CN109030702A - A kind of quality determining method that blood stasis-eliminating and stagnation-dissipating enema is new - Google Patents
A kind of quality determining method that blood stasis-eliminating and stagnation-dissipating enema is new Download PDFInfo
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- CN109030702A CN109030702A CN201811304036.3A CN201811304036A CN109030702A CN 109030702 A CN109030702 A CN 109030702A CN 201811304036 A CN201811304036 A CN 201811304036A CN 109030702 A CN109030702 A CN 109030702A
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- G—PHYSICS
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- 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
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Abstract
The invention discloses a kind of quality determining method that blood stasis-eliminating and stagnation-dissipating enema is new, the quality determining method includes: the inspection of 1) aristolochic acid；2) assay of Paeoniflorin and chlorogenic acid；Assay is the following steps are included: (1) chromatographic condition and system suitability are tested；(2) preparation of reference substance solution；(3) preparation of test solution；(4) it measures.In the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema of the invention, the assay and its measuring method of one of effective component of a herb honeysuckle chlorogenic acid in the inspection and the Chinese prescription of aristolochic acid are increased.Regulation efficient liquid-phase chromatography method measures in prescription the content of effective component Paeoniflorin and chlorogenic acid in two taste Chinese medicine of radix paeoniae rubra and honeysuckle simultaneously (every milliliter of medical fluid is containing Paeoniflorin not less than 1.2mg in new standard, chlorogenic acid is not less than 0.5mg), this method is easy to operate, specificity is strong, favorable reproducibility makes the quality of said preparation control detection more safely, effectively.
Description
Technical field
The present invention relates to the technical field of quality detection of drug more particularly to one kind of Chinese patent drug " blood stasis-eliminating and stagnation-dissipating enema "
New quality determining method.
Background technique
Blood stasis-eliminating and stagnation-dissipating enema is a kind of auspicious Chinese patent drug produced at pharmaceutical Co. Ltd, authentication code [national drug standard
Z20025840], by Radix Angelicae Sinensis, radix paeoniae rubra, glutinous rehmannia, rhizome of chuanxiong, peach kernel, safflower, Radix Salviae Miltiorrhizae, radix cyathulae, trigone, curcuma zedoary, turtle shell, tortoise plastron, pass
Totally 15 taste Chinese medicines are prepared for caulis akebiae, Fructus Forsythiae, honeysuckle, have activating microcirculation and removing stasis medicinal, resolving hard lump, promoting the circulation of qi promoting, clearing heat and detoxicating
The effect of, it can be used for chronic pelvic inflammatory disease, the adjuvant treatment of acute pelvitis of pelvic cavity, to inflammatory masses of ovarian appendages, pelvic tuberculosis mass, basin
The diseases such as chamber blood stasis mass, fibroid, endometriosis, adenomyoma of uterus, ovarian cyst, dysmenorrhea have significant treat
Effect.Existing quality testing mainly includes three parts: (1) using thin layer chromatography identification ferulic acid and catechu in identification project
Aldehyde；It (2) include measurement pH and relative density in inspection item；(3) assay project: high performance liquid chromatography (Chinese Pharmacopoeia
The one annex VID of version in 2010) measurement.Chromatographic condition and system suitability: being to fill out with octadecylsilane chemically bonded silica
Agent is filled, with methanol -0.05moL/L potassium dihydrogen phosphate (24:76) for mobile phase, Detection wavelength 230nm, number of theoretical plate presses Chinese herbaceous peony
Glycosides peak, which calculates, should be not less than 2500；The preparation of reference substance solution: taking Paeoniflorin reference substance appropriate, accurately weighed, and methanol is added to be made
Solution of every 1ml containing 0.1mg to get；The preparation of test solution: precision measures this product 3ml, passes through neutral alumina column (100
~200 mesh, 5g, internal diameter 1cm, with 50% methanol 10ml prewashing), it is eluted with 50% methanol, collects eluent to 50ml capacity
It is appropriate in bottle, add 50% methanol dilution to shake up, filter to scale, take subsequent filtrate to get；Measuring method: accurate respectively to draw control
Product solution and each 10ul of test solution, inject liquid chromatograph, measurement to get.The paeoniflorin content of every 1mL this product must not lack
In the 1.20mg[national drug standards WS-10612 (ZD-0612) -2002-2012Z].
