A method for determining the impurity content in 2-bromobutyric acid by gas phase derivatization chromatography

Abstract

The present invention relates to a method for determining the impurity content in 2-bromobutyric acid by gas phase derivatization chromatography. The present invention uses a derivatization reagent to carry out derivatization treatment on a substance to be tested, and then uses gas chromatography to quantitatively analyze the derivative substance to calculate the content of each impurity in 2-bromobutyric acid. Using the above analysis method, the linearity is good, and the correlation coefficient R 2 It can reach above 0.9995; the accuracy of the derivatization method (calculated by sample recovery rate) can reach above 97.2%. The method of the present invention has the advantages of simple operation, fast derivatization, accuracy and sensitivity, etc. It well solves the shortcomings of conventional methods such as weak specificity, large interference and poor accuracy, and can be quickly promoted and used, so as to effectively and quickly control the distribution of impurities and improve product quality.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical and chemical analysis, and relates to a method for determining the impurity content in 2-bromobutyric acid by using a gas phase derivatization chromatography method, and particularly relates to a method for determining the content by using a gas phase derivatization chromatography method. Background Art

[0002] 2-Bromobutyric acid is a colorless oily liquid, generally prepared by heating bromine and butyric acid. It is mainly used in organic synthesis and is often used in the synthesis of pesticides, pharmaceutical intermediates, etc. Its detection technology usually uses acid-base titration to determine the acid content.

[0003] As a widely used chemical intermediate, 2-bromobutyric acid has been widely used in the synthesis of anticonvulsant drugs, cardiovascular drugs, metabolic and inflammatory disease drugs in recent years. Therefore, 2-bromobutyric acid is regulated as a registered intermediate (RI). Its production management and quality management need to be implemented and managed in accordance with the requirements of CGMP, and its impurity requirements are also high. Common detection methods for 2-bromobutyric acid include titration, gas chromatography, liquid chromatography, etc., which mainly use color reaction or direct determination, resulting in unsatisfactory specificity and accuracy, large measurement errors; in gas chromatography, the sample acidity is high, the peak shape is poor, and the column life is short; in liquid chromatography, the impurity response is small, and the detection limit does not meet the requirements. Therefore, an innovative detection method must be adopted to accurately, quickly and conveniently detect impurities in 2-bromobutyric acid.

[0004] Impurities in 2-bromobutyric acid include butyric acid, crotonic acid, bromoacetic acid, 2-bromopropionic acid, 3-bromopropionic acid, 2-bromo-2-methylpropionic acid, bromopropane, propiolactone, butyrolactone, etc. There are many types and small contents, and the test is very difficult. There are reports at home and abroad that indirectly determine it by derivatization into methyl ester or ethyl ester. However, the derivatization process requires the use of an acidified alcohol solution, the derivatization time is long, and the pH of the solution needs to be adjusted with a weak base after derivatization; and some halogen-containing organic acids will undergo incomplete esterification, or need to be corrected by a calibration curve to compensate for acid methyl esterification with a conversion rate lower than 100%. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for determining the impurity content in 2-bromobutyric acid by gas phase derivatization chromatography, which can achieve simple, rapid and complete derivatization, and each substance can be completely separated and accurately quantified.

[0006] The technical solution of the present invention:

[0007] (1) Sample pretreatment: Accurately measure 200 μL of 2-bromobutyric acid sample, weigh it, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve it for 1 minute, heat it at 70°C for 15 minutes, cool it to room temperature, and mix it.

[0008] (2) Preparation of reference solution: Accurately weigh 500 mg of each impurity reference substance and place it in a 50 mL volumetric flask, add methyl tert-butyl ether to dissolve and dilute to the mark, and shake well; Accurately transfer 3.0 mL of the above solution to a 100 mL volumetric flask, add methyl tert-butyl ether to dilute to the mark, and shake well to obtain the standard stock solution. Accurately measure 2.5 mL of the standard stock solution, add 10 mL of the derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70°C for 15 minutes, cool to room temperature, and mix well.

[0009] (3) Content calculation: Content (%) =;

[0010] Where: A sample : The peak area of ​​impurities in the test sample solution;

[0011] A std : Peak area of ​​impurities in reference solution;

[0012] C sample : Concentration of test sample solution, mg / mL;

[0013] Cstd: concentration of reference solution, mg / mL;

[0014] P: Content of reference substance, %;

[0015] The derivatization reagent in step (1) and step (2) is a methyl tert-butyl ether solution of N,O-bis(trimethylsilyl)trifluoroacetamide, with a concentration of 15% (v / v). The derivatization reagent can also be one of N,O-bis(trimethylsilyl)acetamide, N-methyltrifluoroacetamide, and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide; or used in combination with trimethylchlorosilane.

