Detection method of clonazepam related substances

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of clonazepam related substances, the detection method adopts a high performance liquid chromatography, and chromatographic conditions comprise that a phosphate buffer solution and an acetonitrile solution are adopted as mobile phases for gradient elution, and by adopting the detection method provided by the invention, the detected impurities are more, and the detection sensitivity is high. According to the present invention, the retention ability of the main components and the impurities in the mobile phase system is strong, the peak response is high, the compounds can be effectively separated, and the related substances in the clonazepam can be well monitored.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting clonazepam-related substances. Background Art

[0002] Clonazepam injection was first developed by F. Hoffmann-La Roche. It is an anhydrous preparation with clonazepam as the active ingredient and propylene glycol, anhydrous ethanol, benzyl alcohol and appropriate amount of glacial acetic acid as the inactive ingredients. Clonazepam injection is mainly used to control various types of epilepsy, especially for absence seizures, infantile spasms, myoclonic seizures, akinetic seizures and Lennox-Gastaut syndrome.

[0003] At present, there are few literature reports on the detection methods of related substances in clonazepam injection. Only the detection methods of related substances in clonazepam injection included in the Chinese Pharmacopoeia (ChP) and the British Pharmacopoeia (BP) are provided for reference. However, the detection method of water injection containing clonazepam included in the Chinese Pharmacopoeia is the detection method; the detection method of the British Pharmacopoeia is thin layer chromatography, and the detection method of the Pharmacopoeia has major defects and cannot separate and detect all related substances of clonazepam injection. In addition, the impurity retention time is short and the response is weak, which cannot guarantee the quality and safety of clonazepam injection.

[0004] The study of related substances is an extremely important project in drug quality research. Its content not only directly reflects the purity of the drug, but also has important safety significance. In order to ensure the safety and effectiveness of clonazepam injection, it is necessary to study, detect and control its related substances. According to the synthetic process route of clonazepam and the prescription process of clonazepam preparations, the related substances are mainly process impurities in the production process of raw materials, reaction byproducts and degradation products of excipients of clonazepam preparations, etc.

[0005] The present invention develops a new detection method for clonazepam-related substances based on the existing technology, which can realize the localization and quantitative research on clonazepam impurities and provide a basis for the quality evaluation of clonazepam preparations. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting clonazepam-related substances on the basis of the prior art. The method detects many impurities, the retention capacity of the main component and each impurity in the mobile phase system is strong, the peak response is high, and each compound can be effectively separated, so that the content of related substances in clonazepam injection can be better monitored.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for detecting related substances of clonazepam injection, the method adopts high performance liquid chromatography, the chromatographic column is C 18The column is gradient eluted with phosphate buffer-acetonitrile as the mobile phase, the pH value of the phosphate buffer is 6 to 8, preferably the pH value is 6.8 to 7.2, and the related substances include the following substances:

[0009]

[0010]

[0011] Furthermore, the C18 chromatographic column described in the present invention is a SHIMADZU Shim-pack Scepter HD-C18-80 chromatographic column with a specification of 4.6 mm×150 mm and 3 μm.

[0012] Further, the chromatographic conditions include: detection wavelength: 250-260 nm; preferably detection wavelength: 252-256 nm; more preferably detection wavelength: 254 nm;

[0013] The flow rate: 0.4-1.0 ml / min; preferably 0.5-0.7 ml / min; more preferably 0.6 ml / min;

[0014] The column temperature is 30-40°C; preferably 33-37°C; more preferably 35°C;

[0015] The injection volume: 5 to 50 μl; preferably the injection volume: 5 to 15 μl; more preferably the injection volume is 10 μl;

[0016] The injector temperature is 2-15°C; preferably, the injector temperature is 5-8°C; more preferably, the injection temperature is 6°C.

[0017] Furthermore, the pH value of the phosphate buffer is 7.0; the phosphate is selected from a mixture of one or more of diammonium hydrogen phosphate and dipotassium hydrogen phosphate, preferably diammonium hydrogen phosphate.

[0018] Furthermore, the volume ratio of phosphate buffer to acetonitrile in the mobile phase A is (80-100):(0-20), preferably (88-92):(8-12), and more preferably 90:10;

[0019] The volume ratio of phosphate buffer to acetonitrile in mobile phase B is (20-40):(60-80), preferably (28-32):(68-72); more preferably 30:70.

[0020] A method for detecting clonazepam-related substances, the detection steps comprising:

[0021] (1) Solution preparation: prepare the positioning solution, system suitability solution, test sample solution, reference sample stock solution and reference sample solution respectively;

[0022] (2) Determination method: After the system is stable, inject the system suitability solution, positioning solution, test solution and reference solution respectively, and record the chromatogram.