Summary of the invention
The present invention provides a kind of quality determining method that blood stasis-eliminating and stagnation-dissipating enema is new, can preferably control blood stasis-eliminating and stagnation-dissipating bowel lavage
The quality of liquid formulation guarantees the stability and validity of said preparation, and the existing quality standard of product is revised and improved.Base
In the assay that content determination item includes Paeoniflorin, therefore the inspection of Paeoniflorin is eliminated in identification project, be based on this product
Contain caulis akebiae medicinal material in prescription, contain aristolochic acid I in caulis akebiae, in view of aristolochic acid I there is the Research Literature of renal toxicity to report,
The inspection to aristolochic acid I is increased in this quality standard in check item；It is increased in assay project one in a prescription
The assay and its measuring method of the highly seasoned active ingredient chlorogenic acid for wanting Flos lonicerae.This method uses high performance liquid chromatography
The effective component Paeoniflorin and chlorogenic acid in prescription in two taste Chinese medicine of radix paeoniae rubra and honeysuckle are measured simultaneously.
[identification] takes 1 bottle of this product, ultrasonic 30min, and centrifugation takes supernatant, is extracted 2 times, each 50ml, is closed with ether shaking
And ether solution, it volatilizes, residue adds ethyl alcohol 1ml to dissolve, as test solution.Separately take protocatechualdehyde reference substance that ethyl alcohol is added to be made
The reference substance solution of 1mg/ml.It is tested according to thin-layered chromatography (2015 editions pharmacopeia, four general rules 0502), draws above two solution
Each 10 μ L puts respectively on same silica gel g thin-layer plate, with chloroform-acetone-formic acid (8:1:1) for solvent, is unfolded, takes out, dry in the air
It is dry, it sprays with 10% ferric trichloride ethanol solution, it is clear to be heated to spot development at 105 DEG C.In sample chromatogram, with reference substance
On the corresponding position of chromatography, the spot of same color is shown.
[inspection] aristolochic acid I: it is measured according to high performance liquid chromatography (2015 editions pharmacopeia, four general rules 0512).Chromatographic condition
With system suitability using octadecylsilane chemically bonded silica as filler；Mobile phase be 0.1% phosphoric acid (A) and acetonitrile (B),
Using gradient elution, the volume ratio of (A) and (B) is 59:41 when the gradient elution is 0min.~40min., 40min.~
The volume ratio of (A) and (B) are 59 → 20:41 → 80 when 50min., and the volume ratio of (A) and (B) is 20 when 50min.~55min.
The volume ratio of (A) and (B) are 59:41 when → 59:80 → 41,55min.~60min.；Detection wavelength is 260nm, column temperature 30
℃.Theoretical cam curve is calculated by the peak aristolochic acid I should be not less than 3000.The preparation of reference substance solution: aristolochic acid I is taken to compare
Product, it is accurately weighed, set in brown volumetric flask, add methanol be made the solution of every 1ml 12 μ g containing aristolochic acid to get.Test sample is molten
The preparation of liquid takes one bottle of this product, is ultrasonically treated 30min, and precision pipettes 20ml and sets in triangular flask, and methanol 80ml is added in precision,
Close plug, is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 50ml, adds proper amount of methanol to dissolve and be transferred to 10ml volumetric flask
In, methanol constant volume is added to scale, shake up to get.
[assay] Paeoniflorin and chlorogenic acid: taking 20 μ l of reference substance solution to inject liquid chromatograph, and it is sensitive to adjust detection
Degree, makes the peak height of chromatographic peak reach the 20% of full scale, then 20 μ l of test solution is taken to measure, and should not obtain in sample chromatogram
Now chromatographic peak identical with reference substance chromatographic retention.If there is the identical chromatographic peak of retention time, diode battle array is used
For the more corresponding chromatographic peak of column detector in the uv-visible absorption spectra of 190-300nm wave-length coverage, absorption spectrum should not phase
Together.Chromatographic condition: chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；Mobile phase is
0.1% phosphoric acid (A) and acetonitrile (B), using gradient elution, the body of (A) and (B) when the gradient elution is 0min.~26min.
