Method for detecting related substance 4-methylamino-butyric acid of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloric acid

Abstract

The invention belongs to the technical field of pharmaceutical analysis, particularly relates to a method for detecting a related substance 4-methylamino-butyric acid in 4-methylamino-1-(pyridine-3-yl) butanone hydrochloride, and aims to solve the problem of detection of the content of the related substance 4-methylamino-butyric acid in the 4-methylamino-1-(pyridine-3-yl) butanone hydrochloride. The invention provides a convenient, efficient and accurate detection method, the method can detect the content of the related substance 4-methylamino-butyric acid in the 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloric acid, so that the medication safety is effectively guaranteed, the quality control of the 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloric acid is facilitated, the method is convenient, efficient and accurate, and the method is suitable for industrial production. The method completely accords with the guidance principle verified by a Chinese pharmacopoeia method in the aspects of system applicability, repeatability, specificity and accuracy, and can be used for quality control of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting 4-methylamino-butyric acid, a related substance in 4-methylamino-1-(pyridin-3-yl)butanone hydrochloride. Background Art

[0002] Nicotine, commonly known as nicotine, is an organic compound found in Solanaceae plants (Solanum), and is a representative of N-cholinergic receptor agonists. It has a paralyzing effect on N1 and N2 receptors and the central nervous system. Its sources are mainly extracted from tobacco and artificially synthesized. Artificially synthesized nicotine can effectively solve the problem of limited supply of raw materials for extracting nicotine. Nicotine not only has considerable market space in the field of e-cigarettes, but also has clinical value and market value in the pharmaceutical field.

[0003] 4-(Methylamino)-1-(3-pyridyl)-1-butanone hydrochloride is a key intermediate of nicotine, hereinafter referred to as N3, 4-methylamino-butyric acid, hereinafter referred to as N3-1, 4-methylamino-butyric acid is the product of the hydrolysis of the solvent N-methylpyrrolidone under acidic conditions. It will encounter nitroso radicals in subsequent reactions and may produce the impurity N-nitroso-N-methyl-4-aminobutyric acid (NMBA), which is a known animal and potential human carcinogen. It is the third nitrosamine-type genotoxic impurity detected in marketed ARChemicalbookBs drugs after N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA). Therefore, it is necessary to formulate quality standards during detection and control the content of 4-methylamino-butyric acid in N3 to further control the generation of toxic impurities in subsequent reactions.

[0004] At present, most of the publicly available documents are about the detection method of nicotine, and there are few documents about the detection method of 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride. The present invention discloses for the first time a detection method of 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride, and provides a convenient, efficient and accurate detection method to solve the detection problem of 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride. The method can detect the content of 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride, and is convenient for the quality control of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride. Summary of the invention

[0005] The invention provides a method for detecting 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride. In order to solve the problem of detecting 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride, a convenient, efficient and accurate detection method is provided. The method can detect the content of 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride, and is convenient for quality control of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride. The method is convenient, efficient and accurate, and can be used for quality control of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride.

[0006] To achieve the above object, the present invention provides the following technical solutions: A method for detecting 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride, wherein 4-methylamino-butyric acid is derivatized by a derivatization method, and then the content of the related substance 4-methylamino-butyric acid in 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride is determined by high performance liquid chromatography. The detection method comprises the following steps: (1) Prepare solutions, including blank solution, reference solution and test solution.

[0007] (2) Determination method: After 4-methylamino-butyric acid is derivatized by the derivatization method, the content of the related substance 4-methylamino-butyric acid in 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride is determined by high performance liquid chromatography. After the system is stable, blank solution, reference solution and test solution are added respectively, and the chromatogram is recorded; The chromatographic conditions are as follows: the high performance liquid chromatograph is equipped with an ultraviolet detector, the chromatographic column: octadecylsilane bonded silica gel as the filler, the injection volume: 10-20µl, the column temperature: 35±5°C, the flow rate: 1.0±0.2ml / min, the detection wavelength: 240nm, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is phosphate buffer, the mobile phase B is acetonitrile, and the gradient elution is performed.

