by dilution step.
Dilution step described in above-mentioned steps (2) refers to that taking interior mark storing solution is placed in measuring bottle, add methanol or mobile phase is configured in 100 �� g/ml to mark secondary stock solution, the accurate secondary storing solution of interior mark of drawing is placed in measuring bottle, adds methanol or mobile phase is configured to 2 �� g/ml inner mark solutions.
Further, step (2) takes naringin reference substance and is about 10mg, accurately weighed, is placed in 10ml measuring bottle, adds methanol and dissolves and be diluted to scale, is configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds mobile phase and be diluted to scale, shake up, obtain interior mark working solution (2 �� g/ml).
Prepared by step of the present invention (3) described test sample, further, dilute CHUANKEZHI ZHUSHEYE with mobile phase, and precision measures 1000-2000 �� l and adds 1/5 times of inner mark solution mix homogeneously, filter membrane, takes subsequent filtrate, obtains need testing solution.
Further, step (3) mobile phase dilutes CHUANKEZHI ZHUSHEYE 2000 times, and precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, takes subsequent filtrate, obtain need testing solution.
Step of the present invention (4) detection method, wherein said chromatographic condition: C18Chromatographic column (50-150) �� 2.1mm, particle diameter 3.0��5.0 ��m; Mobile phase: acetonitrile: 0.1��1% aqueous formic acid, isocratic elution (30-40:60-70) sample size: 5��20 �� L, column temperature: 35��45 DEG C, flow velocity 0.2��0.3mL/min.Described Mass Spectrometry Conditions: auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7000��8000CC/min, atomization gas flow: 6��10L/min, gas curtain throughput: 5��8L/min, ion source spray voltage :-3800��-4500V, ion source atomization temperature: 350-400 DEG C.
Mobile phase described in above-mentioned steps (1)-(4) is acetonitrile-0.1��1% aqueous acid, described acid include but not limited to in formic acid, acetic acid one or more. Preferred mobile phase is acetonitrile-0.1% aqueous formic acid.
The detection method that the present invention is best, comprises the steps:
(1) preparation of reference substance solution: Epimedin A, Epimedin B, epimedin and icariin add methanol and dissolve and be diluted to scale, is configured to 4 kinds of 1mg/ml reference substance storing solutions. Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution;
(2) preparation of inner mark solution: take naringin reference substance, accurately weighed, it is placed in measuring bottle, adds methanol and dissolve and be diluted to scale, be configured in 1mg/ml and mark storing solution. It is appropriate that precision measures interior mark storing solution, is placed in measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. The accurate secondary storing solution of interior mark of drawing is put in measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, obtains interior mark working solution (2 �� g/ml);
(3) prepared by need testing solution: with acetonitrile: 0.1% aqueous formic acid (35:65) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution;
(4) testing conditions:
Chromatographic condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 0.1% aqueous formic acid 35:65, isocratic elution 3.5min, sample size 10 �� l, column temperature 40 DEG C, flow velocity 0.22ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7500CC/min, atomization gas flow: 8L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
Beneficial effect
In order to better set forth beneficial effects of the present invention, by following it have been experienced that
Test example one, methodological study
1 material, reagent and instrument
1.1 standard substance
Fig. 1 be shown in their chemical structural formula.
1.2 reagent
Methanol (FisherScientific company of the chromatographic grade U.S.), acetonitrile (FisherScientific company of the chromatographic grade U.S.) formic acid Guangzhou Chemical Reagent Factory, pure water, ultra-pure water (Milli-Elix, Milli-Q water manufacturing system)
1.3 test samples
CHUANKEZHI ZHUSHEYE (lot number: 13052501,13052801,13052601,11121001,13022502,13052702,12081502,13052902,13042702,12072001,12092301,12071902,13053002,13030102,20110305) provided by the positive pharmaceutcal corporation, Ltd in Guangzhou ten thousand.