[0023] Furthermore, a method for detecting clonazepam-related substances comprises the following steps:

[0024] The positioning solution is a benzyl alcohol solution;

[0025] The reference substance stock solution is prepared by weighing appropriate amounts of clonazepam, impurity I, impurity II, impurity VI, impurity VII, impurity VIII, impurity X, impurity XI and benzaldehyde, placing them in a suitable volumetric bottle, and dissolving and diluting them with methanol to prepare a stock solution; weighing an appropriate amount of impurity IX, placing it in a suitable volumetric bottle, and dissolving and diluting it with acetonitrile to prepare a stock solution;

[0026] The system suitability solution: take appropriate amount of benzaldehyde and impurity I, II, VI, VII, VIII, IX, X, XI reference substance stock solutions, place them in a volumetric bottle, add clonazepam injection to dissolve and dilute to the scale, and shake well;

[0027] The test solution: Take this product and get it;

[0028] The reference substance solution: take an appropriate amount of the reference substance stock solution, place it in a volumetric bottle, add methanol to dilute to the scale, and shake well;

[0029] Further, the mobile phase gradient elution procedure is as follows

[0030]

[0031]

[0032] Preferably, the mobile phase gradient elution procedure is as follows

[0033] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0 85 15 35 40 60 45 15 85 50 15 85 60 0 100 60.5 85 15 70 85 15 The present invention also provides a compound represented by impurity X and its Z isomer:

[0034]

[0035] The present invention also provides a compound represented by impurity XI:

[0036]

[0037] Furthermore, the detection method is used to detect related substances in clonazepam raw materials or preparations.

[0038] Beneficial Effects

[0039] The technical solution of the present invention has the following advantages:

[0040] (1) The mobile phase system used in the present invention can effectively improve the retention capacity of the main components and various impurities, and the response of each component is relatively high and the peak shape is relatively good, which solves the problem that the retention capacity of clonazepam and its impurities is weak, the separation degree and the peak shape are poor in the general buffer salt mobile phase system, and can effectively detect the related substances in clonazepam injection, which is of great significance for the quality control of clonazepam injection.

[0041] (2) The present invention adopts a method of controlling the temperature of the sample injector under chromatographic conditions, thereby solving the problem of impurities being unstable at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a solution map for positioning the present invention.

[0043] Figure 2 This is a solution spectrum of the system applicability of the present invention. DETAILED DESCRIPTION

[0044] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technical schemes implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0045] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0046] Example 1: Specificity test

[0047] (1) Instruments: high performance liquid chromatograph, electronic analytical balance, pH meter, etc.

[0048] (2) Solution preparation:

[0049] Diluent: Methanol

[0050] Test solution: Take this product and you'll get it.

[0051] Blank excipient solution: Take blank excipient and obtain the same below.

[0052] Clonazepam reference substance concentrated solution: Weigh an appropriate amount of clonazepam reference substance, dissolve it in methanol and dilute it to make a solution containing approximately 1 mg of clonazepam per 1 ml.

[0053]

[0054]

[0055]

[0056] Concentrated solution of impurity Ⅰ, II, IV, V, VI, VII, VIII, X, XI reference substances: Accurately weigh appropriate amounts of impurity IV, impurity V, impurity VI, impurity VII, impurity VIII, impurity X, and impurity XI reference substances, place them in different volumetric flasks, dissolve and dilute them with methanol to prepare solutions containing approximately 0.5 mg each of impurities Ⅰ, II, IV, V, VI, VII, VIII, XI and 1 mg of impurity X per 1 ml.

[0057] Concentrated solution of impurity III and IX reference substances: Accurately weigh appropriate amounts of impurity III and IX reference substances respectively, place them in different volumetric flasks, dissolve and dilute them with acetonitrile to make solutions containing approximately 0.5 mg of impurity III and IX per 1 ml.

[0058] Benzaldehyde reference concentrated solution: Accurately weigh an appropriate amount of benzaldehyde reference, place it in a suitable volumetric flask, dissolve it in methanol and dilute it to make a solution containing approximately 1.5 mg of benzaldehyde in 1 ml.

[0059] Impurity reference substance stock solution: Accurately measure appropriate amounts of impurity I, II, VI, VII, VIII, X, XI, benzaldehyde, and clonazepam reference substance concentrated solutions, place them in the same volumetric flask, and dilute them with methanol to make a solution containing approximately 20 μg of impurity I, 20 μg of impurity II, 100 μg of impurity X, 50 μg each of impurities VI, VII, VIII, and XI, 150 μg of benzaldehyde, and 10 μg of clonazepam per 1 ml.

[0060] Clonazepam reference substance stock solution: Accurately measure an appropriate amount of the concentrated solution of clonazepam reference substance, place it in a volumetric flask, and dilute it with methanol to make a solution containing approximately 10 μg of clonazepam per 1 ml.

