阅读说明：本技术 利用hplc检测硫酸氢氯吡格雷片溶出度的方法 (Utilize the method for HPLC detection clopidogrel bisulfate tablet dissolution rate ) 是由 沙薇 张兴爽 马宗普 朱盼盼 于 2019-06-28 设计创作，主要内容包括：本发明提供一种利用HPLC检测硫酸氢氯吡格雷片溶出度的方法,包括以下步骤：标准溶液的配制、标准曲线的绘制、硫酸氢氯吡格雷片供试品溶液的制备、供试品溶液的测定,通过峰面积及标准曲线计算出供试品溶液中硫酸氢氯吡格雷的浓度,然后计算出硫酸氢氯吡格雷片的溶出度；其中,所用的溶出介质均为含0.1-0.3％吐温80的pH4.5醋酸缓冲液。本发明的方法可以克服空白介质的吸光度不稳定的问题,而且该方法的溶出介质含吐温80,在避免使用有机溶剂的同时增加硫酸氢氯吡格雷的溶解度,同时经过对该方法的一系列验证,其专属性强、结果准确、稳定性好,可以准确地测定硫酸氢氯吡格雷片在pH4.5溶出介质中的溶出度。(The present invention provides a kind of method using HPLC detection clopidogrel bisulfate tablet dissolution rate, the following steps are included: the preparation of standard solution, the drafting of standard curve, the preparation of clopidogrel bisulfate tablet test solution, the measurement of test solution, the concentration of bisulfate clopidogrel in test solution is calculated by peak area and standard curve, then calculates the dissolution rate of clopidogrel bisulfate tablet；Wherein, dissolution medium used is the pH4.5 acetate buffer solution of the Tween 80 containing 0.1-0.3%.Method of the invention can overcome the problems, such as that the absorbance of blank medium is unstable, and the dissolution medium tween 80 of this method, increase the solubility of bisulfate clopidogrel while avoiding using organic solvent, simultaneously by a series of verifyings to this method, its specificity is strong, result is accurate, stability is good, can accurately measure dissolution rate of the clopidogrel bisulfate tablet in pH4.5 dissolution medium.)

1. a kind of method using HPLC detection clopidogrel bisulfate tablet dissolution rate, which comprises the following steps:

(1) it the preparation of standard solution: after taking bisulfate clopidogrel standard items that methanol is added to dissolve, then is successively diluted with dissolution medium And it is configured to a series of standard solution of concentration gradients respectively；

(2) drafting of standard curve: carrying out HPLC detection for step (1) prepared standard solution respectively, set testing conditions, Standard curve is established respectively according to the corresponding relationship of peak area and concentration of standard solution；

(3) preparation of clopidogrel bisulfate tablet test solution: according to 2015 editions 4 general rules, 0,931 second methods of Chinese Pharmacopoeia according to Method operation, takes clopidogrel bisulfate tablet to be placed in dissolution medium, and revolving speed is 50 turns per minute, selects in the time of 5-60min Selecting each time point takes liquid and is filtered with 0.45 μm of miillpore filter, as test solution；

(4) test solution prepared by step (3) the measurement of test solution: is subjected to HPLC detection, setting detection respectively Condition, then the concentration of bisulfate clopidogrel in test solution is calculated by peak area and standard curve respectively, then count Calculate the dissolution rate of clopidogrel bisulfate tablet；

Wherein, dissolution medium used in step (1) and step (3) is the pH4.5 acetate buffer of the Tween 80 containing 0.1-0.3% Liquid.

2. utilizing the method for HPLC detection clopidogrel bisulfate tablet dissolution rate according to claim 1, which is characterized in that step Suddenly the HPLC testing conditions of (2) and step (4) are as follows: chromatographic column is ovomucin bonded silica gel chiral chromatographic column；Mobile phase body The mixed solution of long-pending 0.01mol/L potassium dihydrogen phosphate and acetonitrile than being 75-82:25-18,20-30 DEG C of column temperature；Detect wave Long 240nm；Flow velocity 0.8-1.0mL/min.

3. utilizing the method for HPLC detection clopidogrel bisulfate tablet dissolution rate according to claim 2, which is characterized in that institute The volume ratio for stating 0.01mol/L potassium dihydrogen phosphate and acetonitrile is 80:20.

