A method for detecting impurities in 2-methylpyridine

Abstract

The present invention relates to the technical field of drug detection, and specifically discloses a detection method for impurities in 2-picoline. The present invention adopts a DB-WAX chromatographic column with a specification of 60m×0.32mm×1.0μm to perform gas chromatography analysis on 2-picoline samples, and realizes accurate detection of 3 kinds of impurities and their contents of pyridine, 3-picoline and 4-picoline present in 2-picoline at the same time. The detection method has strong specificity, low detection limit and quantitative limit, good linear relationship, high recovery rate, good repeatability, strong stability, good durability, and simple and fast operation, low detection cost, accurate and reliable detection result, and can provide data support for the effective control of its quality in the process of producing 2-picoline, and provides guarantee for monitoring the quality stability and clinical drug safety of 2-picoline as betahistine mesylate intermediate.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting impurities in 2-methylpyridine. Background Art

[0002] 2-Methylpyridine, also known as α-methylpyridine, is an organic compound with a chemical formula of C6H7N. It is a colorless liquid, miscible with water, and soluble in most organic solvents. It is mainly used in the synthesis of chemicals such as medicines, dyes, and rubbers, and is also used as a solvent or experimental reagent. 2-Methylpyridine is synthesized from formaldehyde, acetaldehyde, and ammonia water under a cobalt catalyst. The chemical reaction formula of the synthesis process is shown below. In the synthesis process of 2-methylpyridine, related substances such as pyridine, 3-methylpyridine or 4-methylpyridine are inevitably present. These related substances (impurities) directly affect the synthesis reaction of 2-methylpyridine, and then affect the purity of the finished product. When 2-methylpyridine is used as the starting material in the synthesis of betahistine mesylate (or other drugs), its purity directly affects the quality of the subsequent finished product. Therefore, the content of impurities in 2-methylpyridine needs to be strictly controlled.

[0003]

[0004] There is no report about the detection method of related substances (pyridine, 3-picoline or 4-picoline) in 2-picoline. In order to accurately detect the above-mentioned related substances, it is necessary to perform multiple detections, and the operation process is cumbersome, time-consuming and laborious. Therefore, it is necessary to provide a detection method that can accurately detect related substances in 2-picoline. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for detecting impurities in 2-methylpyridine, which can simultaneously and accurately detect the impurities pyridine, 3-methylpyridine and 4-methylpyridine in 2-methylpyridine by gas chromatography.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A method for detecting impurities in 2-methylpyridine comprises the following steps:

[0008] Preparation of test solution: Dissolve 2-methylpyridine sample in solvent to obtain test solution;

[0009] Preparation of reference solution: dissolve pyridine reference, 3-methylpyridine reference and 4-methylpyridine reference in a solvent to obtain a reference solution;

[0010] The reference solution and the test solution are detected by gas chromatography, and the chromatographic conditions of the gas chromatography include:

[0011] A DB-WAX column was used, with specifications of 60 m × 0.32 mm and a filler diameter of 1.0 μm;

[0012] The heating program is as follows: starting temperature 90-110°C, maintaining for 8-12 min, heating to 190-210°C at a rate of 8-12°C / min, maintaining for 4-8 min;

[0013] The injection port temperature is 210-230°C;

[0014] The detector temperature is 240-260°C.

[0015] Compared with the prior art, the detection method of impurities in 2-methylpyridine provided by the present invention adopts a DB-WAX chromatographic column with a specification of 60m×0.32mm×1.0μm to perform gas chromatography analysis on a 2-methylpyridine sample, thereby realizing accurate detection of three impurities and their contents of pyridine, 3-methylpyridine and 4-methylpyridine in 2-methylpyridine at the same time. The present invention improves the peak response intensity of the test solution and the reference solution by limiting the temperature rise program of the gas chromatography and the temperature of the injection port and the detector, thereby improving the detection accuracy. The detection method has strong specificity, low detection limit and quantitative limit, good linear relationship, high recovery rate, good repeatability, strong stability, good durability, simple and fast operation, low detection cost, accurate and reliable detection results, and can provide data support for the effective control of its quality in the process of producing 2-methylpyridine, and provides a guarantee for monitoring the quality stability and clinical drug safety of 2-methylpyridine as an intermediate of betahistine mesylate.

[0016] Preferably, the solvent is methanol.

[0017] Preferably, the concentration of the test solution is 8-12 mg / mL.

[0018] Preferably, the concentration of pyridine in the reference solution is 30-80 μg / mL.

[0019] Preferably, the concentration of 3-methylpyridine in the reference solution is 30-80 μg / mL.

[0020] Preferably, the concentration of 4-methylpyridine in the reference solution is 30-80 μg / mL.