Product is than being 87:13, and the volume ratio of (A) and (B) is 87 → 20:13 → 80 when 26min.~27min., when 27min.~32min.
(A) and the volume ratio of (B) is 20:80；The volume ratio of (A) and (B) are 20 → 87:80 → 13 when 32min.~32.1min.,
The volume ratio of (A) and (B) are 87:13 when 32.1min.~36min..Detection wavelength is 230nm, and column temperature is 30 DEG C.Theoretical tray
Number is calculated by chlorogenic acid peak should be not less than 3000.The preparation of reference substance solution: taking chlorogenic acid, Paeoniflorin reference substance, accurately weighed,
Set in brown volumetric flask, add 50% methanol be made mixed solution of every 1ml containing 15 μ g of chlorogenic acid, the 30 μ g containing Paeoniflorin to get.For
The preparation of test sample solution: taking one bottle of this product, is ultrasonically treated 30 minutes, shakes up, and filters, takes subsequent filtrate, precision pipettes subsequent filtrate 1ml
Set in 50ml volumetric flask, be diluted with water to scale, shake up to get.Measuring method: accurate respectively to draw reference substance solution, for examination
Each 10 μ l of product solution.Inject liquid chromatograph, measurement to get.The every 1ml of this product (C containing Paeoniflorin23H28O11) must not be less than
1.2mg；Containing chlorogenic acid (C16H18O9) 0.5mg must not be less than.
The invention has the advantages that:
1, the HPLC chromatogram condition and method used in this method to the inspection of aristolochic acid I, testing result is accurate, can
Row；To the two kinds of effective component Paeoniflorins and chlorogenic acid in said preparation, used HPLC chromatogram condition and method, testing result
Accurately, feasible.Quality determining method of the invention is reliable and stable, specificity is strong, favorable reproducibility.
2, the identification for increasing aristolochic acid I can preferably guarantee the safety of drug, select Paeoniflorin and green original in prescription
Acid, which carries out assay, can fully and effectively control the quality of blood stasis-eliminating and stagnation-dissipating enema, be conducive to the quality of stable prod, it is ensured that
The safety and validity of clinical application preferably meet the needs of medical treatment and market.
Detailed description of the invention
Attached drawing 1 is aristolochic acid I reference substance HPLC spectrogram in embodiment 1.
Attached drawing 2 is sample (lot number 20170326) HPLC spectrogram in embodiment 1.
Attached drawing 3 is aristolochic acid I reference substance HPLC spectrogram in embodiment 2.
Attached drawing 4 is sample (lot number 20170326) mark-on HPLC spectrogram in embodiment 2.
Attached drawing 5 is aristolochic acid I reference substance HPLC spectrogram in embodiment 3.
Attached drawing 6 is sample (lot number 20170326) mark-on HPLC spectrogram in embodiment 3.
Attached drawing 7 is aristolochic acid I reference substance HPLC spectrogram in embodiment 4.
Attached drawing 8 is sample (lot number 20170326) mark-on HPLC spectrogram in embodiment 4.
Attached drawing 9 is aristolochic acid I reference substance HPLC spectrogram in embodiment 5.
Attached drawing 10 is sample (lot number 20170709) HPLC spectrogram in embodiment 5.
Attached drawing 11 is aristolochic acid I reference substance HPLC spectrogram in embodiment 6.
Attached drawing 12 is sample (lot number 20171018) HPLC spectrogram in embodiment 6.
Attached drawing 13 is 7 Content of Chlorogenic Acid of embodiment, Paeoniflorin reference substance mixed solution HPLC spectrogram.
Attached drawing 14 is sample (lot number 20170326) HPLC spectrogram in embodiment 7.
Attached drawing 15 is 8 Content of Chlorogenic Acid of embodiment, Paeoniflorin reference substance mixed solution HPLC spectrogram.
Attached drawing 16 is sample (lot number 20170326) HPLC spectrogram in embodiment 8.