[0016] The solvent for preparing the derivatization reagent in step (1) and step (2) is one or more of methyl tert-butyl ether, dichloromethane and chloroform.

[0017] The reference substance in the reference solution prepared in step (2) is one or more of 2-bromobutyric acid, butyric acid, crotonic acid, bromoacetic acid, 2-bromopropionic acid, 3-bromopropionic acid, and 2-bromo-2-methylpropionic acid. The amount of the reference substance used is about 0.2% of the test sample, or three different concentrations are prepared to cover a concentration of 0.2%.

[0018] The test sample in step (1) may contain one or more of other substances that do not require derivatization, such as methanol, ethanol, 1-bromopropane, β-propiolactone, and β-butyrolactone.

[0019] The conditions used in the tests of step (1) and step (2) are:

[0020] Chromatograph: Agilent 7890 gas chromatograph with hydrogen flame ionization detector;

[0021] Chromatographic column: Agilent DB-624, 30m×0.53mm, 3μm capillary column;

[0022] Inlet temperature: 220°C;

[0023] Column temperature: Initial column temperature 50°C, increase to 230°C at a rate of 15°C / min, and maintain for 8 min;

[0024] Detector temperature: 250°C;

[0025] Split ratio: 5:1;

[0026] Carrier gas flow rate: 3 mL / min;

[0027] The sample analysis process is to take the test sample solution and the reference solution in the above steps (1) and (2), and use gas chromatography to analyze the sample, with an injection volume of 1 μL.

[0028] The derivatization test method used in the present invention has good linearity in the concentration range of 3.0 μg / mL to 110.0 μg / mL for impurities in 2-bromobutyric acid, and the correlation coefficient R 2 The results are all greater than 0.9995, the spiked recoveries are all greater than 97.2%, and the limits of quantification are all greater than 0.02% (w / w%). This method has fast and complete derivatization, high sensitivity, rapidity and effectiveness, and is very suitable for the detection of impurities in 2-bromobutyric acid.

[0029] The derivatization reaction is shown in the following formula:

[0030] .

[0031] Beneficial effects:

[0032] (1) The present invention uses a common DB-624 gas chromatography column for analysis after derivatization, and realizes the simultaneous detection of impurities such as butyric acid, crotonic acid, bromoacetic acid, 2-bromopropionic acid, 3-bromopropionic acid, and 2-bromo-2-methylpropionic acid in 2-bromobutyric acid. The pretreatment of this method is simple and fast, the analysis results are accurate and reliable, and the quality of the product can be quickly and accurately controlled.

[0033] (2) The present invention uses a 15% (v / v) solution of N,O-bis(trimethylsilyl)trifluoroacetamide in methyl tert-butyl ether as a derivatization reagent. No extraction, transfer, pH adjustment or other operations are required during the process. The conversion rate is high, the recovery rate is good, and the determination result is not affected.

[0034] (3) Compared with traditional technologies, the present invention has obvious advantages: this method can accurately and quantitatively analyze each impurity, which overcomes the shortcomings of conventional methods such as poor specificity, low accuracy, large interference, and low accuracy. It has a certain degree of universality and can be extended to the analysis and detection of most organic carboxylic acids and halogen-containing carboxylic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the gas chromatogram of the derivatization reagent N,O-bis(trimethylsilyl)trifluoroacetamide in methyl tert-butyl ether (BSTFA in MTBE).

[0036] Figure 2 This is the gas phase localization chromatogram of the reference solution (BSTFA derivatized with MTBE solution). The substances ① to ⑥ marked in the figure are trimethylsilane derivatives of crotonic acid, butyric acid, 2-bromopropionic acid, 2-bromo-2-methylpropionic acid, 2-bromobutyric acid, and 3-bromopropionic acid, respectively.

[0037] Figure 3 The gas chromatogram of the test sample solution (BSTFA derivatized with MTBE solution). The substances ①~② marked in the figure are trimethylsilane derivatives of butyric acid and 2-bromobutyric acid, respectively, and ③~④ are unknown impurities. The impurity content is less than 0.03%.

[0038] Figure 4 This is the gas chromatogram of the reference solution (esterified with acidic ethanol solution). The substances ①~⑫ marked in the figure are methanol, ethanol, 1-bromopropane, ethyl butyrate, ethyl bromoacetate, β-propiolactone, ethyl butenoate, β-butyrolactone, ethyl 2-bromopropionate, ethyl 2-bromo-2-methylpropionate, ethyl 3-bromopropionate, and ethyl 2-bromobutyrate, respectively.

