CN115327005A

CN115327005A - Method for detecting related substances of clopidogrel oxide

Info

Publication number: CN115327005A
Application number: CN202210964672.9A
Authority: CN
Inventors: 陆瑶; 张海; 徐霞
Original assignee: Chengdu Shibeikang Biological Medicine Technology Co ltd
Current assignee: Chengdu Shibeikang Biological Medicine Technology Co ltd
Priority date: 2022-08-12
Filing date: 2022-08-12
Publication date: 2022-11-11
Anticipated expiration: 2042-08-12
Also published as: CN115327005B

Abstract

The invention relates to the field of drug detection and analysis, and particularly relates to a method for detecting (7aS, 2' S) -2-O-clopidogrel related substances. The method for detecting the related substances of (7aS, 2') S-2-oxo-clopidogrel adopts high performance liquid chromatography, uses octadecylsilane chemically bonded silica as a filler, has the detection wavelength of 220 +/-2 nm, uses a salt solution with an acidic pH value and containing an acid ion pair as a mobile phase A, and uses a solution containing acetonitrile and/or alcohol as a mobile phase B, and performs gradient elution. The detection method provided by the invention has good specificity and high sensitivity and accuracy, and is suitable for drug registration quality research of (7aS,2') S) -2-oxo-clopidogrel.

Description

Method for detecting related substances of clopidogrel oxide

Technical Field

The invention relates to the field of drug detection and analysis, and particularly relates to a method for detecting (7 aS,2' S) -2-oxo-clopidogrel related substances.

Background

(7 aS,2' S) -2-O-clopidogrel (hereinafter, referred to aS "compound A"), which is a metabolite of clopidogrel in humans, is a more active and safer platelet aggregation inhibitor, and has the chemical name: (S) -methyl 2- (2-chlorophenyl) -2- ((S) -2-oxo-2,6,7,7a-tetrahydrothiophen [3,2-c ] pyridopyridin-5 (4H) yl) acetate:

。

at present, the preparation process of the compound A is mainly a route reported in patent CN 104245826A, R- (-) -o-chloromandelic acid (SM 1) is used as a starting material, an intermediate I is generated after methyl esterification, the intermediate I is condensed with p-nitrobenzenesulfonyl chloride (SM 2) to generate a key intermediate II, and then the key intermediate II is generated through substitution and purification. The general reaction scheme is as follows:

。

according to the molecular characteristics and the preparation process of the compound A, related substances are mainly degradation impurities, isomers and process impurities. Wherein the degradation impurities are hydroxy impurities (P1Z 5), enol impurities (P1Z 12), main isomers such as P1Z2, and key process impurities, namely intermediate II (P1Z 1), and the structural details of each impurity are shown in a table 1.

。

The research on substances is crucial to the quality control of medicines, but the prior art has no complete information on the quality control of the compound A, especially on the quality research of the substances. Although the specification of the prior art (CN 111943958A) discloses a method for detecting a compound of formula I, the method cannot meet the requirement of detecting the above impurities simultaneously, especially cannot meet the requirement of detecting process impurities. In order to better control the related substances of the compound A and meet the requirement of the medicine on the market, the development of a quality control method suitable for the related substances of the compound A is a technical problem to be solved urgently in the quality research of the medicine at present.

Disclosure of Invention

To meet the quality control requirement of (7 aS,2 'S) -2-oxy-clopidogrel, the invention provides a method for detecting a substance related to (7 aS,2' S) -2-oxy-clopidogrel.

According to the method for detecting the related substances of the (7 aS,2' S) -2-oxo-clopidogrel, the high performance liquid chromatography is adopted, so that most impurities including but not limited to impurities P1Z1, P1Z2, P1Z5 and P1Z12 can be detected.

The invention provides a method for detecting related substances of (7 aS,2' S) -2-oxo-clopidogrel, which adopts high performance liquid chromatography and comprises the following operation steps:

(1) Taking (7 aS,2' S) -2-oxy-clopidogrel, and diluting with a solvent to obtain a test solution;

(2) Injecting the sample solution into a high performance liquid chromatograph, and detecting by adopting chromatographic conditions.

In certain embodiments, the chromatographic conditions comprise: using octadecylsilane chemically bonded silica as a chromatographic column of a filler, detecting the wavelength of 220 +/-2 nm, using a salt solution with an acidic pH value and containing an acidic ion pair as a mobile phase A, and using a solution containing acetonitrile and/or alcohol as a mobile phase B, and performing gradient elution.

In certain embodiments, the gradient elution described above comprises at least two steps of: the proportion of the mobile phase A in the first stage procedure is 35-60%, and the proportion of the mobile phase B is 65-40%; in the second stage, the proportion of the mobile phase A is 5-20%, and the proportion of the mobile phase B is 95-80%; and the total time of the first stage procedure is 10-50 minutes, and the total time of the second stage procedure is 10-50 minutes.

