CN108061764A - The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment - Google Patents
The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment Download PDFInfo
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Abstract
The present invention relates to the method for quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, belong to quality control technologies for traditional Chinese medicine field.The method of quality control includes chromatographic condition, four big step of the preparation of reference substance solution, the preparation of test solution and measuring method.The present invention is succinct to sample treatment, effective, despumation interference, can accurate determination sample bullatine A content, the accuracy of medication can be accurately reflected, effectively control the quality of medicinal Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, it ensures that the security, validity, reliability of drug, there is practical significance.
Description
Technical field
The invention belongs to quality control technologies for traditional Chinese medicine fields, and in particular to the quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment
Method processed.
Background technology
The quick-acting antalgesic liniment of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) is recorded at present in hygienic ministerial standard (abbreviation ministry standard) Chinese traditional patent formulation system
The Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment of agent the 20th, the quick-acting antalgesic liniment of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) mainly by Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) and
Other Chinese medicines obtain preparation by impregnating, and major function is relaxing tendons and activating collaterals, swelling and pain relieving, and for traumatic injury, (soft tissue is turned round
Wound, contusion etc.) and various arthralgia.Produce at present the domestic pharmacy producer of the kind have Yunnan alpine yallow herb pharmaceutical Co. Ltd,
Yuxi peacekeeping pharmaceutcal corporation, Ltd of Yunnan Province two, is restricted by historical conditions at that time, no matter from detection method or the product
Kind quality standard, ministry standard has been difficult the requirement for adapting to today's society to drug quality control at present, to ensure many patients
Drug safety, effectively, it is controllable, Yunnan alpine yallow herb pharmaceutical Co. Ltd improves as Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment standard
Careless person, particular under [assay] item, former ministry standard content assaying method avenges upper one for determined by ultraviolet spectrophotometry
Branch wormwood artemisia total alkali.This method is relatively to measure Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) total alkali compared with the absorbance of sample using reference substance absorbance
Content, due to containing the impurity such as polysaccharide, pigment in sample, there is interference greatly in the method, poor reproducibility, and avenge upper one with mixture
Branch wormwood artemisia total alkali control final product quality, does not have specificity, the Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) total alkali in control sample that can not be authentic and valid contains
Amount.Therefore the problem of how overcome the deficiencies in the prior art is current quality control technologies for traditional Chinese medicine field urgent need to resolve.
The content of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide the quality of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment
Control method, this method can with the content of bullatine A in Accurate Determining Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, so as to
The accuracy of medication can be accurately reflected, effectively controls the quality of medicinal Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, and then ensures medicine
Security, validity, the reliability of product have practical significance.
To achieve the above object, the technical solution adopted by the present invention is as follows：
The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment under the method for current standard execution, further includes
The assay of bullatine A, step are as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 60-75: 40-25 first
Alcohol-phosphate buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate is calculated by bullatine A peak should
Not less than 3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added
Water dissolution is simultaneously diluted to 1000mL, and pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：Take bullatine A reference substance that methanol is added to be made every 1ml μ containing 60-100 g's
Solution to get；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment is taken, the ethyl alcohol in liniment is boiled off, then adds in
Weight is the water of taken 1-5 times of liniment weight and dissolving, adds in the ammonia solution that weight is taken 0.2-1 times of liniment weight afterwards,
Then extracted with dichloromethane, obtain dichloromethane extract, dichloromethane extract after washing, then using anhydrous sodium sulfate
Filtrate is collected in dehydration, filtration；Filtrate is done in 60 DEG C of water-bath Back stroke near, is added methanol afterwards and is dissolved, filters, take subsequent filtrate, i.e.,
；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；
This product is per 1ml (C containing bullatine A22H33NO2) for the μ g of 10 μ g~60.
It is further preferred that in step A, the volume ratio of methanol-phosphate buffer solution is 65: 35
It is further preferred that in step C, it is that ethyl alcohol is boiled off in water-bath to boil off the ethyl alcohol in liniment.
It is further preferred that in step C, dichloromethane extraction time is 2 times；Washing times are also 2 times.