1.4 instruments
2, experimental technique
2.1 analyze method
2.1.1 chromatographic condition
Chromatographic column: AgilentEclipseXDB-C18(150 �� 2.1mm, 5.0 ��m). Mobile phase: acetonitrile: 0.1% aqueous formic acid (35:65), isocratic elution 3.5min. Sample size: 10 �� l; Column temperature: 40 DEG C; Flow velocity: 0.22ml/min.
2.1.2 Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source (ESI); Many reactive ions monitoring (MRM) pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 (Epimedin A), m/z853.5 �� 645.3 (Epimedin B), m/z867.4 �� 659.4 (epimedin), m/z721.3 �� 513.2 (icariin), m/z579.2 �� 271.1 (naringin), assisted gas activating QI flow: 7500CC/min, atomization gas flow: 8L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
The preparation of 2.2 working solutions
2.2.1 the preparation of series concentration mixing reference substance solution
Take each about 5mg of 4 flavone reference substances, accurately weighed, it is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions. Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution. 2.2.2 the preparation of inner mark solution
Take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml). 2.2.3 the preparation of need testing solution
With acetonitrile: 0.1% aqueous formic acid (35:65) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, takes subsequent filtrate, obtain need testing solution.
2.2.4 the preparation of standard curve and quality-control sample
Take the standard substance storing solution of 4 kinds of flavone, take accurate volume according to each concrete concentration and be placed in same 10ml measuring bottle, add acetonitrile: 0.1% aqueous formic acid (35:65), to scale, is configured to 4 kinds of flavone and is the hybrid standard product solution of 1000ng/ml. Then according still further under show flow process stepwise dilution, prepare 800,300,100,30,10,3,1ng/ml.
The sample subpackage for preparing is good, put into-80 DEG C of preservations.
For analyzing the quality-control sample of method validation, it is that the standard substance storing solution of 4 kinds of flavone is joined in mobile phase formulated. Its process for preparation also progressively dilutes the quality-control sample being configured to containing basic, normal, high four concentration that concentration is 3,30,300,800ng/ml as stated above with method, and each quality-control sample joins 6 parts. Precision measures 1000 �� l each concentration standard curve samples and quality-control sample adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, takes subsequent filtrate, obtain standard curve and quality-control sample solution. 2.3 methodological studies
2.3.1 the foundation of standard curve
Take the standard curve testing sample of 8 concentration prepared, from low concentration 1ng/ml, 3ng/ml, 10ng/ml, 30ng/ml, 100ng/ml, 300ng/ml, 800ng/ml, 1000ng/ml successively continuous sample introduction, with measured object concentration value (X) for abscissa, the ratio (Y) of measured object and the peak area of internal standard substance is vertical coordinate, obtains standard curve linear equation and linearly dependent coefficient r value.
2.3.2 precision is investigated
It is the quality-control sample of 3,30,300,800ng/ml by the concentration of above-mentioned preparation, measures as stated above. 6 parts of samples of each concentration are prepared respectively in same batch and measure. Quality-control sample METHOD FOR CONTINUOUS DETERMINATION 6 times in withinday precision and accuracy experiment; In day to day precision and accuracy experiment, quality-control sample is in not detecting on the same day, measures 3 days. The ratio of the peak area and interior mark peak area that record compound substitutes into retinue standard curve, calculates and measures concentration.
The RSD (RSD) of precision quality-control sample represents, relative standard deviation requires less than 15%. Accuracy is the mensuration concentration percentage ratio with actual concentration of quality-control sample, it is desirable to accuracy is within the scope of 85-115%.
2.3.3 repeatability is investigated
Take with 6 parts of a collection of CHUANKEZHI ZHUSHEYE sample, prepare by test sample preparation method, and measure the content of 4 kinds of flavonoid glycosides in sample according to above-mentioned chromatograph and Mass Spectrometry Conditions, substitute into retinue biological standard curve, calculate the accuracy and the RSD value that record concentration.
