A pre-column derivatization method and GC detection method for GC detection of hydrazine impurities in carbidopa-containing drugs

Abstract

The invention discloses a pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs and a GC detection method. The pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs is to use acetone as a derivatization reagent to react with hydrazine impurities to generate hydrazone under ultrasonic conditions. The pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs of the invention uses acetone as a derivatization reagent, and can complete the derivatization reaction by ultrasonication for about 5 minutes, without the need to additionally control the reaction temperature or add a buffer system to control the reaction conditions, and is simple to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of drug impurity detection, and in particular to a pre-column derivatization method for GC detection of hydrazine impurity in a carbidopa-containing drug and a GC detection method comprising the pre-column derivatization method. Background Art

[0002] Hydrazine (N2H4), also known as hydrazine, has the following structural formula:

[0003] Hydrazine is a colorless oily liquid with a pungent smell similar to ammonia, and is highly reducing and corrosive. Hydrazine is classified as a Class 2B genotoxic impurity by the International Agency for Research on Cancer (IARC), and is mutagenic and genotoxic both in vivo and in vitro. Therefore, controlling the residue of hydrazine in drugs is of great significance to ensure the safety of taking drugs.

[0004] The synthesis process or degradation of carbidopa is the main source of hydrazine introduction into carbidopa-containing drugs, which may lead to the presence of hydrazine residues in carbidopa-containing drugs. In ICH M7 (ICH Harmonized Guideline Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk M7 (R2)), the acceptable lifetime intake of hydrazine is 0.2ug / day (inhalation) or 39ug / day (other routes).

[0005] Hydrazine is a highly polar compound, which shows weak retention in liquid chromatography, and does not contain ultraviolet absorbing groups, so it is difficult for conventional instruments to detect directly. Therefore, at this stage, most of the detection of hydrazine uses a derivatization reagent, which is derived from hydrazine to form a derivative with ultraviolet absorption or fluorescence absorption, and then detected by gas chromatography-mass spectrometry (GC-MS), differential spectrophotometry, fluorescence detection, chemiluminescence, high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and other methods. However, in the existing detection method, while the derivatization reagent derivatizes hydrazine, the hydrazine group on the chemical structure of carbidopa is also derivatized together, resulting in a significant deviation in the measured hydrazine content, which cannot normally reflect the content of impurity hydrazine in the drug containing carbidopa.

[0006] The present application aims to obtain a method for detecting the impurity hydrazine in a drug containing carbidopa with good accuracy and high sensitivity. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides a pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs, using acetone as a derivatization reagent to specifically react with hydrazine impurities in the drug to generate hydrazones, and almost no reaction with the hydrazine group on the chemical structure of carbidopa, so that the content of hydrazine impurities in carbidopa-containing drugs can be accurately detected. To this end, the present invention also provides a GC detection method for hydrazine impurities in carbidopa-containing drugs.

[0008] In a first aspect of the present invention, a pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs is provided, wherein acetone is used as a derivatization reagent to react with hydrazine impurities under ultrasonic conditions to generate hydrazones.

[0009] As a preferred embodiment, the ultrasonic time is ≥5 min.

[0010] As a preferred embodiment, the concentration of carbidopa in the reaction system is 1.0-2.0 mg / mL.

[0011] As a more preferred embodiment, the concentration of carbidopa in the reaction system is 1.4 mg / mL.

[0012] As a preferred embodiment, the method further comprises the step of adding acetic acid, and the volume ratio of acetone to acetic acid is 500:(0.5-2).

[0013] As a more preferred embodiment, the volume ratio of acetone to acetic acid is 500:0.75.

[0014] A mixed solution formed by acetone and acetic acid is used to disperse the carbidopa-containing drug. Under ultrasonic conditions, the impurity hydrazine in the drug undergoes a derivatization reaction with acetone, wherein acetone serves as a derivatization reagent and acetic acid serves as a catalyst to accelerate the process of the derivatization reaction.

[0015] In a second aspect of the present invention, a GC detection method for hydrazine impurity in carbidopa-containing drugs is provided. After pre-column derivatization of hydrazine impurity in carbidopa-containing drugs by the above method, GC detection is performed using a gas chromatograph with a hydrogen flame ionization detector as the detection instrument.

[0016] As a preferred embodiment, GC detection satisfies one or more of the following conditions:

[0017] a. GC detection adopts split mode, and the split ratio is splitless ~ 2:1;

[0018] b. The injection volume is 1μL~2μL;

[0019] c. The chromatographic column adopts a temperature program mode, the temperature program is to maintain at 40-50°C for 1-3 minutes, increase to 200-250°C at a rate of 30-50°C / min, and maintain for 5-10 minutes;

[0020] d. The chromatographic column used for GC detection is Agilent DB-WAX, with specifications of 30m×0.32mm, 0.5μm;

[0021] e. The oxygen inlet temperature of GC detection is 190-210°C, the carrier gas is nitrogen with a flow rate of 1.2 mL / min, the air flow rate is 300 mL / min, the hydrogen flow rate is 30 mL / min, the tail blow flow rate is 25 mL / min, and the detector temperature is 300°C.

[0022] As a preferred embodiment, the temperature rising program is to maintain at 45°C for 1 min, increase to 240°C at a rate of 40°C / min, and maintain for 8.75 min.