It is further preferred that the method for quality control of the Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, further includes on snow
The assay of bullatine A, step are as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 60-75: 40-25 first
Alcohol-phosphate buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate is calculated by bullatine A peak should
Not less than 3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added
Water dissolution is simultaneously diluted to 1000mL, and pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：Take bullatine A reference substance that methanol is added to be made every 1ml μ containing 60-100 g's
Solution to get；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment 10-50ml is taken, is boiled off in water-bath in liniment
Ethyl alcohol, then add in and 50ml water and dissolve, add in 10ml ammonia solutions afterwards, then extracted 2 times with dichloromethane, used in 2 times
The amount of dichloromethane is 30-60ml, obtains dichloromethane extract, dichloromethane extract after washing, then using anhydrous sulphur
Filtrate is collected in sour sodium dehydration, filtration；Filtrate is done in 60 DEG C of water-bath Back stroke near, and afterwards plus methanol to 10ml and dissolves, and filters,
Take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；
This product is the μ g of 10 μ g~60 containing bullatine A per 1ml.
It is further preferred that the method for quality control of the Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, further includes on snow
The assay of bullatine A, step are as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 65: 35 methanol-phosphoric acid
Salt buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should be not less than by the calculating of bullatine A peak
3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added
Water dissolution is simultaneously diluted to 1000mL, and pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：Take bullatine A reference substance that methanol is added to be made every 1ml μ containing 60-100 g's
Solution to get；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment 50ml is taken, is boiled off in water-bath in liniment
Then ethyl alcohol adds in 50ml water and dissolves, adds in 10ml ammonia solutions afterwards, then extracted 3 times with dichloromethane, two used in 3 times
The amount of chloromethanes is 40ml, obtains dichloromethane extract, dichloromethane extract takes off after washing, then using anhydrous sodium sulfate
Filtrate is collected in water, filtration；Wherein, washing times are 2 times, and total dosage of water is 50ml；Filtrate is in 60 DEG C of water-bath Back stroke near
It is dry, hplc grade methanol is added to make dissolving, and be transferred in 10ml volumetric flasks, methanol is added to shake up to scale, filter, take subsequent filtrate, i.e.,
；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；
This product is per 1ml (C containing bullatine A22H33NO2) for the μ g of 10 μ g~60.
Applicant from documents and materials understand the upper punt-pole of snow mainly contain aconitine, Hypaconitine, the upper punt-pole A prime of snow,
The alkaloids such as B prime, C prime, D prime, element in heptan, in order to preferably control the quality of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, with reference in
The reference substance that state food and medicine calibrating research institute can be provided, by the checking research of the methods of inspection such as specificity, durability, most
Whole applicant selects bullatine A to control product quality.
Bullatine A content assaying method proposed by the invention is mainly several including extracting, washing, evaporating, diluting
A step, the general extraction methods of sample such as make alkaloid in being extracted with organic solvent, used again after alkalescence in being detected using Chinese medicine
It purifies water washing and removes substance etc. outside alkaloid, its main purpose is will to intend the ingredient of detection from sample as fully as possible
It extracts, then the substance outside alkaloid is removed with purifying water washing, the moisture that anhydrous sodium sulfate removing is more than is added in, after being evaporated
Methanol dissolving bullatine A is added to be detected.
Compared with prior art, the present invention its advantage is：
The present invention proposes the method for quality control of new Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, and this method is to sample treatment
Succinctly, effectively, despumation interference, can accurate determination sample bullatine A content, it is existing so as to compensate for
Big, poor reproducibility is disturbed in technology, does not have the problem of specificity.
With efficient liquid-phase chromatography method, the multiple batches of sample size of this kind is measured by us, and has investigated snow upper one
The content of bullatine A, mobile phase use methanol-phosphate buffer solution in branch wormwood artemisia medicinal material.By being to this method
System applicability, linear, precision, reappearance, stability, the rate of recovery are investigated, and show that this method is accurate, reliable, quick, spirit
It is quick.
This method is groped accurate to the processing method of sample survey using bullatine A as reference substance by self experiment
The content of bullatine A in true determination sample.This method is carried out using quick, accurate, sensitive high performance liquid chromatography
Assay can accurately reflect the accuracy of medication, effectively control the quality of medicinal Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, so as to
It ensures the security, validity, reliability of drug, there is practical significance.
