Detection method for related substances of n-butyl bromide and application

Abstract

The invention provides a method for detecting related substances of bromo-n-butane, which adopts a gas chromatography detection method, and the chromatographic conditions are as follows: a chromatographic column is a DB-624 quartz capillary column, and the specification is 30m * 0.32 mm * 1.8 [mu] m; the column temperature is 40 DEG C; the stationary phase is 6% of cyanopropyl phenyl-94% of dimethyl polysiloxane; the temperature of a sample injector is 250 DEG C; the sample size is 1 [mu] L; the split ratio is 20: 1; the carrier gas is nitrogen; the flow rate of nitrogen is 2.1 mL / min; a detector is a hydrogen ion spark detector, and the temperature of the detector is 250 DEG C. The invention further provides application of the method. The method can be used for rapidly and accurately detecting the related substances of the n-butyl bromide, realizes effective separation of the n-butyl bromide and impurities thereof, and is of great significance in improving the effective quality of the n-butyl bromide, controlling the content of the impurities brought into the synthesis reaction of the bupivacaine hydrochloride by the starting material and improving the quality stability of the bupivacaine hydrochloride.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting related substances of bupivacaine hydrochloride starting material n-butyl bromide and application thereof. Background Art

[0002] Bupivacaine hydrochloride, chemical formula is C 18 H 29 ClN2O is a local anesthetic, mainly used for local anesthesia and postoperative analgesia. Bupivacaine hydrochloride has different synthetic routes and methods according to different reaction raw materials. Among them, n-butyl bromide is a starting material for bupivacaine hydrochloride. If there are many impurities, it will inevitably be introduced into the bupivacaine hydrochloride product, affecting the quality of bupivacaine hydrochloride.

[0003] Based on this, it is necessary to control the effective quality of the starting material "n-butyl bromide" of bupivacaine hydrochloride, which is of great significance for the preparation of bupivacaine hydrochloride and improving the quality stability of bupivacaine hydrochloride. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting n-butyl bromide related substances and an application thereof. The present invention can quickly and accurately detect n-butyl bromide related substances and realize effective separation of n-butyl bromide and its impurities, which is of great significance for improving the effective quality of n-butyl bromide, controlling the impurity content of the starting material brought into the synthesis reaction of bupivacaine hydrochloride, and improving the quality stability of bupivacaine hydrochloride.

[0005] The n-butyl bromide related substances of the present invention mainly refer to n-butyl bromide and its main impurity - n-butyl alcohol. The basic information is shown in Table 1.

[0006] Table 1 Information on n-butyl bromide and its impurities

[0007]

[0008]

[0009] The present invention adopts the following technical solutions to solve the above technical problems:

[0010] A method for detecting related substances of n-butyl bromide, using gas chromatography detection method, the chromatographic conditions are:

[0011] The chromatographic column is a DB-624 quartz capillary column, with specifications of 30m×0.32mm×1.8μm; the column temperature is 40°C; the stationary phase is 6% cyanopropylphenyl-94% dimethylpolysiloxane;

[0012] The injector temperature was 250°C; the injection volume was 1 μL; the split ratio was 20:1; the carrier gas was nitrogen; the nitrogen flow rate was 2.1 mL / min;

[0013] The detector is a hydrogen ion spark detector, and the detector temperature is 250°C.

[0014] As one of the preferred embodiments of the present invention, the chromatographic conditions also include using programmed temperature rise: initial temperature 40°C, maintained for 3 minutes; increased to 60°C at a rate of 2°C per minute, maintained for 3 minutes; increased to 240°C at a rate of 30°C per minute, maintained for 3 minutes.

[0015] As one of the preferred embodiments of the present invention, the method comprises the following specific steps:

[0016] (1) Solution preparation

[0017] Diluent: Use organic solvent as diluent;

[0018] System suitability solution: prepare a solution containing n-butyl bromide and n-butyl alcohol as the system suitability solution;

[0019] Test solution: prepare a solution containing the target analyte as the test solution;

[0020] Reference solution: prepare a solution containing n-butanol as the reference solution;

[0021] (2) Gas chromatography analysis

[0022] The gas chromatograph was used for detection. The gas chromatographic conditions were as follows: the chromatographic column was a DB-624 quartz capillary column with a specification of 30m×0.32mm×1.8μm; the column temperature was 40°C; the stationary phase was 6% cyanopropylphenyl-94% dimethylpolysiloxane; the temperature was programmed; the injector temperature was 250°C; the injection volume was 1μL; the split ratio was 20:1; the carrier gas was nitrogen; the nitrogen flow rate was 2.1mL / min; the detector was a hydrogen ion spark detector, and the detector temperature was 250°C;

[0023] Separately inject the system suitability solution, test solution and reference solution into the gas chromatograph, record the chromatogram, and calculate the content of each component.