2.3.4 study on the stability
Take above-mentioned need testing solution, 0,2,4 is placed respectively under room temperature (25 DEG C), 8, the content of 4 kinds of compositions in sample is measured according to above-mentioned identical chromatograph and Mass Spectrometry Conditions respectively after 12,18,24h, substitute into accompanying standard curve, calculate the accuracy and the RSD value that record concentration, investigate room temperature (25 DEG C) shelf-stability, the required times to be analyzed such as single batch sample should be exceeded standing time.
2.3.5 average recovery is investigated
Take 6 parts of the CHUANKEZHI ZHUSHEYE sample recording content, accurate addition is about equal to 4 kinds of flavonoid glycoside chemical reference substances of analysing pharmaceutical samples content respectively, substitute into retinue standard curve, prepare by need testing solution preparation method, measure according to above-mentioned chromatograph and Mass Spectrometry Conditions and calculate average recovery.
3 experimental results
The determination of 3.1 analysis conditions
Application full scan and daughter ion scan pattern obtain full scan and the daughter ion scanning mass spectrum of Epimedin A, Epimedin B, epimedin, icariin and naringin, select highly sensitive mother and sons' ion pair to carry out quantitative analysis, and mass spectrum relevant parameter is in Table 1. By the liquid chromatograph separation condition optimized, the retention time of Epimedin A, Epimedin B, epimedin, icariin and naringin respectively 1.86 in CHUANKEZHI ZHUSHEYE, 1.95,2.02,2.37 and 1.65min, each mensuration material and interior mark do not interfere with each other, and can complete the mensuration of 4 compositions in a sample in 3.5min. Each material scanning of the mass spectrum figure is shown in that Fig. 2, MRM mass chromatogram is shown in Fig. 3.
14 kinds of flavone of table and interior target mother and sons' ion pair and associated voltage parameter
3.2 ranges of linearity and quantitative limit, detection limit
Take the series concentration mixing reference substance solution containing 4 flavone, detect according to above-mentioned chromatograph and Mass Spectrometry Conditions. Using the ratio of each Component peak area and interior mark peak area as vertical coordinate (Y), each constituent concentration is abscissa (X), carries out linear regression, obtains Epimedin A, B, C, the regression equation of icariin and lower limit of quantitation data in Table 2.
The standard curve of 24 kinds of flavone of table and lower limit of quantitation
4 kinds of flavone components are within the scope of 1.0-1000.0ng/ml, and linear relationship is good, and when 4 kinds of material lower limit of quantitation are 1ng/ml, precision and accuracy are all better.
3.3 precision
Take four concentration respectively 3,30,300 and the Quality Control solution of 800ng/ml carry out investigating with withinday precision in the daytime. Withinday precision, the quality-control sample continuous sample introduction of each concentration 6 times;Day to day precision, adopts and is measured in the same method 3 days, calculate the relative standard deviation (RSD) of its content. In a few days with day to day precision result in Table 3, withinday precision RSD 5.57%, day to day precision RSD 5.76%.
34 kinds of flavonoid glycosides of table in the daytime and withinday precision
3.4 repeatability
Take with 6 parts of a collection of CHUANKEZHI ZHUSHEYE sample, prepare by test sample preparation method, and measure the content of 4 kinds of flavonoid glycosides in sample according to above-mentioned chromatograph and Mass Spectrometry Conditions, be 84.8 �� g/ml (RSD2.73%, n=6) with Epimedin A content in batch sample; Epimedin B content is 88.2 �� g/ml (RSD1.06%, n=6); ; Epimedin content is 895.6 �� g/ml (RSD0.83%, n=6); Icariin content is 156.4 �� g/ml (RSD0.70%, n=6).
3.5 stability
Take above-mentioned need testing solution, measure respectively at 0,2,4,8,12,18,24h sample introduction, investigate stability, its measurement result Epimedin A (RSD3.81%, n=7); Epimedin B (RSD2.95%, n=7); Epimedin (RSD2.95%, n=7); Icariin (RSD2.71%, n=7), it was shown that stable in sample solution 24h.