[0008] Furthermore, the chromatographic column is Waters Xbridge RP18, 4.6×150 mm, 3.5 µm.

[0009] Further, the blank solution is purified water; The reference substance solution: take an appropriate amount of 4-methylamino-butyric acid standard, place it in a volumetric flask, add an appropriate amount of phenyl isothiocyanate solution and triethylamine-acetonitrile solution, mix well, store at room temperature, dilute to volume with diluent, shake well, then add an appropriate amount of n-hexane, mix well, let stand and separate, discard the upper layer, remove the lower layer solution, and dilute to volume with diluent to prepare a solution with an appropriate concentration of; The phenyl isothiocyanate solution: take an appropriate amount of phenyl isothiocyanate, put it in a volumetric bottle, dissolve it with acetonitrile and dilute it to the scale, and shake it well; The test solution: take an appropriate amount of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloric acid sample, place it in a volumetric flask, add an appropriate amount of phenyl isothiocyanate solution and triethylamine-acetonitrile solution, mix well, store at room temperature, dilute to volume with diluent, shake well, then add an appropriate amount of n-hexane, mix well, stand and separate, discard the upper layer, remove the lower layer solution, and dilute to volume with diluent to prepare a solution of appropriate concentration; The mobile phase A is a phosphate buffer at pH 6.8.

[0010] Further, the gradient elution is: .

[0011] Furthermore, the volume ratio of the triethylamine-acetonitrile solution is 14:86.

[0012] The related substance 4-methylamino-butyric acid of 4-methylamino-1-(pyridin-3-yl)butanone hydrochloride has the problems of weak response and poor retention in the HPLC detection method, and the problem of thermal decomposition in the GC detection method. In order to solve the problems existing in the traditional HPLC and GC detection, the present invention provides a convenient, efficient and accurate detection method. After N3-1 is derivatized with phenyl isothiocyanate, the content of the related substance N3-1 in N3 is determined by high performance liquid chromatography. The method can detect the related substance 4-methylamino-1-(pyridin-3-yl)butanone hydrochloride, 4-methylamino-butyric acid, so as to effectively ensure the safety of drug use and facilitate the quality control of 4-methylamino-1-(pyridin-3-yl)butanone hydrochloride. The method is convenient, efficient and accurate, and fully complies with the guiding principles of method validation of the Chinese Pharmacopoeia in terms of system applicability, repeatability, specificity and accuracy, and can be used for the quality control of 4-methylamino-1-(pyridin-3-yl)butanone hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the blank solution spectrum of the present invention; Figure 2 It is the reference substance solution spectrum of the present invention; Figure 3 is the selective solution spectrum of the present invention; Figure 4LOQ solution spectrum of the present invention; Figure 5 This is the N3-1 spectrum under the phosphate system mobile phase of Comparative Example 1 of the present invention; Figure 6 This is the N3-1 spectrum under the phosphoric acid system mobile phase of Comparative Example 1 of the present invention; Figure 7 This is the N3-1 spectrum under gas phase conditions of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0014] The present invention is further illustrated by the following examples, but are not intended to limit the present invention. Example 1