[0061] Reference solution: Accurately measure appropriate amounts of impurity reference stock solution and clonazepam stock solution, place in the same volumetric flask, and dilute with methanol to make a solution containing approximately 2 μg of impurity I, 2 μg of impurity II, 10 μg of impurity X, 5 μg each of impurities VI, VII, VIII, and XI, 15 μg of benzaldehyde, and 1 μg of clonazepam per 1 ml.

[0062] Impurity IX reference stock solution: Accurately measure an appropriate amount of the impurity IX reference concentrated solution and dilute it with acetonitrile to make a solution containing approximately 50 μg of impurity IX per 1 ml.

[0063] Impurity IX reference solution: Accurately measure an appropriate amount of the Impurity IX reference stock solution and dilute it with acetonitrile to make a solution containing approximately 5 μg of Impurity IX per 1 ml.

[0064] Benzaldehyde localizing solution: Accurately measure an appropriate amount of the concentrated benzaldehyde reference solution and dilute it with methanol to make a solution containing approximately 15 μg of benzaldehyde per 1 ml.

[0065] Benzyl alcohol reference solution: Weigh an appropriate amount of benzyl alcohol reference substance, dissolve it in methanol and dilute it to make a solution containing approximately 30 mg of benzyl alcohol per 1 ml.

[0066] Clonazepam localizing solution: Accurately measure an appropriate amount of the concentrated clonazepam reference solution and dilute it with methanol to make a solution containing approximately 1 μg of clonazepam per 1 ml.

[0067] Impurity I and Impurity II Location Solutions: Accurately measure appropriate amounts of Impurity I and Impurity II concentrated solutions, place them in different volumetric flasks, and dilute with methanol to make solutions containing 2 μg of Impurity I and 2 μg of Impurity II per 1 ml.

[0068] Impurity IV, V, VI, VII, VIII, X, XI reference substance positioning solution: Accurately measure appropriate amounts of impurity IV, impurity V, impurity VI, impurity VII, impurity VIII, impurity XI, and impurity X reference substance concentrated solutions, place them in different volumetric flasks, and dilute them with methanol to make solutions containing 10 μg of impurity X, 5 μg of impurity IV, impurity V, impurity VI, impurity VII, impurity VIII, and impurity XI per 1 ml.

[0069] Impurity III localization solution: Accurately measure an appropriate amount of the impurity III reference concentrated solution and dilute it to the scale with acetonitrile to prepare a solution containing 5 μg of impurity III per 1 ml.

[0070] System suitability solution: Take appropriate amounts of impurity I, II, VI, VII, VIII, IX, X, XI and benzaldehyde reference concentrated solutions, place them in the same volumetric flask, dilute to the mark with the test sample to make a solution containing approximately 2 μg of impurity I, 2 μg of impurity II, 10 μg of impurity X, 5 μg each of impurities VI, VII, VIII, IX, XI, 15 μg of benzaldehyde and 1 mg of clonazepam per 1 ml.

[0071] Separation solution: Take appropriate amount of impurity I, II, III, IV, V, VI, VII, VIII, IX, X, XI and benzaldehyde reference concentrated solution respectively, place them in the same volumetric flask, dilute to the scale with the test sample to make a solution containing approximately 2μg of impurity I, 2μg of impurity II, 10μg of impurity X, 5μg each of impurities III, IV, V, VI, VII, VIII, IX, XI, 15μg of benzaldehyde and 1mg of clonazepam per 1ml.

[0072] Accurately measure 10 μl each of the diluent, reference solution, impurity IX reference, test solution, blank excipient solution, reference positioning solution, system suitability solution, and resolution solution, inject into the liquid chromatograph, and record the chromatogram.

[0073] Acceptable criteria: When injecting blank excipient solution, there should be no interfering peaks; when continuously injecting 6 needles of reference solution, the RSD of the peak areas of the 6 needles shall be ≤10%; when injecting system suitability solution, the separation between the peaks shall meet the requirements; after the system meets the requirements, inject the test solution, system suitability solution, separation solution and each positioning solution; when the sequence is completed, inject 1 needle of reference solution again, and the RSD of the peak areas of the 7 needles shall be ≤10%.

[0074] Table 1 Specificity test results

[0075]

[0076] The diluent and the auxiliary material peaks before the relative retention time of 0.1 do not interfere with the determination of related substances. In the resolution solution, the resolution between the clonazepam peak and the adjacent peaks and the known impurity peaks meets the requirements; the theoretical plate number is not less than 2000 based on the clonazepam peak, and the peak shape of each impurity is good, indicating that this chromatographic condition is suitable for the determination of related substances.