3.6 average recoveries
Take 6 parts of the CHUANKEZHI ZHUSHEYE sample recording content, accurate addition is about equal to 4 kinds of flavonoid glycoside chemical reference substances of analysing pharmaceutical samples content respectively, preparing by need testing solution preparation method, measure and calculate average recovery according to above-mentioned chromatograph and Mass Spectrometry Conditions, result is in Table 4.
The average recovery of 4 kinds of flavonoid glycosides in table 4 CHUANKEZHI ZHUSHEYE
3.7 sample determination results
15 batches of CHUANKEZHI ZHUSHEYE being prepared by need testing solution preparation method, measure according to above-mentioned chromatograph and Mass Spectrometry Conditions, measurement result is in Table 5 and Fig. 4.
The assay of 4 kinds of flavone components in 515 batches of CHUANKEZHI ZHUSHEYE of table
This research establishes and measures the LC-MS/MS quantitative approach of 4 kinds of flavonoid glycosides in CHUANKEZHI ZHUSHEYE simultaneously, perfect injection quality evaluation system. Experiments show that through system suitability, the method is reliable and stable, sensitive and accurate, and 3.5min can complete the mensuration of a sample, and sample pre-treatments is simple, can be used for the quality monitoring of CHUANKEZHI ZHUSHEYE in actual production. State Food and Drug Administration's national drug standards specify that the CHUANKEZHI ZHUSHEYE every 1ml total amount containing epimedin and icariin is at 0.50-1.00mg. This experiment have detected 15 batches of CHUANKEZHI ZHUSHEYE, and result shows that four kinds of flavonoid glycosides comparision contents in 15 batches of injection is stable, and the total amount of every batch of contained epimedin and icariin is all in the scope that the national drug standards require.
Test example two, mobile phase selection screening test
Flow phase system is optimized by this research, compare methanol-water (35:65), impact that in CHUANKEZHI ZHUSHEYE 4 kinds of flavonoid glycosides are separated by acetonitrile-water (35:65) system, result shows that acetonitrile-water (35:65) system can obtain good peak shape and higher sensitivity; Consider the characteristic of surveyed compound, this research adds appropriate organic acid to improve peak shape and to improve sensitivity in flow phase system, research compares addition formic acid and the acetic acid impact on result of the test, result shows in flow phase system addition formic acid, and add the percentage ratio of formic acid in 0.1% scope time each compound peak shape is symmetrical, sensitivity is higher. When mobile phase (acetonitrile-formic acid water) is in 30:70��40:60 proportion, each composition can reach good separation, and analysis time is suitable for.
Accompanying drawing explanation
Figure 14 plants flavone and interior target structural formula (A is Epimedin A, Epimedin B, epimedin, icariin structural formula, B naringin structural formula)
Figure 24 plants flavone and interior target full scan and daughter ion scanning figure, and (A-1 to A-5 is Epimedin A respectively, Epimedin B, epimedin, icariin and interior target full scan mass spectrum; B-1 to B-5 is Epimedin A respectively, Epimedin B, epimedin, and icariin and interior target daughter ion scan mass spectrum)
Figure 34 plants the mass chromatogram of flavone and internal standard substance, and (A is blank solvent, and B-1 to B-5 is Epimedin A respectively, Epimedin B, epimedin, icariin and interior mark reference substance peak; Epimedin A in C-1 to C-4 respectively sample, Epimedin B, epimedin and icariin)
Figure 44 plants flavonoid glycoside distribution in 15 batches of CHUANKEZHI ZHUSHEYE
Detailed description of the invention
Further illustrate the present invention by the following examples, but scope of the presently claimed invention is not limited.
Embodiment 1
(1) preparation of reference substance solution: each about 5mg of Epimedin A, Epimedin B, epimedin and icariin, accurately weighed, it is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions. Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution.
(2) preparation of inner mark solution: take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml).
(3) prepared by need testing solution: with acetonitrile: 0.1% aqueous formic acid (35:65) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution.