[0015] Prepare the solution: Potassium dihydrogen phosphate: AR and above Potassium hydroxide: AR and above Acetonitrile: HPLC Phenyl isothiocyanate: AR and above Triethylamine: AR and above n-Hexane: AR and above Dilution: Purified water Blank solution: diluent Derivatization agent: Take 250μl of phenyl isothiocyanate, place it in a 20ml volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well. (Concentration: about 0.1mol / L) Reference substance stock solution: Take about 20 mg of N3-1 reference substance, weigh accurately, place in a 20 ml volumetric bottle, add diluent to dissolve and dilute to scale, shake well; then accurately measure 5.0 ml, place in a 50 ml volumetric bottle, dilute to scale with diluent, shake well. (Concentration: 0.1 mg / ml) Reference substance solution: accurately measure 1.0 ml of reference substance stock solution, place in a 10 ml volumetric flask, then add 0.5 ml of derivatizing agent and 0.5 ml of triethylamine-acetonitrile (14:86) solution, mix well, store at room temperature for 20 min, dilute to volume with diluent, shake well; then add 1 ml of n-hexane, mix well, let stand to separate, discard the upper layer; accurately measure 1.0 ml again, place in a 10 ml volumetric flask, dilute to volume with diluent, shake well. (Concentration: 1 μg / ml) Test solution: Take about 20 mg of this product, weigh it accurately, put it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well; then accurately measure 1.0 ml, put it in a 10 ml volumetric flask, add 0.5 ml of derivatizing agent, 0.5 ml of triethylamine-acetonitrile (14:86) solution, mix well, store at room temperature for 20 minutes, dilute to the mark with diluent, shake well; then add 1 ml of n-hexane, mix well, let stand and separate, discard the upper layer; (concentration: about 0.1 mg / ml).

[0016] Chromatographic conditions: Instruments: High performance liquid chromatograph, electronic analytical balance.

[0017] Chromatographic column: Octadecyl bonded silica as filler: (such as Waters Xbridge RP18, 4.6×150mm, 3.5µm or chromatographic column with equivalent performance) Injection volume: 20μl Column temperature: 35℃ Flow rate: 1.0ml / min Wavelength: 240nm Mobile phase A: phosphate buffer pH 6.8 (weigh 2.7 g potassium dihydrogen phosphate, add to 1 L ultrapure water, adjust pH to 6.8 with 5% potassium hydroxide solution, filter, and sonicate) Mobile phase B: acetonitrile Gradient elution program:

[0018] Determination method: After the system is stable, inject blank solution, reference solution and test solution respectively and record the chromatograms.

[0019] Calculation formula: Content = 100% Where: A i ——Peak area of ​​N3-1 in the test solution; A S ——Peak area of ​​N3-1 in reference solution.

[0020] C i ——Concentration of test solution, mg / ml; C S ——Concentration of reference solution, mg / ml.

[0021] Result judgment:

[0022] The detection method of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride related substance 4-methylamino-butyric acid also includes method validation before detection. The method validation is based on the chromatographic conditions of the formal detection, and the measurement results are as follows:

[0023] Example 2: System Applicability System suitability is achieved by measuring the RSD of the N3-1 peak area in 6 or more consecutive injections of reference solution, which requires that the RSD of the N3-1 peak area in 6 or more consecutive injections of reference solution meet the acceptable standard.

[0024]

[0025] Example 3: Specificity The specificity of the method is achieved by determining whether the blank solution interferes with the detection; the separation between N3-1 and adjacent peaks in the selective solution; and the recovery rate of N3-1 in the selective solution. It is required that the blank solution should not interfere with the detection, the separation between N3-1 and adjacent peaks in the selective solution should be ≥1.5; and the recovery rate of N3-1 in the selective solution should be between 90.0% and 110.0%.

[0026]

[0027]

[0028] Example 4: Limit of Detection and Limit of Quantitation The limit of quantification is obtained by detecting the ratio of its response signal to noise S / N ≥ 10; the limit of detection is obtained by detecting the ratio of its response signal to noise S / N ≥ 3. Required LOQ N3-1 ≤0.05%, S / N≥10; RSD of the peak area per unit concentration of N3-1 in 6 LOQ solutions ≤20.0%; LOD≈3(1)LOQ, S / N≥3.

[0029]

[0030]

[0031]

[0032] Example 5: Linearity and Range The impurity concentration is within the range of LOQ to 150% and the concentration is used as the horizontal axis and the peak area as the vertical axis to draw a curve. The impurity concentration is required to be linear within the range of LOQ to 150%. The square of the linear correlation coefficient R (R 2 ) ≥ 0.99, the absolute value of the Y-axis intercept relative to the response value of 100% concentration shall not exceed 25.0%.