[0021] Preferably, the chromatographic conditions of the gas chromatography also include:

[0022] The detector is a hydrogen flame ionization detector;

[0023] The carrier gas was nitrogen with a flow rate of 2.5 to 3.5 mL / min;

[0024] The injection volume is 0.8-1.2 μL;

[0025] The split ratio is (35~45):1.

[0026] For example, the stationary liquid of the DB-WAX chromatographic column is cross-linked polyethylene glycol. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the gas chromatogram of the blank solution in Example 1 of the present invention;

[0028] Figure 2 is the gas chromatogram of the reference substance solution in Example 1 of the present invention;

[0029] Figure 3 The gas chromatogram of the test solution in Example 1 of the present invention;

[0030] Figure 4 The gas chromatogram of the spiked test solution in Example 1 of the present invention;

[0031] Figure 5 The gas chromatogram of the spiked test solution in Comparative Example 1 of the present invention;

[0032] Among them, chromatographic peak 1 is pyridine, chromatographic peak 2 is 3-methylpyridine, chromatographic peak 3 is 4-methylpyridine, and chromatographic peak 4 is 2-methylpyridine. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1

[0035] This embodiment provides a method for detecting impurities in 2-methylpyridine, comprising the following steps:

[0036] (1) Solution preparation

[0037] Blank solution: methanol.

[0038] Reference solution: Take appropriate amounts of pyridine reference, 3-methylpyridine reference and 4-methylpyridine reference respectively, weigh accurately, dilute and dissolve quantitatively with methanol to make a mixed solution containing 50 μg of each in 1 mL to obtain the reference solution. That is, in the reference solution, the concentrations of pyridine, 3-methylpyridine and 4-methylpyridine are all 50 μg / mL.

[0039] Test solution: Take 100 mg of 2-methylpyridine sample, accurately weigh it, place it in a 10 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well to obtain the test solution.

[0040] Reference substance stock solution: Take appropriate amounts of pyridine reference substance, 3-methylpyridine reference substance and 4-methylpyridine reference substance, weigh them accurately, dilute and dissolve them quantitatively with methanol, and make a mixed solution containing 500 μg of each in 1 mL to obtain the reference substance stock solution. That is, in the reference substance stock solution, the concentrations of pyridine, 3-methylpyridine and 4-methylpyridine are all 500 μg / mL.

[0041] Spiked test solution: Take 100 mg of 2-methylpyridine sample, accurately weigh it, put it in a 10 mL volumetric flask, accurately add 1 mL of the above-mentioned reference substance stock solution, dissolve it with methanol and make up to the scale, shake well, and obtain the spiked test solution.

[0042] (2) The blank solution, reference solution, test solution and spiked test solution are tested by gas chromatography. The specific chromatographic conditions of the gas chromatography include:

[0043] A DB-WAX column (60 m × 0.32 mm, 1.0 μm) was used;

[0044] The temperature program of the gas chromatography was as follows: starting temperature 100 °C, maintained for 10 min, increasing to 200 °C at a rate of 10 °C / min, maintained for 6 min;

[0045] The detector was a hydrogen flame ionization (FID) detector with an inlet temperature of 220°C and a detector temperature of 250°C;

[0046] The carrier gas was nitrogen with a flow rate of 3 mL / min;

[0047] The injection volume was 1 μL;

[0048] The split ratio is 40:1.

[0049] The gas chromatogram results of blank solution, reference solution, test solution and spiked test solution are as follows Figures 1 to 4 As shown, the separation degree of each impurity in the reference solution and the spiked test solution was tested, and the test results are shown in Tables 1 and 2. Figures 1 to 4 As can be seen from Tables 1 and 2, in the method for detecting impurities in 2-methylpyridine provided in this embodiment, the blank solvent does not interfere with the detection of each component, the separation degree between the components meets the requirements, and the method has good specificity.

[0050] Table 1 Retention time and separation of each impurity in the reference solution

[0051] Element Retention time min Resolution USP Methanol 3.646 -- Pyridine 9.899 43.446 3-Methylpyridine 13.636 28.83 4-Methylpyridine 13.841 1.915

[0052] Table 2 Retention time and separation of each impurity in the spiked test solution

[0053]

[0054]

[0055] Methodological validation

[0056] 1. Durability test

[0057] The reference solution was prepared according to the method of Example 1, and only the starting temperature, heating rate, injection port temperature and detector temperature were changed, and the single factor variables were controlled. The detection was performed according to the detection method of Example 1, and the detection results are shown in Tables 3 to 6. It can be seen from Tables 3 to 6 that the separation degree of each impurity meets the requirements when the conditions are changed, indicating that the detection method of impurities in 2-methylpyridine provided by the present invention has good durability.