[0077] Example 2: Destructive test

[0078] Take blank excipients and clonazepam injection for acid, alkali, oxidation, high temperature and light tests respectively. The contents are as follows: a. The blank excipient solution and the test solution are not destroyed.

[0079] Acid-base undamaged test sample solution: Take a 10ml brown volumetric flask, add 2ml of water, and dilute to volume with the test sample.

[0080] Acid-base undamaged auxiliary material solution: Take a 10ml brown volumetric bottle, add 2ml water, and dilute to volume with blank auxiliary material.

[0081] Oxidation-undamaged test sample solution: Take a 10ml brown volumetric flask, add 1ml of water, and dilute to volume with the test sample.

[0082] Solution of auxiliary materials that have not been destroyed by oxidation: Take a 10ml brown volumetric flask, add 1ml of water, and dilute to volume with blank auxiliary materials.

[0083] Blank excipient solution and test sample solution that are not destroyed by high temperature and light: Take this product and blank excipient directly as the undestroyed solution.

[0084] b. High temperature damage

[0085] High temperature destruction test solution: Place this product in a 130℃ oven for 48h, and take this sample as the high temperature destruction test solution. High temperature destruction blank auxiliary material solution: Place the blank auxiliary material solution in a 130℃ oven for 48h, and take this sample as the high temperature destruction blank auxiliary material solution.

[0086] c. Acid-base damage

[0087] Acid-base blank: Take 1 ml of 1 mol / L hydrochloric acid in a 10 ml brown volumetric flask, place it at room temperature for 24 h, add 1 mol / L sodium hydroxide solution, adjust to neutral, and dilute to the scale with methanol.

[0088] Acid destruction of blank excipient solution: take about 1 ml of blank excipient into a 10 ml brown volumetric flask, add 1 ml of 1 mol / L hydrochloric acid, shake well, leave at room temperature for 24 hours, add 1 mol / L sodium hydroxide solution, adjust to neutral, and dilute to the scale with blank excipient.

[0089] Acid destruction of test solution: Take about 1 ml of the product in a 10 ml brown volumetric flask, add 1 ml of 1 mol / L hydrochloric acid, shake well, leave at room temperature for 24 hours, add 1 mol / L sodium hydroxide solution, adjust to neutral, and dilute to the scale with the test sample.

[0090] Alkali destruction of blank auxiliary material solution: take about 1 ml of blank auxiliary material into a 10 ml brown volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, shake well, leave at room temperature for 24 hours, add 1 mol / L hydrochloric acid, adjust to neutral, and dilute to the scale with blank auxiliary material.

[0091] Alkali destruction test solution: Take about 1 ml of the product in a 10 ml brown volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, shake well, leave at room temperature for 24 hours, add 1 mol / L hydrochloric acid, adjust to neutral, and dilute to the scale with the test sample.

[0092] d. Oxidative damage

[0093] Oxidative destruction blank: Take a 10ml brown volumetric bottle, add 1ml of 3% hydrogen peroxide solution, shake well, leave at room temperature for 24h, and dilute to the scale with diluent;

[0094] Oxidative destruction of blank auxiliary material solution: take about 1 ml of blank auxiliary material into a 10 ml brown volumetric flask, add 1 ml of 3% hydrogen peroxide solution, shake well, place at room temperature for 24 hours, and dilute to the scale with blank auxiliary material.

[0095] Oxidative damage: Take about 1 ml of this product in a 10 ml brown volumetric bottle, add 1 ml of 3% hydrogen peroxide solution, shake well, leave at room temperature for 24 hours, and dilute to the scale with the test sample;

[0096] e. Light damage

[0097] Light damage to the test solution: Take the product and place it in a strong light test box (illuminance 5000±500lx, near-ultraviolet energy 90μW / cm 2 ) for 48 h, and take this sample directly as the test solution for light damage;

[0098] Light destruction of blank auxiliary material solution: Take blank auxiliary material and place it in a strong light irradiation test box (illuminance 5000±500lx, near-ultraviolet energy 90μW / cm 2 ) for 48 h, and take this sample directly as the blank auxiliary material solution for light destruction.

[0099] Reference solution, impurity IX reference solution, benzyl alcohol positioning solution and system suitability solution: the preparation method is the same as that of "Example 1 Specificity Test".

[0100] Take 10 μl of each of the system suitability solution, reference solution, undamaged solution and the above-mentioned damaged test solutions and inject them into the liquid chromatograph. Use the determined chromatographic conditions to determine the degradation products. The content of each known impurity is calculated by the external standard method. The content of unknown impurities is calculated by the peak area of ​​clonazepam in the reference solution. The content of clonazepam in each damaged solution is calculated by the external standard method with the undamaged test solution as the reference.