Further, the total time of the second stage process includes a proportional falling time of the mobile phase a, a proportional rising time of the mobile phase B, and a maintaining time, wherein the proportional falling time of the mobile phase a is equal to the proportional rising time of the mobile phase B and is 5 to 30 minutes, and the maintaining time is 5 to 30 minutes.

In certain embodiments, the above detection method comprises a system suitability test: respectively weighing an impurity reference substance and a proper amount of (7 aS,2 'S) -2-oxy-clopidogrel, and dissolving and diluting the impurity reference substance and the (7 aS,2' S) -2-oxy-clopidogrel to a mixed solution with a target concentration by a solvent in a one-time preparation mode or a mode of firstly preparing in a concentrated mode and then preparing in a diluted mode.

Further, the system applicability test adopts a first concentrated preparation and then diluted preparation mode: taking a proper amount of impurity reference substances, dissolving the reference substances by using a solvent and quantitatively diluting to prepare mixed solutions containing 5-250 ug of each 1ml of the reference substances, taking a proper amount of (7 aS,2 'S) -2-oxygen-clopidogrel aS impurity stock solutions, putting the mixed solutions into a measuring flask, adding a proper amount of the impurity stock solutions, dissolving the mixed solutions by using the solvent and quantitatively diluting to prepare 0.5-10 ug of each impurity and 0.5-10 mg of (7 aS,2' S) -2-oxygen-clopidogrel in each 1ml of the reference substances.

In certain embodiments, the gradient elution procedure of chromatographic conditions described above comprises: the proportion of the mobile phase A is 35 to 60 percent and the proportion of the mobile phase B is 65 to 40 percent at 0 minute, and the mobile phase A and the mobile phase B are maintained for 20 to 40 minutes; when the time is 30-60 minutes, the proportion of the mobile phase A is reduced to 5% -20%, the proportion of the mobile phase B is increased to 95% -80%, and the time is maintained for 50-70 minutes.

In certain embodiments, the gradient elution procedure of chromatographic conditions described above comprises: the proportion of the mobile phase A is 35 to 60 percent and the proportion of the mobile phase B is 65 to 40 percent at 0 minute, and the mobile phase A and the mobile phase B are maintained for 25 to 35 minutes; the proportion of the mobile phase A is reduced to 5-20% in 40-60 minutes, the proportion of the mobile phase B is increased to 95-80% in 95-65 minutes, and the time is maintained for 55-65 minutes.

Further, the gradient elution procedure of the chromatographic conditions described above comprises: the proportion of the mobile phase A is 35 to 60 percent and the proportion of the mobile phase B is 65 to 40 percent at 0 minute, and the mobile phase A and the mobile phase B are maintained for 30 minutes; the proportion of the mobile phase A is reduced to 5-20% in 50 minutes, the proportion of the mobile phase B is increased to 95-80%, and the mobile phase A is maintained for 60 minutes.

Further, the gradient elution procedure of the chromatographic conditions described above comprises: the proportion of mobile phase a at 0 min was 46% and the proportion of mobile phase B was 54%, and was maintained for 30 min; at 50 minutes the proportion of mobile phase a dropped to 10% and mobile phase B rose to 90% and was maintained for 60 minutes.

In certain embodiments, the gradient elution procedure described above further comprises a system equilibration procedure comprising a recovery time and a maintenance time.

Further, the recovery time is 0.1 to 20 minutes, preferably 0.1 to 5 minutes; the holding time is 0.1 to 20 minutes, preferably 10 minutes.

Further, the gradient elution procedure further comprises a system equilibrium procedure: the proportion of the mobile phase A is increased to 35 to 60 percent and the proportion of the mobile phase B is decreased to 65 to 40 percent at 50.1 to 71 minutes, and the mixture is maintained for 5 to 20 minutes.

Further, the gradient elution procedure described above also includes a system equilibration procedure, illustratively with a 35% to 60% increase in mobile phase a proportion and a 65% to 40% decrease in mobile phase B proportion at 61 minutes, and maintained for up to 70 minutes.

Still further, the system balance procedure described above, for example 61 minutes with the proportion of mobile phase a rising to 46% and the proportion of mobile phase B falling to 54%, was maintained for up to 70 minutes.

In some embodiments, the flow rate of the chromatographic conditions is from 0.5 to 2.0ml/min; preferably 0.8 to 1.5ml/min.

In certain embodiments, the chromatographic conditions described above have a column temperature of 0 to 40 ℃; preferably 20 to 40 ℃.

In certain embodiments, the related substances comprise one or more of P1Z5, P1Z2, P1Z1, P1Z12.

In some embodiments, the solvent comprises acetonitrile, THF, alcohol or a mixed solvent of mobile phases B and a in a volume ratio of 40 to 100; acetonitrile is preferred.

Further, the solvent is a mixed solvent in which the volume ratio of mobile phases B and a is 50 to 95.

In certain embodiments, the acidic ion pair comprises sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, or sodium dodecane sulfonate.

