Method for detecting hydrazine content in carbidopa-containing medicine

Abstract

The invention discloses a method for detecting the hydrazine content in a medicine containing carbidopa. The invention provides a method for detecting hydrazine content in a medicine containing carbidopa, which comprises the following steps: (1) mixing a sample to be detected and an aldehyde substance in a solvent under the action of acid, deriving hydrazine in the sample to be detected and the aldehyde substance to obtain a test solution, and (2) detecting the content of hydrazine in the test solution. Compared with the prior art, the method has the advantages that the carbidopa does not need to be removed through pretreatment, hydrazine introduced in the determination process can be ignored, operation is simple, derivatization time and sample injection time do not need to be accurately controlled, repeatability is good, and quantification is accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and relates to a method for detecting the hydrazine content in a drug containing carbidopa. Background Art

[0002] 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, which can avoid the higher risk of cancer caused by the treatment of Parkinson's disease.

[0003] According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), the limit of genotoxic impurities in this type of preparation is approximately 20 to 50 ppm. Therefore, establishing a hydrazine detection method with high sensitivity, accurate quantitative analysis, good repeatability, convenience and effectiveness is critical to controlling the hydrazine content in carbidopa preparations, which in turn is of great significance to ensuring the health of patients.

[0004] Since hydrazines have the characteristics of lack of chromophore, strong activity, volatility, low relative molecular mass, high polarity, strong reducibility, etc., the detection method presents special requirements in terms of sensitivity, selectivity, stability of the substance to be tested, 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 derived by the amino group of the active group of hydrazine to generate a derivative 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 amino groups and aldehyde groups undergo condensation reactions to generate corresponding hydrazone compounds, which have strong ultraviolet absorption ability, good stability and high sensitivity. However, this method is greatly affected by the matrix of the analyzed sample.

[0005] When the existing technology is used to measure the hydrazine content in the carbidopa raw material, the hydrazine group in the carbidopa structure will also participate in the reaction during the derivatization reaction, thereby causing a significant increase in the hydrazine content and affecting the accuracy of the measurement result.

[0006] There is no control on the hydrazine content of carbidopa preparations in the pharmacopoeias of various countries, and the control on the hydrazine content in carbidopa raw materials is only found in the European Pharmacopoeia (European Pharmacopoeia EP10.0 Carbidopa raw materials text.). In the prior art, there is a method for detecting hydrazine in carbidopa raw materials in the European Pharmacopoeia, which uses ion exchange resin to remove carbidopa first, and then reacts with salicylaldehyde for derivatization, but the method has cumbersome processing steps, and requires avoiding the degradation of the sample in the stage of removing carbidopa with resin to cause the generation of hydrazine, and the sample detection uses thin layer chromatography, which has low sensitivity. Literature reports (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.) have manufacturers complained that the method is difficult to implement and is not satisfactory, so a method using gas chromatography has been developed for detection, but it is not specifically described in the literature. The document also compared the removal of carbidopa with and without ion exchange resin, and detected it with liquid chromatography after derivatization reaction with benzaldehyde. It was found that the hydrazine content after the removal of 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 caused by the interference of carbidopa during the determination. Although this method is simpler and easier than the European Pharmacopoeia method, it requires ion exchange to remove carbidopa, has many operating steps, is time-consuming, and has many influencing factors, which places high demands on the operating ability of personnel. Moreover, the above methods are only applicable to the detection of raw materials, and the preparations usually contain a large amount of excipients and may not be applicable. In particular, the sustained-release preparations containing polymer excipients are easy to agglomerate during the sample processing, so the above methods are difficult to apply.