[0101] Acceptable standard: The degradation products produced by this product after being destroyed by acid, alkali, oxidation, high temperature and light can be well detected, and the separation degree between each degradation product and the main component peak should meet the requirements; the purity of the main peak should meet the requirements, and the percentage of total material should be between 90% and 110%.

[0102] Table 2 Summary of destructive test results

[0103]

[0104] Table 3 Destruction test material balance

[0105]

[0106] Conclusion: 1. It is unstable under acid, alkali, high temperature and light damage conditions, but relatively stable under oxidation damage conditions.

[0107] 2. The benzaldehyde content increased slightly under each destruction condition, and the benzaldehyde content increased most significantly under light conditions. The main impurities degraded by acid destruction were impurities II, IX and VIII; the main impurities degraded by alkali destruction were impurities VIII; no new impurities were found in oxidation destruction. Under high temperature conditions, impurities X and XI were mainly degraded; light destruction was the most severe; in the test solution, the degradation products produced under each destruction condition could be well separated from clonazepam, and the peak purity factor of the main peak met the requirements. After destruction, the materials were basically balanced.

[0108] 3. Under the destruction conditions, each impurity can be well separated from the main component, and the materials before and after destruction are basically balanced; the main peak purity of each destroyed sample and undestroyed sample was analyzed, and the results all met the requirements.

[0109] Example 3: Sensitivity

[0110] Concentrated solutions of each reference substance: Preparation method is the same as that of "Example 1: Specificity test".

[0111] Reference substance stock solution: Take appropriate amounts of impurity I, II, VI, VII, VIII, X, XI, benzaldehyde, and clonazepam reference substance concentrated solutions, respectively, place them in the same volumetric flask, and dilute to the scale with methanol to prepare a solution containing approximately 20 μg of impurity I, 20 μg of impurity II, 100 μg of impurity X, 50 μg each of impurities VI, VII, VIII, and XI, 150 μg of benzaldehyde, and 10 μg of clonazepam per 1 ml.

[0112] Impurity IX reference stock solution: Measure an appropriate amount of the impurity IX reference concentrated solution, place it in a suitable volumetric flask, and dilute to the mark with acetonitrile to make a solution containing approximately 50 μg of impurity IX per 1 ml.

[0113] Quantitation limit solution: Accurately measure an appropriate amount of reference stock solution and dilute it step by step with methanol (impurity IX acetonitrile dilution). If the signal-to-noise ratio S / N is not less than 10, the amount of sample added is the quantitation limit.

[0114] Detection limit solution: accurately measure an appropriate amount of quantitative limit solution, dilute it step by step with methanol (impurity IX acetonitrile dilution), when the signal-to-noise ratio S / N is not less than 3, the amount of sample injected is the detection limit. Repeat the injection of quantitative limit solution for 6 times, and the RSD of the peak area is required to be ≤10%.

[0115] Table 4 Quantitation limit determination results

[0116]

[0117]

[0118]

[0119] Table 5 Detection limit determination results

[0120]

[0121]

[0122] The test results show that the quantitative limit concentration and detection limit concentration of each impurity and clonazepam are low, and can be well detected and accurately quantified; the quantitative limit solution was repeatedly injected 6 times, and the RSD of the peak area was less than 10%, indicating that the method has a high sensitivity.

[0123] Example 4: Linearity and Range

[0124] Take 6 concentration points within the range of the quantitative limit and the 200% limit, perform linear regression on the peak area y against the concentration x, calculate the linear equation and correlation coefficient, and the correlation coefficient r should be greater than 0.995.

[0125] Table 6 Impurity I and Impurity II Linearity Test Results

[0126]

[0127] Table 7 Benzaldehyde and clonazepam linearity test results

[0128]

[0129] Table 8 Impurity VI and impurity VII linearity test results

[0130]

[0131]

[0132] Table 9 Impurity VIII and impurity IX linearity test results

[0133]

[0134] Table 10 Impurity X and impurity XI linearity test results

[0135]

[0136] Impurity I has a good linear relationship in the concentration range of 0.101μg / ml to 4.048μg / ml (n=6); the correlation coefficient r is 1.000; Impurity II has a good linear relationship in the concentration range of 0.100μg / ml to 4.008μg / ml (n=6); the correlation coefficient r is 1.000; Benzaldehyde has a good linear relationship in the concentration range of 0.797μg / ml to 31.900μg / ml (n=6); the correlation coefficient r is 1.000; Clonazepam has a good linear relationship in the concentration range of 0.051μg / ml to 2.050μg / ml (n=6); the correlation coefficient r is 1.000; Impurity VI has a good linear relationship in the concentration range of 0.181μg / ml to 7.225μg / ml (n=6); the correlation coefficient r is 0.999; Impurity VII The linear relationship is good in the concentration range of 0.244μg / ml to 9.742.μg / ml (n=6); the correlation coefficient r is 1.000; the linear relationship of impurity VIII is good in the concentration range of 0.226μg / ml to 9.045.μg / ml (n=6); the correlation coefficient r is 1.000; the linear relationship of impurity IX is good in the concentration range of 0.279μg / ml to 11.175.μg / ml (n=6); the correlation coefficient r is 1.000; the linear relationship of impurity X is good in the concentration range of 0.482μg / ml to 19.299.μg / ml (n=6); the correlation coefficient r is 1.000; the linear relationship of impurity XI is good in the concentration range of 0.265μg / ml to 10.609.μg / ml (n=6); the correlation coefficient r is 1.000.