[0039] Figure 5 The figure is a curve showing the change in concentration and peak area of ​​impurities (trimethylsilane derivatives of impurities) that may appear in 2-bromobutyric acid, wherein the abscissa represents the impurity concentration and the ordinate represents the peak area. DETAILED DESCRIPTION

[0040] The following is a further detailed description of the embodiments of the present invention in conjunction with the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given. However, the protection scope of the present invention should include improvements and modifications made based on the principles of the present invention, and is not limited to the embodiments described below.

[0041] Example 1: Linear range test

[0042] 1) Preparation of standard stock solution: Accurately weigh 500 mg of each impurity such as 2-bromobutyric acid, butyric acid, crotonic acid, 2-bromopropionic acid, 3-bromopropionic acid, and 2-bromo-2-methylpropionic acid, and place them in a 50 mL volumetric flask. Add methyl tert-butyl ether to dissolve and dilute to the scale, and shake well. Accurately pipette 3.0 mL of the above solution into a 100 mL volumetric flask, add methyl tert-butyl ether to dilute to the scale, and shake well.

[0043] 2) Preparation of derivatization reagent: Accurately measure 15.0 mL of N,O-bis(trimethylsilyl)trifluoroacetamide and place it in a 100 mL volumetric flask. Add methyl tert-butyl ether to dilute to the mark and shake well.

[0044] 3) Preparation of reference solution: Accurately measure 2.5 mL of standard stock solution, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70°C for 15 minutes, cool to room temperature, and mix.

[0045] 4) Preparation of linear range solution: Accurately measure 6.0mL, 4.0mL, 2.5mL, 1.0mL, 0.5mL, 0.2mL, and 0.1mL of standard stock solution, respectively, add 10mL of derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70℃ for 15 minutes, cool to room temperature, mix, and inject respectively.

[0046] 5) The test results are shown in Table 1:

[0047] Table 1

[0048]

[0049] The curve is drawn by measuring the results. Figure 5 shown.

[0050] Example 2: Accuracy (recovery) test

[0051] 1) Preparation of test sample solution: Accurately measure 200 μL of 2-bromobutyric acid sample, weigh it, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve it for 1 minute, heat it at 70°C for 15 minutes, cool it to room temperature, and mix it.

[0052] 2) Preparation of accuracy test solution (50% concentration level): accurately measure 200 μL of 2-bromobutyric acid sample to be tested, weigh it, accurately measure 1.0 mL of standard stock solution, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70°C for 15 minutes, cool to room temperature, and mix. Prepare 3 copies in parallel.

[0053] 3) Preparation of accuracy test solution (100% concentration level): accurately measure 200 μL of 2-bromobutyric acid sample to be tested, weigh it, accurately measure 2.5 mL of standard stock solution, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70°C for 15 minutes, cool to room temperature, and mix. Prepare 3 copies in parallel.

[0054] 4) Preparation of accuracy test solution (150% concentration level): accurately measure 200 μL of 2-bromobutyric acid sample to be tested, weigh it, accurately measure 5.0 mL of standard stock solution, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70°C for 15 minutes, cool to room temperature, and mix. Prepare 3 copies in parallel.

[0055] 5) The above solutions were injected separately, and the test results are shown in Table 2:

[0056] Table 2

[0057]

[0058] The accuracy of the method was investigated by adding different concentrations of impurities to the sample. The concentrations investigated were 0.1%, 0.2% and 0.3% of the sample concentration. Each concentration was repeated 3 times, and the recovery rate was greater than 97.2%, and the relative standard deviation was ≤2.6%. The accuracy was calculated by measured value / true value×100%.

[0059] Example 3: Derivatization conditions

[0060] 3.1: Choice of derivatization reagent

[0061] Referring to relevant literature, it was found that the derivatization reagent used in the literature for carboxylic acid derivatization was basically acidified methanol. This experiment investigated three derivatization reagents: derivatization reagent ① was 10% sulfuric acid methanol solution (10 mL sulfuric acid was slowly dripped into a 100 mL volumetric flask containing about 50 mL methanol placed in an ice water bath, and after returning to room temperature, it was diluted to the mark with methanol and mixed evenly.); derivatization reagent ② was 15% BSTFA in MTBE solution (15.0 mL of N, O-bis (trimethylsilyl) trifluoroacetamide was accurately measured and placed in a 100 mL volumetric flask, methyl tert-butyl ether was added to dilute to the mark, and shaken well.); derivatization reagent ③ was MSTFA in dichloromethane solution (10.0 mL of N-methyl-N-(trimethylsilyl) trifluoroacetamide was accurately measured and added with 0.2 ml of trimethylchlorosilane and placed in a 100 mL volumetric flask, dilute to the mark with dichloromethane, and shaken well.).