Further, the concentration of the acid ion pair is 0.001 mol/L-2.0 mol/L; preferably 0.005mol/L to 0.5mol/L.

In certain embodiments, the salt of the above-described salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate, or potassium acetate.

Further, the concentration of the salt solution is 0.001 mol/L-2.0 mol/L; preferably 0.005mol/L to 0.5mol/L.

In some embodiments, the salt solution has a pH of 1.0 to 6.0; preferably 2.0 to 5.0.

Further, the pH value is adjusted by a pH adjusting agent, and the pH adjusting agent comprises phosphoric acid, hydrochloric acid, sulfuric acid, glacial acetic acid or formic acid.

In some embodiments, the mobile phase B is alcohol, acetonitrile or a mixed solution of alcohol and acetonitrile at a volume ratio of 80-100; the volume ratio of the alcohol to the acetonitrile is preferably 90 to 98.

Further, the above alcohol includes methanol, ethanol, propanol or isopropanol.

In some embodiments, the mobile phase a is a salt solution (pH 1.0-6.0) containing sodium octane sulfonate. The salt solution is as defined above. Further, the pH is preferably 2.0 to 5.0.

Further, the mobile phase A is a salt solution (pH value is 1.0-6.0) containing 0.001 mol/L-2.0 mol/L sodium octane sulfonate solution. The salt solution is as defined above.

Furthermore, the mobile phase A is 0.001 mol/L-2.0 mol/L salt solution (pH value is 1.0-6.0) containing 0.001 mol/L-2.0 mol/L sodium octane sulfonate solution. The salt solution is as defined above.

Furthermore, the mobile phase A is 0.001 mol/L-2.0 mol/L potassium dihydrogen phosphate solution (pH value is 1.0-6.0) containing 0.001 mol/L-2.0 mol/L sodium octane sulfonate solution.

In certain embodiments, the mobile phase B is acetonitrile.

In certain embodiments, the mobile phase B is an alcohol. The alcohol is as defined above.

In some embodiments, the mobile phase B is a mixed solution of acetonitrile and alcohol at a volume ratio of 80 to 100; the volume ratio of the alcohol to the acetonitrile is preferably 90 to 98. The alcohol is as defined above.

In certain embodiments, in the above system suitability test, peaks are generated in the following order: P1Z5, P1Z2, compound a, P1Z1, P1Z12.

In some embodiments, the chromatogram of the test solution exhibits peaks in the following order: P1Z5, P1Z2, compound a, P1Z1, P1Z12.

In some embodiments, the chromatogram of the test solution contains the relevant substance in a peak area calculated by an external standard method and/or a peak area calculated by a self-control method and/or a peak area normalization method.

In some embodiments, the above chromatogram of the test solution contains no more than 0.1% of impurities P1Z1 and P1Z5, if any; and/or impurity P1Z2 is not more than 1.0%; and/or impurity P1Z12 is not more than 0.1%; and/or no more than 0.1% of other individual unknown impurities; and/or total impurities of not more than 2.0%.

In some embodiments, the chromatogram of the sample solution contains, if any, no more than 0.1% of impurities P1Z1 and P1Z5, calculated as peak areas by external standard method; and/or impurity P1Z2 is not more than 1.0% calculated by peak area normalization method; and/or impurity P1Z2 is not more than 1.0% calculated by peak area according to self control method; and/or other single impurities are not more than 0.1% calculated by a peak area normalization method; and/or other individual impurities should not exceed 0.1% by peak area as per the control.

In some embodiments, the chromatogram of the sample solution contains, if any, no more than 0.1% of impurities P1Z1 and P1Z5, calculated as peak areas by external standard method; and/or impurity P1Z2 is not more than 1.0% calculated by peak area normalization method; and/or other individual impurities should not exceed 0.1% by peak area normalization.

In some embodiments, the chromatogram of the test solution contains no more than 2.0% of the peak area of each impurity, if any, as calculated by peak area normalization.

Advantageous effects: the method for detecting the (7 aS,2' S) -2-oxo-clopidogrel related substances has the advantages of good peak types of all peaks, high separation degree of all peaks larger than 1.5, good separation degree, good method specificity and high sensitivity and accuracy, meets the quality analysis and detection requirements, and is suitable for the quality research of related substances of the products.

Drawings

FIG. 1: system applicability solution chromatograms.

Detailed Description

The scheme of the present invention will be explained below with reference to experimental examples and examples. It will be appreciated by those skilled in the art that the following examples are illustrative only and should not be taken as limiting the scope of the invention. The specific techniques or conditions not specified in each case are performed according to the techniques or conditions described in the literature in the field or according to the product description. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products obtained commercially.

Compound a was soluble in acetonitrile, slightly soluble in methanol, and practically insoluble in water. The compound a working control (lot No. 201113-D, 99.6% pure) and the test sample (lot No. 39201001) used in the following experimental examples and examples were both from kyushekang biomedical science and technology ltd, and the P1Z5 control was purchased from TRC; the isomer impurity P1Z2 and the impurity P1Z12 cannot be synthesized due to instability, but can be detected in the main drug, and LC-MS detection is used for positioning.