Attached drawing 17 is 9 Content of Chlorogenic Acid of embodiment, Paeoniflorin reference substance mixed solution HPLC spectrogram.
Attached drawing 18 is sample (lot number 20170326) HPLC spectrogram in embodiment 9.
Attached drawing 19 is 10 Content of Chlorogenic Acid of embodiment, Paeoniflorin reference substance mixed solution HPLC spectrogram.
Attached drawing 20 is sample (lot number 20170326) HPLC spectrogram in embodiment 10.
Attached drawing 21 is 11 Content of Chlorogenic Acid of embodiment, Paeoniflorin reference substance mixed solution HPLC spectrogram.
Attached drawing 22 is sample (lot number 20170709) HPLC spectrogram in embodiment 11.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.It should be understood that these embodiments
It is only illustrative of the invention and is not intended to limit the scope of the invention.In addition, it should also be understood that, having read present disclosure
Later, those skilled in the art can make various modifications or changes to the present invention, and such equivalent forms equally fall within the application institute
Attached claims limited range.
The detection (one) of 1 aristolochic acid I of embodiment
The detection embodiment of aristolochic acid I:
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 260nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as table 1:
1 mobile phase gradient of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0~40 | 59 | 41 |
| 40~50 | 59→20 | 41→80 |
| 50~55 | 20→59 | 80→41 |
| 55~60 | 59 | 41 |
The preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made
The solution of every 2 μ g of 1ml I containing aristolochic acid to get；
The preparation of test solution: taking one bottle of this product (lot number 20170326), is ultrasonically treated 30min, and precision pipettes 20ml
It sets in triangular flask, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, adds
Proper amount of methanol is dissolved and is transferred in 10ml volumetric flask, and methanol constant volume is added to scale, shake up to get；
Measuring method: taking 20 μ l of reference substance solution to inject liquid chromatograph, adjusts detection sensitivity, reaches the peak height of chromatographic peak
To the 20% of full scale, then 20 μ l of test solution is taken to measure, aristolochic acid I must not be detected.
As a result as shown in Figure 1 and Figure 2, aristolochic acid I is not detected in sample.
The inspection (two) of 2 aristolochic acid I of embodiment
The detection embodiment of aristolochic acid I:
Chromatographic condition: chromatographic column: crowd C18,4.6 × 250mm, 5 μm are praised in Changzhou, column temperature: 30 DEG C, flow velocity: and 1.0ml/min,
Detection wavelength: 260nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elution program such as table 2.
2 mobile phase gradient of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0~40 | 59 | 41 |
| 40~50 | 59→20 | 41→80 |
| 50~55 | 20→59 | 80→41 |
| 55~60 | 59 | 41 |
The preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made
The solution of every 2 μ g of 1ml I containing aristolochic acid to get.
The preparation of test solution: taking one bottle of this product (lot number 20170326), is ultrasonically treated 30min, and precision pipettes 20ml
It sets in triangular flask, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, adds
Proper amount of methanol is dissolved and is transferred in 10ml volumetric flask, and methanol constant volume is added to scale, shake up to get.
Measuring method takes 20 μ l of reference substance solution to inject liquid chromatograph, adjusts detection sensitivity, reaches the peak height of chromatographic peak
To the 20% of full scale, then 20 μ l of test solution is taken to measure, aristolochic acid I must not be detected.
As a result as shown in Figure 3, Figure 4.
The inspection (three) of 3 aristolochic acid I of embodiment
The detection embodiment of aristolochic acid I:
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 25 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 260nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as table 3:
3 mobile phase gradient of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0~40 | 59 | 41 |
| 40~50 | 59→20 | 41→80 |
| 50~55 | 20→59 | 80→41 |
| 55~60 | 59 | 41 |
The preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made
The solution of every 2 μ g of 1ml I containing aristolochic acid to get.
The preparation of test solution: taking one bottle of this product (lot number 20170326), is ultrasonically treated 30min, and precision pipettes 20ml
It sets in triangular flask, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, adds
Proper amount of methanol is dissolved and is transferred in 10ml volumetric flask, and methanol constant volume is added to scale, shake up to get.
