Method for measuring content of hydrazine in carbidopa-containing drug

Abstract

A method for measuring the content of hydrazine in a carbidopa-containing drug, comprising the following steps: (1) in a solvent, under the action of acid, mixing a sample under test with an aldehydes substance, said sample being a carbidopa-containing drug, and hydrazine in said sample and the aldehydes substance being derivatized to obtain a test solution; and (2) measuring the content of the hydrazine in the test solution. Compared with the prior art, the method for measuring the content of hydrazine in a carbidopa-containing drug does not require pretreatment to remove carbidopa, introduces negligible hydrazine during measurement, is easy to operate, does not need to accurately control derivatization time and sample introduction time, and exhibits good repeatability and accurate measurements.

Description

A method for detecting hydrazine content in drugs containing carbidopa

[0001] This application claims priority to Chinese Patent Application No. 2023110640677, filed on August 22, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The invention belongs to the field of analytical technology and relates to a method for detecting the hydrazine content in a medicine containing carbidopa. Background Art

[0003] Parkinson's patients need to take a combination preparation of levodopa and carbidopa (hereinafter referred to as carbidopa-levodopa preparation), especially early Parkinson's patients and patients with hereditary dopa-responsive dystonia (DRD). They need to take this preparation for a long time, and as time goes by, the dosage increases year by year. Therefore, the lower the hydrazine content in the preparation, the better, so as to avoid the higher cancer risk caused by the treatment of Parkinson's disease.

[0004] According to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for genotoxic impurity limits, the limit for genotoxic impurities in this type of preparation is approximately 20 to 50 ppm. Therefore, establishing a highly sensitive, quantitatively accurate, reproducible, convenient and effective hydrazine detection method is critical for controlling the hydrazine content in carbidopa preparations, and is therefore of great significance for ensuring the health of patients.

[0005] Because hydrazines have the characteristics of lacking chromophores, strong activity, volatility, low relative molecular mass, high polarity, and strong reducibility, the detection method presents special requirements in terms of sensitivity, selectivity, stability of the analyte, matrix complexity, etc., so it has different characteristics from other drug impurity analysis and detection in the development and selection of analytical methods. In addition, hydrazine compounds easily interact with the silanyl groups on the chromatographic column, resulting in severe tailing of the peak shape, which is a difficulty in the establishment of analytical methods. Hydrazine is difficult to detect by direct chromatography and spectroscopy, but it can be derivatized by the active group amino group of hydrazine to generate derivatives with strong ultraviolet absorption ability, and within a certain concentration range, the absorption value of the derivative is linearly related to the hydrazine content, and the hydrazine content can be measured by liquid chromatography. In the prior art, aldehydes are often used as derivatization reagents, and the amino group and the aldehyde group undergo condensation reaction to generate corresponding hydrazone compounds, which have strong ultraviolet absorption ability, good stability, and high sensitivity. However, this method is greatly affected by the analysis sample matrix.

[0006] When the existing technology is used to determine the hydrazine content in carbidopa raw materials, the hydrazine group in the carbidopa structure will also participate in the reaction during the derivatization reaction, resulting in a significant increase in the hydrazine content, affecting the accuracy of the determination result.

[0007] There is no control for the hydrazine content of carbidopa preparations in any of the pharmacopoeias of various countries. Control of the hydrazine content in carbidopa raw materials is only found in the European Pharmacopoeia (European Pharmacopoeia EP10.0 Carbidopa Raw Material Text). The European Pharmacopoeia has a method for detecting hydrazine in carbidopa raw materials in the prior art. This method uses an ion exchange resin to first remove carbidopa and then undergoes a derivatization reaction with salicylaldehyde. However, this method has complicated processing steps and requires avoiding sample degradation during the resin removal phase, which leads to the production of hydrazine. Sample detection uses thin-layer chromatography, which has low sensitivity. A literature report (Kean, Thomas & Miller, J & Skellern, G & Snodin, David. (2007). Acceptance criteria for levels of hydrazine in substances for pharmaceutical use and analytical methods for its determination. Pharmeuropa scientific notes. 2006. 23-33.) reported that some manufacturers complained that this method was difficult to implement and was not satisfactory. Therefore, a method using gas chromatography was developed for detection, but this is not described in detail in the literature. The document also compared the use and non-use of ion exchange resin to remove carbidopa, and then carried out derivatization reaction with benzaldehyde and then detected by liquid chromatography. It was found that the hydrazine content after removing carbidopa with ion exchange resin was basically consistent with the result measured by the European Pharmacopoeia method (3ppm), but the result measured without ion exchange resin was abnormally high (239ppm), which was mainly due to the interference of carbidopa during the determination. Although this method is simpler and easier to use than the European Pharmacopoeia method, it requires ion exchange to remove carbidopa, has many steps, is time-consuming, and has many influencing factors, which places high demands on the operator's operational ability. Moreover, the above methods are only applicable to the detection of raw materials. The preparation products usually contain a large amount of excipients and are not necessarily applicable. In particular, the sustained-release preparations containing polymer excipients are easy to agglomerate during sample processing, making the above methods difficult to apply.