[0007] Therefore, a method for detecting the 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. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a detection method for determining the hydrazine content in a drug containing carbidopa. The currently disclosed prior art lacks a detection method for the hydrazine content in a preparation containing carbidopa, and there is only a determination method for hydrazine in a carbidopa raw material, and the method needs to remove carbidopa with an ion exchange resin first, which is cumbersome and time-consuming to operate. The preparation product contains a large amount of auxiliary materials, especially polymer auxiliary materials, 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 need to remove carbidopa, and does not need to deduct the hydrazine background (not more than 2ppm) introduced during the measurement process. It uses a highly sensitive and widely used liquid chromatography for detection, and the experimental steps are simple, repeatable, and accurate, and can be used for quality control of commercially produced products.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a method for detecting the hydrazine content in a drug containing carbidopa, which comprises the following steps:

[0011] (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;

[0012] (2) Detecting the hydrazine content in the test solution;

[0013] The solvent is an alcohol solvent, or a mixed solvent of an alcohol solvent and water;

[0014] 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;

[0015] The alcohol solvent is one or more of methanol, ethanol and isopropanol;

[0016] The aldehyde substance is one or more of benzaldehyde, salicylaldehyde and dimethylaminobenzaldehyde.

[0017] 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.

[0018] In one embodiment, in step (1), the alcohol solvent is methanol and / or ethanol, preferably methanol.

[0019] 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).

[0020] In one embodiment, in step (1), the acid is conventional in the art, preferably HCl, HNO 3 and TFA (trifluoroacetic acid), more preferably HCl.

[0021] 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.

[0022] In a certain embodiment, in step (1), the drug containing carbidopa is preferably a carbidopa API (preferably a monohydrate carbidopa API) or a preparation containing carbidopa; preferably, the preparation is an immediate 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.

[0023] In one embodiment, in step (1), the drug containing carbidopa is preferably carbidopa, carbidopa-levodopa or entacapone-levodopa.

[0024] In one embodiment, in step (1), the aldehyde substance is benzaldehyde.

[0025] 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).

[0026] 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.

[0027] 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.

[0028] In one embodiment, in step (1), the derivatization may include conventional mixing operations, such as vortexing, shaking and stirring, preferably vortexing.

[0029] In one embodiment, in step (1), the derivatization reaction system consists of the solvent, the acid, the drug containing carbidopa and the aldehyde substance.

[0030] 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.

[0031] 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.

[0032] In one 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 more preferably within 3 minutes, for example, the obtained sample solution is immediately injected and tested.

[0033] In one embodiment, in step (2), the detection is performed by conventional detection methods in the art, and the detection is preferably performed by liquid chromatography, gas chromatography, thin layer chromatography or ultraviolet spectrophotometry, more preferably by liquid chromatography.

[0034] 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 (such as hydrazine dihydrochloride) and preparing it by the same method as the test solution.

[0035] 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.

[0036] In one embodiment, in step (2), when detection is performed by liquid chromatography, 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).

[0037] In a certain scheme, in step (2), when 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.

[0038] In one embodiment, in step (2), when detection is performed by liquid chromatography, the detection wavelength is 310 nm.

[0039] In one embodiment, in step (2), when detection is performed by liquid chromatography, the flow rate is 1.5 mL / min.

[0040] In one embodiment, in step (2), when detection is performed by liquid chromatography, the column temperature is 35°C.

[0041] In a certain scheme, 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.

[0042] In a certain embodiment, the method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps:

[0043] (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;

[0044] (2) Liquid chromatography was used to detect the hydrazine content in the test solution.

[0045] In a certain embodiment, the method for detecting the hydrazine content in the drug containing carbidopa comprises the following steps:

[0046] (1) A drug containing carbidopa is mixed with a methanol solution of hydrochloric acid (e.g., 1 mL of a 2.5 mmol / L methanol solution of hydrochloric acid) (e.g., for 10 to 15 seconds), and then derivatized with a methanol solution of benzaldehyde (e.g., 1 mL of a methanol solution of 0.25%, 1.0%, or 2.0% benzaldehyde) to obtain a test solution;

[0047] (2) The hydrazine content in the test solution is immediately tested by liquid chromatography.

[0048] 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.

[0049] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0050] The reagents and raw materials used in the present invention are all commercially available, and the reagents used for sample processing are all analytically pure.

[0051] The positive and progressive effects of the present invention are:

[0052] 1. Compared with the prior art, the present invention does not require pretreatment to remove carbidopa, the hydrazine introduced during the determination process can be ignored, the operation is simple, there is no need to accurately control the derivatization time and the injection time, the repeatability is good, and the quantitative accuracy is accurate. In the prior art, due to the presence of carbidopa during the experiment, the determination result of the hydrazine content will be higher than the true value, so it is necessary to deduct the hydrazine content generated during the reaction, but the repeatability of the determination result is poor. The present invention can reduce the hydrazine content introduced during the reaction process to a negligible amount (not exceeding 2ppm), the repeatability is significantly improved, and it is easier to popularize the quality control and method of the preparation product containing carbidopa.