Experimental example 1: methodological investigations of methods for detecting substances related to Compound A

Preparation of various reference substance solutions and test substance solutions

Test solution: an appropriate amount of a sample (Compound A, batch No. 39201001) was precisely weighed, dissolved in acetonitrile and quantitatively diluted to prepare a solution containing about 1mg per 1ml (fresh preparation for clinical use).

Impurity control solution: appropriate amounts of the P1Z5 control and the P1Z1 control were taken, dissolved in acetonitrile, and quantitatively diluted to give mixed solutions each containing about 1. Mu.g per 1 ml.

System applicability solution: respectively taking a proper amount of a P1Z5 reference substance and a proper amount of a P1Z1 reference substance, dissolving the reference substances with acetonitrile, quantitatively diluting to prepare mixed solutions containing 10ug of each 1ml of the reference substances, taking the mixed solutions as impurity stock solutions, taking about 10mg of a compound A, putting the compound A into a 10ml measuring flask, precisely adding 1ml of the impurity stock solutions, diluting the compound A to the scale with the acetonitrile, and shaking up.

Description of the drawings: the solution can also be prepared by dissolving with a mixed solvent comprising but not limited to THF, alcohol or mobile phase B and A in a volume ratio of 40 to 100.

Condition of capsule wall chromatography

According to the chemical structural formula of the compound A, the inventor first selects the chromatographic conditions disclosed in the specification test 1 of the patent CN111943958A, but when the impurity P1Z1 is added, the impurity P1Z1 is overlapped with the impurity P1Z12, and the detection method of related substances of the product is finally determined by searching, adding an ion pair reagent and matching with an optimization mode such as adjusting a gradient elution program and the like.

(1) Selection of mobile phase and gradient elution procedure

The mobile phase was screened as follows, with other conditions unchanged:

(a) Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution containing 0.01mol/L sodium octane sulfonate solution (pH value adjusted to 3.0 with phosphoric acid), mobile phase B: methanol;

(b) Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution containing 0.01mol/L sodium octane sulfonate solution (pH value adjusted to 3.0 with phosphoric acid), mobile phase B: methanol: acetonitrile (94;

(c) Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution containing 0.01mol/L sodium octane sulfonate solution (pH value adjusted to 3.0 with phosphoric acid), mobile phase B: and (3) acetonitrile.

Gradient elution procedure 1 is described in tables 1-1:

。

the gradient elution procedure is described in tables 1-2:

。

and (3) respectively adopting a chromatographic condition a and gradient elution procedures 1-2, and respectively adopting a chromatographic condition b and c and a gradient elution procedure 2, respectively carrying out sample injection detection on the system applicability solution, and collecting a chromatogram map.

The results show that: the chromatographic condition a and the gradient elution procedure 1 have good peak patterns and poor separation between impurities. Under the chromatographic condition a and the condition of the gradient elution program 2, the peak shape is good, the main peak and each impurity can be well separated, and the impurity peaks can also be well separated. The chromatographic conditions b and c are respectively the same as those of the gradient elution procedure 2, the peak patterns are good, the main peak and each impurity can be well separated, and the impurity peaks can also be well separated.

(2) Selection of pH of the aqueous phase of the Mobile phase

Under otherwise identical conditions, seven mobile phase a pH's were selected for the test, pH1 (1.0), pH2 (2.0), pH3 (3.0), pH4 (4.0), pH5 (5.0), pH6 (6.0), pH7 (7.0), and the pH adjusting agent was any one of phosphoric acid, hydrochloric acid, sulfuric acid, glacial acetic acid, or formic acid. And (4) respectively carrying out sample injection detection on the system applicability solution and collecting a chromatogram map by adopting the conditions.

The results show that: under the conditions of the first six pH values, no matter what pH regulator is adopted, the peak type is good, the main peak and each impurity can be well separated, the impurity peaks can also be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the impurity number have no obvious change. However, under neutral conditions of pH7.0, some impurities were not separated efficiently, and the peak patterns of some impurity peaks were asymmetric. The pH value of the acidic mobile phase has no influence on the measurement result of related substances.

(3) Selection of column temperature

Four column temperatures were selected for the test under otherwise identical conditions, column temperature 1 (0 ℃), column temperature 2 (20 ℃), column temperature 3 (30 ℃) and column temperature 4 (40 ℃). And (4) respectively carrying out sample injection detection on the system applicability solution and collecting a chromatogram map by adopting the conditions.

The results show that: under the four column temperature conditions, the peak types are good, the main peak and each impurity can be well separated, the impurity peaks can be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the impurity number have no obvious change. The column temperature is shown to have no influence on the measurement result of the related substances.

(4) Selection of flow rate

Four chromatographic flow rates were selected for testing, flow 1 (0.5 ml/min), flow 2 (1.0 ml/min), flow 3 (1.5 ml/min), and flow 4 (2.0 ml/min) with other conditions unchanged. And (3) respectively carrying out sample injection detection on the system applicability solution and collecting a chromatogram map by adopting the four conditions.