[0023] As a preferred embodiment, GC detection comprises the following steps:

[0024] S1. Prepare blank solution, sensitivity solution, resolution solution, reference solution and test solution;

[0025] S2. Inject the blank solution, sensitivity solution, resolution solution, reference solution and test solution in step S1 into the chromatograph in sequence, and record the chromatograms of the blank solution, sensitivity solution, resolution solution, reference solution and test solution in sequence;

[0026] S3. According to the peak area of ​​the chromatogram, the content of impurity hydrazine in the test solution was calculated by external standard method, and the calculation method was as follows:

[0027]

[0028] Among them, A x : The peak area of ​​the derivative to be tested in the test solution; A r : Average peak area of ​​hydrazine derivatives in reference solution; C r : The theoretical concentration of hydrazine impurity in the reference solution; C x : Concentration of carbidopa in the test solution.

[0029] As a preferred embodiment, the blank solution is prepared by mixing acetone and acetic acid in a volume ratio (V / V) of 500:0.75; the reference solution is prepared by dissolving hydrazine monohydrate reference with the blank solution, and the concentration of impurity hydrazine in the reference solution is 0.083 μg / mL; the sensitivity solution is prepared by diluting the reference solution with the blank solution, and the concentration of the sensitivity solution is 0.025 μg / mL; the resolution solution is prepared by dissolving a carbidopa-containing drug with the reference solution, and the concentration of carbidopa in the resolution solution is 1.4 mg / mL, and the concentration of impurity hydrazine is 0.083 μg / mL; the test solution is prepared by dissolving a carbidopa-containing pharmaceutical preparation to be tested with the blank solution, and the concentration of carbidopa in the test solution is 1.4 mg / mL.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs of the present invention uses acetone as a derivatization reagent and can complete the derivatization reaction by ultrasonication for about 5 minutes. There is no need to additionally control the reaction temperature or add a buffer system to control the reaction conditions, and the operation is simple.

[0032] (2) The pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs of the present invention has strong specificity. Acetone mainly undergoes a derivatization reaction with the hydrazine impurity in the drug, and hardly reacts with the hydrazine group on the chemical structure of carbidopa, which is conducive to obtaining relatively accurate detection results in subsequent GC detection.

[0033] (3) The GC detection method of the impurity hydrazine in the carbidopa-containing drug of the present invention has high detection sensitivity, which can reach 18 ppm, and the detection limit is as low as 6 ppm.

[0034] (4) The GC detection method of the impurity hydrazine in the carbidopa-containing drug of the present invention can complete the detection by GC, has strong economic and popularization, and can meet the release detection requirements of the impurity hydrazine in the carbidopa-containing drug.

[0035] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 GC chromatograms of the blank solution, resolution solution, reference solution and test solution in Example 1 of the present application;

[0037] Figure 2 yes Figure 1 A partial enlarged view of

[0038] Figure 3 This is a linear result diagram obtained in Example 1 of the present application;

[0039] Figure 4 This is a GC chromatogram of detecting the impurity hydrazine in the carbidopa-containing drug in Example 2 of the present application;

[0040] Figure 5 GC chromatograms of the hydrazine reference solution and the carbidopa test solution in Comparative Example 1;

[0041] Figure 6 GC chromatograms corresponding to different derivatization diluents used in Comparative Example 1;

[0042] Figure 7 GC chromatograms of blank solution, reference solution, test solution and acetonitrile (ACN) in Comparative Example 2;

[0043] Figure 8 This is a comparison chart of GC-FID and GC-MS chromatograms in Comparative Example 3. DETAILED DESCRIPTION

[0044] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0045] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0046] Carbidopa was confirmed in 1969 to be the levorotatory isoform of α-methyldopa hydrazine, which has a strong inhibitory activity on L-aromatic amino acid decarboxylase and is difficult to pass through the blood-brain barrier. Carbidopa is used in combination with levodopa to treat Parkinson's disease and Parkinson's syndrome. In addition, carbidopa is also used to inhibit tumor growth. The application in this field suggests that carbidopa can inhibit the proliferation and migration of vascular smooth muscle cells, which may be a new direction for the treatment of cardiovascular diseases, and also provides new treatment ideas for the prevention and treatment of vascular hyperplasia. Therefore, carbidopa is widely used.

[0047] Carbidopa is a compound containing a hydrazine structure. During the synthesis process, the impurity hydrazine may remain in the raw material or drug preparation due to the substitution of starting materials or compound degradation, which may pose a safety hazard to the human body when taken. Therefore, the detection and reasonable control of hydrazine is a very important part of the development process of this type of drug.

[0048] However, hydrazine is a highly polar compound, has weak retention in liquid chromatography, and does not contain ultraviolet absorbing groups, making it difficult to detect directly with conventional instruments. Therefore, at present, the detection of hydrazine is mostly carried out by derivatization with a derivatization reagent and then detection.

[0049] There are many types of derivatization reagents, the most common of which are alkylation reagents, silanation reagents, acylation reagents, fluorescent derivatization reagents, ultraviolet derivatization reagents, etc.

[0050] The alkylating agents include 2,2-dimethylpropane, 18-crown-6, n-butyl borate, O-methoxyamine hydrochloride, pentafluorobenzyl bromide, N-methyl-N-nitroso-p-toluenesulfonamide, N,N-dimethylformamide di-tert-acetaldehyde, N,N-dimethylformamide di-acetaldehyde, N,N-dimethylformamide di-formal, N,N-dimethylformamide di-propionaldehyde, 1-methyl-3-nitro-1-nitrosoguanidine, and trimethylaniline.