4. utilizing the method for HPLC detection clopidogrel bisulfate tablet dissolution rate according to claim 2, which is characterized in that institute Stating column temperature is 25 DEG C.

5. utilizing the method for HPLC detection clopidogrel bisulfate tablet dissolution rate according to claim 2, which is characterized in that institute Stating flow velocity is 1.0mL/min.

6. utilizing the method for HPLC detection clopidogrel bisulfate tablet dissolution rate according to claim 1, which is characterized in that institute The weight ratio for stating pH4.5 acetate buffer solution tween 80 is 0.2%.

Technical field

The present invention relates to clopidogrel bisulfate tablet dissolution determination technical fields, and in particular to detects sulfuric acid using HPLC The method of clopidogrel hydrogen tablet dissolution.

Background technique

Clopidogrel bisulfate tablet is by a kind of platelet aggregation inhibitor of match Norfin, Inc, France exploitation, in 1997 Ratify to list through FDA for the first time.The main component of clopidogrel bisulfate tablet is bisulfate clopidogrel, can selectively be inhibited The combination of ADP and platelet receptor then inhibit activation ADP and II b/ of glycoprotein GP, III a compound, to inhibit blood platelet Aggregation, is mainly used for the circulation disorder of the heart, brain and other arteries caused by preventing and treating because of the high coherent condition of blood platelet. Determined by ultraviolet spectrophotometry is used in Chinese Pharmacopoeia 2015 editions, but during doing Conformance Assessment, according to Chinese Pharmacopoeia Method configuration pH4.5 dissolution medium, using ultraviolet spectrophotometry in pharmacopeia 240nm condition measure when, blank medium Absorbance it is unstable always, lead to not the dissolution rate of each sample point of Accurate Determining.

Summary of the invention

Technology existing for the ultraviolet spectrophotometry clopidogrel bisulfate tablet dissolution rate provided for Chinese Pharmacopoeia is asked Topic, the present invention provide a kind of method using HPLC detection clopidogrel bisulfate tablet dissolution rate, and this method can overcome blank The unstable problem of the absorbance of medium, and the dissolution medium tween 80 of this method, are avoiding using the same of organic solvent The solubility of Shi Zengjia bisulfate clopidogrel, while by a series of verifyings to this method, specificity is strong, result is quasi- Really, stability is good, can accurately measure dissolution rate of the clopidogrel bisulfate tablet in pH4.5 dissolution medium.

Technical solution provided by the present invention is specific as follows:

A method of clopidogrel bisulfate tablet dissolution rate is detected using HPLC, comprising the following steps:

(1) it the preparation of standard solution: after taking bisulfate clopidogrel standard items that methanol is added to dissolve, then is successively diluted with dissolution medium And it is configured to a series of standard solution of concentration gradients respectively；

(2) drafting of standard curve: carrying out HPLC detection for step (1) prepared standard solution respectively, set testing conditions, Standard curve is established respectively according to the corresponding relationship of peak area and concentration of standard solution；

(3) preparation of clopidogrel bisulfate tablet test solution: according to 2015 editions 4 general rules, 0,931 second methods of Chinese Pharmacopoeia according to Method operation, takes clopidogrel bisulfate tablet to be placed in dissolution medium, and revolving speed is 50 turns per minute, respectively at the time of 5-60min Interior selection each time point takes liquid and is filtered with 0.45 μm of miillpore filter, as test solution；

(4) test solution prepared by step (3) the measurement of test solution: is subjected to HPLC detection, setting detection respectively Condition, then the concentration of bisulfate clopidogrel in test solution is calculated by peak area and standard curve respectively, then count Calculate the dissolution rate of clopidogrel bisulfate tablet；

Wherein, dissolution medium used in step (1) and step (3) is the pH4.5 acetate buffer of the Tween 80 containing 0.1-0.3% Liquid.

Preferably, the HPLC testing conditions of step (2) and step (4) are as follows: chromatographic column is ovomucin bonded silica gel hand Property chromatographic column；Mobile phase is that the mixing of 0.01mol/L potassium dihydrogen phosphate and acetonitrile that volume ratio is 75-82:25-18 is molten Liquid, 20-30 DEG C of column temperature；Detection wavelength 240nm；Flow velocity 0.8-1.0mL/min.