Measuring method: taking 20 μ l of reference substance solution to inject liquid chromatograph, adjusts detection sensitivity, reaches the peak height of chromatographic peak
To the 20% of full scale, then 20 μ l of test solution is taken to measure, aristolochic acid I must not be detected.
As a result as shown in Figure 5, Figure 6.
The inspection (four) of 4 aristolochic acid I of embodiment
The detection embodiment of aristolochic acid I:
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
0.8ml/min, Detection wavelength: 260nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as table 4.
4 mobile phase gradient of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0~40 | 59 | 41 |
| 40~50 | 59→20 | 41→80 |
| 50~55 | 20→59 | 80→41 |
| 55~60 | 59 | 41 |
The preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made
The solution of every 2 μ g of 1ml I containing aristolochic acid to get.
The preparation of test solution: taking one bottle of this product (lot number 20170326), is ultrasonically treated 30min, and precision pipettes 20ml
It sets in triangular flask, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, adds
Proper amount of methanol is dissolved and is transferred in 10ml volumetric flask, and methanol constant volume is added to scale, shake up to get；
Measuring method: taking 20 μ l of reference substance solution to inject liquid chromatograph, adjusts detection sensitivity, reaches the peak height of chromatographic peak
To the 20% of full scale, then 20 μ l of test solution is taken to measure, aristolochic acid I must not be detected.
As a result as shown in Figure 7, Figure 8.
The inspection (five) of 5 aristolochic acid I of embodiment
The detection embodiment of aristolochic acid I:
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 260nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as table 5:
5 mobile phase gradient of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0~40 | 59 | 41 |
| 40~50 | 59→20 | 41→80 |
| 50~55 | 20→59 | 80→41 |
| 55~60 | 59 | 41 |
The preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made
The solution of every 2 μ g of 1ml I containing aristolochic acid to get.
The preparation of test solution: taking one bottle of this product (lot number 20170709), is ultrasonically treated 30min, and precision pipettes 20ml
It sets in triangular flask, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, adds
Proper amount of methanol is dissolved and is transferred in 10ml volumetric flask, and methanol constant volume is added to scale, shake up to get.
Measuring method: taking 20 μ l of reference substance solution to inject liquid chromatograph, adjusts detection sensitivity, reaches the peak height of chromatographic peak
To the 20% of full scale, then 20 μ l of test solution is taken to measure, aristolochic acid I must not be detected.
As a result as shown in Figure 9, Figure 10.
The inspection (six) of 6 aristolochic acid I of embodiment
The detection embodiment of aristolochic acid I:
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 260nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as table 6.
6 mobile phase gradient of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0~40 | 59 | 41 |
| 40~50 | 59→20 | 41→80 |
| 50~55 | 20→59 | 80→41 |
| 55~60 | 59 | 41 |
The preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made
The solution of every 2 μ g of 1ml I containing aristolochic acid to get；
The preparation of test solution: taking one bottle of this product (lot number 20171018), is ultrasonically treated 30min, and precision pipettes 20ml
It sets in triangular flask, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, adds
Proper amount of methanol is dissolved and is transferred in 10ml volumetric flask, and methanol constant volume is added to scale, shake up to get；
Measuring method: taking 20 μ l of reference substance solution to inject liquid chromatograph, adjusts detection sensitivity, reaches the peak height of chromatographic peak
To the 20% of full scale, then 20 μ l of test solution is taken to measure, aristolochic acid I must not be detected.
As a result as shown in Figure 11, Figure 12.