The results show that: under the four flow velocity conditions, the peak patterns are good, the main peak and each impurity can be well separated, the impurity peaks can also be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the measured impurity number have no obvious change. Indicating that the flow rate has no influence on the measurement result of the related substances.

(5) Selection of chromatography column model

Three different chromatography column models were selected, with other conditions unchanged: type 1: c18 column, 250 × 4.6mm,5 μm, model 2: c18 column, 250 × 4.6mm,3.5 μm, model 3: and (4) a C18 column, 250 × 3.0mm and 3.0 μm, respectively carrying out sample injection detection on the system adaptive solution, and collecting a chromatogram.

The results show that: under the conditions of the three types, the peak types are good, the main peak and each impurity can be well separated, the impurity peaks can be well separated, the separation degree of each peak is more than 1.5, and the measured known impurity content and the measured impurity number have no obvious change. The model of the C18 column is shown to have no influence on the determination result of related substances. In addition, the inventor basically tries the common C18 column, the separation degree can be more than 1.5, and each peak type is good, which proves that the chromatographic method of the invention is not influenced by the model number and the brand of the column.

Experimental example 2: methodology validation

Transmitter specific verification

Chromatographic conditions are as follows: a high performance liquid chromatography is adopted, a chromatographic column (Chromcore 120C 18.4.6 mm multiplied by 250mm 5 mu m) with octadecylsilane chemically bonded silica as a filling agent is adopted, the detection wavelength is 220nm, the mobile phase and gradient elution program is the mobile phase C and gradient elution program 3 in the experimental example 1, the flow rate is 1.0ml/min, and the column temperature is 30 ℃.

The preparation method of each solution was the same as in Experimental example 1.

Appropriate amounts of known impurities P1Z1, P1Z5 and compound A are taken, and sample injection and determination are carried out according to the formulated chromatographic conditions. The analysis results are shown in Table 2-1, and the chromatogram of the system applicability solution is shown in FIG. 1.

According to test results, under the proposed chromatographic conditions, the solvent does not interfere with the detection of each impurity in the test solution, and the separation degrees among the impurities and between the impurities and a main peak are all more than 1.5; the peak purity indexes all meet the analysis requirements; the order of the peak appearance of each substance is as follows: P1Z5, P1Z2, main peak, P1Z1 and P1Z12, and the result shows that the specificity of the method is good.

Test for mechanical destruction of components

The sample preparation method comprises the following steps: weighing 3 parts of a proper amount of a compound A sample (batch number: 39201001), dissolving the sample with 2ml of acetonitrile, respectively adding 0.1ml of 0.1mol/L hydrochloric acid or 70ul of 0.1mol/L sodium hydroxide or 1ml of 3% hydrogen peroxide, respectively, standing strong acid and strong base at room temperature for 2 minutes, standing at oxidation room temperature for 24 hours, dissolving with acetonitrile after strong destruction, and diluting to prepare a solution containing about 1mg of compound A per 1ml, thus obtaining the compound A. The results of the force failure test are shown in tables 2-2.

Under the conditions of acid, alkali and oxidation forced destruction tests, the separation degree of a main peak and adjacent peaks thereof is greater than 1.5, the degraded impurities do not interfere with the detection of known impurities, and the purity parameters of the main peak meet the specification.

\9354

Taking appropriate amounts of impurities P1Z5 and P1Z1 and a working reference substance of the compound A, respectively adding acetonitrile to dissolve and gradually dilute, and taking the solution as the detection limit of each substance when the peak height of each substance is about 3 times of the baseline noise, namely the signal-to-noise ratio is about 3; the limit of quantitation for this material is when the peak height is about 10 times the baseline noise, i.e., the signal-to-noise ratio is about equal to 10. The results are shown in tables 2 to 3.

The test result shows that: the detection limit of the impurity P1Z5 is 1.080ng, the detection limits of other impurities are lower than the detection limit of P1Z5, the sample injection concentration planned by the relevant substance inspection of the product is 1mg/ml, the sample injection amount is 10 mu l, and the absolute sample injection amount is 10000ng. The absolute sample injection amount is more than 9000 times of the detection limit of P1Z5, which indicates that the proposed sample injection concentration can ensure the effective detection of the impurities in the product.

\9355

Taking appropriate amount of reference substance stock solutions of impurities P1Z5 and P1Z1 under a 'special' test item, precisely measuring, diluting with acetonitrile to prepare solutions with the limit concentrations of 10%, 20%, 50%, 80%, 100% and 200%, taking 1 μ l of each linear reference substance solution according to the preset chromatographic conditions, injecting into a gas chromatograph, recording a chromatogram, and performing linear regression by taking the concentration as abscissa and the peak area as ordinate, wherein the results are shown in tables 2-4.

