Method for detecting related substances in 3-isobutylglutaric anhydride

Abstract

The invention provides a method for detecting related substances in 3-isobutylglutaric anhydride, which adopts a gas chromatographic method for detection, and the chromatographic conditions of the gas chromatographic method are as follows: a detector is a hydrogen flame ionization detector; a chromatographic column adopts a capillary chromatographic column with polyethylene glycol 20M as a stationary liquid; according to the method, the peak response intensity of the test solution and the reference solution is improved by setting the temperature rising program of the gas chromatography, the temperature of the sample inlet, the temperature of the detector and other conditions, so that the detection precision is improved. The detection method is high in specificity, good in repeatability, good in linear relation, high in recovery rate, low in detection limit and quantitation limit, good in durability, rapid in operation and identification, low in detection cost and accurate and reliable in detection result, can provide data support for effective quality control of 3-isobutylglutaric anhydride in the production process of 3-isobutylglutaric anhydride, and has wide application prospects. And a guarantee is provided for monitoring the quality stability and clinical medication safety of a product taking 3-isobutylglutaric anhydride as a starting material.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting related substances in 3-isobutylglutaric anhydride. Background Art

[0002] 3-Isobutylglutaric anhydride (the structural formula is shown in Formula 1) is a light yellow to yellow liquid and can be used as a pharmaceutical intermediate. It is easily decomposed into 3-isobutylglutaric acid when exposed to water. During the storage of 3-isobutylglutaric anhydride, related substances such as 3-isobutylglutaric acid (the structural formula is shown in Formula 2) are inevitably degraded.

[0003]

[0004] As a starting material and key intermediate for synthesizing pharmaceutical products, the purity of 3-isobutylglutaric anhydride directly affects the quality of the synthesized products. Therefore, the content of related substances in 3-isobutylglutaric anhydride needs to be strictly controlled.

[0005] Currently, there is no report on the method for detecting related substances in 3-isobutylglutaric anhydride. Therefore, there is a need in the art to provide a method for accurately detecting related substances in 3-isobutylglutaric anhydride. Summary of the Invention

[0006] In view of the above problems, the present invention provides a method for detecting related substances in 3-isobutylglutaric anhydride. The 3-isobutylglutaric acid in 3-isobutylglutaric anhydride can be accurately detected by gas chromatography.

[0007] To solve the above technical problems, the technical solution provided by the present invention is: a method for detecting related substances in 3-isobutylglutaric anhydride, which is detected by gas chromatography. The chromatographic conditions of the gas chromatography include: the detector is a flame ionization detector;

[0008] The chromatographic column is a capillary chromatographic column with polyethylene glycol 20M as the stationary liquid;

[0009] Temperature programming: the initial temperature is 80°C to 82°C, maintained for 4.5 min to 5.5 min, heated at a rate of 3°C / min to 7°C / min to 90°C to 110°C, maintained for 0 min, and then heated at a rate of 35°C / min to 45°C / min to 290°C to 310°C, maintained for 2.5 min to 3.5 min.

[0010] The temperature of the injection port is 240°C to 260°C;

[0011] The temperature of the detector is 290°C to 310°C.

[0012] Preferably, the chromatographic column is Agilent DB-WAX, with a specification of 30m×0.53mm and a packing diameter of 1.0μm.

[0013] Preferably, the chromatographic conditions of the gas chromatography further include: the carrier gas is nitrogen.

[0014] Preferably, the flow rate of the nitrogen is 2.8 ml / min to 3.2 ml / min.

[0015] Preferably, the chromatographic conditions of the gas chromatography further include: the injection volume is 0.8 μl to 1.2 μl.

[0016] Preferably, the chromatographic conditions of the gas chromatography further include: the split ratio is 5:1 to 15:1.

[0017] Preferably, the split ratio is 8:1 to 12:1.

[0018] Preferably, it includes the following steps: Preparation of the test solution: Dissolve the 3-isobutylglutaric anhydride sample in a solvent to obtain the test solution; Preparation of the reference solution: Dissolve the 3-isobutylglutaric anhydride sample and the 3-isobutylglutaric acid reference substance in a solvent to obtain the reference solution.

[0019] Preferably, the solvent is acetonitrile.