[0058] Table 3 Separation of impurities in reference solution at different starting temperatures

[0059] Initial temperature 100℃ 90℃ 110℃ Pyridine 43.928 47.802 42.449 3-Methylpyridine 28.963 29.065 30.055 4-Methylpyridine 1.882 1.873 1.897

[0060] Table 4 Separation of impurities in reference solution at different heating rates

[0061]

[0062]

[0063] Table 5 Separation of impurities in reference solution at different injection port temperatures

[0064] Inlet temperature 220℃ 210℃ 230℃ Pyridine 43.928 43.763 43.743 3-Methylpyridine 28.963 28.683 28.799 4-Methylpyridine 1.882 1.880 1.896

[0065] Table 6 Separation of impurities in reference solution at different detector temperatures

[0066] Detector temperature 250℃ 240℃ 260℃ Pyridine 43.928 43.824 44.079 3-Methylpyridine 28.963 28.936 29.077 4-Methylpyridine 1.882 1.883 1.886

[0067] 2. Detection limit and quantification limit test

[0068] The reference solution was prepared according to the method of Example 1, and the detection was carried out according to the chromatographic conditions in Example 1, and the gas chromatogram was recorded. The peak height was 10 times the baseline noise as the quantitative limit, and the peak height was 3 times the baseline noise as the detection limit. The test results of the quantitative limit and the detection limit are shown in Table 7, and the repeatability test results of the quantitative limit are shown in Table 8. It can be seen from Tables 7 to 8 that the detection limit and the quantitative limit of the detection method provided by the present invention are low for detecting impurities in 2-methylpyridine, and the detection of impurities in 2-methylpyridine has the characteristics of high sensitivity; the relative standard deviation (RSD) of the peak area of ​​each impurity repeated 6 times is 4.34% at most, indicating that the detection method provided by the present invention has good repeatability of the quantitative limit.

[0069] Table 7 Test results of limit of quantitation and limit of detection

[0070]

[0071] Table 8 Results of repeatability test of limit of quantitation

[0072] Element 1 2 3 4 5 6 RSD% Pyridine 1813 1730 1909 1782 1845 1826 3.31 2-Methylpyridine 1905 1820 1910 2030 2005 1849 4.34 3-Methylpyridine 1685 1732 1763 1764 1685 1767 2.25 4-Methylpyridine 2024 2028 2064 1972 1960 2070 2.26

[0073] 3. Linear relationship test

[0074] Accurately weigh appropriate amounts of pyridine reference substance, 3-methylpyridine reference substance, 4-methylpyridine reference substance and 2-methylpyridine reference substance, prepare sample solutions of 7 to 8 concentration levels respectively, detect the sample solutions according to the chromatographic conditions in Example 1, determine the peak area of ​​the gas chromatogram, plot the standard working curve and establish a linear equation with the component concentration in the sample solution as the abscissa and the peak area as the ordinate, and the final test results are shown in Tables 9 to 12. It can be seen from the data in Tables 9 to 12 that in the detection method provided by the present invention, the concentration of 2-methylpyridine and its impurities has a good linear relationship with the peak area measured by gas chromatography.

[0075] Table 9 Linear relationship test results of pyridine

[0076]

[0077] Table 10 Linear relationship test results of 3-methylpyridine

[0078]

[0079] Table 11 Linear relationship test results of 4-methylpyridine

[0080]

[0081] Table 12 Linear relationship test results of 2-methylpyridine

[0082]

[0083] 4. Recovery rate test

[0084] Prepare the reference stock solution according to the method of Example 1. Take 100 mg of 2-methylpyridine sample, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol to dissolve it, and prepare 9 copies in parallel; then add 0.2 mL, 1 mL, and 1.2 mL of the reference stock solution respectively, so that the concentration of each impurity is low, medium, and high, respectively, and prepare 3 copies in parallel; use methanol to make the volume to the scale, shake well, and use it as the recovery test solution. Detection was performed according to the detection method and chromatographic conditions in Example 1. The recovery rates of each impurity in the 2-methylpyridine sample were finally measured as shown in Tables 13 to 15. It can be seen from Tables 13 to 15 that the recovery rates of each impurity at 3 different addition amounts were between 96% and 106%, and the maximum RSD was 3.46%, indicating that the detection method provided by the present invention has good accuracy.