Preferably, the volume ratio of the 0.01mol/L potassium dihydrogen phosphate and acetonitrile is 80:20.

Preferably, the column temperature is 25 DEG C.

Preferably, the flow velocity is 1.0mL/min.

Preferably, the weight ratio of the pH4.5 acetate buffer solution tween 80 is 0.2%.

For the ultraviolet spectrophotometry that Chinese Pharmacopoeia provides when 240nm condition measures, the absorbance of blank medium is always not Stablize, lead to not the dissolution rate of each sample point of Accurate Determining bisulfate clopidogrel, the present invention looks for another way, selection The method of HPLC, in the method, used dissolution medium tween 80 improve the solubility of bisulfate clopidogrel, Can true, Accurate Determining clopidogrel bisulfate tablet in the dissolution rate of pH4.5 dissolution medium, more effectively drug quality is carried out Comparison control, and bisulfate clopidogrel measuring method specificity provided by the present invention is good, linear, precision, stabilization Property test etc. meet analysis require.

Detailed description of the invention

Fig. 1 is that bisulfate clopidogrel blank, standard items and test solution are superimposed chromatogram；

Fig. 2 is the chromatogram of bisulfate clopidogrel standard solution；

Fig. 3 is the chromatogram of clopidogrel bisulfate tablet test solution；

Fig. 4 is the dissolution curve of clopidogrel bisulfate tablet.

Specific embodiment

Technical solution of the present invention is further described combined with specific embodiments below, while to method of the invention A series of verifyings are done.

1 instrument and reagent

Thermo U3000 type high performance liquid chromatograph；

Agilent digestion instrument (Agilent)；

Clopidogrel bisulfate tablet (specification 75mg/ piece, lot number 20170923)；

Bisulfate clopidogrel standard items (are purchased from Zhong Jian institute, content 99.5%)；

Methanol, acetonitrile are chromatographically pure (Merk).

The dissolution rate of 2 HPLC methods measurement clopidogrel bisulfate tablet

The detection chromatographic condition of dissolution determination: chromatographic column is ovomucin bonded silica gel chiral chromatographic column ULTRONES-OVM； Mobile phase be 0.01mol/L potassium dihydrogen phosphate: acetonitrile (80:20), 25 DEG C of column temperature；Detection wavelength 240nm；Flow velocity 1.0mL/ min；

The step of dissolution determination, is as follows, and below with w/v containing 0.2%(, unit is g/mL) pH4.5 of Tween 80 Acetate buffer solution 1000mL is dissolution medium:

(1) bisulfate clopidogrel standard items 16.3mg is taken, it is accurately weighed, it is placed in 50mL measuring bottle, adds methanol dissolution and constant volume, It shaking up, precision measures solution 10mL and is placed in 50mL measuring bottle, and solubilization goes out medium to scale, it shakes up, as stock solution, according to Secondary dilution is configured to every 1mL containing 5.0 μ g/mL of clopidogrel, 12.5 μ g/mL, 20 μ g/mL, 25 μ g/mL, 30 μ g/mL respectively Series of standards solution；Precision measures each 10 μ l of above-mentioned standard solution and injects liquid chromatograph, records chromatogram, one of color Spectrogram is as shown in Figure 2；

The result shows that clopidogrel is when concentration is the 5.0 μ g/mL of μ g/mL ~ 30, concentration is linear with peak area, regression equation It is 1.0000 for y=20.56x+0.0042, linearly dependent coefficient r, linear relationship is good；

(2) clopidogrel bisulfate tablet test sample 12 are taken, according to 2015 editions 4 general rules, 0,931 second methods of Chinese Pharmacopoeia, to contain 0.2%(w/v, unit are g/mL) the pH4.5 acetate buffer solution 1000mL of Tween 80 is dissolution medium, revolving speed is every It 50 turns of minute, operates according to methods, takes solution (fluid infusion mould respectively at 5min, 12min, 15min, 20min, 30min, 45min, 60min Formula) 10mL, it is filtered with 0.45 μm of miillpore filter, as test solution；

(3) take bisulfate clopidogrel standard items appropriate, it is accurately weighed, add dissolution medium to dissolve and quantify dilution every 1mL is made In contain 25 μ g bisulfate clopidogrels solution, as standard solution.Take standard solution and test solution with colouring Analysis is measured under spectral condition, precision measures test solution, each 10 μ l of standard solution, is injected separately into liquid chromatograph, Test solution chromatogram is recorded, one of chromatogram is as shown in Figure 3.The accumulative the amount of dissolution of each point, knot are calculated by external standard method Fruit sees Fig. 4 and table 1.

