CN106706789B - With the method in relation to substance in high effective liquid chromatography for measuring drotaverine hydrochloride injection - Google Patents
With the method in relation to substance in high effective liquid chromatography for measuring drotaverine hydrochloride injection Download PDFInfo
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Abstract
The invention discloses a kind of with the method in relation to substance in high effective liquid chromatography for measuring drotaverine hydrochloride injection, the described method comprises the following steps: a. prepares test sample solution: sample to be tested is configured to test sample solution with solvent；B. it detects: using octadecylsilane chemically bonded silica as chromatographic column filler, using phosphate buffer as mobile phase A, using acetonitrile as Mobile phase B, using methanol as mobile phase C, using linear gradient elution method, the test sample solution, Detection wavelength 244nm are detected.Use the method for high performance liquid chromatography provided by the present application detection drotaverine hydrochloride bulk pharmaceutical chemicals ingredient, can quick, effective, accurate and reliable separation detection go out in drotaverine hydrochloride bulk pharmaceutical chemicals related substance, the product quality for being conducive to improve drotaverine hydrochloride injection, improves patient medication safety.
Description
Technical field
The present invention relates to Pharmaceutical Analysis technical fields, bend him with high effective liquid chromatography for measuring hydrochloric acid more particularly, to a kind of
Tie up the method in relation to substance in woods injection.
Background technique
Drotaverine hydrochloride (Drotaverine hydrochloride), chemical name are as follows: 1- { (3,4- diethoxybenzenes
Base) methylene -6,7- diethoxy -1,2,3,4- four hydrogen isoquinoline hydrochloric acid salts, trade name No-SPA (RNO-SPA), be by
A kind of novel solution spasm medicine that match Norfin, Inc of France develops in the pharmaceutical factory Hungarian co-partnership company Chinoin.The medicine is
Isoquinoline class derivate, with traditional anisodamine, the anticholinergics such as atropine release spasm medicine difference, it is directly acted on smoothly
Myocyte, the mechanism of action of diastole smooth muscle are as follows: act on smooth muscle cells, change cell membrane potential and permeability；
Inhibit phosphodiesterase, it is horizontal to increase adenosine cyclophosphate (cAMP) in myocyte；Inhibit intracellular initial calcium ion reaction, smoothly
Flesh diastole releases spasm.Its main feature is that: (1) effect comprehensively, curative effect lasting, and drotaverine hydrochloride shrinks smooth muscle spasmodic
Each link all work, especially high-tension smooth muscle has a special efficacy, and elimination half-life period is 9-11h, therefore validity period is long；
(2) adverse reaction is small, and drotaverine hydrochloride does not influence autonomic nerves system, on the normal smooth muscle of gastrointestinal tract without influence, without M
The adverse reaction of receptor antagonist.The indication of the medicine includes coronary artery insufficiency, endarteritis obliterans, angina pectoris, stomach
Intestinal smooth spasm, irritable bowel syndrome, cholecystalgia, renal colic and the urinary tract spasm, hysterotrismus, dysmenorrhea etc..Its advantage
Be the reaction of no serious cardiovascular, without parasympathetic blockade, acorea amplification, the acute attack without glaucoma, without urethra wing
The expansion of Guang, rapid-action, adverse reaction is few.
The ingredient of drotaverine hydrochloride bulk pharmaceutical chemicals is specific, mainly includes drotaverine hydrochloride and 2 kinds of related impurities (second
Base papaverine, Drotaverine ketone), this 2 kinds of substances are thoroughly quickly separated particularly significant.
The related substance high efficient liquid phase analysis method reference of drotaverine hydrochloride injection State Bureau standard (YBH17602006)
Drotaverine hydrochloride injection checks standard JX20000051, drotaverine hydrochloride tablet quality standard WS-2127 (X-184)-
2002 formulate.
State Bureau's standard (YBH17602006), using phosphate buffer (pH3.3 ± 0.05)-, acetonitrile-methanol is isocratic washes
De-, analysis time period is shorter, and about 5~7 minutes appearances of main peak have rear trailing phenomenon, it is understood that there may be unknown impuritie and main peak are not
The case where efficiently separating, each known impurities separation is preferable, but drotaverine hydrochloride injection has 3 through improving sterilization process condition
A unknown impuritie degradation is obvious, and separation is undesirable, and certain impurity are overlapped between retention time about 3~5 minutes, and measurement is not
Accurately, Related substances separation requirement cannot be reached, be unfavorable for practical popularization and application.
Therefore, it is necessary to be improved to drotaverine hydrochloride injection Related substance method, establish one quickly, have
The detection method of effect is sterilized 20 minutes in sterilization process by 100 DEG C, after becoming 121 DEG C of sterilizings 15 minutes, can be distinguished and be detected
All related substances in drotaverine hydrochloride bulk pharmaceutical chemicals are conducive to the product quality for improving drotaverine hydrochloride injection, mention
High patient medication safety.