[0024] As one of the preferred embodiments of the present invention, in step (1), the organic solvent includes but is not limited to methanol, which is used to dilute and prepare the system suitability solution, the test solution, and the reference solution.

[0025] As one of the preferred embodiments of the present invention, in the system suitability solution of step (1), the concentration of n-butane bromide is 25 mg / mL, and the concentration of n-butanol is 0.125 mg / mL.

[0026] As one of the preferred embodiments of the present invention, in the test solution of step (1), the concentration of the target detection substance in the test solution is 25 mg / mL.

[0027] As one of the preferred embodiments of the present invention, in the reference solution of step (1), the concentration of n-butanol is 0.125 mg / mL.

[0028] As one of the preferred embodiments of the present invention, in the step (2), the gas chromatograph is an Angen gas chromatograph model 8860.

[0029] As one of the preferred embodiments of the present invention, in step (2), the content of each component is calculated by the main component external standard method with the addition of a correction factor.

[0030] An application of the above-mentioned method for detecting related substances of n-butyl bromide is to use the n-butyl bromide detected and separated by the detection method as a starting material for the synthesis reaction of bupivacaine hydrochloride.

[0031] The advantages of the present invention compared to the prior art are:

[0032] (1) The present invention successfully separates various impurities in the n-butyl bromide product by selecting appropriate gas chromatography detection conditions. The known impurities are clearly separated from the main component in the GC chromatogram, and the impurities and their contents can be accurately tested. The system has good applicability and specificity. The impurities are calculated by the main component external standard method with a correction factor, which is no more than 0.5%, and the total amount of impurities shall not exceed 1.0%, which meets the detection limit requirements and the quality control requirements of the starting material.

[0033] (2) In the existing detection methods for halogenated alkanes, electron capture detectors are often used to improve their response and achieve the purpose of detection; the present invention uses a more conventional flame ionization detector to achieve the same purpose, which is more widely used;

[0034] (3) The present invention provides a bupivacaine hydrochloride starting material with clearly controllable impurities and impurity contents, thereby facilitating the quality control of bupivacaine hydrochloride.

[0035] In summary, the present invention can quickly and accurately detect the related substances of n-butyl bromide, and realize the effective separation of n-butyl bromide and its impurities, which is of great significance for improving the effective quality of n-butyl bromide, controlling the impurity content of the starting materials brought into the synthesis reaction of bupivacaine hydrochloride, and improving the quality stability of bupivacaine hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the system suitability chromatogram in Example 1;

[0037] Figure 2 is the chromatogram of the test solution in Example 1;

[0038] Figure 3 is the linear relationship diagram of n-butyl bromide in Example 2;

[0039] Figure 4 It is the linear relationship diagram of n-butanol in Example 2. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the raw materials, reagents, equipment and methods used in the following embodiments are conventional raw materials, reagents, equipment and methods in the art unless otherwise specified, and will not be repeated.

[0041] Example 1

[0042] A method for detecting related substances of n-butyl bromide in this embodiment adopts gas chromatography detection method, comprising the following steps:

[0043] (1) Solution preparation

[0044] Diluent: Methanol (used to dilute and prepare the following system suitability solutions, test solution, and reference solution).

[0045] Blank solution: methanol.

[0046] System suitability solution: Accurately weigh n-butyl bromide and n-butyl alcohol, and dilute with diluent to a solution containing 25 mg / mL n-butyl bromide and 0.125 mg / mL n-butyl alcohol.

[0047] Test solution: Accurately weigh the n-butyl bromide product to be tested and dilute it with diluent to a solution with a concentration of 25 mg / mL.

[0048] Reference solution: Accurately weigh n-butanol and dilute it with diluent to a solution containing 0.125 mg / mL n-butanol.

[0049] (2) Gas chromatography analysis

[0050] The samples were detected by gas chromatograph (Angen, model: 8860). The gas chromatographic conditions were as follows: the chromatographic column was a DB-624 quartz capillary column with a specification of 30m×0.32mm×1.8μm; the column temperature was 40°C; the stationary phase was 6% cyanopropylphenyl-94% dimethylpolysiloxane; the temperature was programmed: the initial temperature was 40°C, maintained for 3 min; the temperature was increased to 60°C at a rate of 2°C per minute, maintained for 3 min; the temperature was increased to 240°C at a rate of 30°C per minute, maintained for 3 min; the injector temperature was 250°C; the injection volume was 1μL; the split ratio was 20:1; the carrier gas was nitrogen; the nitrogen flow rate was 2.1mL / min; the detector was a hydrogen ion spark detector, and the detector temperature was 250°C.