[0033]

[0034] Example 6: Precision Repeatability is achieved by testing the RSD or absolute value of the change difference of the impurity content in 6 repetitive solutions. The impurity content in the 6 repetitive solutions is required to meet the following requirements: 0.5%≤ when the content is <5.0%, RSD≤5.0%; 0.1%≤ when the content is <0.5%, RSD≤10.0%.

[0035]

[0036] Example 7: Solution Stability The reference solution, test solution and selective solution were placed at room temperature for 0 days, 1 day, 2 days and 3 days before being sampled. The change pattern of the test results over time was investigated to provide a basis for the placement time of the solution during the test.

[0037] Require: Reference solution: Compared with the fresh reference solution, the recovery rate of N3-1 should be between 90.0% and 110.0%.

[0038] Test solution: Compared with 0 days, the impurity content of the test solution after being placed at room temperature for a period of time should meet the following requirements: when the content is ≥0.1%, the absolute value of the change difference shall not exceed 20% of the limit; when the content is <0.1%, the absolute value of the change difference shall not exceed 0.03%; impurities below LOQ are not included in the calculation.

[0039] Selective solution: Compared with day 0, after the selective solution is left at room temperature for a period of time, the recovery rate of N3-1 should be between 90.0% and 110.0%.

[0040]

[0041]

[0042]

[0043] After verification, this method was proved to have good system applicability, specificity, quantification limit and detection limit, linearity and range, precision and solution stability, and was suitable for the analysis and detection of related substances (N3-1) in 4-methylamino-1-(pyridin-3-yl)butanone hydrochloride (N3).

[0044] The following comparative examples illustrate the screening process of chromatographic conditions such as chromatographic columns and mobile phases in this patent.

[0045] Comparative Example 1: Investigating the liquid phase response of 4-methylamino-butyric acid in the phosphoric acid system and the salt system 1.1 Chromatographic conditions Chromatographic conditions① Instruments: High performance liquid chromatograph, electronic analytical balance.

[0046] Chromatographic column: Octadecyl bonded silica as filler: (such as YMC Tiart C18 EXRS, 4.6 mm × 150 mm, 3 μm or chromatographic column with equivalent performance) Injection volume: 10μl Column temperature: 30℃ Flow rate: 1.0ml / min Mobile phase A: phosphoric acid: water = 1:1000 (V / V) Wavelength: 220nm Mobile phase B: acetonitrile Mobile phase: Mobile phase A: Mobile phase B = 95:5 Running time: 30 minutes Chromatographic conditions② Instruments: High performance liquid chromatograph, electronic analytical balance.

[0047] Chromatographic column: Octadecyl bonded silica as filler: (such as YMC Tiart C18 EXRS, 4.6 mm × 150 mm, 3 μm or chromatographic column with equivalent performance) Injection volume: 10μl Column temperature: 45℃ Flow rate: 1.0ml / min Mobile phase A: 20 mM K 2 HPO 4 (pH10.0) Wavelength: 220nm Mobile phase B: acetonitrile Mobile phase: Mobile phase A: Mobile phase B = 95:5 Running time: 30 minutes 1.2 Reagents