In view of the above-mentioned problems, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is exploring various conditions of the drotaverine hydrochloride in relation to substance efficient liquid phase chromatographic analysis, provide
The related substance HPLC analytical method of one simplicity, reliable drotaverine hydrochloride.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A method of with substance related in high effective liquid chromatography for measuring drotaverine hydrochloride injection, this method includes
Following steps:
(1) test sample solution is prepared:
Drotaverine hydrochloride is taken to be configured to test sample solution with solvent；
(2) reference substance solution is prepared:
Drotaverine hydrochloride, ethaquin and Drotaverine ketone is taken to be configured to reference substance solution with solvent；
(3) compounding system applicability solution:
Drotaverine hydrochloride, ethaquin and Drotaverine ketone is taken to be configured to system suitability solution with solvent；
(4) system suitability experiment is carried out；
(5) detection sensitivity experiment is carried out；
The detection sensitivity that high performance liquid chromatograph is adjusted with reference substance solution measures the peak height of principal component chromatographic peak completely
The 10-25% of journey；
(6) it is measured according to following high-efficient liquid phase chromatogram condition:
Mobile phase: using phosphate buffer as mobile phase A, using acetonitrile as Mobile phase B, using methanol as mobile phase C；It is described
Phosphate buffer is adjusted to pH=3.3 ± 0.05 with triethylamine for 0.1% phosphate aqueous solution；
UV detector: Detection wavelength 200-400nm；
Chromatographic column: using octadecylsilane chemically bonded silica as filler；
Flow velocity: 0.5-2.0mL/min；
Column temperature: 20-60 DEG C；
(7) external standard method is pressed, the content in relation to substance is calculated.
Preferably, the Detection wavelength of the UV detector is 200-300nm, more preferably 244nm.
Preferably, the flow velocity is 1.0mL/min；The column temperature is 30-50 DEG C, more preferably 40 DEG C.
Preferably, the phosphate buffer is made after adjusting pH3.3 ± 0.05 with triethylamine by 0.1% phosphate aqueous solution
At.
Preferably, the preparation of the test sample solution, which specifically includes, prepares the molten of hydrochloric Drotaverine 0.8mg/mL
Liquid, the solvent of the solution are the mixed solvent that mobile phase A, methanol and acetonitrile are formed, and the mixed solvent is by methanol and second
1:9 is mixed the mixed mixed liquor of 2:1 nitrile by volume with mobile phase A again by volume.
Preferably, the preparation of the reference substance solution, which specifically includes, prepares hydrochloric Drotaverine, ethaquin and bends
The solution of Ta Weilin ketone, the concentration of drotaverine hydrochloride is 4.0 μ g/mL in the solution；The concentration of ethaquin is 1.6 μ
g/mL；The concentration of Drotaverine ketone is 4.0 μ g/mL；Drotaverine hydrochloride, ethaquin and Drotaverine ketone are first used into methanol
With the mixed liquor dissolution that acetonitrile volume ratio is 2:1, the mixed solvent that mobile phase A, methanol and acetonitrile are formed then is added, it is described mixed
Bonding solvent is that 1:9 is mixed the mixed mixed liquor of 2:1 by volume with mobile phase A again by volume by methanol and acetonitrile.
Preferably, the preparation of the system suitability solution, which specifically includes, prepares hydrochloric Drotaverine, ethaquin
With the solution of Drotaverine ketone, the concentration of drotaverine hydrochloride is 0.8mg/mL in the solution；The concentration of ethaquin is
1.6μg/mL；The concentration of Drotaverine ketone is 4.0 μ g/mL；Drotaverine hydrochloride, ethaquin and Drotaverine ketone are first used
The mixed liquor that methanol and acetonitrile volume ratio are 2:1 dissolves, and the mixed solvent that mobile phase A, methanol and acetonitrile are formed, institute is then added
Stating mixed solvent is that 1:9 is mixed the mixed mixed liquor of 2:1 by volume with mobile phase A again by volume by methanol and acetonitrile.
Preferably, the system suitability, which refers to, takes 20 μ L of system suitability solution to inject liquid chromatograph, hydrochloric acid
The separating degree of Drotaverine and ethaquin is greater than 3.0, and separating degree meets the requirements between each impurity peaks, and theoretical cam curve presses salt
Sour Drotaverine calculates, and is not less than 3000.