[0051] Silylation agents include bis(trimethylsilyl)acetamide, N,Obis(trimethylsilyl)acetamide, bis(trimethylsilyl)trifluoroacetamide, dimethyldioxysilane, hexamethyldisilazane, 1,1,1,3,3,3 hexamethylsilazane, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide, N-methyltrifluoroacetamide, trifluoroacetic acid, trimethylchlorosilane, trimethylsilylimidazole, dimethyldichlorosilane, and N-methyl-n-(trimethylsilyl)trifluoroacetamide.

[0052] The acylating agent includes acetic anhydride, trifluoroacetic anhydride, pentafluoropropionic anhydride, heptafluorobutyric anhydride, N-methylbis(trifluoroacetic anhydride)imidazole, 1-(trifluoroacetyl)imidazole.

[0053] Fluorescent derivatization reagents include dansyl chloride, dansyl hydrazide, fluorescamine, o-phthalaldehyde, 4-bromomethyl-7-methoxycoumarin, fluorenylmethoxy chloride, fluorescein isothiocyanate, and 4-chloro-7-nitrobenzene monoxide.

[0054] UV derivatization reagents include 2,4-dinitrofluorobenzene, p-nitrobenzoyl chloride, p-toluenesulfonyl chloride, phenyl isothiocyanate, p-nitrophenyl bromide, p-bromobenzoyl bromide, naphthoyl bromide, N,N-nitrobenzylisopropylisourea, 3,5-dinitrobenzoyl chloride, p-methoxybenzoyl chloride, 2,4-dinitrophenylhydrazine, and p-nitrobenzylmethoxyamine hydrochloride.

[0055] Derivatization is a common method before gas or liquid phase detection. However, for the specific reaction system of drugs containing carbidopa, how to choose a suitable derivatization reagent is an important problem. On the one hand, the derivatization reagents themselves are of various types and complex; on the other hand, when the derivatization reagents react with hydrazine, they also react with the hydrazine group in the chemical structure of carbidopa, which affects the accuracy of the test. In order to solve the above technical problems, technicians in this field repeatedly screened the derivatization reagents and accidentally discovered that in the carbidopa system, when acetone was used as a derivatization reagent, acetone reacted with the impurity hydrazine alone, and almost did not react with the hydrazine group in the chemical structure of carbidopa, which had a strong specificity.

[0056] The relevant terms involved in this application are explained as follows:

[0057] Carbidopa is an organic compound with the chemical formula C 10 H 14 N2O4.

[0058] Carbidopa-containing drugs are drug preparations containing carbidopa raw materials or carbidopa.

[0059] GC detection is gas chromatography detection.

[0060] Acetic acid is anhydrous acetic acid, also known as glacial acetic acid, abbreviated as HAc.

[0061] ppm means parts per million.

[0062] DMF: N,N-dimethylformamide.

[0063] Derivatization is a chemical transformation that converts a compound into a substance with a similar chemical structure. Generally speaking, when a compound with a specific function participates in a derivatization reaction, the solubility, boiling point, melting point, aggregation state or chemical composition will deviate. The resulting new chemical properties can be used for quantification or separation. The main function of derivatization is to convert difficult-to-analyze substances into substances with similar chemical structures but easy to analyze, which is convenient for quantification and separation.

[0064] Pre-column derivatization refers to the derivatization of the sample before it enters the chromatographic column. That is, the sample is derivatized with a derivatization reagent before entering the chromatographic column for chromatographic analysis.

[0065] Ultrasonic conditions refer to the use of an ultrasonic cleaning machine or similar equipment to ultrasonically treat the sample, thereby accelerating the mixing or reaction of materials through ultrasound.

[0066] Hydrogen flame ionization detector (FID), used to test hydrogen flame ionization machines.

[0067] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0068] In a first aspect of the present invention, a pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs is provided. Under ultrasonic conditions, acetone is used as a derivatization reagent to react with hydrazine impurities to generate hydrazones. The ultrasonic time is ≥5 min, and the concentration of carbidopa in the reaction system is 1.0-2.0 mg / mL. Preferably, the concentration of carbidopa is 1.4 mg / mL.

[0069] The above-mentioned pre-column derivatization method further comprises the step of adding acetic acid, where the acetic acid acts as a catalyst, and the volume ratio of acetone to acetic acid is 500:(0.5-2), preferably, the volume ratio of acetone to acetic acid is 500:0.75.

[0070] The second aspect of the present invention provides a GC detection method for hydrazine impurities in drugs containing carbidopa. After the impurity hydrazine in the drug containing carbidopa is derivatized before the column by the above method, a gas chromatograph with a hydrogen flame ionization detector is used as a detection instrument for GC detection. The GC detection adopts a split mode, the split ratio is no split ~ 2: 1, the injection volume is 1μL ~ 2μL, and the chromatographic column adopts a temperature program mode. The temperature program is maintained at 40-50°C for 1-3min, and increased to 200-250°C at a rate of 30-50°C / min, and maintained for 5-10min. Preferably, the temperature program is maintained at 45°C for 1min, increased to 240°C at a rate of 40°C / min, and maintained for 8.75min.

[0071] Among them, the chromatographic column is Agilent DB-WAX, with specifications of 30m×0.32mm, 0.5μm.

[0072] Among them, the oxygen inlet temperature is 190-210°C, the carrier gas is nitrogen with a flow rate of 1.2mL / min, the air flow rate is 300mL / min, the hydrogen flow rate is 30mL / min, the tail blow flow rate is 25mL / min, and the detector temperature is 300°C.