Description of the drawings
Fig. 1 is bullatine A uv absorption spectra；
Fig. 2 is using volume ratio as 65：Bullatine A reference substance HPLC color of the 35 acetonitrile-water as mobile phase
Spectrogram is as shown；
Fig. 3 is using volume ratio as 65：35 acetonitrile-water is as mobile phase Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC colors
Spectrogram；
Fig. 4 is using volume ratio as 70：The phosphoric acid solution of 30 acetonitrile -0.1% is compareed as mobile phase bullatine A
Product HPLC chromatogram；
Fig. 5 is using volume ratio as 70：The phosphoric acid solution of 30 acetonitrile -0.1% is as mobile phase Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic
Liniment HPLC chromatogram；
Fig. 6 is using volume ratio as 72：28 acetonitrile-water is as mobile phase bullatine A reference substance HPLC chromatogram
Figure；
Fig. 7 is using volume ratio as 72：28 acetonitrile-water is as mobile phase Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC colors
Spectrogram；
Fig. 8 is using volume ratio as 60：40 acetonitrile-water is as mobile phase bullatine A reference substance HPLC chromatogram
Figure；
Fig. 9 is using volume ratio as 60：40 acetonitrile-water is as mobile phase Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC colors
Spectrogram is as shown.
Specific embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright scope.In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition
Or it is carried out according to product description.Production firm person is not specified in material therefor or equipment, and being can be by buying what is obtained
Conventional products.
Bullatine A reference substance of the present invention is provided by National Institute for Food and Drugs Control, lot number：
110895-200404。
Instrument includes：High performance liquid chromatograph Agilent 1100, high performance liquid chromatograph Thermo ultimate
3000, ultraviolet-uisible spectrophotometer (Shanghai You Ke instrument and meters Co., Ltd UV759CRT), ultraviolet-uisible spectrophotometer
(Shanghai Precision Scientific Apparatus Co., Ltd UV757CRT), electronic balance (sartorius CPA225D, ten a ten thousandths).
Embodiment 1
The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment further includes the assay of bullatine A,
Step is as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 60: 40 methanol-phosphoric acid
Salt buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should be not less than by the calculating of bullatine A peak
3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added
Water dissolution is simultaneously diluted to 1000mL, and pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：Bullatine A reference substance is taken to add methanol that the solution that every 1ml contains 60 μ g is made,
To obtain the final product；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment is taken, the ethyl alcohol in liniment is boiled off, then adds in
Weight is the water of taken 1 times of liniment weight and dissolving, adds in the ammonia solution that weight is taken 0.2 times of liniment weight afterwards, then
It is extracted with dichloromethane, obtains dichloromethane extract, dichloromethane extract takes off after washing, then using anhydrous sodium sulfate
Filtrate is collected in water, filtration；Filtrate is done in 60 DEG C of water-bath Back stroke near, is added methanol afterwards and is dissolved, filters, take subsequent filtrate, i.e.,
；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；
This product is the μ g of 10 μ g~60 containing bullatine A per 1ml.
Embodiment 2
The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment further includes the assay of bullatine A,
Step is as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 75: 25 methanol-phosphoric acid
Salt buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should be not less than by the calculating of bullatine A peak
3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added
Water dissolution is simultaneously diluted to 1000mL, and pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：Bullatine A reference substance is taken to add methanol that every 1ml is made molten containing 100 μ g
Liquid to get；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment is taken, the ethyl alcohol in liniment is boiled off, then adds in
Weight is the water of taken 5 times of liniment weight and dissolving, adds in the ammonia solution that weight is taken 1 times of liniment weight afterwards, then uses
Dichloromethane extracts, and obtains dichloromethane extract, and dichloromethane extract is dehydrated after washing, then using anhydrous sodium sulfate,
Filtrate is collected in filtration；Filtrate near dry, afterwards plus methanol and dissolved, is filtered in 60 DEG C of water-bath Back stroke, take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；
This product is the μ g of 10 μ g~60 containing bullatine A per 1ml.