Preferably, gradient elution is carried out with mobile phase A, Mobile phase B and mobile phase C；The gradient are as follows:
Beneficial effects of the present invention are as follows:
The present invention is based on HPLC analytical method, for separation of the existing technology is not thorough, peak shape is dragged
The disadvantages of tail is serious, packet peak, using new buffer solution system and mobile phase ratio, enables impurity to be kept completely separate, solves
The relevant issues of the prior art；And method provided by the invention learns research and verifying, verifying through the methods of specificity, sensitivity
As a result in tolerance interval；The present invention carries out specificity comparative study with current standard, it was demonstrated that its method more can be controlled effectively
The related substance of drotaverine hydrochloride injection processed；The method of the present invention also has following beneficial effects,
1. mobile phase is prepared relatively simple easy to operate；
2. impurity can be efficiently separated, principal component is above State Bureau's standard without hangover, dopant species, the quantity of detection
(YBH17602006) method, accuracy in detection are high, improve the safety of product, methodological science, reliable, controllably；
3. it is easy to operate, it is suitable for practical application and popularization.
Detailed description of the invention
Fig. 1 shows the HPLC map of the testing result of the offer of the application comparative example 1；
Fig. 2 shows the HPLC maps for the testing result that the application comparative example 2 provides；
Fig. 3 shows the HPLC map of the testing result of the offer of the embodiment of the present application 1；
Fig. 4 shows the HPLC map of the testing result of the offer of the embodiment of the present application 2；
Fig. 5 shows the HPLC map of the testing result of the offer of the embodiment of the present application 3；
Fig. 6 A shows the HPLC map of the testing result of the offer of the application comparative example 3；
Fig. 6 B shows the HPLC map of the testing result of the offer of the embodiment of the present application 4；
Fig. 7 A shows the HPLC map of the testing result of the offer of the application comparative example 4；
Fig. 7 B shows the HPLC map of the testing result of the offer of the embodiment of the present application 5；
Fig. 8 A shows the HPLC map of the testing result of the offer of the application comparative example 5；
Fig. 8 B shows the HPLC map of the testing result of the offer of the embodiment of the present application 6；
Fig. 9 A shows the HPLC map of the testing result of the offer of the application comparative example 6；
Fig. 9 B shows the HPLC map of the testing result of the offer of the embodiment of the present application 7；
Figure 10 A shows the HPLC map of the testing result of the offer of the application comparative example 7；
Figure 10 B shows the HPLC map of the testing result of the offer of the embodiment of the present application 8；
Figure 11 shows the HPLC map of the testing result of the offer of the embodiment of the present application 14；
Figure 12 shows the HPLC map of the testing result of the offer of the embodiment of the present application 14；
Figure 13 shows the HPLC map of the testing result of the offer of the embodiment of the present application 14；
Figure 14 shows the HPLC map of the testing result of the offer of the embodiment of the present application 14.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
According to drotaverine hydrochloride Material synthesis process route and test, possible related substance can be determined are as follows:
(1) starting material: 3,4- diethoxy phenylacetic acids and 3,4- diethoxy phenyl ethylamine；
(2) synthetic intermediate (condensation product): N- (3,4- diethoxy phenylacetyl)-β-(3,4- diethoxybenzene) ethamine；
(3) catabolite: ethaquin, Drotaverine ketone.
Starting material and intermediate have been controlled in feed preparation process, and formulate inner quality standard.This method weight
Point pays close attention to 3 unknown impurities to become larger of degrading in 2 known catabolite ethaquins, Drotaverine ketone and sterilization process.
Step 1. prepares test sample solution: precision measurement drotaverine hydrochloride injection is appropriate, and solubilizer 2 quantitatively dilutes
It is made containing about the solution of drotaverine hydrochloride 0.8mg in every 1mL, as test solution；
Step 2. prepares reference substance solution: it is each suitable that precision weighs drotaverine hydrochloride, ethaquin and Drotaverine ketone
Amount, solubilizer 1 dissolve in right amount, then every 1mL is made containing about 4.0 μ g of drotaverine hydrochloride, 1.6 μ of ethaquin with the dilution of solvent 2
The mixed solution of g and 4.0 μ g of Drotaverine ketone, as reference substance solution；
Step 3. compounding system applicability solution: precision weighs drotaverine hydrochloride, ethaquin and Drotaverine ketone
Each appropriate, solubilizer 1 dissolves in right amount, then is diluted to every 1mL containing about 1.6 μ g of ethaquin, 4.0 μ of Drotaverine ketone with solvent 2
The mixed solution of g and drotaverine hydrochloride 0.8mg, as system suitability solution；
Step 4. detection: using octadecylsilane chemically bonded silica as chromatographic column filler, with phosphate buffer (0.1%
Phosphate aqueous solution use triethylamine adjust pH3.3 ± 0.05) as mobile phase A, using acetonitrile as Mobile phase B, using methanol as flow
Dynamic phase C detects the test sample solution, Detection wavelength 244nm using linear gradient elution method；Column temperature is 40 DEG C, and flow velocity is
1.0mL/ minute.Gradient condition is shown in Table 1；
Table 1
The accurate 20 μ L of system suitability solution that measures of step 5. injects liquid chromatograph, records chromatogram.Number of theoretical plate is pressed
Drotaverine hydrochloride calculates, should be not less than 3000, and the separating degree of drotaverine hydrochloride peak and ethaquin should be greater than 3.0, respectively
Separating degree meets the requirements between impurity peaks；
Step 6. is accurate to measure 20 μ L of reference substance solution, injects liquid chromatograph, adjusts detection sensitivity, make principal component color
The peak height of spectral peak is the 10%-25% of full scale；
Step 7. presses external standard method, calculates the content of ethaquin, Drotaverine ketone, other single impurity；
In above-mentioned steps 1,2 and 3, the solvent 1 is methanol acetonitrile mixed solution (Jia Chun ︰ acetonitrile=2 ︰ 1)；Solvent 2 is
Mobile phase A ︰ methanol acetonitrile mixed solution (Jia Chun ︰ acetonitrile=2 ︰ 1)=9 ︰ 1.