[0008] Therefore, a method for detecting hydrazine content that is simple to operate, has good repeatability, and can be used for quality control of commercial products is needed, and how to solve the introduction of hydrazine content during the determination process is very important.

[0009] Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a detection method for measuring the hydrazine content in a drug containing carbidopa. Currently, the disclosed prior art lacks a detection method for the hydrazine content in a preparation containing carbidopa. There is only a method for determining hydrazine in a carbidopa raw material, and this method requires first removing carbidopa with an ion exchange resin. The operation is cumbersome and time-consuming. The preparation product contains a large amount of excipients, especially polymer excipients, which are more difficult to extract in an aqueous solution. If carbidopa is not removed, the measurement result will be abnormally high and the repeatability is poor. The present invention does not require the removal of carbidopa, and does not require the deduction of the hydrazine background (no more than 2 ppm) introduced during the measurement process. The method uses a highly sensitive and widely used liquid chromatography for detection. The experimental steps are simple, the repeatability is good, the accuracy is high, and it can be used for quality control of commercially produced products.

[0011] In order to achieve the above object, the present invention provides the following technical solutions:

[0012] The present invention provides a method for detecting the hydrazine content in a drug containing carbidopa, comprising the following steps:

[0013] (1) In a solvent, under the action of an acid, a sample to be tested and an aldehyde substance are mixed, wherein the sample to be tested is a drug containing carbidopa, and hydrazine in the sample to be tested is derivatized with the aldehyde substance to obtain a test solution;

[0014] (2) Detecting the hydrazine content in the test solution;

[0015] The solvent is an alcohol solvent, or a mixed solvent of an alcohol solvent and water;

[0016] In the solvent, the volume fraction of the alcohol solvent is 90% to 100%, and the volume fraction refers to the percentage of the alcohol solvent in the total volume of the solvent;

[0017] The alcohol solvent is one or more of methanol, ethanol and isopropanol;

[0018] The aldehyde substance is one or more of benzaldehyde, salicylaldehyde and dimethylaminobenzaldehyde.

[0019] In one embodiment, in step (1), the volume fraction of the alcohol solvent in the solvent is 90%, 92%, 94%, 96%, 98% or 100%, where the volume fraction refers to the percentage of the alcohol solvent in the total volume of the solvent.

[0020] In one embodiment, in step (1), the alcohol solvent is methanol and / or ethanol, preferably methanol.

[0021] In one embodiment, in step (1), the concentration of the acid is preferably 0.01 to 5 mmol / L, such as 0.02 mmol / L, 0.5 mmol / L, 1 mmol / L or 5 mmol / L, preferably 0.5 mmol / L; the concentration of the acid is the concentration of the acid in the reaction system of step (1).

[0022] In one embodiment, in step (1), the acid is conventional in the art, preferably one or more of HCl, HNO3 and TFA (trifluoroacetic acid), more preferably HCl.

[0023] In one embodiment, in step (1), the sample to be tested preferably contains 2 mg to 50 mg of carbidopa, more preferably 5 mg to 15 mg of carbidopa, for example, 5 mg, 10 mg, 12.5 mg or 15 mg of sample weight.

[0024] In a certain embodiment, in step (1), the drug containing carbidopa is preferably a carbidopa bulk drug (preferably a monohydrate carbidopa bulk drug) or a preparation containing carbidopa; preferably, the preparation is a rapid-release preparation or a controlled-release preparation (e.g., a sustained-release preparation); further preferably, the preparation containing carbidopa is a carbidopa tablet, a carbidopa controlled-release tablet, a carbidopa sustained-release capsule, a carbidopa sustained-release tablet, a carbidopa tablet, or an entacapone carbidopa tablet.

[0025] In one embodiment, in step (1), the drug containing carbidopa is preferably carbidopa, carbidopa-levodopa or entacapone-carbidopa-levodopa.