[0053] 2. Compared with the prior art, the present invention is applicable to a significantly wider range of products. The prior art can only detect carbidopa APIs, while 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

[0054] Figure 1 is a chromatogram of a blank solvent in the specificity investigation of the present invention;

[0055] Figure 2 The chromatogram of the reference solution in the specificity investigation of the present invention;

[0056] Figure 3 The chromatogram is a blank mixed powder solution in the specificity investigation of the present invention;

[0057] Figure 4 It is the chromatogram of the test solution in the specificity investigation of the present invention. DETAILED DESCRIPTION

[0058] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0059] 50% methanol is a methanol aqueous solution with a volume fraction of 50%

[0060] 1% benzaldehyde methanol solution is a 1% benzaldehyde methanol solution by volume

[0061] Hydrochloric acid methanol solution is prepared by mixing concentrated hydrochloric acid and methanol to the corresponding concentration.

[0062] The batch numbers of the samples to be tested used in the present invention are as follows:

[0063] Samples to be tested batch number Carbidopa API 2109218 Sinemet IR (Merck) W019112 Sinemet CR (Merck) W005272 WD-1603 (Handu Pharmaceutical) 2022120601 WD-2010 (Handu Pharmaceutical) 2022021201

[0064] Example 1: Determination of the hydrazine content of carbodiol-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using 2.5 mmol / L hydrochloric acid methanol solutions with different methanol ratios (50% / 100% methanol)

[0065] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0066] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid methanol solution (50% / 100% methanol) to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, and vortex vigorously on a vortexer for 3 minutes to use as the reference substance solution.

[0067] Preparation of test solution: Take 10 carbidopa 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), quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal the tube tightly, place it on a vortex machine 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).

[0068] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are detailed in the table below.

[0069] Table 1. Determination results of hydrazine content in methanol solution using different ratios

[0070]

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

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

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

[0074] Preparation of test solution: Take 10 carbidopa 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), put it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution, quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of benzaldehyde (0.25% / 1.0% / 2.0%) methanol solution, seal the tube, place it on a vortex machine 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).

[0075] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are shown in the table below.

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

[0077]

[0078] Example 3: Determination of hydrazine content in carbidopa-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different sample amounts (equivalent to about 5 mg / 10 mg / 15 mg of carbidopa)

[0079] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0080] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid methanol solution to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, and vortex vigorously on a vortexer for 3 minutes to use as the reference substance solution.

[0081] Preparation of test solution: Take 10 carbidopa 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), put it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution, quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal the tube, place it on a vortex machine 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).

[0082] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are shown in the table below.

[0083] Table 3. Determination results of hydrazine content using different sample amounts

[0084]

[0085] Example 4: Determination of hydrazine content in carbocycline-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different acid types (hydrochloric acid / nitric acid / trifluoroacetic acid)

[0086] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0087] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid / nitric acid / trifluoroacetic acid methanol solution to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, and vortex vigorously on a vortexer for 3 minutes to use as the reference substance solution.

[0088] Preparation of test solution: Take 10 carbidopa 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), put it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid / nitric acid / trifluoroacetic acid methanol solution, quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal 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).

[0089] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are shown in the table below.

[0090] Table 4. Determination results of hydrazine content using different types of acids

[0091]

[0092] 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)

[0093] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0094] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 0.1 / 2.5 / 5 mmol / L hydrochloric acid methanol solution to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, and vortex vigorously on a vortexer for 3 minutes to use as the reference substance solution.

[0095] Preparation of test solution: Take 10 carbidopa 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), put it in a stoppered test tube, accurately add 1 mL of 0.1 / 2.5 / 5 mmol / L hydrochloric acid methanol solution, quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal the tube tightly, place it on a vortex machine 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).

[0096] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are shown in the table below.