The laboratory apparatus used in the following examples and comparative examples is Agilent high performance liquid chromatograph；
The chromatographic column used is COSMOSIL 5C18-MS-Ⅱ250mm×4.6mm 5μm；In each embodiment, with phosphate
Buffer (0.1% phosphate aqueous solution use triethylamine adjust pH3.3 ± 0.05) as mobile phase A, using acetonitrile as Mobile phase B,
Using methanol as mobile phase C, solvent 1 is methanol acetonitrile mixed solution (Jia Chun ︰ acetonitrile=2 ︰ 1)；Solvent 2 is mobile phase A ︰ methanol
Acetonitrile mixed solution (Jia Chun ︰ acetonitrile=2 ︰ 1)=9 ︰ 1；Detect the test sample solution, Detection wavelength 244nm；Column temperature is
40 DEG C, flow velocity is 1.0mL/ minutes, sample volume: 20 μ L.Gradient elution carries out linear elution according to table 2:
Table 2
Impurity information see the table below 3:
3 impurity information of table summarizes
Embodiment 1-3
System suitability
Embodiment 1, system suitability solution are prepared:
Take drotaverine hydrochloride, known impurities Drotaverine ketone, known impurities ethaquin each appropriate respectively, solubilizer
Every 1mL is being made containing about 4.0 μ g (0.5%) of Drotaverine ketone, 1.6 μ g of ethaquin with the dilution of solvent 2 in 1 appropriate dissolution
(0.2%), the mixed solution of drotaverine hydrochloride 0.8mg, as system suitability solution；
Embodiment 2, test solution are prepared:
It is appropriate that precision measures drotaverine hydrochloride injection, and quantitatively dilution is made and about hydrochloric in every 1mL bends him solubilizer 2
The solution for tieing up woods 0.8mg, as test solution；
Embodiment 3, selective solution are prepared:
Drotaverine hydrochloride, known impurities Drotaverine ketone, known impurities ethaquin, Drotaverine condensation are taken respectively
Object (intermediate), starting material 3,4- diethoxy phenylacetic acid and 3, each appropriate solubilizer 1 of 4- diethoxy phenyl ethylamine are appropriate molten
Solution, then every 1mL is made containing about Drotaverine ketone, Drotaverine condensation product, 3,4- diethoxy phenylacetic acid, 3 with the dilution of solvent 2,
The mixing of each 4.0 μ g (0.5%) of 4- diethoxy phenyl ethylamine, ethaquin 1.6 μ g (0.2%), drotaverine hydrochloride 0.8mg
Solution, it is alternatively that property solution；Sample introduction simultaneously records chromatogram.
Comparative example 1-2
System suitability
It is tested by drotaverine hydrochloride injection quality standard YBH17602006；
Chromatographic condition:
Chromatographic column: octadecylsilane chemically bonded silica is filler；
Mobile phase: phosphate buffer (takes diammonium hydrogen phosphate 13.2g, water 200mL is added to make to dissolve, add 85% phosphoric acid
7.6mL is diluted with water to 1000mL, adjusts pH to 3.3 ± 0.05)-acetonitrile-methanol (800: 400: 800)；
Solvent: 0.1mol/LHCl- methanol-acetonitrile (8: 1: 1)；
Flow velocity: 1.0mL/min；Wavelength: 244nm；Column temperature: 40 DEG C；Sample volume: 20 μ L；
Comparative example 1, system suitability solution are prepared: taking drotaverine hydrochloride, known impurities Drotaverine ketone, known respectively
Every 1mL is made containing about 4.0 μ g (0.5%) of Drotaverine ketone, ethyl small-mouthed jar in each appropriate solubilizer dissolved dilution of impurity ethaquin
The mixed solution of 1.6 μ g (0.2%) of pavine, drotaverine hydrochloride 0.8mg, as system suitability solution；
Compare 2, test solution is prepared: precision measurement drotaverine hydrochloride injection is appropriate, and solubilizer quantitatively dilutes system
At the solution in every 1mL containing about drotaverine hydrochloride 0.8mg, as test solution；
Sample introduction simultaneously records chromatogram.