7 chlorogenic acid of embodiment, paeoniflorin content measurement (one)
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 230nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as the following table 7:
7 mobile phase gradient of table
| Time (min) | 0.1% phosphate aqueous solution (%) | Acetonitrile (%) |
| 0 | 87 | 13 |
| 26 | 87 | 13 |
| 27 | 20 | 80 |
| 32 | 20 | 80 |
| 32.1 | 87 | 13 |
| 36 | 87 | 13 |
The preparation of reference substance solution: it is appropriate to weigh chlorogenic acid, Paeoniflorin reference substance, adds methanol to be configured to concentration and is respectively
0.0150375, the standard solution of 0.03366mg/ml；
The preparation of test solution: precision absorption blood stasis-eliminating and stagnation-dissipating enema (lot number: it is 20170326) ultrasonic, it mixes, filtering,
It takes subsequent filtrate 1.0ml into 50ml volumetric flask, suitable quantity of water is added to dilute, ultrasound makes sufficiently to dissolve, and adds water to graduation mark, filters, takes
Subsequent filtrate to obtain the final product；
Measuring method: 10 μ l of accurate pipette samples and reference substance, into hplc determination.
As a result as shown in figs. 13 and 14, chlorogenic acid, paeoniflorin content are respectively 0.76mg/ml, 2.08mg/ml.
8 chlorogenic acid of embodiment, paeoniflorin content measurement (two)
Chromatographic condition: chromatographic column: crowd C18,4.6 × 250mm, 5 μm are praised in Changzhou, column temperature: 30 DEG C, flow velocity: and 1.0ml/min,
Detection wavelength: 230nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elution program such as the following table 8:
8 mobile phase gradient of table
| Time (min) | 0.1% phosphate aqueous solution (%) | Acetonitrile (%) |
| 0 | 87 | 13 |
| 26 | 87 | 13 |
| 27 | 20 | 80 |
| 32 | 20 | 80 |
| 32.1 | 87 | 13 |
| 36 | 87 | 13 |
The preparation of reference substance solution: it is appropriate to weigh chlorogenic acid, Paeoniflorin reference substance, adds methanol to be configured to concentration and is respectively
0.0150375, the standard solution of 0.03366mg/ml；
The preparation of test solution: precision absorption blood stasis-eliminating and stagnation-dissipating enema (lot number: it is 20170326) ultrasonic, it mixes, filtering,
It takes subsequent filtrate 1.0ml into 50ml volumetric flask, suitable quantity of water is added to dilute, ultrasound makes sufficiently to dissolve, and adds water to graduation mark, filters, takes
Subsequent filtrate to obtain the final product；
Measuring method: 10 μ l of accurate pipette samples and reference substance, into hplc determination.
As a result as shown in Figure 15, Figure 16, chlorogenic acid, paeoniflorin content are respectively 0.78mg/ml, 2.09mg/ml.
9 chlorogenic acid of embodiment, paeoniflorin content measurement (three)
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 25 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 230nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as the following table 9.
9 mobile phase gradient of table
| Time (min) | 0.1% phosphate aqueous solution (%) | Acetonitrile (%) |
| 0 | 87 | 13 |
| 26 | 87 | 13 |
| 27 | 20 | 80 |
| 32 | 20 | 80 |
| 32.1 | 87 | 13 |
| 36 | 87 | 13 |
The preparation of reference substance solution: it is appropriate to weigh chlorogenic acid, Paeoniflorin reference substance, adds methanol to be configured to concentration and is respectively
0.0150375, the standard solution of 0.03366mg/ml.
The preparation of test solution: precision absorption blood stasis-eliminating and stagnation-dissipating enema (lot number: it is 20170326) ultrasonic, it mixes, filtering,
It takes subsequent filtrate 1.0ml into 50ml volumetric flask, suitable quantity of water is added to dilute, ultrasound makes sufficiently to dissolve, and adds water to graduation mark, filters, takes
Subsequent filtrate to obtain the final product.
Measuring method: 10 μ l of accurate pipette samples and reference substance, into hplc determination.
As a result as shown in Figure 17, Figure 18, chlorogenic acid, paeoniflorin content are respectively 0.81mg/ml, 2.14mg/ml.
10 chlorogenic acid of embodiment, paeoniflorin content measurement (four)
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
1.2ml/min, Detection wavelength: 230nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as the following table 10.