[0137] Example 5: Correction Factor

[0138] Different people used different instruments and different detectors (ultraviolet, diode array) to perform linear determination of clonazepam and each impurity. The slope of the clonazepam linear equation was divided by the slope of the impurity linear equation to calculate the correction factor of each impurity relative to clonazepam.

[0139] Table 11 Determination results of each impurity correction factor

[0140]

[0141] The correction factors of known impurities in clonazepam injection measured by three different liquid phases were basically consistent. The correction factors of impurities VI, VII, VIII, I, IX, X, II, and XI were 0.89, 0.75, 2.35, 2.30, 1.18, 2.40, 1.18, and 2.19, respectively.

[0142] Example 6: Accuracy

[0143] Accuracy is achieved by measuring the recovery rate between the actual concentration and theoretical concentration of each component. It is required that the recovery rate of each component in the solution with the quantitative limit concentration, 50% limit concentration, 100% limit concentration and 150% limit concentration recovery rate should be between 50% and 150%.

[0144] Table 12 Benzaldehyde average recovery test results

[0145]

[0146]

[0147] Table 13 Impurity I average recovery test results

[0148]

[0149] Table 14 Impurity II average recovery test results

[0150]

[0151] Table 15 Impurity VI average recovery test results

[0152]

[0153]

[0154] Table 16 Impurity VII average recovery test results

[0155]

[0156] Table 17 Impurity VIII average recovery test results

[0157]

[0158]

[0159] Table 18 Impurity IX average recovery test results

[0160]

[0161] Table 19 Impurity X average recovery test results

[0162]

[0163] Table 20 Impurity XI average recovery test results

[0164]

[0165]

[0166] The recovery rates of the quantitative limit levels of each impurity were all within 50%-150%; the recovery rates of other concentration levels were all within 80%-120% and the RSDs were all less than 10% (n=9). This indicates that the recovery rate of the related substance content of this product determined using this chromatographic condition is high and the accuracy is good.

[0167] Example 7: Precision

[0168] Repeatability is achieved by measuring the RSD of the test results of 6 sample solutions, and the RSD should be less than 15%.

[0169] The intermediate precision is tested by different experimenters at different times using different instruments on 12 samples of the test product, and the RSD should be less than 15%.

[0170] Table 21 Repeatability and intermediate precision test results

[0171]

[0172]

[0173] In the repeatability test, the RSDs of the known impurities, maximum unknown impurities and total impurities in the 6 samples were all less than 3.35%, which met the requirements, and the contents of the known impurities, maximum unknown impurities and total impurities were basically consistent; in the intermediate precision test, the RSDs of the known impurities, maximum unknown impurities and total impurities in the 12 samples were all less than 5.86%, and the contents of the known impurities, maximum unknown impurities and total impurities were basically consistent, indicating that the method has good precision.

[0174] Example 8: Solution Stability

[0175] Place the system suitability solution, reference solution, sample solution and test solution in an injector controlled at 6°C, accurately measure 10 μl at different time points and inject into the liquid chromatograph, and record the chromatogram. Statistic the changes in the peak area of ​​each impurity, and calculate the content and RSD of known impurities, maximum unknown impurities and total impurities in the sample solution, requiring the ratio of the peak area to 0 hours to be 80% to 120%.

[0176] Table 22 Results of peak area determination of relevant substance reference solution at 6°C storage stability test -1

[0177]

[0178] Table 23 Results of peak area determination of relevant substance reference solution at 6°C storage stability test -2

[0179]

[0180]

[0181] Table 24 Peak area determination results of the 6°C storage stability test of the reference substance solution -3

[0182]

[0183] Table 25 Results of separation degree determination of solution suitability for related substances at 6°C -1

[0184]

[0185] Table 26 Results of separation degree determination of solution suitability for related substances at 6°C -2

[0186]

[0187] *Note: Fill in the main peak area here.