The dissolution determination result of 1 12 clopidogrel bisulfate tablet test samples of table

3 method validations

3.1 chromatographic condition

Chromatographic column is ovomucin bonded silica gel chiral chromatographic column ULTRONES-OVM；Mobile phase is that volume ratio is 80:20's The mixed solution of 0.01mol/L potassium dihydrogen phosphate and acetonitrile, 25 DEG C of column temperature；Detection wavelength 220nm；Flow velocity 1.0mL/min.

The test of 3.2 interference

Clopidogrel bisulfate tablet sample and blank each 1 are taken, is respectively placed in stripping rotor, according to above-mentioned dissolution determination Method operation separately takes bisulfate clopidogrel standard items to add flowing phased soln and the standard solution of 0.025mg/mL is made.It presses " 3.1 chromatographic condition " difference 10 μ L of sample introduction, the results showed that at bisulfate clopidogrel appearance, blank solution is without any chromatographic peak Occur, shows that blank auxiliary and dissolution medium are noiseless to the measurement of standard solution and test solution, the result is shown in Figure 1.

3.3 linear test

Bisulfate clopidogrel standard items 16.3mg is taken, it is accurately weighed, it is placed in 50mL measuring bottle, adds methanol dissolution and constant volume, shake Even, precision measures solution 10mL and is placed in 50mL measuring bottle, then solubilization goes out medium to scale, shakes up, as stock solution, according to Secondary dilution is configured to every 5.0 μ g/mL of 1mL containing clopidogrel hydrogen sulfate, 12.5 μ g/mL, 20 μ g/mL, 25 μ g/mL, 30 μ respectively The series of standards solution of g/mL.Precision measures each 10 μ l of above-mentioned standard solution and injects liquid chromatograph, records chromatogram.As a result Show bisulfate clopidogrel when concentration is the 5.0 μ g/mL of μ g/mL ~ 30, concentration is in a linear relationship with peak area, recurrence side Journey is y=20.56x+0.0042, and linearly dependent coefficient r is 1.0000, and linear relationship is good.

3.4 precision test

Taking concentration is the standard solution of 0.025mg/mL, is repeated sample introduction 6 times, RSD 0.38% meets precision requirement.

3.5 stability test

The test solution under " 3.2 " is taken, places 0,2,4,6,8h difference sample introduction measurement peak areas, RSD=at room temperature 0.30%, show that test solution is basicly stable in 8h.

3.6 recovery test

Bisulfate clopidogrel standard items 19.6,26.1,32.7mg are taken respectively, it is accurately weighed, it is placed in 100mL measuring bottle, respectively Blank auxiliary is added by recipe quantity, and goes out medium to scale with solubilization after proper amount of methanol ultrasonic dissolution, shakes up, filtration, essence Close measurement subsequent filtrate 1mL is placed in 10mL measuring bottle, then solubilization goes out medium to scale, shakes up, by chromatostrip under " 3.1 " item Part measurement calculates the rate of recovery by external standard method.The result shows that average recovery rate 99.4%, RSD=0.70% (n=9).

4 discuss

In view of bisulfate clopidogrel, solubility is very low in the medium of pH4.5, and dissolution medium of the invention contains 0.2% (w/v) Tween 80 increases the solubility of bisulfate clopidogrel while avoiding using organic solvent, it can be made to reach in 30min 80% or more, convenient for the comparison with former triturate；This method specificity is good, stablizes, can true, Accurate Determining hydrogen sulfate chlorine pyrrole lattice Dissolution rate of the thunder piece in each sample time of pH4.5 dissolution medium, it is bent convenient for drawing its dissolution in pH4.5 dissolution medium Line more effectively can compare research to drug quality.

It should be noted last that: technical solution of the present invention that the above embodiments are only illustrative and not limiting is any right The equivalent replacement and do not depart from the modification of spirit and scope of the invention or locally replace that the present invention carries out, should all cover in this hair Within bright protective scope of the claims.