In conclusion, when the concentration of P1Z5 is between 0.1002 mu g/ml and 2.0040 mu g/ml, the linear relation between the concentration and the peak area is good; the concentration of the impurity P1Z1 is in the range of 0.1080 mug/ml-2.1600 mug/ml, and the concentration and the peak area have good linear relation; the concentration of the compound A is in the range of 0.1169 mu g/ml to 2.3380 mu g/ml, and the concentration and the peak area have good linear relation; the ratio of the Y-axis intercept to the 100% peak area is within 25%.

\9356

Taking 100% impurity mixed reference solution under the linear test item, continuously injecting for 6 times, recording peak area, and inspecting injection precision. The results show that: RSD of the impurity P1Z5 peak area is as follows: 0.9%, RSD of impurity P1Z1 peak area is: 2.1 percent, meets the requirements and meets the measurement requirements of liquid chromatography precision.

6 parts of test sample solutions are prepared in parallel, the reference sample solution is subjected to the same sample injection precision test, the content of each impurity in the test sample is measured according to the proposed chromatographic conditions, the impurities P1Z5 and P1Z1 are calculated according to an external standard method by using the peak area, the impurities P1Z2 and P1Z12 and the total impurities are calculated according to a self-reference method and a surface area normalization method respectively, and the results are shown in tables 2-5.

From the above results, it is understood that the impurities P1Z5 and P1Z1 were not detected, and the impurities P1Z2 and P1Z12 were detected in 6 samples, and that the detection results of the impurities P1Z2 and P1Z12 and total impurities in the 6 samples were not significantly different when calculated by the self-control method and the area normalization method, and RSD of the 6 samples by the area normalization method was better than that by the self-control method, so the area normalization method was selected as the method for calculating impurities. The area normalization method detects 6 samples, the average value of the impurities P1Z2 is 0.25%, the RSD is 0.7%, the average value of the impurities P1Z12 is 0.02%, the RSD is 17.0%, the average value of the total impurities is 0.27%, and the RSD is 1.6%, which indicates that the method has good repeatability.

\9357recoveryrate

In order to examine the degree of closeness between the detection results of the impurities P1Z5 and P1Z1 and the true values by the proposed detection method, the accuracy is verified by adopting a sample-adding and recycling mode. Taking a proper amount of a test sample with known impurity content, preparing a test sample solution according to a formulated method, adding known impurities with limit concentrations of 80%, 100% and 120% in the test sample solution respectively, then carrying out sample injection measurement, and calculating the recovery rate. The test results are shown in tables 2 to 6.

From the results, the sample recovery rate of P1Z5 is 97.36-105.60%, the average recovery rate is 102.4%, and the RSD is 2.6%; the sample recovery rate of P1Z1 is 91.12-102.08%, the average recovery rate is 97.2%, and the RSD is 3.2%, which shows that the method can accurately detect impurities of P1Z5 and P1Z1 in the product, and has good accuracy.

\ 9358

In order to verify the degree of accuracy of the measurement results when the proposed liquid phase conditions slightly varied, the durability of the proposed chromatographic conditions was examined.

Taking the system applicability solution, the test sample solution and the self control solution, and determining according to the proposed chromatographic conditions.

Test results show that when the flow rate, the column temperature, the chromatographic column and the mobile phase are slightly changed, for example, the flow rate is changed by +/-0.2 ml/min, the column temperature is changed by +/-5 ℃, chromatographic columns of different types are changed (the filler is not changed), the proportion of the mobile phase B is changed by +/-2, and the pH value of the mobile phase A is changed by +/-2, the measured known impurity content and the impurity number are not obviously changed under various conditions. The column temperature, flow rate, chromatographic column and mobile phase pH value have no influence on the measurement result of related substances.

Example 1: examination of related substances 1 (Mobile phase 1)

The preparation and chromatographic conditions of each solution were the same as in experimental example 2, wherein the chromatographic condition mobile phase B was methanol, and the other conditions were unchanged.

The determination method comprises the following steps: and injecting each solution into a chromatographic system, and collecting a chromatogram.

Example 2: examination of related substances 2 (Mobile phase 2)

The preparation and chromatographic conditions of each solution were the same as those of Experimental example 2, wherein the chromatographic condition mobile phase B was acetonitrile, and other conditions were unchanged.

Example 3: measurement of related substance 3 (mobile phase 3)

The preparation and chromatographic conditions of each solution are the same as those of experimental example 2, wherein the chromatographic condition mobile phase B is a mixed solution of methanol and acetonitrile with the volume ratio of 80.

Example 4: measurement of related substance 4 (mobile phase 4)

The preparation and chromatographic conditions of each solution were the same as those of Experimental example 2, wherein the acidic ion pair in the mobile phase A under the chromatographic conditions was changed from 0.01mol/L sodium octane sulfonate to 2.0mol/L sodium pentane sulfonate, and the other conditions were unchanged.