[0026] In one embodiment, in step (1), the aldehyde substance is benzaldehyde.

[0027] In one embodiment, in step (1), the volume percentage concentration of the aldehyde substance is 0.1% to 5%, for example, 0.2%, 0.8%, 1.6% or 2%, preferably 0.8%; the concentration of the aldehyde substance is the volume percentage concentration of the aldehyde substance in the reaction system of step (1).

[0028] In one embodiment, in step (1), the derivatization is a condensation reaction between the aldehyde substance and the hydrazine in the drug containing carbidopa; preferably, the derivatization is a condensation reaction between benzaldehyde and the hydrazine in the drug containing carbidopa to generate a hydrazone substance.

[0029] In one embodiment, in step (1), the derivatization time is preferably 1-10 minutes, such as 1 minute, 2 minutes, 3 minutes, 5 minutes or 10 minutes, preferably 3 minutes.

[0030] In one embodiment, in step (1), the derivatization may include conventional mixing operations, such as vortexing, shaking and stirring, preferably vortexing.

[0031] In one embodiment, in step (1), the derivatization reaction system consists of the solvent, the acid, the drug containing carbidopa, and the aldehyde substance.

[0032] In a certain embodiment, in step (1), the derivatization may further include a filtration step; the filtration is preferably filtration through a nylon filter membrane; the nylon filter membrane is preferably a nylon filter membrane with a pore size of 0.45 μm.

[0033] In one embodiment, in step (2), the detection determines the content of hydrazine in the drug containing carbidopa by detecting the content of the derivatization product in the test solution.

[0034] In a certain embodiment, in step (2), the time from the sample solution to the injection test does not need to be specially controlled, and is preferably within 10 minutes, further preferably within 5 minutes, and even more preferably within 3 minutes. For example, the obtained sample solution is immediately injected and tested.

[0035] In one embodiment, in step (2), the detection is performed by conventional detection methods in the art, preferably liquid chromatography, gas chromatography, thin layer chromatography or ultraviolet spectrophotometry, more preferably liquid chromatography.

[0036] In one embodiment, the detection method further includes the step of preparing a reference solution. The reference solution can be prepared by conventional methods, for example, by replacing the drug containing carbidopa with a hydrazine reference solution (e.g., hydrazine dihydrochloride) and preparing it by the same method as the test solution.

[0037] In one embodiment, in step (2), when the detection is performed by liquid chromatography, the chromatographic column in the liquid chromatography is a Waters Xbridge C18 chromatographic column, preferably a Waters Xbridge C18 4.6-mm×10-cm 5 μm chromatographic column.

[0038] In one embodiment, in step (2), when liquid chromatography is used for detection, mobile phase A is acetonitrile, mobile phase B is an aqueous solution of 0.01-0.1% disodium ethylenediaminetetraacetate, and the volume ratio of mobile phase A to mobile phase B is (25:75)-(75:25).

[0039] In one embodiment, in step (2), when liquid chromatography is used for detection, mobile phase A is acetonitrile, mobile phase B is an aqueous solution of 0.03% disodium ethylenediaminetetraacetate, and the volume ratio of mobile phase A to mobile phase B is 55:45.

[0040] In one embodiment, in step (2), when liquid chromatography is used for detection, the detection wavelength is 310 nm.

[0041] In one embodiment, in step (2), when detection is performed by liquid chromatography, the flow rate is 1.5 mL / min.

[0042] In one embodiment, in step (2), when detection is performed by liquid chromatography, the column temperature is 35°C.

[0043] In a certain embodiment, in step (2), when the detection is performed by liquid chromatography, the test solution and the reference solution are respectively injected into the liquid chromatograph, preferably, the injection volume is 20 μL.

[0044] In one embodiment, the method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps:

[0045] (1) In methanol, under the action of hydrochloric acid, a test sample and benzaldehyde are mixed, wherein the test sample is a drug containing carbidopa, and hydrazine in the test sample is derivatized with the aldehyde substance to obtain a test solution;

[0046] (2) Liquid chromatography was used to detect the hydrazine content in the test solution.

[0047] In one embodiment, the method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps:

[0048] (1) A drug containing carbidopa is mixed with a methanolic hydrochloric acid solution (e.g., 1 mL of a 2.5 mmol / L methanolic hydrochloric acid solution) (e.g., for 10 to 15 seconds), and then derivatized with a methanolic benzaldehyde solution (e.g., 1 mL of a 0.25%, 1.0%, or 2.0% benzaldehyde methanol solution) to obtain a test solution;

[0049] (2) The hydrazine content in the test solution is immediately detected by liquid chromatography.