[0097] Table 5. Determination results of hydrazine content using different acid concentrations

[0098]

[0099] Example 6: Determination of hydrazine content in carbocycline-levodopa controlled-release tablets (WD-1603, Handu Pharmaceutical) using different types of alcohol (methanol / ethanol)

[0100] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0101] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid methanol (ethanol) solution to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol (ethanol) solution, stopper, place on a vortex instrument and vortex vigorously for 3 minutes, as the reference substance solution.

[0102] Preparation of test solution: Take 10 carbidopa 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), put it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol (ethanol) solution, quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol (ethanol) solution, seal the tube, place it on a vortex machine 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).

[0103] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are shown in the table below.

[0104] Table 6. Determination results of different types of alcohol hydrazine content

[0105]

[0106] 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)

[0107] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0108] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid methanol solution to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, and vortex vigorously on a vortexer for 3 minutes to use as the reference substance solution.

[0109] Preparation of test solution: Take 10 carbidopa 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), put it in a stoppered test tube, accurately add 1 mL of 2.5 mmol / L hydrochloric acid methanol solution, quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal the tube, place it on a vortex machine and vortex vigorously for 1 / 2 / 3 / 5 / 10 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).

[0110] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The results are shown in the table below.

[0111] Table 7. Determination results of hydrazine content at different reaction times

[0112]

[0113] It can be seen from the results in the above table that when the methanol solution is used, even as the reaction time becomes longer, 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.

[0114] Example 8: Determination of hydrazine content in different products containing carbidopa

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

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

[0117] Preparation of test solution: 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, rapidly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal 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).

[0118] Accurately measure 20 μL of the test solution and reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the hydrazine content by the peak area according to the external standard method.

[0119] Table 8. Results of hydrazine assays for different drugs containing carbidopa

[0120]

[0121] Example 9: Determination of hydrazine content in carbodiol controlled-release tablets (WD-1603, Handu Pharmaceuticals)

[0122] Chromatographic conditions: Waters Xbridge C18 4.6-mm×10-cm 5μm column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; detection wavelength at 310nm; flow rate at 1.5mL / min; column temperature at 35°C.

[0123] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid methanol solution to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, and vortex vigorously on a vortexer for 3 minutes to use as the reference substance solution.

[0124] Preparation of test solution: Take 10 carbidopa 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), put them into 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, seal the tube tightly, vortex vigorously on a vortex machine for 3 minutes, filter 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).

[0125] Accurately measure 20 μL of the test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the hydrazine content by peak area according to the external standard method. The determination results are shown in the table below.

[0126] Table 9. Carbohydrate-dependent double-dopa controlled-release tablets hydrazine assay results

[0127]

[0128] Example 10

[0129] The hydrazine content of carbidopa raw material was determined using 2.5mmol / 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: Waters Xbridge C18 4.6-mm×10-cm5μm chromatographic column; acetonitrile-water (containing 0.03% disodium ethylenediaminetetraacetate) (55:45) as the mobile phase; the detection wavelength was 310nm; the flow rate was 1.5mL / min; and the column temperature was 35℃.

[0130] Preparation of reference substance: Take about 25.6 mg of hydrazine dihydrochloride reference substance, weigh accurately, dissolve in water and dilute to make a solution containing about 6.25 μg of hydrazine per 1 mL, accurately measure an appropriate amount, dilute with 2.5 mmol / L hydrochloric acid methanol (water) solution (0% / 50% / 60% / 70% / 80% / 90% / 100% methanol) to make a solution containing about 0.625 μg of hydrazine per 1 mL, as the reference substance stock solution. Accurately measure 1 mL of the reference substance stock solution, place in a stoppered test tube, accurately add 4 mL of 1% benzaldehyde methanol solution, stopper tightly, place on a vortex instrument and vortex vigorously for 3 minutes, as the reference substance solution.

[0131] Preparation of test solution: Take 13.5 mg of carbidopa monohydrate bulk drug (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), quickly vortex for 10 to 15 seconds to moisten, then accurately add 4 mL of 1% benzaldehyde methanol solution, seal, place on a vortex instrument and vortex vigorously for 1 / 2 / 3 / 5 / 10 minutes, filter 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).

[0132] Accurately measure 20 μL of the test solution and reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the hydrazine content by the peak area according to the external standard method.

[0133] The test results of 0% and 100% methanol are shown in the table below.