[0085] Table 13 Recovery of pyridine

[0086]

[0087]

[0088] Table 14 Recovery of 3-methylpyridine

[0089]

[0090] Table 15 Recovery of 4-methylpyridine

[0091]

[0092] 5. Repeatability test

[0093] Six groups of 2-picoline samples from the same batch were taken, and the test solutions were prepared according to the method in Example 1, and the samples were injected according to the chromatographic conditions in Example 1, and the contents of various impurities in different groups of 2-picoline samples were detected respectively. The test results are shown in Table 16. As can be seen from Table 16, the detection results of the detection method provided by the present invention for repeated detection of impurities in the same batch of 2-picoline samples are basically consistent, and have good repeatability.

[0094] Table 16 Repeatability test results of each impurity content (%)

[0095]

[0096] 6. Solution stability test

[0097] The reference solution and the test solution were prepared according to the method in Example 1, and after being placed at room temperature for 0 h, 2 h, 4 h, 8 h and 16 h, gas chromatography was performed under the chromatographic conditions in Example 1. The stability test results of the solution are shown in Tables 17 and 18. As can be seen from Tables 17 and 18, the same method was used for testing within 16 h at room temperature, and the results obtained were basically the same - the relative standard deviation (RSD) of the peak area of ​​each impurity of the reference solution was less than 2%, and the relative standard deviation (RSD) of the impurity content of the test solution was less than 0.7%, and there was no significant difference in the test results, indicating that the reference solution and the test solution prepared by the present invention have good stability.

[0098] Table 17 Stability of reference solution (peak area)

[0099] Impurities 0h 2h 4h 8h 16h RSD% Pyridine 25951 25746 25675 25341 25311 1.07 3-Methylpyridine 23782 23713 23417 23341 23321 0.92 4-Methylpyridine 27978 27982 27583 27421 27365 1.08

[0100] Table 18 Stability of test solution (peak area)

[0101]

[0102] Comparative Example 1

[0103] This comparative example provides a method for detecting impurities in 2-methylpyridine, which is similar to Example 1, except that the chromatographic column is replaced with a DB-WAX chromatographic column (30m×0.25mm, 0.25μm; the stationary liquid is cross-linked polyethylene glycol), and other conditions are the same as in Example 1 and will not be repeated.

[0104] The gas chromatogram of this comparative example is as follows Figure 5 As shown, the peak response values ​​of each impurity in the spiked test solution were significantly reduced, 3-methylpyridine and 4-methylpyridine did not achieve baseline separation, and the peak responses of the main component and each impurity were relatively concentrated.

[0105] Comparative Example 2

[0106] This comparative example provides a method for detecting impurities in 2-methylpyridine, which is similar to Example 1, except that: ① the injection port temperature is 250°C and the detector temperature is 250°C; ② the carrier gas is nitrogen and the flow rate is 50 mL / min; the other conditions are the same as those in Example 1 and will not be repeated here.

[0107] In this comparative example, the peak response of the spiked test solution was significantly reduced, and the detection accuracy could not reach the level of Example 1.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting impurities in 2-methylpyridine, characterized in that: The following steps are involved: Preparation of test solution: Dissolve 2-methylpyridine sample in solvent to obtain test solution; Preparation of reference solution: dissolve pyridine reference, 3-methylpyridine reference and 4-methylpyridine reference in a solvent to obtain a reference solution; The solvent is methanol; The reference solution and the test solution are detected by gas chromatography, and the chromatographic conditions of the gas chromatography include: A DB-WAX column was used, with specifications of 60 m × 0.32 mm and a filler diameter of 1.0 μm; The heating program is as follows: starting temperature 90~110℃, maintaining for 8~12min, heating to 190~210℃ at a rate of 8~12℃ / min, maintaining for 4~8min; The injection port temperature is 210~230℃; The temperature of the detector is 240-260°C; the detector is a hydrogen flame ionization detector.

2. The method for detecting impurities in 2-picoline according to claim 1, characterized in that: The concentration of the test solution is 8-12 mg / mL.

3. The method for detecting impurities in 2-methylpyridine according to claim 1, characterized in that: The concentration of pyridine in the reference solution is 30-80 μg / mL.

4. The method for detecting impurities in 2-methylpyridine according to claim 1, characterized in that: The concentration of 4-methylpyridine in the reference solution is 30-80 μg / mL.

5. The method for detecting impurities in 2-methylpyridine according to claim 1, characterized in that: The concentration of 3-methylpyridine in the reference solution is 30-80 μg / mL.

6. The method for detecting impurities in 2-methylpyridine according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography also include: the carrier gas is nitrogen, and the flow rate of the nitrogen is 2.5-3.5 mL / min.

7. The method for detecting impurities in 2-methylpyridine according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography also include: the injection volume is 0.8-1.2 μL.

8. The method for detecting impurities in 2-methylpyridine according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography method also include: a split ratio of (35~45):1.

Images