10 mobile phase gradient of table
| Time (min) | 0.1% phosphate aqueous solution (%) | Acetonitrile (%) |
| 0 | 87 | 13 |
| 26 | 87 | 13 |
| 27 | 20 | 80 |
| 32 | 20 | 80 |
| 32.1 | 87 | 13 |
| 36 | 87 | 13 |
The preparation of reference substance solution: it is appropriate to weigh chlorogenic acid, Paeoniflorin reference substance, adds methanol to be configured to concentration and is respectively
0.0150375, the standard solution of 0.03366mg/ml；
The preparation of test solution: precision absorption blood stasis-eliminating and stagnation-dissipating enema (lot number: it is 20170326) ultrasonic, it mixes, filtering,
It takes subsequent filtrate 1.0ml into 50ml volumetric flask, suitable quantity of water is added to dilute, ultrasound makes sufficiently to dissolve, and adds water to graduation mark, filters, takes
Subsequent filtrate to obtain the final product；
Measuring method: 10 μ l of accurate pipette samples and reference substance, into hplc determination.
As a result as shown in Figure 19, Figure 20, chlorogenic acid, paeoniflorin content are respectively 0.80mg/ml, 2.19mg/ml.
11 chlorogenic acid of embodiment, paeoniflorin content measurement (five)
Chromatographic condition: chromatographic column: Dikma Platisil C18,4.6 × 250mm, 5 μm, column temperature: 30 DEG C, flow velocity:
1.0ml/min, Detection wavelength: 230nm, mobile phase: 0.1% phosphate aqueous solution is mobile phase A, acetonitrile is Mobile phase B, elutes journey
Sequence such as the following table 11.
11 mobile phase gradient of table
| Time (min) | 0.1% phosphate aqueous solution (%) | Acetonitrile (%) |
| 0 | 87 | 13 |
| 26 | 87 | 13 |
| 27 | 20 | 80 |
| 32 | 20 | 80 |
| 32.1 | 87 | 13 |
| 36 | 87 | 13 |
The preparation of reference substance solution: it is appropriate to weigh chlorogenic acid, Paeoniflorin reference substance, adds methanol to be configured to concentration and is respectively
0.0150375, the standard solution of 0.03366mg/ml；
The preparation of test solution: precision absorption blood stasis-eliminating and stagnation-dissipating enema (lot number: it is 20170709) ultrasonic, it mixes, filtering,
It takes subsequent filtrate 1.0ml into 50ml volumetric flask, suitable quantity of water is added to dilute, ultrasound makes sufficiently to dissolve, and adds water to graduation mark, filters, takes
Subsequent filtrate to obtain the final product；
Measuring method: 10 μ l of accurate pipette samples and reference substance, into hplc determination.
As a result as shown in Figure 21, Figure 22, chlorogenic acid, paeoniflorin content are respectively 0.80mg/ml, 1.97mg/ml.
Show that the HPLC chromatogram condition and method that use in this method to the inspection of aristolochic acid can by above-mentioned embodiment
Row, obtained qualification result are accurate；To the two kinds of effective component Paeoniflorins and chlorogenic acid in said preparation, used HPLC chromatogram
Condition and method are feasible, and obtained qualification result is accurate.The present invention helps to promote the quality of this product and guarantees having for product
Effect property and safety.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (7)
1. a kind of quality determining method that blood stasis-eliminating and stagnation-dissipating enema is new, which is characterized in that the described method includes:
(1) aristolochic acid is detected with HPLC；The condition of the HPLC is as follows:
A) preparation of test solution: taking one bottle of given the test agent, is ultrasonically treated 30min, and precision pipettes 20ml and sets triangular flask
In, methanol 80ml is added in precision, and close plug is ultrasonically treated 30min, filtering, and precision measures subsequent filtrate 5ml, proper amount of methanol is added to dissolve
And be transferred in 10ml volumetric flask, methanol constant volume is added to scale, shake up to get；
B) preparation of reference substance solution: taking aristolochic acid I reference substance, accurately weighed, sets in brown volumetric flask, methanol is added to be made often
The solution of 2 μ g of 1ml I containing aristolochic acid to get；
C) using octadecylsilane chemically bonded silica as filler；Mobile phase is 0.1% phosphoric acid (A) and acetonitrile (A), is washed using gradient