[0050] The detection method of the present invention can also be applied to benserazide; the benserazide can be a benserazide bulk drug or a preparation containing benserazide.

[0051] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0052] The reagents and raw materials used in the present invention are all commercially available, and the reagents used for sample treatment are all analytically pure.

[0053] The positive progress effect of the present invention is:

[0054] 1. Compared with the prior art, the present invention does not require pretreatment to remove carbidopa, the hydrazine introduced during the determination process is negligible, the operation is simple, there is no need to precisely control the derivatization time and injection time, the results are reproducible, and the quantitative results are accurate. In the prior art, due to the presence of carbidopa during the experiment, the hydrazine content measurement result will be higher than the true value, so the hydrazine content generated during the reaction needs to be deducted, but the measurement results are poorly reproducible. The present invention can reduce the hydrazine content introduced during the reaction process to a negligible level (no more than 2 ppm), significantly improving reproducibility and facilitating the quality control of carbidopa-containing preparations and the popularization of the method.

[0055] 2. Compared with existing technologies, the present invention is applicable to a significantly wider range of products. Existing technologies can only detect carbidopa APIs. The present invention is applicable not only to carbidopa APIs, but also to various common preparations and sustained-release preparations containing carbidopa. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG1 is a chromatogram of a blank solvent in the specificity investigation of the present invention;

[0057] FIG2 is a chromatogram of a reference solution in the specificity investigation of the present invention;

[0058] FIG3 is a chromatogram of a blank mixed powder solution in the specificity investigation of the present invention;

[0059] FIG4 is a chromatogram of the test solution in the specificity investigation of the present invention. DETAILED DESCRIPTION

[0060] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0061] 50% methanol is a methanol aqueous solution with a volume fraction of 50%

[0062] 1% benzaldehyde methanol solution is a 1% benzaldehyde methanol solution by volume

[0063] Hydrochloric acid methanol solution is prepared by mixing concentrated hydrochloric acid and methanol to the corresponding concentration.

[0064] The batch numbers of the samples to be tested used in the present invention are as follows:

[0065] Example 1: Determination of the hydrazine content of carbohydride-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using 2.5 mmol / L hydrochloric acid-methanol solutions with different methanol ratios (50% / 100% methanol)

[0066] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0067] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L methanolic hydrochloric acid solution (50% / 100% methanol) to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0068] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution (50% / 100% methanol), vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of 1% benzaldehyde methanol solution, stopper it tightly, place it on a vortex instrument and vortex vigorously for 3 minutes, filter it with a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0069] Accurately measure 20 μL of each test solution and reference solution and inject them into a liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area using the external standard method. The results are detailed in Table 1 below.

[0070] Table 1. Determination results of hydrazine content in methanol solution with different ratios

[0071] Example 2: Determination of the hydrazine content of carbohydaldehyde-containing controlled-release tablets (WD-1603, Handu Pharmaceutical) using benzaldehyde solutions of different volume percentage concentrations (0.25% / 1.0% / 2.0%)

[0072] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0073] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L methanolic hydrochloric acid to a solution containing approximately 0.625 μg of hydrazine per 1 mL to prepare this reference substance stock solution. Accurately measure 1 mL of this reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a methanolic solution of benzaldehyde (0.25% / 1.0% / 2.0%), stopper tightly, and vortex vigorously for 3 minutes on a vortexer to prepare this reference substance solution.

[0074] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution, vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of benzaldehyde (0.25% / 1.0% / 2.0%) methanol solution, stopper the tube tightly, place it on a vortex instrument and vortex vigorously for 3 minutes, filter it through a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0075] Accurately measure 20 μL of each test solution and reference solution and inject them into a liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area using the external standard method. The results are shown in Table 2 below.

[0076] Table 2. Determination results of hydrazine content in benzaldehyde solutions of different concentrations

[0077] Example 3: Determination of hydrazine content in carbidopa-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different sample amounts (equivalent to approximately 5 mg / 10 mg / 15 mg of carbidopa)

[0078] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0079] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L methanolic hydrochloric acid to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0080] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 5 mg / 10 mg / 15 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution, vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of 1% benzaldehyde methanol solution, stopper the tube tightly, place it on a vortex instrument and vortex vigorously for 3 minutes, filter it through a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0081] Accurately measure 20 μL of each test solution and reference solution and inject them into a liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area using the external standard method. The results are shown in Table 3 below.