[0048] 1.3 Solution preparation Diluent: water; Reference solution: weigh 10 mg N3-1 into a 10 ml volumetric flask, dilute to scale with diluent, and shake well (concentration: 1 mg / ml) Test solution: weigh 20 mg N3 into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well (concentration: 1 mg / ml) Disadvantages of method 1: Low response and weak retention in phosphoric acid and phosphate mobile phases; Comparative Example 2: The boiling point of 4-methylamino-butyric acid is 237°C, and the gas phase method can be used for detection to examine the gas phase response 2.1 Chromatographic conditions Instrument: Gas chromatograph equipped with FID detector, autosampler, analytical balance; GC chromatographic conditions: Chromatographic column: (such as CP-Volamin 30m×0.32mm or other chromatographic columns with equivalent performance) Column temperature: 40°C, hold for 3 min, increase the temperature to 240°C at 15°C / min, hold for 3 min Inlet temperature: 240°C Detector temperature: 250°C Split ratio: 10:1 Flow rate: 3mL / min Air flow rate: 300ml / min Hydrogen flow rate: 30ml / min Make-up gas flow rate: 25ml / min Injection volume: 10ul 2.2 Reagents Methanol: HPLC and above; 2.3 Solution preparation Diluent: methanol; Reference solution: Take an appropriate amount of N3-1, place it in a volumetric flask, and dilute it with diluent to make a solution with a concentration of 1 mg / ml; Test solution: Take an appropriate amount of N3 sample, place it in a volumetric flask, and dilute it with diluent to make a solution with a concentration of 1 mg / ml.

[0049] Disadvantages of method 2: Under this condition, N3-1 decomposes into four peaks, which may be caused by the high temperature conditions. By comparing Examples 1-2, this solution is determined to be the final analysis method.

Claims

1. A method for detecting 4-methylamino-butyric acid, a related substance of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride, characterized in that: After 4-methylamino-butyric acid is derivatized by a derivatization method, the content of the related substance 4-methylamino-butyric acid in 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride is determined by high performance liquid chromatography. The detection method comprises the following steps: (1) Prepare solutions, including blank solution, reference solution and test solution; (2) Determination method: After 4-methylamino-butyric acid is derivatized by the derivatization method, the content of the related substance 4-methylamino-butyric acid in 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloride is determined by high performance liquid chromatography. After the system is stable, blank solution, reference solution and test solution are added respectively, and the chromatogram is recorded; The chromatographic conditions are as follows: the high performance liquid chromatograph is equipped with an ultraviolet detector, the chromatographic column: octadecylsilane bonded silica gel as the filler, the injection volume: 10-20µl, the column temperature: 35±5°C, the flow rate: 1.0±0.2ml / min, the detection wavelength: 240nm, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is phosphate buffer, the mobile phase B is acetonitrile, and the gradient elution is performed.

2. The method according to claim 1, characterized in that: The chromatographic column is Waters Xbridge RP18, 4.6×150 mm, 3.5 µm.

3. The method according to claim 2, characterized in that: The blank solution is purified water; The reference substance solution: take an appropriate amount of 4-methylamino-butyric acid standard, place it in a volumetric flask, add an appropriate amount of phenyl isothiocyanate solution and triethylamine-acetonitrile solution, mix well, store at room temperature, dilute to volume with diluent, shake well, then add an appropriate amount of n-hexane, mix well, let stand and separate, discard the upper layer, remove the lower layer solution, and dilute to volume with diluent to prepare a solution with an appropriate concentration of; The phenyl isothiocyanate solution: take an appropriate amount of phenyl isothiocyanate, put it in a volumetric bottle, dissolve it with acetonitrile and dilute it to the scale, and shake it well; The test solution: take an appropriate amount of 4-(methylamino)-1-(3-pyridyl)-1-butanone hydrochloric acid sample, place it in a volumetric flask, add an appropriate amount of phenyl isothiocyanate solution and triethylamine-acetonitrile solution, mix well, store at room temperature, dilute to volume with diluent, shake well, then add an appropriate amount of n-hexane, mix well, stand and separate, discard the upper layer, remove the lower layer solution, and dilute to volume with diluent to prepare a solution of appropriate concentration; The mobile phase A is a phosphate buffer at pH 6.

8.

4. The method according to claim 3, characterized in that: The gradient elution is: 。 5. The method according to claim 3, characterized in that: The volume ratio of the triethylamine-acetonitrile solution is 14:86.

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