Embodiment 3
The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment further includes the assay of bullatine A,
Step is as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 65: 35 methanol-phosphoric acid
Salt buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should be not less than by the calculating of bullatine A peak
3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added
Water dissolution is simultaneously diluted to 1000mL, and pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：Take bullatine A reference substance that methanol is added to be made every 1ml μ containing 60-100 g's
Solution to get；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment 50ml is taken, is boiled off in water-bath in liniment
Then ethyl alcohol adds in 50ml water and dissolves, adds in 10ml ammonia solutions afterwards, then extracted 3 times with dichloromethane, two used in 3 times
The amount of chloromethanes is 40ml, obtains dichloromethane extract, dichloromethane extract takes off after washing, then using anhydrous sodium sulfate
Filtrate is collected in water, filtration；Wherein, washing times are 2 times, and total dosage of water is 50ml；Filtrate is in 60 DEG C of water-bath Back stroke near
It is dry, hplc grade methanol is added to make dissolving, and be transferred in 10ml volumetric flasks, methanol is added to shake up to scale, filter, take subsequent filtrate, i.e.,
；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；
This product is per 1ml (C containing bullatine A22H33NO2) for the μ g of 10 μ g~60.
1st, Detection wavelength is definite
Bullatine A reference substance is taken to be dissolved in right amount with methanol, with methanol blank control, in 200~400nm wavelength
Range scans draw abosrption spectrogram (see Fig. 1), as a result have absorption maximum at 210nm wavelength.Therefore 210nm is selected as snow
The assay wavelength of upper bullatine A.
2nd, the preparation of test solution
By literature query, extracting the solvent of bullatine A has methanol, chloroform, dichloromethane, therefore I
Investigated recovery rate of three kinds of solvents to Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal material.It weighs with a collection of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal material (lot number：
16071001) 4.0g adds in ammonia solution 5ml, is separately added into methanol, chloroform, dichloromethane 60ml, stands overnight, and filters,
Filter residue and conical flask are washed 2 times, each 20ml respectively with methanol, chloroform, dichloromethane, and washing lotion and filtrate merge, 60 DEG C
Be evaporated, residue adds methanol solution transfer into 10ml measuring bottles, and methanol is added to shake up to scale, filters, take subsequent filtrate to get.On snow
Recovery rate result such as the following table 1 of bullatine A in alpine yallow herb medicinal material.
Table 1
| Solvent | Methanol | Chloroform | Dichloromethane |
| The content of bullatine A | 113.47μg/g | 140.69μg/g | 150.89 |
By experiment, dichloromethane can more preferably extract bullatine A, and toxicity is less than chloroform, so
We select Extraction solvent of the dichloromethane as bullatine A in Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment.
3rd, the selection of mobile phase
The present invention selects the mobile phase of 4 different proportions to screen.It is as follows：
1. methanol-phosphate buffer (65：35), bullatine A reference substance HPLC chromatogram is as shown in Figure 2；
Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC chromatogram is as shown in Figure 3；
2. methanol-phosphate buffer (70：30), bullatine A reference substance HPLC chromatogram is as shown in Figure 4；
Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC chromatogram is as shown in Figure 5；
3. methanol-phosphate buffer (72：28), bullatine A reference substance HPLC chromatogram is as shown in Figure 6；
Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC chromatogram is as shown in Figure 7；
4. methanol-phosphate buffer (60：40), bullatine A reference substance HPLC chromatogram is as shown in Figure 8；
Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment HPLC chromatogram as shown in Figure 9
The result shows that：Using mobile phase methanol-phosphate buffer (60:40) when, bullatine A appearance time
It is partially late.Compare mobile phase methanol-phosphate buffer (65:35)、(70：And (72 30):28), methanol-phosphate buffer
(72:28) peak shape is symmetrical, methanol-phosphate buffer (65:35) reappearance is preferable, can meet containing for bullatine A
It is fixed to measure.
4th, blank assay
This product for not adding Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal material is taken, blank solution is made by test solution preparation method, as a result blank
It tests in the identical retention time position of snow bullatine A reference substance, no other Interference Peaks occur.