[0082] Table 3. Determination results of hydrazine content using different sample amounts

[0083] Example 4: Determination of the hydrazine content of carbocycline-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different acid types (hydrochloric acid / nitric acid / trifluoroacetic acid)

[0084] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5 μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0085] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L hydrochloric acid / nitric acid / trifluoroacetic acid methanol solution to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0086] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid / nitric acid / trifluoroacetic acid methanol solution, vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of 1% benzaldehyde methanol solution, stopper the tube tightly, place it on a vortex instrument and vortex vigorously for 3 minutes, filter it through a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (to be injected immediately).

[0087] Accurately measure 20 μL of each test solution and reference solution and inject them into a liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area using the external standard method. The results are shown in Table 4 below.

[0088] Table 4. Determination results of hydrazine content using different types of acid

[0089] Example 5: Determination of hydrazine content in carbocycline-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different acid concentrations (0.1 / 2.5 / 5 mmol / L)

[0090] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0091] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 0.1 / 2.5 / 5 mmol / L methanolic hydrochloric acid solutions to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0092] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 0.1 / 2.5 / 5 mmol / L hydrochloric acid methanol solution, vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of 1% benzaldehyde methanol solution, stopper the tube tightly, place it on a vortex instrument and vortex vigorously for 3 minutes, filter it through a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0093] Accurately measure 20 μL of each test solution and reference solution and inject them into the liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area according to the external standard method. The results are shown in Table 5 below.

[0094] Table 5. Determination results of hydrazine content using different acid concentrations

[0095] Example 6: Determination of the hydrazine content of carbohydride-dopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different types of alcohol (methanol / ethanol)

[0096] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0097] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L hydrochloric acid in methanol (ethanol) to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol (ethanol), stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0098] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol (ethanol) solution, vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of 1% benzaldehyde methanol (ethanol) solution, stopper the tube tightly, place it on a vortex instrument and vortex vigorously for 3 minutes, filter it with a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0099] Accurately measure 20 μL of each test solution and reference solution and inject them into the liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area according to the external standard method. The results are shown in Table 6 below.

[0100] Table 6. Determination results of hydrazine content using different types of alcohols

[0101] Example 7: Determination of hydrazine content in carbocycline-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) at different reaction times (1 / 2 / 3 / 5 / 10 minutes)

[0102] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0103] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L methanolic hydrochloric acid to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0104] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution, vortex rapidly for 10 to 15 seconds to moisten it, then accurately add 4 mL of 1% benzaldehyde methanol solution, stopper the tube tightly, place it on a vortex instrument and vortex vigorously for 1 / 2 / 3 / 5 / 10 minutes, filter it through a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0105] Accurately measure 20 μL of each test solution and reference solution and inject them into the liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area according to the external standard method. The results are shown in Table 7 below.

[0106] Table 7. Determination results of hydrazine content at different reaction times

[0107] As can be seen from the results in the table above, when using methanol solution, even as the reaction time increases, the hydrazine group in the carbidopa structure basically does not participate in the reaction during the derivatization reaction. There is no need to remove carbidopa, and there is no need to deduct the hydrazine background introduced during the determination process. The stability and accuracy of the determination results are good.

[0108] Example 8: Determination of hydrazine content in different products containing carbidopa

[0109] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0110] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L methanolic hydrochloric acid to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0111] Preparation of test solution: Accurately weigh an appropriate amount of fine powder (equivalent to approximately 12.5 mg of carbidopa) and place it in a stoppered test tube. Accurately add 1 mL of 2.5 mmol / L hydrochloric acid in methanol. Vortex rapidly for 10 to 15 seconds to moisten the tube. Accurately add 4 mL of 1% benzaldehyde in methanol. Seal the tube tightly. Vortex vigorously on a vortex machine for 3 minutes. Filter through a nylon filter membrane with a pore size of 0.45 μm. The filtrate is used as the test solution (to be injected immediately).

[0112] Accurately measure 20 μL of each test solution and reference solution and inject them into a liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area using the external standard method. The results are shown in Table 8 below.