[0188] Table 27 Related substance determination results of the test solution with sample addition at 6°C

[0189]

[0190] Table 28 Related substances Test results of the stability of the test solution at 6°C

[0191]

[0192]

[0193] At 6°C, the impurity reference solution is stable for 44 hours, the impurity IX reference solution is stable for 101 hours, the system suitability solution is stable for 85 hours, and the test solution and the spiked test solution are stable for 85 hours.

[0194] Example 9: Durability

[0195] Purpose of the durability test: To determine whether the determination of sample-related substances is affected by slight changes in chromatographic conditions.

[0196] The determination conditions are: slight changes in wavelength, column temperature, flow rate, mobile phase ratio, pH value of the salt solution and the use of different chromatographic columns.

[0197] Table 29 Parameters of chromatographic condition changes

[0198]

[0199] Investigate the separation between the main peak, known impurity peak and adjacent impurity peak in the system suitability solution under various conditions, the retention time, tailing factor and theoretical plate number of the main peak and known impurity peak; calculate the impurity determination results in the test solution, use the external standard method for known impurities and the main component external standard method for unknown impurities. The total impurities are the sum of all impurities except benzaldehyde, and calculate the ratio of the content of related substances under various change conditions to the initial conditions.

[0200] 1. The influence of wavelength change on the determination results of related substances

[0201] Accurately measure 10 μl of system suitability solution, test solution, and reference solution, and inject them into the liquid chromatograph at wavelengths of 252 nm, 254 nm, and 256 nm, respectively, and record the chromatograms.

[0202] Table 30 Effect of the change of detection wavelength of related substances on the measurement results

[0203]

[0204]

[0205] From the data in the table above, we can see that the content of related substances in this product is determined using three different wavelengths of 252nm, 254nm and 256nm. The retention time and theoretical plate number of each component peak do not change significantly with the change of wavelength. The separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in wavelength basically does not affect the determination results of related substances in this product.

[0206] 2 Effect of pH value change of phosphate solution in mobile phase on the determination results of related substances

[0207] Accurately measure 10 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph under the condition of mobile phase phosphate pH 6.8, 7.0, and 7.2, and record the chromatograms.

[0208] Table 31 Effect of pH changes of buffer solutions on the determination results

[0209]

[0210]

[0211] From the data in the above table, it can be seen that when the mobile phases prepared by three different pH phosphate solutions were used to determine the related substances of this product, the retention time of each component peak and the number of theoretical plates changed slightly, the separation of each chromatographic peak met the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions did not change significantly. This shows that a slight change in the pH value of the phosphate solution in the mobile phase basically does not affect the determination results of the related substances of this product.

[0212] 3. The influence of slight changes in the proportion of mobile phase on the determination results of related substances

[0213] Accurately measure 10 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph under the condition of changing the mobile phase ratio, and record the chromatogram.

[0214] Table 32 Effect of changes in mobile phase A of related substances on the determination results

[0215]

[0216]

[0217] Table 33 Effect of changes in mobile phase B of related substances on the determination results

[0218]

[0219]

[0220] From the data in the above table, we can see that when different proportions of mobile phase B are used to determine the content of related substances in this product, the retention time and theoretical plate number of each component peak slightly change with the change of the mobile phase ratio. The separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in the proportion of mobile phase B does not basically affect the determination results of the content of related substances in this product. However, when increasing the organic phase ratio of mobile phase A, that is, mobile phase A2, to determine the content of related substances in this product, the ratio of the determination result of impurity VII to the initial conditions is low, so it is necessary to strictly control the proportion of mobile phase A.

[0221] 4. The influence of flow rate change on the determination results of related substances

[0222] Accurately measure 10 μl of system suitability solution, test solution, and reference solution, and inject them into the liquid chromatograph at flow rates of 0.5 ml / min, 0.6 ml / min, and 0.7 ml / min, respectively, and record the chromatograms.

[0223] Table 34 Effect of the flow rate change of related substances on the measurement results

[0224]

[0225]

[0226] From the data in the table above, we can see that when three different flow rates of 0.5ml / min, 0.6ml / min and 0.7ml / min were used to determine the content of related substances in this product, the retention time of each component peak slightly advanced with the increase of flow rate, the number of theoretical plates slightly changed, the separation of each chromatographic peak met the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions did not change significantly. This shows that a slight change in flow rate basically does not affect the determination results of related substances in this product.

[0227] 5. The influence of column temperature change on the determination results of related substances

[0228] Accurately measure 10 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph at column temperatures of 33°C, 35°C, and 37°C, and record the chromatograms.

[0229] Table 35 Effect of column temperature changes on determination results of related substances

[0230]

[0231]

[0232] Conclusion: From the data in the above table, we can see that when three different column temperatures of 33℃, 35℃ and 37℃ are used to determine the content of related substances in this product, the retention time of each component peak is slightly advanced as the column temperature increases, the number of theoretical plates does not change significantly, the separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in column temperature basically does not affect the determination results of related substances in this product.