[0134] Table 10. Determination results of hydrazine content in mmol / L hydrochloric acid aqueous solution

[0135]

[0136] Table 11. Determination results of hydrazine content in mmol / L hydrochloric acid methanol solution (100% methanol)

[0137]

[0138] It can be seen from the results in the above table that, 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 becomes longer, the degree to which the hydrazine group in the carbidopa structure participates in the reaction during the derivatization reaction becomes higher, resulting in a significant increase in the hydrazine content, and the stability and accuracy of the measurement results are poor.

[0139] Methodological investigation:

[0140] 1. Exclusivity

[0141] The blank excipient and blank solvent have no interference with the detection of hydrazine. For details, please refer to the attached figure of the specification ( Figure 1-Figure 4 ).

[0142] 2. System Applicability

[0143] The reference solution was injected 6 times in succession (three injections for STD1 and three injections for STD2), and the response factor RSD and tailing factor were calculated. The results are shown in Table 12. The system suitability test results met the requirements.

[0144] Table 12. Results of suitability of the hydrazine system for carbodiol controlled-release tablets

[0145] Sample name Response Factor STD1 <![CDATA[1.531537×10 5 ]]> STD1 <![CDATA[1.530268×10 5 ]]> STD1 <![CDATA[1.529422×10 5 ]]> STD2 <![CDATA[1.540544×10 5 ]]> STD2 <![CDATA[1.536535×10 5 ]]> STD2 <![CDATA[1.541482×10 5 ]]> average <![CDATA[1.534965×10 5 ]]> RSD% 0.35 Tailing Factor 0.9

[0146] 3. Linearity and range

[0147] Six limit concentration levels of reference solution were prepared, one portion for each concentration, and one portion was measured. The results are shown in Table 13. The hydrazine content had a good linear relationship in the concentration range of 0.0624 μg / mL to 1.2490 μg / mL.

[0148] Table 13. Linearity and range determination results of hydrazine in carbodiol controlled-release tablets

[0149]

[0150]

[0151] 4. Accuracy test

[0152] An appropriate amount of raw material and auxiliary material mixed powder (equivalent to 12.5 mg of carbidopa) was accurately weighed, and hydrazine reference substances equivalent to 40%, 80%, 100% and 120% of the limit concentration levels were added respectively, and three copies of each concentration were prepared in parallel according to the method described in Example 9. The results are shown in Table 14. The average recovery rates of hydrazine at concentration levels of 40%, 80%, 100% and 120% were all between 70.0% and 130.0%, and the RSD of the determination results of 9 samples was less than 15.0%, and the accuracy met the requirements.

[0153] Table 14. Carbohydrate-based controlled-release tablets hydrazine accuracy results

[0154]

[0155] 5. Repeatability

[0156] Six spiked (40% limit concentration level) test sample solutions were prepared in parallel. The results are shown in Table 15. The repeatability results were good.

[0157] Table 15. Repeatability results of carbohydrate-dopa controlled-release tablets (n=6)

[0158]

[0159] 6. Limit of Quantitation and Limit of Detection

[0160] Preparation of quantitative limit solution: dilute downward according to the peak of the linear solution with the lowest concentration until the signal-to-noise ratio of the main peak is about 10, and inject 6 times continuously; preparation of detection limit solution: dilute downward according to the peak of the quantitative limit solution until the signal-to-noise ratio of the main peak is about 3. The concentrations of the quantitative limit and the detection limit are 0.0624μg / mL and 0.0187μg / mL, respectively, and the concentration levels are 5ppm and 1.5ppm, respectively. The results are detailed in Table 16. The results show that the detection sensitivity of the present invention is high.

[0161] Table 16. Results of limit of quantification (LOQ) and limit of detection (LOD) of carbohydrazine for levodopa controlled-release tablets

[0162]

[0163] Table 17. Repeatability test results of quantitation limit of hydrazine for carbodiol controlled-release tablets (n=6)

[0164]

[0165] The results showed that the specificity, system suitability, linearity and range, limit of quantification and limit of detection, accuracy and repeatability of this method all met the validation requirements and were 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) The drug containing carbidopa was 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 the test solution; (2) Liquid chromatography was used to detect the hydrazine content in the test solution.

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