(4) testing conditions:
High-efficient liquid phase chromatogram condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 0.1% aqueous formic acid 35:65, isocratic elution 3.5min, sample size 10 �� l, column temperature 40 DEG C, flow velocity 0.22ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7500CC/min, atomization gas flow: 8L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
Take reference substance, inner mark solution and need testing solution respectively according to above-mentioned testing conditions, measure Epimedin A, Epimedin B, epimedin and Icariin content respectively 66.11 �� g/ml, 84.37 �� g/ml, 724.5 �� g/ml, 160.6 �� g/ml.
Embodiment 2
(1) preparation of reference substance solution: each about 5mg of Epimedin A, Epimedin B, epimedin and icariin, accurately weighed, it is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions.Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (65:35) is diluted to scale, shakes up, and must mix reference substance storing solution.
(2) preparation of inner mark solution: take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml).
(3) prepared by need testing solution: with acetonitrile: 0.1% aqueous formic acid (35:65) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution.
(4) testing conditions:
High-efficient liquid phase chromatogram condition
Chromatographic column C18100 �� 2.1mm, 3.0 ��m, mobile phase acetonitrile: 0.1% aqueous formic acid 40:60, isocratic elution 3.5min, sample size 5 �� l, column temperature 35 DEG C, flow velocity 0.2ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7000CC/min, atomization gas flow: 6L/min, gas curtain throughput: 5L/min, ion source spray voltage :-3800V, ion source atomization temperature: 350 DEG C.
Take reference substance, inner mark solution and need testing solution respectively according to above-mentioned testing conditions, measure Epimedin A, Epimedin B, epimedin and Icariin content respectively 63.47 �� g/ml, 80.35 �� g/ml, 725.1 �� g/ml, 156.3 �� g/ml.
Embodiment 3
(1) preparation of reference substance solution: each about 5mg of Epimedin A, Epimedin B, epimedin and icariin, accurately weighed, it is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions. Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (65:35) is diluted to scale, shakes up, and must mix reference substance storing solution.
(2) preparation of inner mark solution: take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml).
(3) prepared by need testing solution: with acetonitrile: 0.1% aqueous formic acid (35:65) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution.
(4) testing conditions:
High-efficient liquid phase chromatogram condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 0.1% aqueous formic acid 35:65, isocratic elution 3.5min, sample size 20 �� l, column temperature 45 DEG C, flow velocity 0.3ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 8000CC/min, atomization gas flow: 10L/min, gas curtain throughput: 8L/min, ion source spray voltage :-4500V, ion source atomization temperature: 400 DEG C.
Take reference substance, inner mark solution and need testing solution respectively according to above-mentioned testing conditions, measure Epimedin A, Epimedin B, epimedin and icariin respectively 65.33 �� g/ml, 78.95 �� g/ml, 716.4 �� g/ml, 151.7 �� g/ml.
Embodiment 4
(1) preparation of reference substance solution: each about 5mg of Epimedin A, Epimedin B, epimedin and icariin, accurately weighed, it is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions. Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution.
(2) preparation of inner mark solution: take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (25:75) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml).
(3) prepared by need testing solution: with acetonitrile: 1% aqueous formic acid (35:65) dilutes CHUANKEZHI ZHUSHEYE 1000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution.
(4) testing conditions:
High-efficient liquid phase chromatogram condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 0.5% aqueous formic acid 30:70, isocratic elution 5.5min, sample size 20 �� l, column temperature 45 DEG C, flow velocity 0.2ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7500CC/min, atomization gas flow: L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
Take reference substance, inner mark solution and need testing solution respectively according to above-mentioned testing conditions, measure Epimedin A, Epimedin B, epimedin and Icariin content respectively 62.18 �� g/ml, 81.12 �� g/ml, 718.4 �� g/ml, 152.6 �� g/ml.