[0073] Among them, GC detection includes the following steps:

[0074] S1. Prepare blank solution, sensitivity solution, resolution solution, reference solution and test solution;

[0075] Specifically, the blank solution is prepared by mixing acetone and acetic acid in a volume ratio (V / V) of 500:0.75; the reference solution is prepared by dissolving hydrazine monohydrate reference with the blank solution, and the concentration of impurity hydrazine in the reference solution is 0.083 μg / mL; the sensitivity solution is prepared by diluting the reference solution with the blank solution, and the concentration of the sensitivity solution is 0.025 μg / mL; the resolution solution is prepared by dissolving a carbidopa-containing drug with the reference solution, and the concentration of carbidopa in the resolution solution is 1.4 mg / mL, and the concentration of impurity hydrazine is 0.083 μg / mL; the test solution is prepared by dissolving a carbidopa-containing drug preparation to be tested with the blank solution, and the concentration of carbidopa in the test solution is 1.4 mg / mL.

[0076] S2. Inject the blank solution, sensitivity solution, resolution solution, reference solution and test solution in step S1 into the chromatograph in sequence, and record the chromatograms of the blank solution, sensitivity solution, resolution solution, reference solution and test solution in sequence;

[0077] S3. Calculate the content of hydrazine impurity in the test solution by external standard method according to the peak area of ​​the chromatogram.

[0078] The content of impurity hydrazine in the test solution was calculated by the following method:

[0079]

[0080] Among them, A x : The peak area of ​​the derivative to be tested in the test solution; A r : Average peak area of ​​hydrazine derivatives in reference solution; C r : The theoretical concentration of hydrazine impurity in the reference solution; C x : Concentration of carbidopa in the test solution.

[0081] Example 1 Validation of analytical methodology for detection of hydrazine impurity in carbidopa-containing drugs (batch number of carbidopa-levodopa sustained-release tablets used: PG240628, specification: levodopa 360 mg, carbidopa 90 mg)

[0082] Determination method:

[0083] 1. Instrument: Agilent 7890B gas chromatograph;

[0084] 2. Chromatographic conditions:

[0085] 1) Chromatographic column: Agilent DB-WAX (30 m × 0.32 mm, 0.5 μm), starting temperature at 45 °C, maintained for 1 min, increased to 240 °C at a rate of 40 °C / min, maintained for 8.75 min;

[0086] 2) Detector: hydrogen flame ionization detector (FID), temperature is 300℃;

[0087] 3) Inlet temperature: 200°C;

[0088] 4) Carrier gas: high-purity nitrogen, flow rate is 1.2 mL / min, split ratio is 2:1;

[0089] 5) The air flow rate is 300 mL / min, the hydrogen flow rate is 30 mL / min, and the tail gas flow rate is 25 mL / min;

[0090] 6) Direct injection, the injection volume is 1 μL.

[0091] 3. Reagents

[0092] 1) Blank solution (diluent): acetone-acetic acid (volume ratio 500:0.75).

[0093] 2) Reference solution: Weigh the hydrazine monohydrate reference substance, place it in a volumetric flask, add diluent to dissolve and quantitatively dilute to a solution containing about 0.083 μg / mL of impure hydrazine.

[0094] 3) Separation solution: Take carbidopa sustained-release tablets, grind them, place them in a volumetric flask, add 30% of the volume of the reference solution in the volumetric flask, sonicate for 5 minutes, cool to room temperature, add the reference solution to quantitatively dilute to a solution containing 1.4 mg / mL of carbidopa, and filter to obtain.

[0095] 4) Sensitivity solution: dilute the reference solution with the blank solution until the reference solution contains about 0.025 μg / mL of impure hydrazine.

[0096] 5) Test solution: Take carbidopa-containing sustained-release tablets, grind them, place them in a volumetric flask, add 30% of the volume of the volumetric flask with diluent, sonicate for 5 minutes, cool to room temperature, add diluent to quantitatively dilute to a solution containing 1.4 mg / mL of carbidopa, filter, and obtain.

[0097] The blank solution (injection number ≥ 1 needle), sensitivity solution (injection number 1 needle), resolution solution (injection number 1 needle), reference solution (injection number 6 needles), test solution (injection number ≥ 1 needle, one needle of reference solution is required for 6 injections of test solution) and reference solution (injection number ≥ 1 needle) are directly injected into the gas chromatograph, and the chromatogram is recorded. The content of impurity hydrazine is calculated by peak area according to the external standard method.

[0098] The maximum daily dose of carbidopa is 612.5 mg. Carbidopa-levodopa sustained-release tablets are taken orally, with a maximum daily intake of 39 ug / day. The calculated limit of hydrazine is approximately 60 ppm.

[0099] System suitability requirements: The blank solution (diluent) should have no interference except the solvent peak; the signal-to-noise ratio of the hydrazine derivative peak in the sensitivity solution should be ≥10; the separation between the derivative peak to be tested and the adjacent peak in the resolution solution should not be less than 1.5; the RSD% of the hydrazine derivative peak area in the first 6 injections and all reference solutions should not be greater than 10.

[0100] The calculation formula of hydrazine content is:

[0101]

[0102] Where: Ax is the peak area of ​​the derivative to be tested in the test solution;

[0103] Ar is the peak area of ​​the hydrazine derivative in the reference solution;

[0104] Cx is the concentration of carbidopa in the test solution;

[0105] Cr is the theoretical concentration of impurity hydrazine in the reference solution.