[0020] Preferably, the concentration of the test solution is 20 mg / ml to 30 mg / ml; the concentration of the 3-isobutylglutaric anhydride sample in the reference solution is 20 mg / ml to 30 mg / ml, and the concentration of the 3-isobutylglutaric acid reference substance is 20 μg / ml to 30 μg / ml.

[0021] Beneficial effects

[0022] Compared with the prior art, the method for detecting related substances in 3-isobutylglutaric anhydride provided by the present invention uses a capillary chromatographic column with polyethylene glycol 20M as the stationary liquid to perform gas chromatography analysis on the 3-isobutylglutaric anhydride sample, realizing the accurate detection of 3-isobutylglutaric acid existing in 3-isobutylglutaric anhydride. By setting conditions such as the temperature programming of the gas chromatography, the temperature of the injection port and the detector, etc., the present invention improves the peak response intensity of the test solution and the reference solution, thereby improving the detection accuracy. This detection method has strong specificity, good repeatability, good linear relationship, high recovery rate, low detection limit and quantification limit, good durability, and is fast and convenient to operate and identify, with low detection cost and accurate and reliable detection results. It can provide data support for the effective control of the quality of 3-isobutylglutaric anhydride during the production process, and provide a guarantee for monitoring the quality stability of products using 3-isobutylglutaric anhydride as the starting material and the safety of clinical medications.

[0023] It should be noted that the reference solution of the present invention adopts the standard solution addition method, which can eliminate the matrix effect of the sample. Description of the Drawings

[0024] Figure 1 It is the gas chromatogram of the blank solution in Example 1 of the present invention;

[0025] Figure 2 It is the gas chromatogram of the reference solution in Example 1 of the present invention;

[0026] Figure 3 It is the gas chromatogram of the test solution in Example 1 of the present invention; Detailed Embodiment

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments.

[0028] Example 1

[0029] This example provides a method for detecting related substances in 3-isobutylglutaric anhydride, including the following steps:

[0030] 1. Chromatographic conditions

[0031] Instrument: Agilent 7890N gas chromatograph

[0032] Chromatographic column: Agilent DB-WAX (30m × 0.53mm, 1μm);

[0033] Temperature programming: initial temperature 80°C, hold for 5 min, increase the temperature at a rate of 5°C / min to 100°C, hold for 0 min, then increase the temperature at a rate of 40°C / min to 300°C, hold for 3 min.

[0034] Injection port temperature is 250°C;

[0035] The detector is a flame ionization detector (FID detector), and the detector temperature is 300°C;

[0036] The carrier gas is nitrogen, and the flow rate is 3 ml / min;

[0037] Direct injection, injection volume is 1 μl; split ratio is 10:1.

[0038] 2. Solution preparation.

[0039] Blank solution: acetonitrile.

[0040] Preparation of reference stock solution: Take an appropriate amount of 3-isobutylglutaric anhydride reference substance, weigh accurately, quantitatively dilute and dissolve with acetonitrile to obtain a reference stock solution with a concentration of 200 μg / ml.

[0041] Preparation of reference solution: Accurately weigh 200 mg of 3-isobutylglutaric acid sample, place it in a 10-ml volumetric flask, accurately add 1 ml of the above-mentioned reference stock solution, dissolve it with acetonitrile and dilute to the mark to obtain the reference solution. Test solution: Accurately weigh 200 mg of 3-isobutylglutaric anhydride sample, place it in a 10-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark to obtain the test solution.

[0042] 3. Method validation

[0043] (1) System suitability test

[0044] Detect the above-mentioned blank solvent, reference solution and test solution according to the chromatographic conditions in Example 1. The gas chromatograms of the blank solution, reference solution and test solution are as Figures 1 - 3 shown. The retention times and resolution results of the impurities in the test solution are shown in Table 1. It can be seen from Table 1 that the blank solvent does not interfere with the detection of each component, the resolution between each component meets the requirements, and the method has good specificity.

[0045] Table 1 Retention times and resolution of impurities in the test solution

[0046] Component Retention time (min) Resolution Acetonitrile 1.693 - 3 - Isobutylglutaric anhydride 5.688 10.786 3 - Isobutylglutaric acid 9.915 12.654 Unknown impurity 1 12.965 14.567

[0047] (2) Repeatability test

[0048] Take 6 portions of the test sample, dilute them with acetonitrile respectively to prepare test solutions with a concentration of 20 mg / ml, detect them according to the chromatographic conditions in Example 1, and record the chromatograms. The results of the repeatability test are shown in Table 2. It can be seen from Table 2 that the determination results of the 6 test solutions are basically the same, indicating that the method has good repeatability.