Example 5: measurement of related substance 5 (mobile phase 5)

The preparation and chromatographic conditions of each solution were the same as in Experimental example 2, wherein the salt solution in mobile phase A under chromatographic conditions was changed from 0.01mol/L potassium dihydrogen phosphate to 0.001mol/L ammonium acetate, and the other conditions were unchanged.

Example 6: measurement of related substance 6 (mobile phase 6)

The solution preparation and chromatographic conditions were the same as in Experimental example 2, in which mobile phase A was adjusted to pH 5.0 with glacial acetic acid, and the other conditions were unchanged.

Example 7: measurement of the relevant substance 7 (gradient program 3)

The solution formulation and chromatographic conditions were the same as in Experimental example 2, wherein the gradient elution procedure is shown in Table 3-1, and other conditions were unchanged.

Example 8: measurement of the relevant substance 8 (gradient program 4)

The solution formulation and chromatographic conditions were the same as in experimental example 2, with the gradient elution procedure shown in tables 3-2, and other conditions being unchanged.

Example 9: measurement of the relevant substance 9 (gradient program 5)

The solution formulation and chromatographic conditions were the same as in experimental example 2, with the gradient elution procedure shown in tables 3-3, and other conditions being unchanged.

Example 10: measurement of the relevant substance 10 (gradient program 6)

The solution formulation and chromatographic conditions were the same as in Experimental example 2, with the gradient elution procedure shown in tables 3-4, and other conditions being unchanged.

Example 11: measurement of the relevant substance 11 (gradient program 7)

The solution formulation and chromatographic conditions were the same as in experimental example 2, with the gradient elution procedure shown in tables 3-5, and other conditions being unchanged.

Example 12: measurement of the relevant substance 12 (gradient program 8)

The solution formulation and chromatographic conditions were the same as in Experimental example 2, with the gradient elution procedure shown in tables 3-6, and other conditions being unchanged.

Example 13: measurement of relevant substance 13 (pH of mobile phase A1.0)

The preparation and chromatographic conditions of each solution were the same as those of Experimental example 2, in which the pH of mobile phase A was changed from 4.0 to 1.0, and the other conditions were unchanged.

And (3) determination: and injecting each solution into a chromatographic system, and collecting a chromatogram.

Example 14: measurement of relevant substance 14 (pH of mobile phase A6.0)

The preparation and chromatographic conditions of each solution were the same as those in Experimental example 2, in which the pH of mobile phase A was changed from 4.0 to 6.0, and the other conditions were unchanged.

Example 15: measurement of related substance 15

The preparation and chromatographic conditions of each solution were the same as those in Experimental example 2, wherein the flow rate was changed from 1.0ml/min to 2.0ml/min, and the other conditions were unchanged.

Example 16: measurement of related substance 16

The preparation and chromatographic conditions of each solution were the same as those in Experimental example 2, wherein the flow rate was changed from 1.0ml/min to 0.5ml/min, and the other conditions were unchanged.

Example 17: measurement of related substance 17 (mobile phase 7)

The preparation and chromatographic conditions of each solution are the same as those in Experimental example 2, wherein the acidic ion pair in the mobile phase A under the chromatographic conditions is changed from 0.01mol/L sodium octane sulfonate to 0.001mol/L sodium pentane sulfonate, and other conditions are not changed.

Example 18: measurement of related substance 18 (Mobile phase 8)

The preparation and chromatographic conditions of each solution were the same as in Experimental example 2, wherein the salt solution in mobile phase A under chromatographic conditions was changed from 0.01mol/L potassium dihydrogen phosphate to 2.0mol/L ammonium acetate, and the other conditions were unchanged.

It is noted that the last 10 minutes of examples 1 to 10, 13 to 18 and the last 20 minutes of examples 11 to 12 are the system equilibration times and that there are no more impurity elution peaks for this part of the procedure.

Impurities P1Z1 and P1Z5 are calculated by peak areas according to an external standard method, P1Z2 and P1Z12 are calculated by peak areas according to a self-contrast method or an area normalization method, and other single impurities and total impurities are calculated by the peak area normalization method.

Data statistics and summarization: the chromatogram of experiment example 2 "under specificity" and examples 1 to 18 above were summarized, and the peak area, retention time and resolution were counted, and the results showed: (1) in each system applicability solution chromatogram: the peak type of each peak is better, the separation degree between each main peak and the impurity peak and between the impurity peak and the impurity peak is between 1.5 and 15.0 and is more than 1.5, the separation degree of each peak is better, each method is good in exclusive property, and the method meets the quality analysis requirement. (2) in the chromatogram of each test sample solution: the content difference of each main peak compound A calculated by peak area according to an external standard method and the compound A in the experimental example 2 is within 0.1 percent; the impurities P1Z5 and P1Z1 are not detected, the impurities P1Z2 and P1Z12 are detected, the content of the impurity P1Z2 is 0.24-0.27 percent, the content of the impurity P1Z12 is 0.01-0.04 percent and the total impurities are not more than 0.30 percent according to the area normalization method, which indicates that the accuracy and the reproducibility of the proposed related substance determination method are good. Accordingly, the quality control standard of the product can be determined, namely, if an impurity peak exists in a test sample solution, the impurities P1Z1 and P1Z5 are calculated according to an external standard method by the peak area and are not more than 0.1%; the impurity P1Z2 is not more than 1.0 percent calculated by a peak area calculation or peak area normalization method according to a self-contrast method; the other single unknown impurities are not more than 0.1 percent calculated according to a peak area normalization method; the sum of the peak areas of all impurities is not more than 2.0 percent. Wherein the impurity P1Z12 is not more than 0.1% calculated by peak area according to self contrast method or area normalization method; can be controlled according to unknown impurities.