De-, the volume ratio of (A) and (B) is 59:41 when the gradient elution is 0min~40min, when 40min~50min (A) and (B)
Volume ratio be 20:80, the volume ratio of (A) and (B) is 59:41 when 50min~60min；Detection wavelength is 240-280nm, column
Temperature is 25-35 DEG C；The number of plates is calculated by the peak aristolochic acid I should be not less than 3000；
(2) content of Paeoniflorin and chlorogenic acid is measured simultaneously with HPLC；The condition of the HPLC is as follows:
A) preparation of test solution: taking one bottle of given the test agent, is ultrasonically treated 30 minutes, shakes up, and filters, takes subsequent filtrate, accurate
Pipette subsequent filtrate 1ml to set in 50ml volumetric flask, be diluted with water to scale, shake up to get；
B) preparation of reference substance solution: taking chlorogenic acid, Paeoniflorin reference substance, accurately weighed, sets in brown volumetric flask, adds 50% first
Alcohol be made mixed solution of every 1ml containing 15 μ g of chlorogenic acid, the 30 μ g containing Paeoniflorin to get；
C) chromatographic condition: using octadecylsilane chemically bonded silica as filler；Using 0.1% phosphate aqueous solution as mobile phase (A), with
Acetonitrile is mobile phase (A), and using gradient elution, the volume ratio of (A) and (B) is 87 when the gradient elution is 0min~26min:
The volume ratio of (A) and (B) are 87 → 20:13 → 80 when 13,26min~27min, the volume of (A) and (B) when 27min~32min
Than for 20:80；The volume ratio of (A) and (B) are 80 → 87:80 → 13 when 32min~32.1min, when 32.1min~36min (A)
The volume ratio of (B) is 87:13；Detection wavelength is 210nm~250nm, and column temperature is 20 DEG C~35 DEG C；The number of plates presses chlorogenic acid peak
3000 should be not less than by calculating；
D) measuring method: it is accurate respectively to draw reference substance solution, each 10 μ l of test solution, liquid chromatograph is injected, is measured, i.e.,
?；The every 1ml of given the test agent (C containing Paeoniflorin23H28O11) 1.2mg must not be less than；Containing chlorogenic acid (C16H18O9) 0.5mg must not be less than.
2. the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema according to claim 1, which is characterized in that increase in the method
The inspection of aristolochic acid is added.
3. the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema according to claim 1, which is characterized in that increase in the method
The assay of chlorogenic acid is added.
4. the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema according to claim 1, which is characterized in that detect horse in HPLC
In pocket bell acid, using reversed C18 chromatographic column, mobile phase is 0.1% phosphoric acid (A) and acetonitrile (B), using gradient elution, the ladder
Degree elution when being 0min.~40min. the volume ratio of (A) and (B) be 59:41, the volume of (A) and (B) when 40min.~50min.
Than for 20:80, the volume ratio of (A) and (B) is 59:41 when 50min.~60min..
5. the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema according to claim 1, which is characterized in that surveyed simultaneously in HPLC
In the content for determining Paeoniflorin and chlorogenic acid, using reversed C18 chromatographic column, mobile phase is 0.1% phosphoric acid (A) and acetonitrile (B), is used
Gradient elution, the volume ratio of (A) and (B) is 87:13,26min.~27min. when the gradient elution is 0min.~26min.
When (A) and (B) volume ratio be 87 → 20:13 → 80, the volume ratio of (A) and (B) is 20:80 when 27min.~32min.；
The volume ratio of (A) and (B) are 20 → 87:80 → 13 when 32min.~32.1min., when 32.1min.~36min. (A) and (B)
Volume ratio be 87:13；Detection wavelength is 230nm, and column temperature is 30 DEG C.
6. the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema according to claim 1, which is characterized in that detect horse in HPLC
In pocket bell acid, Detection wavelength 260nm, column temperature is 30 DEG C, flow rate of mobile phase 0.8-1.2ml/min.
7. the new quality determining method of blood stasis-eliminating and stagnation-dissipating enema according to claim 1, which is characterized in that surveyed simultaneously in HPLC
In the content for determining Paeoniflorin and chlorogenic acid, column Mobile phase flow velocity is 0.8-1.2ml/min.
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