[0106] 1. Specificity experiment:

[0107] Take blank solution, resolution solution, reference solution and test solution, inject them into gas chromatograph by direct injection, and record the chromatogram, such as Figure 1 , 2 shown.

[0108] from Figure 1 , 2 It can be seen that there is no interference between the blank solution and the test solution at the peak of the derivative to be measured, and the separation degree between the peak of the derivative to be measured and the adjacent peaks in the separation solution meets the requirements (separation degree is greater than or equal to 1.5). The results show that the specificity is good.

[0109] 2. Linearity and range experiments:

[0110] Linear stock solution: Accurately weigh 26.5 mg of hydrazine monohydrate reference substance, place in a 50 mL volumetric flask, add diluent to volume, and shake well. Accurately pipette 0.25 mL of the above solution, place in a 50 mL volumetric flask, add diluent to volume, shake well, and obtain a linear stock solution. According to Table 1, pipette different volumes of linear stock solution into different volumetric flasks, add diluent to volume, shake well, and obtain hydrazine solutions of corresponding concentrations, which are 0.025 μg / mL, 0.042 μg / mL, 0.083 μg / mL, 0.125 μg / mL, and 0.166 μg / mL, respectively.

[0111] Table 1

[0112]

[0113]

[0114] Linearity determination: Take 1 μL of the linear solution at each concentration level and inject it into the gas chromatograph by direct injection. Make one injection at each concentration level and record the chromatogram.

[0115] Inject samples in order from low to high concentrations, use solution concentration as the horizontal axis and peak area as the vertical axis to make a linear recovery equation. The correlation coefficient r is required. 2 Should not be less than 0.990, see Figure 3 The linear results are shown in Table 2:

[0116] Table 2

[0117]

[0118] from Figure 3 As shown in Table 2, the method has a good linear relationship within the concentration range of 0.0246 μg / mL to 0.1643 μg / mL of hydrazine.

[0119] 3. Accuracy experiment:

[0120] Blank sample solution: Take an appropriate amount of carbidopa sustained-release tablets, grind them, place them in a volumetric flask, add a diluent that is 30% of the volume of the volumetric flask, ultrasonicate for 5 minutes, cool to room temperature, add the diluent to quantitatively dilute to a solution containing 1.4 mg / mL of carbidopa, and filter to obtain the solution.

[0121] Accuracy stock solution: Prepare accuracy stock solutions equivalent to the LOQ (L1) level (corresponding to a concentration of 0.025 μg / mL), 100% (L3) level (corresponding to a concentration of 0.083 μg / mL), and 150% (L4) level (corresponding to a concentration of 0.125 μg / mL) of the reference solution according to the method in Table 1.

[0122] Accuracy sample solution: Take an appropriate amount of carbidopa sustained-release tablets, grind them, place them in a suitable volumetric flask, add 30% of the volume of the volumetric flask's accuracy reserve solution, ultrasonicate for 5 minutes, cool to room temperature, add the corresponding accuracy reserve solution to quantitatively dilute to a solution containing 1.4 mg / mL of carbidopa, filter, and obtain. (Prepare three copies in parallel for each level)

[0123] The above solutions were respectively injected into the gas chromatograph by direct injection, the chromatogram was recorded, and the recovery rate of hydrazine was calculated by peak area according to the external standard method. The results are shown in Table 3:

[0124] Table 3

[0125]

[0126] As can be seen from Table 3, the recoveries of hydrazine at the three concentration levels were all between 80% and 120%, and the RSD% was ≤10, indicating that the accuracy of the method was good.

[0127] 4. Precision experiment:

[0128] 4.1 Repeatability Experiment

[0129] The 100% level accuracy stock solution in the accuracy experiment was used as the repeatability reference solution, and the same analyst prepared the repeatability solution (same as the 100% accuracy sample solution), and repeated preparation was performed 6 times. 1 μL of each repeatability reference solution and repeatability solution was injected into the gas chromatograph by direct injection, and the sample was analyzed and the chromatogram was recorded. The content of impurity hydrazine was calculated by peak area according to the external standard method. The results are shown in Table 4.

[0130] Table 4

[0131]

[0132] The results showed that the RSD% of the impurity hydrazine content (ppm) detected in 6 replicate solutions prepared in parallel by the same analyst was less than 10, indicating that the method had good repeatability.

[0133] 4.2 Intermediate precision experiment

[0134] According to the preparation method of repeatability solution, another analyst prepared the intermediate precision solution of the same batch of products in the repeatability experiment, repeated the preparation 6 times, and measured on different days using different instruments. The results are shown in Table 5.

[0135] Table 5

[0136]

[0137]

[0138] The results showed that the RSD% of the impurity hydrazine content detected in 12 test solutions in the intermediate precision experiment and repeatability experiment was less than 15, which showed that different personnel and different instruments had little influence on the determination results of this method and the precision of this method was good.

[0139] 5. Limit of Quantitation and Limit of Detection Experiments

[0140] Limit of quantitation (LOQ) solution: The reference solution was diluted stepwise with the goal of achieving a signal-to-noise ratio of 10 or higher for the hydrazine derivative peak.

[0141] Limit of detection (LOD) solution: Dilute the limit of quantitation solution 3 times. Inject the above solutions into the gas chromatograph by direct injection, record the chromatogram, and calculate the signal-to-noise ratio of the hydrazine derivative. The results are shown in Tables 6 and 7. Table 6 corresponds to the results of the limit of quantitation experiment, and Table 7 corresponds to the results of the limit of detection experiment.