5th, the investigation of linear relationship and the range of linearity
Precision weighs bullatine A reference substance 7.60mg, puts in 100ml measuring bottles, and methanol is added to dissolve and is diluted to quarter
Degree, shakes up, accurate respectively to draw 1 μ l of reference substance solution, 2 μ l, 5 μ l, 10 μ l, 20 μ l, 30 μ l injection liquid as reference substance solution
Chromatography, measurement result are shown in Table 2
Table 2
| Sample size | Bullatine A peak area |
| 1μl | 51.76193 |
| 2μl | 100.81599 |
| 5μl | 245.68393 |
| 10μl | 507.40195 |
| 20μl | 1014.30756 |
| 30μl | 1528.23975 |
Using peak area as ordinate, the concentration of reference substance is abscissa, draws standard curve, obtains regression equation, y=
6702.21728x-2.21354 r=0.99998, bullatine A is in the range of the μ g/ml of 7.6 μ g/ml~228 in good
Linear relationship.
6th, precision investigates (repetitive test)
Same sample lots (06052701) is taken to prepare test solution according to the method under assay item, inject liquid phase color
Spectrometer, replication 6 times calculate precision.It the results are shown in Table 3,
The repeated measurement result of table 3
The result shows that：6 times sample introduction repeatability is good, and test error is small.
7th, serviceability test
1. study on the stability
Same sample lots (06052701) is taken to prepare test solution according to the method under assay item, inject liquid phase color
Spectrometer, at interval of 2 it is small when measure once, investigate bullatine A stability at room temperature.Test result is shown in Table 4.
4 Stability Determination result of table
Illustrate that sample is interior basicly stable when 10 is small at ambient temperature.
2. different manufacturers chromatographic column is investigated
It the results are shown in Table 5.
Table 5
8th, accuracy test (recovery test)
Take this product 5ml (lot numbers：06052701), it is accurate add in reference substance solution (73.5 μ g/ml of concentration, 105.1 μ g/ml,
114.6 μ g/ml) 5ml, test solution is made according to assay item method, calculates the rate of recovery, the results are shown in Table 6.
Table 6
9th, the determination data of sample
Assay is carried out to 10 batches of samples in accordance with the law, the results are shown in Table 7.
The assay result of 70 batches of samples of table
Contain bullatine A in 11.48~31.78 (μ g/ml) per 1ml according to 10 batches of sample measurement results, it is contemplated that
The factor of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal material and production technology etc., so regulation is the μ of 10 μ g~60 containing bullatine A per 1ml
g。
It, can by the methodological study to the assay of bullatine A in Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment
To find out high performance liquid chromatography is used to have the characteristics that strong specificity, high sensitivity, favorable reproducibility, accuracy are high.Use the party
Method carries out assay to 10 batches of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniments, achieves satisfied result.Experiments have shown that this method can
Assay accurately effectively is carried out to Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) first in Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (6)
1. the method for quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, which is characterized in that further include bullatine A
Assay, step are as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 60-75: 40-25 methanol-phosphorus
Hydrochlorate buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should not be low by the calculating of bullatine A peak
In 3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added water-soluble
It solves and is diluted to 1000mL, pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：The solution that bullatine A reference substance is taken to add methanol that every 1ml μ containing 60-100 g are made,
To obtain the final product；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment is taken, the ethyl alcohol in liniment is boiled off, then adds in weight
It is the water of taken 1-5 times of liniment weight and dissolving, adds in the ammonia solution that weight is taken 0.2-1 times of liniment weight afterwards, then
It is extracted with dichloromethane, obtains dichloromethane extract, dichloromethane extract takes off after washing, then using anhydrous sodium sulfate
Filtrate is collected in water, filtration；Filtrate is done in 60 DEG C of water-bath Back stroke near, is added methanol afterwards and is dissolved, filters, take subsequent filtrate, i.e.,
；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get；
This product is the μ g of 10 μ g~60 containing bullatine A per 1ml.
2. the method for quality control of according to claim a kind of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, which is characterized in that step
In rapid A, the volume ratio of methanol-phosphate buffer solution is 65: 35.