[0233] 6. The influence of different batches of chromatographic columns of the same model on the relevant measurement results

[0234] Accurately measure 10 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph under the same model but different batches of chromatographic columns to record the chromatograms.

[0235] Table 36 Effect of replacing chromatographic column on the determination results of related substances

[0236]

[0237]

[0238]

[0239] From the data in the table above, we can see that when the same model but different batches of chromatographic columns are used to determine the related substances of this product, the retention time of each component peak and the number of theoretical plates change slightly, the separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that the use of the same model but different batches of chromatographic columns basically does not affect the determination results of the related substances of this product.

[0240] In summary, the system applicability and specificity, sensitivity, linearity and range, accuracy, precision, solution stability and durability of this method all meet the requirements, indicating that this method is suitable for the detection of related substances in clonazepam injection.

[0241] The above is a description of a specific embodiment of the present invention. It should be understood that the present invention is not limited to the above embodiment, and the above embodiment and description are only for illustrating the principle of the present invention.

[0242] Those skilled in the art may make various non-substantial changes and improvements to the present invention without departing from the concept of the present invention, all of which fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting related substances in clonazepam injection, the method using high performance liquid chromatography, the chromatographic column being C 18 The column is gradient eluted with phosphate buffer-acetonitrile as the mobile phase, the pH value of the phosphate buffer is 6 to 8, preferably the pH value is 6.8 to 7.2, and the related substances include the following substances:

2. The detection method according to claim 1, characterized in that: The C18 chromatographic column is a SHIMADZU Shim-pack Scepter HD-C18-80 chromatographic column with a specification of 4.6 mm×150 mm and 3 μm.

3. The detection method according to claim 1, characterized in that: The chromatographic conditions include: detection wavelength: 250-260 nm; preferably detection wavelength: 252-256 nm; more preferably detection wavelength: 254 nm; More preferably, the flow rate is 0.4-1.0 ml / min; preferably 0.5-0.7 ml / min; more preferably 0.6 ml / min; More preferably, the column temperature is 30-40°C; preferably, the column temperature is 33-37°C; more preferably, the column temperature is 35°C; More preferably, the injection volume is 5 to 50 μl; preferably, the injection volume is 5 to 15 μl; more preferably, the injection volume is 10 μl; More preferably, the injector temperature is 2-15°C; preferably the injector temperature is 5-8°C; more preferably the injection temperature is 6°C.

4. The detection method according to claim 1, characterized in that: The pH value of the phosphate buffer is 7.0; the phosphate is selected from a mixture of one or more of diammonium hydrogen phosphate and dipotassium hydrogen phosphate, preferably diammonium hydrogen phosphate.

5. The detection method according to claim 1, characterized in that: The volume ratio of phosphate buffer to acetonitrile in the mobile phase A is (80-100):(0-20), preferably (88-92):(8-12), and more preferably 90:10; the volume ratio of phosphate buffer to acetonitrile in the mobile phase B is (20-40):(60-80), preferably (28-32):(68-72); more preferably 30:

70.

6. The detection method according to any one of claims 1 to 5, characterized in that: The detection steps include: (1) Solution preparation: Prepare the positioning solution, system suitability solution, test sample solution, reference sample stock solution and reference sample solution respectively; (2) Determination method: After the system is stable, inject the system suitability solution, positioning solution, test solution and reference solution respectively, and record the chromatogram.

7. The detection method according to claim 6, characterized in that: The detection steps include: The positioning solution is a benzyl alcohol solution; The reference substance stock solution is prepared by weighing appropriate amounts of clonazepam, impurity I, impurity II, impurity VI, impurity VII, impurity VIII, impurity X, impurity XI and benzaldehyde, placing them in a suitable volumetric bottle, and dissolving and diluting them with methanol to prepare a stock solution; weighing an appropriate amount of impurity IX, placing it in a suitable volumetric bottle, and dissolving and diluting it with acetonitrile to prepare a stock solution; The system suitability solution: take appropriate amount of benzaldehyde and impurity I, II, VI, VII, VIII, IX, X, XI reference substance stock solutions, place them in a volumetric bottle, add clonazepam injection to dissolve and dilute to the scale, and shake well; The test solution: Take this product and get it; The reference substance solution: take an appropriate amount of the reference substance stock solution, place it in a volumetric flask, add methanol to dilute to the scale, and shake well; the mobile phase gradient elution procedure is as follows: ； Preferably, the mobile phase gradient elution procedure is as follows: 。 8. A compound represented by an impurity X and its Z isomer:

9. A compound represented by impurity XI:

10. Use of the detection method according to any one of claims 1 to 7 for detecting related substances in clonazepam raw materials or preparations.

Images