[0113] Table 8. Determination results of different carbidopa-containing drugs

[0114] Example 9: Determination of hydrazine content in carbocycline-levodopa controlled-release tablets (WD-1603, Handu Pharmaceuticals)

[0115] Chromatographic conditions: A Waters Xbridge C18 4.6-mm×10-cm 5μm column was used; the mobile phase was acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45); the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0116] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with 2.5 mmol / L methanolic hydrochloric acid to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0117] Preparation of test solution: Take 10 carbidopa-levodopa controlled-release tablets, accurately weigh them, grind them into powder, accurately weigh an appropriate amount of fine powder (equivalent to about 12.5 mg of carbidopa), place it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution and 4 mL of 1% benzaldehyde methanol solution, stopper it tightly, place it on a vortex machine and vortex vigorously for 3 minutes, filter it through a nylon filter membrane with a pore size of 0.45 μm, and take the filtrate as the test solution (need to be injected immediately).

[0118] Accurately measure 20 μL each of the test solution and the reference solution and inject them into the liquid chromatograph. Record the chromatogram. Calculate the hydrazine content using the peak area using the external standard method. The results are shown in Table 9 below.

[0119] Table 9. Hydrazine determination results of carbendazim and levodopa controlled-release tablets

[0120] Example 10

[0121] The hydrazine content of carbidopa raw material was determined using a 2.5 mmol / L hydrochloric acid methanol (water) solution with different methanol ratios (0% / 50% / 60% / 70% / 80% / 90% / 100% methanol) at different reaction times (1 / 2 / 3 / 5 / 10 minutes). Chromatographic conditions were as follows: a Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetic acid) (55:45) as the mobile phase; the detection wavelength was 310 nm; the flow rate was 1.5 mL / min; and the column temperature was 35°C.

[0122] Preparation of Reference Substance: Accurately weigh approximately 25.6 mg of hydrazine dihydrochloride reference substance, dissolve in water, and dilute to a solution containing approximately 6.25 μg of hydrazine per 1 mL. Accurately measure an appropriate amount and dilute with a 2.5 mmol / L methanolic hydrochloric acid solution (0% / 50% / 60% / 70% / 80% / 90% / 100% methanol) to a solution containing approximately 0.625 μg of hydrazine per 1 mL. This will serve as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution and place it in a stoppered test tube. Accurately add 4 mL of a 1% benzaldehyde solution in methanol, stopper tightly, and vortex vigorously for 3 minutes. This will serve as the reference substance solution.

[0123] Preparation of test solution: Take 13.5 mg of carbidopa monohydrate bulk drug substance (equivalent to 12.5 mg of carbidopa), accurately weigh, place in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol (water) solution (0% / 50% / 60% / 70% / 80% / 90% / 100% methanol), rapidly vortex for 10-15 seconds to wet, then accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, place in a vortex instrument and vortex vigorously for 1 / 2 / 3 / 5 / 10 minutes, filter through a nylon filter with a pore size of 0.45 μm, and take the filtrate as the test solution (to be injected immediately).

[0124] Accurately measure 20 μL each of the test solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the hydrazine content based on the peak area using the external standard method.

[0125] The test results of 0% and 100% methanol are shown in Tables 10-11 below.

[0126] Table 10. Determination results of hydrazine content in mmol / L hydrochloric acid aqueous solution

[0127] Table 11. Determination results of hydrazine content in mmol / L hydrochloric acid methanol solution (100% methanol)

[0128] As can be seen from the results in the table above, when a hydrochloric acid solution with a higher water content is used, without prior removal of carbidopa with an ion exchange resin, as the reaction time increases, the degree to which the hydrazine group in the carbidopa structure participates in the reaction during the derivatization reaction increases, resulting in a significant increase in the hydrazine content and poor stability and accuracy of the measurement results.

[0129] Methodological investigation:

[0130] 1. Exclusivity

[0131] The blank excipient and blank solvent had no interference with the detection of hydrazine, as shown in the accompanying drawings (Figures 1-4) of the specification for details.

[0132] 2. System applicability

[0133] The reference solution was injected six times continuously (three injections for STD1 and three injections for STD2), and the response factor (RSD) and tailing factor (tailing factor) were calculated. The results are shown in Table 12. The system suitability test results met the requirements.

[0134] Table 12. Results of the suitability of the hydrazine system for carbodiol-2-dopa controlled-release tablets

[0135] 3. Linearity and range

[0136] Six reference solutions at six limiting concentration levels were prepared, with one aliquot of each concentration measured. The results are shown in Table 13. Hydrazine content showed good linearity within the concentration range of 0.0624 μg / mL to 1.2490 μg / mL.