[0142] Table 6

[0143]

[0144] Table 7

[0145]

[0146]

[0147] The results showed that the RSD% of the peak area of ​​hydrazine derivatives in the quantitative limit solution for 6 consecutive injections was no more than 15%, and the signal-to-noise ratio was greater than or equal to 10; the signal-to-noise ratio S / N of hydrazine derivatives in the detection limit solution for 3 consecutive injections was greater than or equal to 3. (Based on the acceptable signal-to-noise ratio of 10, it can be concluded that the minimum quantitative limit can reach 18ppm, and the corresponding minimum detection limit can reach 6ppm)

[0148] 6. Solution stability test:

[0149] The reference solution and the test solution were taken and placed at room temperature for measurement at zero day, one day, and two days to examine the change in hydrazine content and to determine the stability of the solution. The results are shown in Table 8.

[0150] Table 8

[0151]

[0152] The results showed that the absolute value of the change in the hydrazine content of the reference solution was less than or equal to 20% within 2 days at room temperature; the impurity hydrazine was not detected in the test solution within 2 days at room temperature, which proved that the reference and test solutions had good stability within 2 days at room temperature.

[0153] 7. Durability test:

[0154] By making slight changes to the injection volume, injection port temperature, and replacement of different chromatographic columns of the same type in the chromatographic conditions, the optimal chromatographic parameters are found and the tolerance of the results is determined to be unaffected by slight changes in the target parameters. The specific investigation items are shown in Table 9. The system suitability solution (the blank solution, sensitivity solution, resolution solution, and reference solution in Example 1 are collectively referred to as the system suitability solution) and the durability solution (same as the repeatability solution) are respectively injected into the gas chromatograph by direct injection, and the chromatogram is recorded. The test results are shown in Table 10.

[0155] Table 9

[0156]

[0157] Table 10

[0158]

[0159] The results show that when the injection volume and injection port temperature change slightly, the determination results of impurity hydrazine are basically consistent, indicating that this method has good durability.

[0160] Example 2

[0161] A method for detecting the impurity hydrazine in carbidopa sustained-release tablets (batch number: PG240701-C, specification: levodopa 360 mg, carbidopa 90 mg), a two-component compound preparation containing carbidopa and levodopa.

[0162] Take 10 compound tablets, grind them, take an appropriate amount (containing about 14 mg of carbidopa) of powder, put it in a 10 mL volumetric flask, add 3 mL of diluent (acetone-acetic acid = 500:0.75), ultrasonicate for 5 minutes, cool to room temperature, add diluent to make up to volume, shake well, and filter. Detect according to the determination method in Example 1, and the obtained chromatogram is as follows Figure 4 As shown, according to the calculation formula in Example 1, the content of impurity hydrazine in the preparation product was calculated by the external standard method using the peak area of ​​the derivative, and the content was measured to be 10.3 ppm, which meets the quality standard of the product (≤60 ppm). Comparative Example 1 HPLC detection of impurity hydrazine in carbidopa-containing drugs using benzaldehyde as a derivatization agent (1) HPLC conditions:

[0163] - Column: Waters HSS T3, 4.6×150 mm, 3.5 μm;

[0164] - Mobile phase A: 0.05% trifluoroacetic acid (TFA); Mobile phase B: acetonitrile;

[0165] - Flow rate: 1.3 mL / min;

[0166] -Injection volume: 5uL;

[0167] - Sample chamber temperature: room temperature;

[0168] - Column temperature: 30°C;

[0169] - Wavelength: 300nm; and

[0170] - Gradient program is shown in Table 11.

[0171] Table 11

[0172] Time (min) A(％) B(％) 0.0 95 5 1.0 95 5 20.0 55 45 23.0 10 90 23.1 95 5 28.0 95 5

[0173] (2) Preparation of solution:

[0174] Thinner:

[0175] DMF: 1 mol / L H2SO4 = 90:10, specifically, 2.6 mL of concentrated sulfuric acid was slowly added dropwise to 100 mL of water, and 100 mL of the solution was slowly added to 900 mL of DMF;

[0176] Benzaldehyde-methanol solution (1%): Measure 1 mL of benzaldehyde in a 100 mL volumetric flask and make up to volume with methanol;

[0177] Borax solution (4 mg / mL): weigh 100 mg of borax into a 25 mL volumetric flask, dissolve it in water and make up to volume;

[0178] Levodopa test solution: weigh an appropriate amount of levodopa bulk drug into a suitable volumetric flask, add diluent to dissolve and quantitatively dilute to a solution containing approximately 5 mg / mL of levodopa;

[0179] Carbidopa test solution: Weigh an appropriate amount of carbidopa API into a suitable volumetric flask, add diluent to dissolve and quantitatively dilute to a solution containing approximately 5 mg / mL of carbidopa;

[0180] Derivatization process: Take 2 mL of the above benzaldehyde-methanol solution and 0.5 mL of borax solution, add 0.5 mL of the test solution, shake well and place at room temperature for 30 minutes.

[0181] Blank solution: Take 0.5 mL of diluent and perform simultaneous derivatization.