[0051] Take blank solution, system suitability solution, test solution and reference solution and inject them into gas chromatograph respectively. Use the above chromatographic conditions for injection, record the chromatogram, and perform calculation according to the principal component external standard method with correction factor.

[0052] The chromatogram of the system suitability solution of this example is shown in Figure 1 , the chromatogram of the test solution is shown in Figure 2 .

[0053] Depend on Figure 1 , Figure 2 It can be seen that in the system suitability solution chromatogram, the separation between n-butyl bromide and n-butyl alcohol peaks is greater than 2.0, and the separation with other nearby unknown impurity peaks also meets the requirements, the number of plates is greater than 3000, and the system suitability results of this method are good. n-butyl alcohol was detected in the test solution, and the result calculated by the main component external standard method with correction factor was 0.01%.

[0054] Example 2

[0055] This example is a methodological verification of the method of the present invention.

[0056] 1. Exclusivity

[0057] The instrument and chromatographic conditions were the same as those in Example 1.

[0058] Investigate the separation between the peaks.

[0059] Take appropriate amounts of n-butyl bromide and n-butyl alcohol, dissolve and dilute with methanol to form a mixed solution containing 25 mg / mL of n-butyl bromide and 0.125 mg / mL of n-butyl alcohol, take 1 μL and inject it into the gas chromatograph. The test results are shown in Table 2.

[0060] Table 2 Separation results

[0061]

[0062] The blank solution does not interfere with the inspection of related substances of this product. The above results show that the separation degree between n-butane bromide and the adjacent impurity peak is 10.87, the separation degree between n-butanol and the adjacent impurity peaks is 3.65 and 10.87, the separation degree between each impurity peak is good, the theoretical plate number is greater than 10000, and the specificity of this method is good.

[0063] 2. Limit of Detection and Limit of Quantitation

[0064] The stock solutions of each reference substance were gradually diluted. The quantification limit was determined when the signal-to-noise ratio was approximately 10:1, and the detection limit was determined when the signal-to-noise ratio was approximately 3:1. The results are shown in Table 3.

[0065] Table 3 Results of detection limit and quantification limit

[0066]

[0067] From the above results, it can be seen that the quantitative limit concentration of each impurity is between 3.15 and 6.25 μg / mL, which is equivalent to the test sample concentration of 0.0125% to 0.0248%. The result is good and meets the detection sensitivity requirements.

[0068] 3. Linearity and Correction Factor

[0069] Take the reference stock solution to prepare solutions with different concentrations of linear relationships, calculate the linear equation, and use the slope value to calculate the impurity correction factor. The results are as follows: Figure 3 , Figure 4 As shown in Table 4.

[0070] Table 4 Linearity and correction factor results

[0071]

[0072] From the above results, it can be seen that the components have a good linear relationship within the range of quantitative limit concentration to 150% limit concentration, and the correlation coefficients are all greater than 0.999. The correction factor of n-butanol is 0.56, which exceeds the range of 0.9 to 1.1. In order to make the experiment simpler and faster and reduce the inspection cost, the main component external standard method with correction factor is used to calculate the impurity content.

[0073] 4. System Precision Experiment

[0074] Test solution: accurately weigh n-butyl bromide and dilute it with solvent to a solution containing 25 mg / mL n-butyl bromide;

[0075] Accurately measure 1 μL of the above test solution, inject it into the gas chromatograph, record the chromatogram, count the peak area and retention time of each component, and calculate the RSD (%). The results are shown in Table 5.

[0076] Table 5 Precision results

[0077]

[0078] From the above results, it can be seen that the test solution was injected 6 times continuously, and the RSD (%) of the peak area and retention time were both less than 2%, and the system precision of this method was good.

[0079] 5. Accuracy Experiment

[0080] Take the impurity reference stock solution, prepare the test solution, 50% limit concentration, 100% limit concentration and 150% limit concentration spiked test solution in three parallel batches, prepare the reference solution separately, accurately measure 1 μL of the above solution, inject it into the gas chromatograph, record the chromatogram, and calculate according to the main component external standard plus correction factor

[0081] The measured amount and added amount of 3 recovery solutions at each concentration were used to calculate the original impurity amount in the test solution, and the recovery rate was calculated based on the original amount, measured amount and added amount, and the average value and relative standard deviation (RSD) of the 9 recovery rates were calculated. The results are shown in Tables 6-1 and 6-2.