Claims

1. A method for detecting related substances of (7 aS,2' S) -2-oxo-clopidogrel is characterized by adopting high performance liquid chromatography, and the operation method comprises the following steps:

taking (7 aS,2' S) -2-oxy-clopidogrel, and diluting with a solvent to obtain a test solution;

injecting the sample solution into a high performance liquid chromatograph, and detecting according to chromatographic conditions.

2. The detection method according to claim 1, wherein the chromatographic conditions comprise:

octadecylsilane chemically bonded silica is used as a filling agent, the detection wavelength is 220 +/-2 nm, a salt solution with an acidic pH value and containing an acidic ion pair is used as a mobile phase A, a solution containing acetonitrile and/or alcohol is used as a mobile phase B, and gradient elution is carried out.

3. The detection method according to any one of claims 1 to 2, wherein the gradient elution comprises at least two steps: the proportion of the mobile phase A in the first stage procedure is 35-60%, and the proportion of the mobile phase B is 65-40%; in the second stage, the proportion of the mobile phase A is 5-20%, and the proportion of the mobile phase B is 95-80%; and the total time of the first stage procedure is 10-50 minutes, and the total time of the second stage procedure is 10-50 minutes.

4. The detection method according to any one of claims 1 to 4, wherein the total time of the second stage process comprises a proportional down time of mobile phase A, a proportional up time of mobile phase B and a holding time, wherein the proportional down time of mobile phase A is equal to the proportional up time of mobile phase B and is 5 to 30 minutes, and the holding time is 5 to 30 minutes.

5. The detection method according to any one of claims 1 to 4, wherein the gradient elution procedure comprises: the proportion of the mobile phase A is 35 to 60 percent and the proportion of the mobile phase B is 65 to 40 percent at 0 minute, and the mobile phase A and the mobile phase B are maintained for 20 to 40 minutes; when the time is 30-60 minutes, the proportion of the mobile phase A is reduced to 5-20%, the proportion of the mobile phase B is increased to 95-80%, and the time is maintained for 50-70 minutes.

6. The detection method according to any one of claims 1 to 5, characterized in that it comprises a system suitability test: respectively weighing an impurity reference substance and a proper amount of (7 aS,2 'S) -2-oxy-clopidogrel, and dissolving and diluting the impurity reference substance and the (7 aS,2' S) -2-oxy-clopidogrel to a mixed solution with a target concentration by a solvent in a one-time preparation mode or a mode of firstly preparing in a concentrated mode and then preparing in a diluted mode.

7. The detection method according to any one of claims 1 to 6, wherein the substance concerned comprises one or more of P1Z1, P1Z2, P1Z5 and P1Z12.

8. The detection method according to any one of claims 1 to 7, wherein the solvent comprises acetonitrile, THF, an alcohol or a mixed solvent of mobile phases B and A in a volume ratio of 40 to 100; acetonitrile is preferred.

9. The detection method according to any one of claims 1 to 8, wherein the acidic ion pair comprises sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, or sodium dodecane sulfonate.

10. The detection method according to any one of claims 1 to 9, wherein the salt of the salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate, or potassium acetate.

11. The detection method according to any one of claims 1 to 10, wherein the salt solution has a pH of 1.0 to 6.0; preferably 2.0 to 5.0.

12. The detection method according to any one of claims 1 to 11, wherein the pH is adjusted using a pH adjuster comprising phosphoric acid, hydrochloric acid, sulfuric acid, glacial acetic acid, or formic acid.

13. The detection method according to any one of claims 1 to 12, wherein the mobile phase B is an alcohol, acetonitrile or a mixed solution of an alcohol and acetonitrile at a volume ratio of 80 to 100; the volume ratio of the alcohol to the acetonitrile is preferably 90 to 98.

14. The detection method according to any one of claims 1 to 13, wherein the alcohol comprises methanol, ethanol, propanol or isopropanol.

15. The detection method according to any one of claims 1 to 14, wherein in the chromatogram of the test solution, if a relevant substance exists, impurities P1Z1 and P1Z5 are not more than 0.1%; and/or impurity P1Z2 is not more than 1.0%; and/or impurity P1Z12 is not more than 0.1%; and/or no more than 0.1% of other individual unknown impurities; and/or total impurities of not more than 2.0%.