3. the method for quality control of according to claim a kind of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, which is characterized in that step
In rapid C, it is that ethyl alcohol is boiled off in water-bath to boil off the ethyl alcohol in liniment.
4. the method for quality control of according to claim a kind of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment, which is characterized in that step
In rapid C, dichloromethane extraction time is 2 times；Washing times are also 2 times.
5. the method for quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment according to claim 1, which is characterized in that also wrap
The assay of bullatine A is included, step is as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 60-75: 40-25 methanol-phosphorus
Hydrochlorate buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should not be low by the calculating of bullatine A peak
In 3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added water-soluble
It solves and is diluted to 1000mL, pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：The solution that bullatine A reference substance is taken to add methanol that every 1ml μ containing 60-100 g are made,
To obtain the final product；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment 10-50ml is taken, the second in liniment is boiled off in water-bath
Then alcohol adds in 50ml water and dissolves, adds in 10ml ammonia solutions afterwards, then extracted 2 times with dichloromethane, dichloro used in 2 times
The amount of methane is 30-60ml, obtains dichloromethane extract, dichloromethane extract after washing, then using anhydrous sodium sulfate
Filtrate is collected in dehydration, filtration；Filtrate is done in 60 DEG C of water-bath Back stroke near, and afterwards plus methanol to 10ml and dissolves, and filters, takes continuous
Filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get；
This product is the μ g of 10 μ g~60 containing bullatine A per 1ml.
6. the method for quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment according to claim 1, which is characterized in that also wrap
The assay of bullatine A is included, step is as follows：
A, chromatographic condition：It is filler with octadecylsilane chemically bonded silica；Using volume ratio as 60-75: 40-25 methanol-phosphorus
Hydrochlorate buffer solution is mobile phase；Detection wavelength is 210 ± 2nm；Number of theoretical plate should not be low by the calculating of bullatine A peak
In 3000；
The preparation method of the phosphate buffer solution is to take disodium hydrogen phosphate 1.97g and potassium dihydrogen phosphate 0.22g, is added water-soluble
It solves and is diluted to 1000mL, pH to 7.3 is adjusted with the phosphoric acid solution that mass concentration is 80%；
B, the preparation of reference substance solution：The solution that bullatine A reference substance is taken to add methanol that every 1ml μ containing 60-100 g are made,
To obtain the final product；
C, the preparation of test solution：Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment 50ml is taken, the ethyl alcohol in liniment is boiled off in water-bath,
Then add in 50ml water and dissolve, add in 10ml ammonia solutions afterwards, then extracted 3 times with dichloromethane, dichloromethane used in 3 times
Amount for 40ml, obtain dichloromethane extract, dichloromethane extract is dehydrated after washing, then using anhydrous sodium sulfate, filter
It crosses, collects filtrate；Wherein, washing times are 2 times, and total dosage of water is 50ml；Filtrate is in 60 DEG C of water-bath Back stroke near dry, additive color
Spectrum level methanol makes dissolving, and is transferred in 10ml volumetric flasks, and methanol is added to shake up to scale, filters, take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get；
This product is the μ g of 10 μ g~60 containing bullatine A per 1ml.
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|---|---|---|---|---|
| CN104739955A (en) * | 2015-02-04 | 2015-07-01 | 贵阳中医学院 | Preparation method of purified short-pedicel aconite root alkaloid by employing cation exchange resin method |
| CN106769964A (en) * | 2016-12-09 | 2017-05-31 | 中国科学院新疆理化技术研究所 | The method of quality control of Herba Achilleae extract |
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2017
- 2017-12-01 CN CN201711252095.6A patent/CN108061764A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104739955A (en) * | 2015-02-04 | 2015-07-01 | 贵阳中医学院 | Preparation method of purified short-pedicel aconite root alkaloid by employing cation exchange resin method |
| CN106769964A (en) * | 2016-12-09 | 2017-05-31 | 中国科学院新疆理化技术研究所 | The method of quality control of Herba Achilleae extract |
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| 李军红: "HPLC法测定骨痛灵巴布剂中雪上一枝蒿甲素含量", 《中国中医药信息杂志》 * |
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Application publication date: 20180522 |