Embodiment 5
(1) preparation of reference substance solution: each about 5mg of Epimedin A, Epimedin B, epimedin and icariin, accurately weighed, it is respectively placed in 5ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions.Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution.
(2) preparation of inner mark solution: take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% acetic acid aqueous solution (25:75) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (25:75) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml).
(3) prepared by need testing solution: with acetonitrile: 0.1% aqueous formic acid (25:75) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution.
(4) testing conditions:
High-efficient liquid phase chromatogram condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 0.1% acetic acid aqueous solution 40:60, isocratic elution 3.5min, sample size 10 �� l, column temperature 40 DEG C, flow velocity 0.22ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7500CC/min, atomization gas flow: 8L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
Take reference substance, inner mark solution and need testing solution respectively according to above-mentioned testing conditions, measure Epimedin A, Epimedin B, epimedin and Icariin content respectively 66.33 �� g/ml, 79.39 �� g/ml, 722.8 �� g/ml, 158.3 �� g/ml.
Embodiment 6
(1) preparation of reference substance solution: each about 10mg of Epimedin A, Epimedin B, epimedin and icariin, accurately weighed, it is respectively placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to 4 kinds of reference substance storing solutions. Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution.
(2) preparation of inner mark solution: take naringin reference substance and be about 10mg, accurately weighed, it is placed in 10ml measuring bottle, adds methanol and dissolve and be diluted to scale, be configured to interior mark storing solution. It is appropriate that precision measures interior mark storing solution, puts in 10ml measuring bottle, adds acetonitrile: 0.1% aqueous formic acid (35:65) is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml. In accurate absorption, the secondary storing solution 200 �� l of mark, puts in 10ml measuring bottle, adds acetonitrile: 0.5% phosphate aqueous solution (35:65) is diluted to scale, shakes up, obtain interior mark working solution (2 �� g/ml).
(3) prepared by need testing solution: with acetonitrile: 0.5% phosphate aqueous solution (35:65) dilutes CHUANKEZHI ZHUSHEYE 2000 times, precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, take subsequent filtrate, obtain need testing solution.
(4) testing conditions:
High-efficient liquid phase chromatogram condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 1% acetic acid aqueous solution 35:65, isocratic elution 4.0min, sample size 15 �� l, column temperature 35 DEG C, flow velocity 0.2ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7500CC/min, atomization gas flow: 8L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
Take reference substance, inner mark solution and need testing solution respectively according to above-mentioned testing conditions, measure Epimedin A, Epimedin B, epimedin and Icariin content respectively 66.18 �� g/ml, 77.95 �� g/ml, 726.0 �� g/ml, 155.6 �� g/ml.
Claims (6)
1. a CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method, it is characterised in that comprise the steps:
1) preparation of reference substance solution: take reference substance Epimedin A, Epimedin B, epimedin and icariin, dissolves with methanol or mobile phase and get final product;
2) preparation of inner mark solution: take naringin reference substance, dissolves with methanol or mobile phase and get final product;
3) preparation of need testing solution: take CHUANKEZHI ZHUSHEYE methanol or mobile phase dilutes and get final product;
4) detection method: high performance liquid chromatography mass spectrometry, described high-efficient liquid phase chromatogram condition, C18Chromatographic column 50��150 �� 2.1mm, 3.0��5.0 ��m; Mobile phase: acetonitrile: 0.1%��1% aqueous formic acid 30:70��40:60, isocratic elution, sample size: 5��20 �� l; Column temperature: 35��45 DEG C; Flow velocity: 0.2��0.3ml/min; Described Mass Spectrometry Conditions, auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7000��8000CC/min, atomization gas flow: 6��10L/min, gas curtain throughput: 5��8L/min, ion source spray voltage: 3800��4500V, ion source atomization temperature: 350��400 DEG C.
2. a kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method as claimed in claim 1, it is characterized in that, the reference substance Epimedin A described in step (1), Epimedin B, epimedin and icariin are each configured to the reference substance storing solution of 1mg/ml; Precision measures reference substance storing solution and is placed in same measuring bottle respectively again, adds methanol dilution and is configured to Epimedin A, Epimedin B, epimedin and icariin and is the mixing reference substance storing solution of 1000ng/ml.