[0137] Table 13. Linearity and range determination results of hydrazine in carbodiol controlled-release tablets

[0138] 4. Accuracy test

[0139] An appropriate amount of raw material and excipient powder mixture (equivalent to 12.5 mg of carbidopa) was accurately weighed, and a hydrazine reference substance at concentration limits of 40%, 80%, 100%, and 120% was added, respectively. Three replicates were prepared for each concentration according to the method described in Example 9. The results are detailed in Table 14. The average recoveries of hydrazine at concentrations of 40%, 80%, 100%, and 120% ranged from 70.0% to 130.0%. The RSDs for the nine sample measurements were less than 15.0%, demonstrating satisfactory accuracy.

[0140] Table 14. Accuracy results of carbohydrazine for levodopa controlled-release tablets

[0141] 5. Repeatability

[0142] Six spiked (40% limit concentration level) test sample solutions were prepared in parallel. The results are shown in Table 15. The repeatability was good.

[0143] Table 15. Repeatability results of carbohydrazine for controlled-release levodopa tablets (n=6)

[0144] 6. Limit of Quantitation and Limit of Detection

[0145] The quantification limit solution was prepared by diluting the solution downward based on the peak elution of the lowest concentration linear solution until the signal-to-noise ratio of the main peak was approximately 10, and this solution was injected six times continuously. The detection limit solution was prepared by diluting the solution downward based on the peak elution of the quantification limit solution until the signal-to-noise ratio of the main peak was approximately 3. The concentrations of the quantification limit and detection limit were 0.0624 μg / mL and 0.0187 μg / mL, respectively, and the concentration levels were 5 ppm and 1.5 ppm, respectively. The results are detailed in Tables 16-17. The results demonstrate that the present invention has high detection sensitivity.

[0146] Table 16. Limit of quantification (LOQ) and limit of detection (LOD) results for carbohydrazine levodopa controlled-release tablets

[0147] Table 17. Repeatability of the limit of quantitation of hydrazine for carbodiol controlled-release tablets (n=6)

[0148] The results showed that the method's specificity, system suitability, linearity and range, limit of quantification and limit of detection, accuracy and repeatability all met the validation requirements and was suitable for the detection of hydrazine in WD-1603 products.

Claims

1. A method for detecting the hydrazine content in a drug containing carbidopa, characterized in that: The steps include: (1) In a solvent, under the action of an acid, a sample to be tested and an aldehyde substance are mixed, wherein the sample to be tested is a drug containing carbidopa, and hydrazine in the sample to be tested is derivatized with the aldehyde substance to obtain a test solution; (2) Detecting the hydrazine content in the test solution; The solvent is an alcohol solvent, or a mixed solvent of an alcohol solvent and water; In the solvent, the volume fraction of the alcohol solvent is 90% to 100%, and the volume fraction refers to the percentage of the alcohol solvent in the total volume of the solvent; The alcohol solvent is one or more of methanol, ethanol and isopropanol; The aldehyde substance is one or more of benzaldehyde, salicylaldehyde and dimethylaminobenzaldehyde.

2. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 1, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one or more of the following conditions: (1) In the solvent, the volume fraction of the alcohol solvent is 90%, 92%, 94%, 96%, 98% or 100%; (2) The alcohol solvent is methanol and / or ethanol; (3) The concentration of the acid is 0.01 to 5 mmol / L; the concentration of the acid is the concentration of the acid in the reaction system of step (1); (4) The acid is one or more of HCl, HNO3 and trifluoroacetic acid; (5) The sample to be tested contains 2 mg to 50 mg of carbidopa; (6) The carbidopa-containing drug is a carbidopa API (preferably a monohydrated carbidopa API) or a preparation containing carbidopa; (7) The aldehyde substance is benzaldehyde; (8) The concentration of the aldehyde substance is 0.1% to 5%; the concentration of the aldehyde substance is the volume percentage concentration of the aldehyde substance in the reaction system of step (1); (9) The derivatization time is 1-10 minutes; (10) The time from the sample solution to the injection test is within 10 minutes; and (11) The detection is carried out by liquid chromatography, gas chromatography, thin layer chromatography or ultraviolet spectrophotometry.

3. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 2, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one or more of the following conditions: (1) The alcohol solvent is methanol; (2) The concentration of the acid is 0.02 mmol / L, 0.5 mmol / L, 1 mmol / L or 5 mmol / L; (3) The acid is HCl; (4) The sample to be tested contains 5 mg to 15 mg of carbidopa; (5) The preparation is an immediate release preparation or a controlled release preparation, and the controlled release preparation is preferably a sustained release preparation; (6) The concentration of the aldehyde substance is 0.2%, 0.8%, 1.6% or 2%; (7) The derivatization time is 1 minute, 2 minutes, 3 minutes, 5 minutes or 10 minutes; (8) The time from the sample solution to the injection test is within 3 minutes; and (9) The detection is performed by liquid chromatography.

4. The method for detecting the hydrazine content in a drug containing carbidopa as claimed in claim 3, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one or more of the following conditions: (1) The concentration of the acid is 0.5 mmol / L; (2) The sample to be tested contains 5 mg, 10 mg, 12.5 mg or 15 mg of carbidopa; (3) The preparation containing carbidopa is carbidopa tablets, carbidopa controlled-release tablets, carbidopa sustained-release capsules, carbidopa sustained-release tablets, carbidopa tablets or entacapone carbidopa tablets; (4) The concentration of the aldehyde substance is 0.8%; (5) the derivatization time is 3 minutes; and (6) The obtained test solution is immediately sampled and tested.

5. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 1, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one or both of the following conditions: (1) the derivatization operation is vortexing, shaking or stirring, preferably vortexing; and (2) The derivatization further comprises a filtration step, such as filtration with a nylon filter membrane, wherein the nylon filter membrane is preferably a nylon filter membrane with a pore size of 0.45 μm.

6. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 5, characterized in that: In step (1), the derivatization reaction system consists of the solvent, the acid, the drug containing carbidopa and the aldehyde substance.

7. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 1, characterized in that: The detection method further includes the step of preparing a reference solution, for example, replacing the drug containing carbidopa with a hydrazine reference solution (such as hydrazine dihydrochloride) by the same method as the test solution in step (1) as claimed in claim 1.

8. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 7, characterized in that: When the detection is performed by liquid chromatography, the test solution and the reference solution are respectively injected into a liquid chromatograph, preferably, the injection volume is 20 μL.

9. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 1, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one or both of the following conditions: (1) When the detection is performed by liquid chromatography, the chromatographic column in the liquid chromatography is a Waters Xbridge C18 chromatographic column; and (2) When the detection is performed by liquid chromatography, the mobile phase A is acetonitrile, the mobile phase B is a 0.01-0.1% aqueous solution of disodium ethylenediaminetetraacetate, and the volume ratio of the mobile phase A to the mobile phase B is 25:75-75:

25.

10. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 1, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one or more of the following conditions: (1) When the detection is performed by liquid chromatography, the chromatographic column in the liquid chromatography is a Waters Xbridge C18 4.6-mm×10-cm 5μm chromatographic column; (2) When the detection is performed by liquid chromatography, mobile phase A is acetonitrile, mobile phase B is an aqueous solution of 0.03% disodium ethylenediaminetetraacetate, and the volume ratio of mobile phase A to mobile phase B is 55:45; (3) When the detection is carried out by liquid chromatography, the detection wavelength is 310 nm; (4) When the detection is by liquid chromatography, the flow rate is 1.5 mL / min; and (5) When the detection is performed by liquid chromatography, the column temperature is 35°C.

11. The method for detecting the hydrazine content in a drug containing carbidopa according to claim 1, characterized in that: The method for detecting the hydrazine content in the drug containing carbidopa satisfies one of the following schemes: Solution 1: The method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps: (1) In methanol, under the action of hydrochloric acid, a sample to be tested and benzaldehyde are mixed, wherein the sample to be tested is a drug containing carbidopa, and hydrazine in the sample to be tested is derivatized with the aldehyde substance to obtain a test solution; (2) Detecting the hydrazine content in the test solution by liquid chromatography; Option 2: The method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps: (1) A drug containing carbidopa is mixed with a methanol solution of hydrochloric acid, and then derivatized with a methanol solution of benzaldehyde to obtain a test solution; (2) Detecting the hydrazine content in the test solution by liquid chromatography; Option 3: The method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps: (1) A drug containing carbidopa is mixed with 1 mL of 0.1 mmol / L, 2.5 mmol / L or 5 mmol / L hydrochloric acid methanol solution for 10 to 15 seconds, and then derivatized with 4 mL of 0.25%, 1.0% or 2.0% benzaldehyde methanol solution to obtain a drug. Test solution; (2) Liquid chromatography was used to detect the hydrazine content in the test solution.

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