Conclusion:
Comparative example 1 and comparative example 2 are shown in Fig. 1, shown in Fig. 2: in system suitability solution, ethaquin, Drotaverine ketone
It is preferable with principal component separating degree, but main peak retention time is shorter, there is trailing phenomenon, there are unknown impuritie with main peak is not yet in effect separates
The case where；In addition, solvent peak rear impurity appearance time is very fast, and unknown impuritie separation is undesirable, at about 3~5 points of retention time
Partial impurities are overlapped between clock, and separating effect cannot reach Related substances separation requirement.
Embodiment 1-3 result is as shown in Fig. 3, Fig. 4: ethaquin, Drotaverine ketone and principal component separating degree are preferable, and
Principal component retention time is moderate, no trailing phenomenon；In addition, each unknown impuritie separating effect is preferable after solvent peak；Shown in Fig. 5: choosing
In selecting property solution, starting material, intermediate, known impurities are added and investigate separating degree, the results show that drotaverine hydrochloride and 3,4-
5 diethoxy phenylacetic acid, 3,4- diethoxy phenyl ethylamine, Drotaverine condensation product, ethaquin, Drotaverine ketone impurity
Separating degree be all larger than 3.0, separating effect can reach Related substances separation requirement.
Embodiment 4-8
Destructive testing
Test solution preparation
Solution before destroying: embodiment 4 takes drotaverine hydrochloride injection 1mL (being approximately equivalent to drotaverine hydrochloride 20mg)
It is placed in 25mL volumetric flask, solubilizer 2 is diluted to scale, shakes up, as not destroying solution.
High temperature solution: embodiment 5 takes drotaverine hydrochloride injection 1mL (to be approximately equivalent to drotaverine hydrochloride
It 20mg) is placed in 25mL volumetric flask, 100 DEG C of heating 8h are cooled to room temperature, and solvent 2 is diluted to scale, are shaken up, broken as high temperature
Bad solution.
Embodiment 6, illumination destroy solution: drotaverine hydrochloride injection 1mL being taken (to be approximately equivalent to drotaverine hydrochloride
20mg), it is placed in 25mL volumetric flask, illumination 15h under ultraviolet lamp, solvent 2 is diluted to scale, destroys solution as illumination.
Embodiment 7, alkali destroy solution: drotaverine hydrochloride injection 1mL (being equivalent to drotaverine hydrochloride about 20mg) is taken,
It is placed in 25mL volumetric flask, adds 1mol/L sodium hydroxide 1mL, after 60 DEG C of destruction 1.5h, add 1mol/L hydrochloric acid 1mL to neutralize, solvent 2
It is diluted to scale, is shaken up, destroys solution as alkali.
Oxidative demage solution: embodiment 8 takes drotaverine hydrochloride injection 1mL (to be approximately equivalent to drotaverine hydrochloride
20mg), it being placed in 25mL volumetric flask, adds 30% hydrogen peroxide 2mL, 60 DEG C of destruction 8h, solvent 2 is diluted to scale, shakes up, as
Oxidative demage solution.
Sample introduction simultaneously records chromatogram.
Comparative example 3-7
Destructive testing
It is tested by drotaverine hydrochloride injection quality standard YBH17602006,
Chromatographic condition:
Chromatographic column: octadecylsilane chemically bonded silica is filler；
Mobile phase: phosphate buffer (takes diammonium hydrogen phosphate 13.2g, water 200mL is added to make to dissolve, add 85% phosphoric acid
7.6mL is diluted with water to 1000mL, adjusts pH to 3.3 ± 0.05)-acetonitrile-methanol (800: 400: 800)；
Solvent: 0.1mol/LHCl solution-methyl alcohol-acetonitrile (8: 1: 1)；Flow velocity: 1.0mL/min；Wavelength: 244nm；Column temperature:
40℃；Sample volume: 20 μ L；
Test solution preparation:
Solution before destroying: comparative example 3 takes drotaverine hydrochloride injection 1mL (being approximately equivalent to drotaverine hydrochloride 20mg)
It is placed in 25mL volumetric flask, solubilizer is diluted to scale, shakes up, as not destroying solution；
High temperature solution: comparative example 4 takes drotaverine hydrochloride injection 1mL (to be approximately equivalent to drotaverine hydrochloride
It 20mg) is placed in 25mL volumetric flask, 100 DEG C of heating 8h are cooled to room temperature, solvent is diluted to scale, shakes up, as high temperature
Solution.
Comparative example 5, illumination destroy solution: drotaverine hydrochloride injection 1mL being taken (to be approximately equivalent to drotaverine hydrochloride
20mg), it is placed in 25mL volumetric flask, illumination 15h under ultraviolet lamp, solvent is diluted to scale, destroys solution as illumination.