[0062] 3.2: Choice of derivatization temperature

[0063] This experiment investigated the effect of different derivatization temperatures on the derivatization effect. The temperatures were 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃ and 90℃ respectively.

[0064] 3.3: Choice of derivatization time

[0065] This experiment investigated the effect of different derivatization times on the derivatization effect. The derivatization times were 5min, 10min, 15min, 20min, 25min, 30min, and 35min respectively.

[0066] 3.4: Discussion of test results

[0067] The results showed that all three derivatization reagents could be used for derivatization. However, considering that derivatization reagent ① had a slightly poorer derivatization effect, a higher degree of operational complexity, and required extraction and deacidification, derivatization reagent ③ had a comparable derivatization effect to derivatization reagent ②, but consumed more reagents; therefore, considering all factors, derivatization reagent ② was the best choice.

[0068] The results showed that considering the effects of derivatization temperature and time, the temperature range was 60℃~80℃ and the time was 10min~20min for complete derivatization. Therefore, considering comprehensively, the derivatization temperature was 70℃ and the derivatization time was 15min.

[0069] Due to the limitations of textual expression and the objective existence of other impurities with similar chemical structures, ordinary technicians in this technical field can make appropriate improvements or changes without departing from the principles of the present invention, and can also combine the above-mentioned technical features in an appropriate manner; these improvements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the impurity content in 2-bromobutyric acid by gas phase derivatization chromatography, characterized in that: The measurement steps include: (1) Sample pretreatment: accurately measure 200 μL of 2-bromobutyric acid sample, weigh it, add 10 mL of derivatization reagent, vortex and ultrasonically dissolve it for 1 minute, heat it at 70°C for 15 minutes, cool it to room temperature, and mix it; (2) Preparation of reference solution: Accurately weigh 500 mg of each impurity reference substance and place it in a 50 mL volumetric flask, add methyl tert-butyl ether to dissolve and dilute to the mark, and shake well; Accurately pipette 3.0 mL of the above solution into a 100 mL volumetric flask, add methyl tert-butyl ether to dilute to the mark, and shake well to obtain a standard stock solution; Accurately measure 2.5 mL of the standard stock solution, add 10 mL of the derivatization reagent, vortex and ultrasonically dissolve for 1 minute, heat at 70°C for 15 minutes, cool to room temperature, and mix well; (3) The test conditions used are: Chromatograph: Agilent 7890 gas chromatograph with hydrogen flame ionization detector; Chromatographic column: Agilent DB-624, 30m×0.53mm, 3μm capillary column; Inlet temperature: 220°C; Column temperature: Initial column temperature 50°C, increase to 230°C at a rate of 15°C / min, and maintain for 8 min; Detector temperature: 250°C; Split ratio: 5:1; Carrier gas flow rate: 3 mL / min; (4) Sample analysis: Take the test sample solution and the reference solution in step (1) and step (2) above, and analyze them by gas chromatography with an injection volume of 1 μL; (5) Content calculation: content / %=; Where: A sample : The peak area of ​​impurities in the test sample solution; A std : Peak area of ​​impurities in reference solution; C sample : Concentration of test sample solution, mg / ml; C std : Concentration of reference solution, mg / ml; P: content of reference substance, %; The reference substances in the reference solution prepared in step (2) are 2-bromobutyric acid, butyric acid, crotonic acid, bromoacetic acid, 2-bromopropionic acid, 3-bromopropionic acid and 2-bromo-2-methylpropionic acid; the amount of the reference substances used is 0.2% of the test sample, or three different concentrations are prepared and cover a concentration of 0.2%.

2. The method for determining the impurity content in 2-bromobutyric acid by gas phase derivatization chromatography according to claim 1, characterized in that: The derivatization reagent in step (1) and step (2) is a methyl tert-butyl ether solution of N,O-bis(trimethylsilyl)trifluoroacetamide, with a volume concentration of 15%; the derivatization reagent is one of N,O-bis(trimethylsilyl)acetamide, N-methyltrifluoroacetamide, and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide; or is used in combination with trimethylchlorosilane.

3. The method for determining the impurity content in 2-bromobutyric acid by gas phase derivatization chromatography according to claim 1, characterized in that: The solvent for preparing the derivatization reagent in step (1) and step (2) is one or more of methyl tert-butyl ether, dichloromethane and chloroform.

4. The method for determining 2-bromobutyric acid and its impurity content by gas phase derivatization chromatography according to claim 1, characterized in that: In the step (1), the test sample contains one or more of other substances that do not need to be derivatized, such as methanol, ethanol, 1-bromopropane, β-propiolactone, and β-butyrolactone.

5. The method for determining 2-bromobutyric acid and its impurity content by gas phase derivatization chromatography according to claim 1, characterized in that: The derivatization reaction is shown below: 。

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