[0182] Sample test results such as Figure 5 As shown, from Figure 5 It can be observed that the derivatization product peak in the carbidopa test solution has a great interference with the detection of hydrazine. Using the same chromatographic conditions, the interference peak in the carbidopa test solution was identified by LCMS. The molecular weight of the derivatization products of carbidopa and hydrazine was 209 [M+H], indicating that under the current derivatization conditions, carbidopa will be destroyed to generate the same substance as hydrazine derivatization, and the specificity of this method is poor.

[0183] Under this chromatographic condition, in order to eliminate interference, the diluents used for derivatization were screened, mainly as follows:

[0184] Diluent 1 (original diluent, strongly acidic): DMF: 1 mol / L H2SO4 = 90:10, v / v;

[0185] Diluent 2 (weakly acidic): DMF:H2O:HAc=1000:500:0.5, v / v;

[0186] Diluent 3 (neutral): DMF:H2O=1000:500, v / v.

[0187] The observation results are as follows: after adjusting the diluent to weak acid and neutral, the hydrazine derivative peak still exists, and the peak area of ​​the hydrazine derivative is slightly reduced under the weak acid derivatization conditions, such as Figure 6 This indicates that the derivatization method is not applicable under the current conditions, and other methods or derivatization reagents will be used for further development.

[0188] Comparative Example 2 HPLC Detection of Hydrazine Impurity in Carbidopa Drugs Using the Sample Pretreatment Method of the European Pharmacopoeia Carbidopa API Detection Method

[0189] (1) HPLC conditions:

[0190] - Column: Agilent Poroshell EC C18, 3.0×150mm, 2.7μm;

[0191] -Mobile phase A: 0.1% phosphoric acid; mobile phase B: methanol: acetonitrile = 50:50;

[0192] - Flow rate: 0.6 mL / min;

[0193] -Injection volume: 5uL;

[0194] - Sample chamber temperature: 5°C;

[0195] - Column temperature: 37°C;

[0196] - Wavelength: 272nm; and

[0197] - Gradient program is shown in Table 12.

[0198] Table 12

[0199] Time (min) A(％) B(％) 0.0 80 20 1.0 80 20 21.0 40 60 25.0 5 95 27.0 5 95 27.1 80 20 33.0 80 20

[0200] (2) Preparation of solution:

[0201] Test solution (a): Accurately weigh 0.50 g of carbidopa API and dissolve it in an appropriate amount of dilute hydrochloric acid, and dilute to 2 mL with dilute hydrochloric acid.

[0202] Test solution (b): Add 25g of strongly basic anion exchange resin to two conical flasks respectively. Add 150mL of freshly boiled cold water (excluding CO2) to each conical flask and shake it every 30 minutes. Pour out the water in the two conical flasks (try to pour out as much water as possible) and repeat this operation twice.

[0203] Take two 100mL measuring cylinders, marked as A and B. Add the first portion of resin washed with water to A (wash the resin into measuring cylinder A with 60mL water), and add the second portion of resin washed with water to B (wash the resin into measuring cylinder B with 20mL water). Insert a gas tube into each measuring cylinder and push it to the bottom. Pass nitrogen for 30 minutes. Add 1mL of the test solution (a) to measuring cylinder A. After 1 minute, stop aerating measuring cylinder A, filter, and transfer the filtrate to measuring cylinder B (measuring cylinder B still keeps aeration); after 1 minute, stop aerating measuring cylinder B, filter B immediately, add the mixed solution (1mL of freshly prepared 200g / L salicylaldehyde methanol solution + 20mL pH5.5 phosphate buffer solution) to the filtrate, and shake it thoroughly for 1 minute. Place in a 60℃ water bath for 15 minutes, the solution becomes clear, and after cooling, add 2mL of toluene (analytical grade) and shake vigorously for 2 minutes.

[0204] Reference solution (a): Accurately weigh 10 mg of hydrazine monohydrate and dilute it 500 times with dilute hydrochloric acid.

[0205] Reference solution (b): Replace 1 mL of test solution (a) with 1 mL of reference solution (a) and proceed in parallel with the preparation of test solution (b).

[0206] The sample test results are as follows: referring to the sample pretreatment method of the hydrazine detection method in the European Pharmacopoeia Carbidopa API, it was found that there were still interference peaks in Carbidopa (test solution b), such as Figure 7 As shown, the pre-processing method is very complicated and difficult to operate.

[0207] A comprehensive comparison of Comparative Example 1, Comparative Example 2 and Example 2 shows that although the derivatization reaction is carried out with a derivatization reagent containing a carbonyl group, benzaldehyde and salicylaldehyde will derivatize the hydrazine group on the chemical structure of carbidopa while derivatizing the impurity hydrazine, thereby interfering with the test of the impurity hydrazine; and when acetone is used as a derivatization reagent, acetone mainly undergoes a derivatization reaction with the impurity hydrazine in the drug, and hardly reacts with the hydrazine on the chemical structure of carbidopa.

[0208] Comparative Example 3 GC-MS Detection of Hydrazine Impurity in Carbidopa-Containing Drugs Using Acetone as Derivatization Reagent (1) GC-MS Parameters

[0209] -Chromatographic column: Agilent DB-WAX, 30m×0.32mm, 0.5μm;

[0210] - Inlet temperature: 200°C;

[0211] -Split ratio: 2:1;

[0212] -Carrier gas flow rate (nitrogen): 1.2 mL / min;

[0213] - Detector temperature: 300°C;

[0214] - Air flow rate: 300mL / min;

[0215] - Hydrogen flow rate: 30 mL / min;

[0216] -Blow-off flow rate: 25 mL / min;

[0217] - Injection volume: 1 μL; and

[0218] -Temperature program: start at 45°C, maintain for 1 min, increase to 240°C at a rate of 40°C / min, and maintain for 8.75 min.