Comparative example 6, alkali destroy solution: drotaverine hydrochloride injection 1mL (being equivalent to drotaverine hydrochloride about 20mg) is taken,
It is placed in 25mL volumetric flask, adds 1mol/L sodium hydroxide 1mL, after 60 DEG C of destruction 1.5h, add 1mol/L hydrochloric acid 1mL to neutralize, solvent
It is diluted to scale, is shaken up, destroys solution as alkali.
Oxidative demage solution: comparative example 7 takes drotaverine hydrochloride injection 1mL (to be approximately equivalent to drotaverine hydrochloride
20mg), it being placed in 25mL volumetric flask, adds 30% hydrogen peroxide 2mL, 60 DEG C of destruction 8h, solvent is diluted to scale, shakes up, as
Oxidative demage solution.
Conclusion:
Comparative example 3-7 result is shown in Fig. 6 A, 7A, 8A, 9A, shown in 10A: under each degradation condition, removing known impurities Harverine
Outside alkali and Drotaverine ketone, other degradation impurities are before main peak appearance time (RT=6.1), and in retention time about 3~5
There is overlapping between minute, separating degree is poor, influences testing result.
Embodiment 4-8 result is shown in Fig. 6 B, 7B, 8B, 9B, shown in 10B: (1) test sample can be learnt in various failure conditions
Under catabolite retention time be all larger than 3.0 minutes, each catabolite that destroys can separate very well with solvent peak and main peak, molten
Related substance-measuring is not interfered in agent；Each impurity peaks separating degree is all larger than 3.0；
(2) drotaverine hydrochloride injection compares through destructive testing with not destroying, ethaquin: high temperature, alkali and
Changes of contents is larger under the conditions of Oxidative demage, and illumination destruction has no significant change；Drotaverine ketone: it is bright that alkali destroys changes of contents
Aobvious, high temperature and illumination destruction changes of contents are larger, and Oxidative demage has no significant change；Unknown impuritie: have in various degree
Degradation generates, and illumination, oxidation are more obvious；
(3) in addition to high temperature solution, solution impurity number is than State Bureau standard before other destroy solution and destroy
(YBH17602006) method checks that impurity number is more, and Drotaverine ketone content, largest single impurity content and total impurities are above country
Office's standard inspection method result.
In conclusion Related substances separation method of the present invention measures solution and destruction solution impurity number before destruction, bends his and tie up
Woods ketone content, other largest single impurity contents and total impurities are above State Bureau's standard (YBH17602006) inspection method result.
Therefore, the specificity of Related substances separation method and separation efficiency are better than existing standard inspection method after modification.
The following are drotaverine hydrochloride injection Related substances separation method validations of the present invention:
Embodiment 9
Detection limit and quantitative limit
It takes drotaverine hydrochloride under specificity item (concentration: 4 μ g/mL), ethaquin (concentration: 1.6 μ g/mL), bend him
The single positioning solution for tieing up woods ketone (concentration: 4 μ g/mL) surveys its quantitative limit (S/N >=10), detection limit (S/N >=3) with dilution method,
It the results are shown in Table 4
The detection of table 4 limit and quantitative limit
Conclusion: under this chromatographic condition, the quantitative limit of drotaverine hydrochloride, ethaquin and Drotaverine ketone and detection are limited
Meet and receives limit.
Embodiment 10
Standard curve and the range of linearity
Precision weighs drotaverine hydrochloride, each 10mg of Drotaverine ketone standard items, is respectively placed in 100mL volumetric flask, adds
1 dissolution solvent 2 of solvent is diluted to scale, shakes up, as drotaverine hydrochloride, Drotaverine ketone stock solution；Precision weighs second
Base papaverine standard items 20mg, sets in 50mL volumetric flask, and 1 dissolution solvent 2 of solubilizer is diluted to scale, shakes up, and precision measures
Above-mentioned solution 5mL is set in 50mL volumetric flask, and solubilizer 2 is diluted to scale, is shaken up, as ethaquin stock solution.Precision amount
Take appropriate hydrochloric acid Drotaverine, ethaquin, Drotaverine ketone stock solution, solubilizer 2, which is made, hydrochloric in every 1mL bend his and tie up
0.16,1.0,2.0,4.0,5.0,6.0 μ g of woods；0.048,0.8,1.2,1.6,2.0,2.4 μ g of ethaquin；Drotaverine ketone
0.32,6 parts of solution of 1.0,2.0,4.0,5.0,6.0 μ g.Precision measures above-mentioned each 20 μ L of solution and is injected separately into high-efficient liquid phase color
Spectrometer records chromatogram, measures peak area, is that abscissa makees linear regression using peak area A as ordinate, concentration C.It the results are shown in Table
5,
5 drotaverine hydrochloride of table and the HPLC of known impurities are linear
| Title | Linear equation | Related coefficient | Range of linearity μ g/mL |
| Drotaverine hydrochloride | A=29.120C+0.5168 | R2=0.9993 | 0.16~6.0 |
| Ethaquin | A=107.38C+0.8246 | R2=0.9999 | 0.048~2.4 |
| Drotaverine ketone | A=23.756C+0.0453 | R2=0.9996 | 0.32~6.0 |
Conclusion: the result shows that, drotaverine hydrochloride is in 0.16~6.0 μ g/mL, ethaquin in 0.048~2.4 μ g/
Within the scope of 0.32~6.0 μ g/mL, peak area and measurement concentration are in good linear relationship for mL, Drotaverine ketone.