3. a kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method as claimed in claim 1, it is characterized in that: the naringin reference substance described in step (2) is configured to the interior mark storing solution of 1mg/ml, be configured to 2 �� g/ml inner mark solutions by dilution step.
4. a kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method as claimed in claim 3, it is characterized in that: described dilution step refers to that taking interior mark storing solution is placed in measuring bottle, add methanol or mobile phase is configured in 100 �� g/ml to mark secondary stock solution, the accurate secondary storing solution of interior mark of drawing is placed in measuring bottle, adds methanol or mobile phase is configured to 2 �� g/ml inner mark solutions.
5. a kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method as claimed in claim 1, it is characterized in that: step (3) dilutes CHUANKEZHI ZHUSHEYE with mobile phase, precision measures 1000-2000 �� l and adds 1/5 times of inner mark solution mix homogeneously, filter membrane, take subsequent filtrate, obtain need testing solution.
6. a kind of CHUANKEZHI ZHUSHEYE flavonoids effective constituent detection method as claimed in claim 1, it is characterised in that comprise the steps:
1) preparation of reference substance solution: Epimedin A, Epimedin B, epimedin and icariin add methanol and dissolve and be diluted to scale, is configured to 4 kinds of 1mg/ml reference substance storing solutions; Precision measures 4 kinds of flavone reference substance storing solutions in right amount respectively, puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, must mix reference substance storing solution;
2) preparation of inner mark solution: take naringin reference substance, accurately weighed, it is placed in measuring bottle, adds methanol and dissolve and be diluted to scale, be configured in 1mg/ml and mark storing solution; It is appropriate that precision measures interior mark storing solution, is placed in measuring bottle, adds acetonitrile: 0.1% aqueous formic acid 35:65 is diluted to scale, shakes up, and obtains the secondary stock solution of mark in 100 �� g/ml; The accurate secondary storing solution of interior mark of drawing is put in measuring bottle, adds acetonitrile: 0.1% aqueous formic acid 35:65 is diluted to scale, shakes up, obtains interior mark working solution 2 �� g/ml;
3) prepared by need testing solution: use acetonitrile: 0.1% aqueous formic acid 35:65 dilutes CHUANKEZHI ZHUSHEYE 2000 times, and precision measures 1000 �� l and adds interior mark working solution 200 �� l, vortex 40s, crosses 0.22 ��m of filter membrane, takes subsequent filtrate, obtain need testing solution;
4) testing conditions:
Chromatographic condition
Chromatographic column C18150 �� 2.1mm, 5.0 ��m, mobile phase acetonitrile: 0.1% aqueous formic acid 35:65, isocratic elution 3.5min, sample size 10 �� l, column temperature 40 DEG C, flow velocity 0.22ml/min;
Mass Spectrometry Conditions
Auxiliary gasification electric spray ion source ESI; Many reactive ions monitoring MRM pattern anionic textiles, monitoring ion pair is respectively as follows: m/z883.4 �� 675.2 Epimedin A, m/z853.5 �� 645.3 Epimedin B, m/z867.4 �� 659.4 epimedin, m/z721.3 �� 513.2 icariin, m/z579.2 �� 271.1 naringin, assisted gas activating QI flow: 7500CC/min, atomization gas flow: 8L/min, gas curtain throughput: 6L/min, ion source spray voltage :-4000V, ion source atomization temperature: 350 DEG C.
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| CN104034815A (en) * | 2014-06-11 | 2014-09-10 | 广州万正药业有限公司 | Method for measuring contents of icariin and bepimedin C in cough-treatable injection |
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| CN104034815A (en) * | 2014-06-11 | 2014-09-10 | 广州万正药业有限公司 | Method for measuring contents of icariin and bepimedin C in cough-treatable injection |
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