[0219] - Mode: MS2 SCAN;

[0220] - Scanning range: 19-200;

[0221] - Ion source temperature: 220°C;

[0222] -Gain: 1;

[0223] - Scan time: 100ms and

[0224] -m / z: 112-97.

[0225] (2) Solution preparation

[0226] Diluent: acetone-acetic acid (500:0.75).

[0227] Reference solution: Accurately weigh an appropriate amount of hydrazine monohydrate reference substance, place it in a suitable volumetric flask, add diluent to dissolve and quantitatively dilute to a solution containing about 1 μg / mL of impure hydrazine.

[0228] Test solution: Take carbidopa-levodopa sustained-release tablets (specifications: levodopa 360 mg, carbidopa 90 mg), grind, weigh an appropriate amount of powder and place it in a suitable volumetric flask, add diluent to dissolve and quantitatively dilute to a solution containing approximately 2.6 mg / mL of carbidopa.

[0229] The sample detection results are as follows: Comparing FID and MS detectors: a. Both detectors have good specificity; b. GC-MS has no significant advantage in sensitivity, such as Figure 8 (GC signal-to-noise ratio was 142.6; GC-MS signal-to-noise ratio was 200.6).

[0230] In view of the following advantages, it is better to choose FID detector: 1) Compared with GC-MS, GC has lower equipment failure rate, easier instrument maintenance, lower probability of incidents, and better repeatability; 2) At higher test sample concentrations, GC has stronger anti-contamination ability than GC-MS; 3) Many factories have GC instruments, and the method is feasible to transfer to factories. This method can be used as a release method for the determination of the impurity hydrazine content in carbidopa-containing drugs.

[0231] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A pre-column derivatization method for GC detection of hydrazine impurities in carbidopa-containing drugs, characterized in that: Under ultrasonic conditions, acetone was used as a derivatization reagent to react with impurity hydrazine to generate hydrazone.

2. The method according to claim 1, characterized in that The ultrasound time was ≥5 min.

3. The method according to claim 1, characterized in that The concentration of carbidopa in the reaction system is 1.0-2.0 mg / mL.

4. The method according to claim 1, characterized in that The method further comprises the step of adding acetic acid, wherein the volume ratio of acetone to acetic acid is 500:(0.5-2).

5. The method according to claim 4, characterized in that The volume ratio of acetone to acetic acid is 500:0.

75.

6. A GC detection method for hydrazine impurity in a drug containing carbidopa, characterized in that: After pre-column derivatization of the hydrazine impurity in the carbidopa-containing drug by the method described in any one of claims 1 to 5, GC detection is performed using a gas chromatograph with a hydrogen flame ionization detector as a detection instrument.

7. The method according to claim 6, characterized in that GC detection meets one or more of the following conditions: a. GC detection adopts split mode, and the split ratio is splitless ~ 2:1; b. The injection volume is 1μL~2μL; c. The chromatographic column adopts a temperature program mode, the temperature program is to maintain at 40-50°C for 1-3 minutes, increase to 200-250°C at a rate of 30-50°C / min, and maintain for 5-10 minutes; d. The chromatographic column used for GC detection is Agilent DB-WAX, with specifications of 30m×0.32mm, 0.5μm; e. The oxygen inlet temperature of GC detection is 190-210°C, the carrier gas is nitrogen with a flow rate of 1.2 mL / min, the air flow rate is 300 mL / min, the hydrogen flow rate is 30 mL / min, the tail blow flow rate is 25 mL / min, and the detector temperature is 300°C.

8. The method according to claim 7, characterized in that The temperature program was to maintain at 45°C for 1 min, increase to 240°C at a rate of 40°C / min, and maintain for 8.75 min.

9. The method according to claim 6, characterized in that GC testing includes the following steps: S1. Prepare blank solution, sensitivity solution, resolution solution, reference solution and test solution; S2. Inject the blank solution, sensitivity solution, resolution solution, reference solution and test solution in step S1 into the chromatograph in sequence, and record the chromatograms of the blank solution, sensitivity solution, resolution solution, reference solution and test solution in sequence; S3. According to the peak area of ​​the chromatogram, the content of impurity hydrazine in the test solution was calculated by external standard method, and the calculation method was as follows: Among them, A x : The peak area of ​​the derivative to be tested in the test solution; A r : Average peak area of ​​hydrazine derivatives in reference solution; C r : The theoretical concentration of hydrazine impurity in the reference solution; C x : Concentration of carbidopa in the test solution.

10. The method according to claim 9, characterized in that The blank solution is prepared by mixing acetone and acetic acid in a volume ratio (V / V) of 500:0.75; the reference solution is prepared by dissolving hydrazine monohydrate reference with the blank solution, and the concentration of impurity hydrazine in the reference solution is 0.083 μg / mL; the sensitivity solution is prepared by diluting the reference solution with the blank solution, and the concentration of the sensitivity solution is 0.025 μg / mL; the resolution solution is prepared by dissolving a carbidopa-containing drug with the reference solution, and the concentration of carbidopa in the resolution solution is 1.4 mg / mL, and the concentration of impurity hydrazine is 0.083 μg / mL; the test solution is prepared by dissolving a carbidopa-containing pharmaceutical preparation to be tested with the blank solution, and the concentration of carbidopa in the test solution is 1.4 mg / mL.

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