Embodiment 11
Test solution stability test
Take drotaverine hydrochloride injection according to Related substances separation method, prepare test solution, respectively at 0,2,4,6,
8, the variation of ethaquin, Drotaverine ketone peak area, other largest single impurities and total impurities is investigated in 10,12h sample introduction.Knot
Fruit is shown in Table 6,
6 test solution stability test result of table
Conclusion: in 12h, Harverine alkali content RSD is 0.89%, and Drotaverine ketone content RSD is 5.43%, other are most
Big single miscellaneous content RSD is 1.78%, and total miscellaneous content RSD is 2.51%.It can be seen that test solution from the above determination data to exist
Each impurity and total miscellaneous no significant change, have good stability in 12h.
Embodiment 12
Repetitive test
Drotaverine hydrochloride injection is taken, according to Related substances separation method, prepares 6 parts of test solutions in parallel, every part each
Measurement 1 time, calculates each impurity content.It the results are shown in Table 7,
7 repetitive test result of table
Conclusion: 6 parts of test solution measurement results meet the requirements, and repeatability is good.
Embodiment 13
Accuracy test
Drotaverine hydrochloride injection 1mL is taken respectively, is set in 25mL volumetric flask, and totally 9 parts, every 3 parts are one group, accurate respectively
Ethaquin, Drotaverine each 0.5mL of ketone impurity stock solution (limit 50%), 1.0mL (limit 100%), 1.5mL is added
(limit 150%), solubilizer 2 are diluted to scale, as basic, normal, high three groups of concentration test solutions, measure, calculate back in accordance with the law
Yield and RSD.It the results are shown in Table 8 and table 9,
8 ethaquin accuracy test result of table
9 Drotaverine ketone accuracy test result of table
Embodiment 14
Serviceability test
In relation to substance durability mainly from mobile phase difference phosphoric acid concentration, difference pH, chromatographic column different in flow rate and different etc.
Several aspect verifyings.It the results are shown in Table 10,
10 serviceability test result of table
Serviceability test conditions correlation map is shown in Figure 11-14,
Conclusion: to sum up serviceability test the result shows that, this method chromatographic condition parameter has drotaverine hydrochloride injection
Material impurities separation is closed to have not significant impact with inspection.
Embodiment 15
Related substances separation
Each three batches of the forward and backward sample of sterilization process of change and original is taken to grind product respectively, according to national standards and the method for the present invention
Related substances separation is carried out, and carries out comparative study, the impurity variation of sample before and after sterilization process, Xin Laofang are changed in comprehensive assessment
The detection performance of method.It the results are shown in Table 11,
11 sterilization process of table before changing after drotaverine hydrochloride injection grind the related material impurities of product with original compared with
Conclusion:
1, rear each three batches of samples and original grind the related substance comparison of product to sterilization process before changing:
(1) there is 1 impurity to grind in product in original in the drotaverine hydrochloride injection after sterilization process change not occur, contain
Amount is 0.02% or so；Original, which is ground in product, has 3 impurity not occur in self-control sample；
(2) dopant species and impurity number are less than original and grind production in the drotaverine hydrochloride injection after sterilization process change
Product, make sample by oneself and original is ground in product containing ethaquin and Drotaverine ketone, make ethaquin in sample by oneself and bend
Ta Weilin ketone content is not higher than original and grinds product, makes other largest single impurity contents and total impurities in sample by oneself and is not higher than original and grinds
Product, identical unknown impuritie are respectively less than original and grind product.Other maximum simple substance are respectively less than 0.1%.Self-control quality is ground not less than original
Product.
2, three batches of samples and three batches of sample comparisons before changing after sterilization process change, dopant species are consistent with number, second
Base papaverine and Drotaverine ketone content are without significant change, other largest single impurities and total impurities are without significant change.