[0082] Table 6-1 Accuracy results (calculated by external standard method)

[0083]

[0084]

[0085] Table 6-2 Accuracy results (calculation of correction factor coefficients)

[0086]

[0087]

[0088] From the above results, it can be seen that the impurity recovery rate of the recovery rate experiment using the method of the present invention is in the range of 90% to 108%, and the recovery rate RSD is within 5%, indicating that the accuracy of this method is good and the impurity detection results are accurate and reliable.

[0089] 6. Durability Test

[0090] The chromatographic conditions of this method were fine-tuned by changing the starting column temperature and the chromatographic column flow rate, respectively, with the separation degree in the system suitability solution as an indicator. The experimental results are shown in Table 7.

[0091] Table 7 Separation results under different column temperatures and column flow rates

[0092]

[0093]

[0094] From the above results, it can be seen that the separation of n-butyl bromide and n-butane is not affected after the chromatographic conditions are changed in a small range, and the method has good durability.

[0095] In summary, the detection method provided by the present invention has strong specificity and good durability. Changing chromatographic conditions such as column flow rate and initial column temperature within a small range will not affect the detection of n-butyl bromide related substances. The method provided by the present invention can accurately detect related substances in n-butyl bromide, and the separation between each impurity and between the main component and the impurity is good. The detection result is accurate and reliable, and the quality standard of n-butyl bromide can be controlled more scientifically, which is of great significance to improving the quality stability of bupivacaine hydrochloride.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances of n-butyl bromide, characterized in that: The gas chromatography detection method was adopted, and the chromatographic conditions were: The chromatographic column is a DB-624 quartz capillary column, with specifications of 30m×0.32mm×1.8μm; the column temperature is 40°C; the stationary phase is 6% cyanopropylphenyl-94% dimethylpolysiloxane; The injector temperature was 250°C; the injection volume was 1 μL; the split ratio was 20:1; the carrier gas was nitrogen; the nitrogen flow rate was 2.1 mL / min; The detector is a hydrogen ion spark detector, and the detector temperature is 250°C.

2. The method for detecting related substances of n-butyl bromide according to claim 1, characterized in that: The chromatographic conditions also include using programmed temperature increase: initial temperature 40°C, maintained for 3 minutes; increased to 60°C at a rate of 2°C per minute, maintained for 3 minutes; increased to 240°C at a rate of 30°C per minute, maintained for 3 minutes.

3. The method for detecting related substances of n-butyl bromide according to claim 1 or 2, characterized in that: The specific steps include: (1) Solution preparation Diluent: Use organic solvent as diluent; System suitability solution: prepare a solution containing n-butyl bromide and n-butyl alcohol as the system suitability solution; Test solution: prepare a solution containing the target analyte as the test solution; Reference solution: prepare a solution containing n-butanol as the reference solution; (2) Gas chromatography analysis The gas chromatograph was used for detection. The gas chromatographic conditions were as follows: the chromatographic column was a DB-624 quartz capillary column with a specification of 30m×0.32mm×1.8μm; the column temperature was 40°C; the stationary phase was 6% cyanopropylphenyl-94% dimethylpolysiloxane; the temperature was programmed; the injector temperature was 250°C; the injection volume was 1μL; the split ratio was 20:1; the carrier gas was nitrogen; the nitrogen flow rate was 2.1mL / min; the detector was a hydrogen ion spark detector, and the detector temperature was 250°C; Separately inject the system suitability solution, test solution and reference solution into the gas chromatograph, record the chromatogram, and calculate the content of each component.

4. The method for detecting related substances of n-butyl bromide according to claim 3, characterized in that: In the step (1), the organic solvent is methanol, which is used to dilute and prepare the system suitability solution, the test solution, and the reference solution.

5. The method for detecting related substances of n-butyl bromide according to claim 3, characterized in that: In the system suitability solution of step (1), the concentration of n-butane bromide is 25 mg / mL, and the concentration of n-butanol is 0.125 mg / mL.

6. The method for detecting related substances of n-butyl bromide according to claim 3, characterized in that: In the test solution of step (1), the target detection substance concentration of the test solution is 25 mg / mL.

7. The method for detecting related substances of n-butyl bromide according to claim 3, characterized in that: In the reference solution of step (1), the concentration of n-butanol is 0.125 mg / mL.

8. The method for detecting related substances of n-butyl bromide according to claim 3, characterized in that: In the step (2), the gas chromatograph is an Angen gas chromatograph model 8860.

9. The method for detecting related substances of n-butyl bromide according to claim 3, characterized in that: In the step (2), the content of each component is calculated by the main component external standard method with the addition of a correction factor.

10. An application of the method for detecting n-butyl bromide-related substances as claimed in any one of claims 1 to 9, characterized in that: The n-butyl bromide separated by the detection method is used as the starting material for the synthesis reaction of bupivacaine hydrochloride.

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