The method of the present invention and the comparison of State Bureau's standard (YBH17602006) method are as shown in table 12 below:
12 the method for the present invention of table and State Bureau's Comparison of standards result
Using HPLC analytical method of the bulk pharmaceutical chemicals drotaverine hydrochloride in relation to substance of the invention, gradient is washed
It is de-, the impurity of drotaverine hydrochloride injection is sufficiently disclosed, the safety of product, methodological science, reliable, operation letter are improved
Just, controllably, it is suitable for practical application and popularization.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (9)
1. a kind of method in relation to substance in high effective liquid chromatography for measuring drotaverine hydrochloride injection, which is characterized in that
This method comprises the following steps:
(1) test sample solution is prepared:
Drotaverine hydrochloride injection is taken to be configured to test sample solution with solvent；
(2) reference substance solution is prepared:
Drotaverine hydrochloride, ethaquin and Drotaverine ketone is taken to be configured to reference substance solution with solvent；
(3) compounding system applicability solution:
Drotaverine hydrochloride, ethaquin and Drotaverine ketone is taken to be configured to system suitability solution with solvent；
(4) system suitability experiment is carried out；
(5) detection sensitivity experiment is carried out；
The detection sensitivity that high performance liquid chromatograph is adjusted with reference substance solution, makes the peak height full scale of principal component chromatographic peak
10-25%；
(6) it is measured according to following high-efficient liquid phase chromatogram condition:
Mobile phase: using phosphate buffer as mobile phase A, using acetonitrile as Mobile phase B, using methanol as mobile phase C；The phosphoric acid
Salt buffer is adjusted to pH=3.3 ± 0.05 with triethylamine for 0.1% phosphate aqueous solution；
UV detector: Detection wavelength 200-400nm；
Chromatographic column: using octadecylsilane chemically bonded silica as filler；
Flow velocity: 0.5-2.0mL/min；
Column temperature: 20-60 DEG C；
(7) external standard method is pressed, the content in relation to substance is calculated；
Gradient elution is carried out with mobile phase A, Mobile phase B and mobile phase C；The gradient are as follows:
2. the method according to claim 1, wherein the Detection wavelength of the UV detector is 200-
300nm。
3. the method according to claim 1, wherein the Detection wavelength of the UV detector is 244nm.
4. the method according to claim 1, wherein the flow velocity is 1.0mL/min；The column temperature is 30-50
℃。
5. according to the method described in claim 4, it is characterized in that, the column temperature is 40 DEG C.
6. the method according to claim 1, wherein the preparation of the test sample solution specifically includes preparation and contains
The solution of drotaverine hydrochloride 0.8mg/mL, the solvent of the solution are the mixed solvent that mobile phase A, methanol and acetonitrile are formed,
The mixed solvent is that 1:9 is mixed the mixed mixed liquor of 2:1 by volume with mobile phase A again by volume by methanol and acetonitrile
It closes.
7. the method according to claim 1, wherein the preparation of the reference substance solution specifically includes preparation saliferous
The solution of sour Drotaverine, ethaquin and Drotaverine ketone, the concentration of drotaverine hydrochloride is 4.0 μ g/ in the solution
mL；The concentration of ethaquin is 1.6 μ g/mL；The concentration of Drotaverine ketone is 4.0 μ g/mL；By drotaverine hydrochloride, ethyl
Papaverine and Drotaverine ketone are first dissolved with the mixed liquor that methanol and acetonitrile volume ratio are 2:1, and mobile phase A, methanol is then added
The mixed solvent formed with acetonitrile, the mixed solvent be by methanol and acetonitrile by volume the mixed mixed liquor of 2:1 again with
1:9 is mixed mobile phase A by volume.
8. the method according to claim 1, wherein the preparation of the system suitability solution specifically includes preparation
The solution of hydrochloric Drotaverine, ethaquin and Drotaverine ketone, the concentration of drotaverine hydrochloride is in the solution
0.8mg/mL；The concentration of ethaquin is 1.6 μ g/mL；The concentration of Drotaverine ketone is 4.0 μ g/mL；Hydrochloric acid is bent him to tie up
Woods, ethaquin and Drotaverine ketone are first dissolved with the mixed liquor that methanol and acetonitrile volume ratio are 2:1, and mobile phase is then added
A, the mixed solvent that methanol and acetonitrile are formed, the mixed solvent are by methanol and the acetonitrile mixed mixing of 2:1 by volume
1:9 is mixed liquid by volume with mobile phase A again.
9. taking system suitability molten the method according to claim 1, wherein the system suitability refers to
The separating degree of 20 μ L of liquid injection liquid chromatograph, drotaverine hydrochloride and ethaquin is greater than 3.0, separates between each impurity peaks
Degree meets the requirements, and theoretical cam curve is calculated by drotaverine hydrochloride, is not less than 3000.
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| CN111796043B (en) * | 2020-08-15 | 2022-02-22 | 山东北大高科华泰制药有限公司 | Papaverine hydrochloride powder injection for injection and quality detection method thereof |
| CN114113375B (en) * | 2021-11-10 | 2023-06-27 | 湖北大学 | Method for detecting content of drotaverine hydrochloride bulk drug by HPLC |
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