[0049] Table 2 Results of the repeatability test

[0050]

[0051] (3) Linearity test

[0052] Take an appropriate amount of the reference stock solution, prepare 7 sample solutions with different concentrations respectively, inject the above sample solutions for detection according to the chromatographic conditions in Example 1, take the concentration in the sample solution as the abscissa and the peak area as the ordinate, plot the standard working curve and establish the linear equation. The final test results are shown in Table 3. It can be seen from Table 3 that for the detection method provided by the present invention, there is a good linear relationship between the concentration of 3-isobutylglutaric acid and the peak area.

[0053] Table 3 Results of the linear relationship test of 3-isobutylglutaric acid

[0054]

[0055] (4) Recovery rate test

[0056] Take the reference stock solution and prepare three concentrations of low, medium and high respectively, with 3 replicates for each concentration; dilute and make up the volume with acetonitrile to obtain the test solution for recovery rate. Detect according to the chromatographic conditions in Example 1. The recovery rates of the related substance 3-isobutylglutaric anhydride in the 3-isobutylglutaric anhydride sample are shown in Table 4. It can be seen from Table 4 that at three different addition amounts of 3-isobutylglutaric anhydride, the average recovery rate is 97.2% and the RSD is 2.8%, indicating that the accuracy of this detection method is good.

[0057] Table 4 Results of the spiked recovery rate test

[0058]

[0059] (5) Detection limit and quantitation limit test

[0060] Precisely measure an appropriate amount of the reference stock solution, perform stepwise dilution, detect according to the chromatographic conditions in Example 1, and record the chromatogram. When S / N = 10, it is the quantitation limit, and when S / N = 3, it is the detection limit. The test results of the quantitation limit and detection limit are shown in Tables 5 - 6. It can be seen from Tables 5 - 6 that the detection limit and quantitation limit of the related substances in 3-isobutylglutaric anhydride detected by the detection method provided by the present invention are relatively low, and the determination of the related substances in 3-isobutylglutaric anhydride has the characteristic of high sensitivity.

[0061] Table 5 Results of the quantitation limit test

[0062]

[0063] Table 6 Results of the detection limit test

[0064]

[0065] (7) Solution stability test

[0066] Prepare the test solution according to the method in Example 1, place it at room temperature for 0h, 4h, 8h, 12h, 24h respectively, inject and determine according to the chromatographic conditions in Example 1. The results of the solution stability test are shown in Table 7. It can be seen from Table 7 that when the test solution is placed at room temperature for 24h, the RSD is 1.7%, indicating that the stability of the test solution is good.

[0067] Table 7 Results of the solution stability test of the test solution

[0068] Time (h) 3 - Isobutylglutaric acid (%) Total impurities (%) 0 0.011 0.033 4 0.011 0.032 8 0.011 0.033 12 0.011 0.032 24 0.012 0.033 RSD (%) 3.9 1.7

[0069] (8) Robustness test

[0070] Take the test sample solution, and separately change the initial temperature, temperature rising rate, injector temperature, and detector temperature of the chromatographic column, and inject the sample for determination according to the chromatographic conditions in Example 1. The results are shown in Tables 8 - 11. It can be seen from Tables 8 - 11 that when each condition is changed, the resolution of each component meets the requirements, indicating that the method provided by this solution has good durability.

[0071] Table 8 Resolution at Different Initial Temperatures

[0072] Condition Initial temperature 80°C Initial temperature 78°C Initial temperature 82°C Acetonitrile - - - 3 - Isobutylglutaric anhydride 10.786 10.998 10.247 3 - Isobutylglutaric acid 12.654 13.436 12.065 Unknown impurity 14.567 15.213 14.568

[0073] Table 9 Resolution at Different Temperature Rising Rates

[0074] Condition Heating rate 40°C Heating rate 35°C Heating rate 45°C Acetonitrile - - - 3 - Isobutylglutaric anhydride 10.786 11.825 10.324 3 - Isobutylglutaric acid 12.654 12.786 12.432 Unknown impurity 14.567 14.628 14.231

[0075] Table 10 Resolution at Different Injector Temperatures

[0076] Condition 250℃ 240℃ 260℃ Acetonitrile - - - 3 - Isobutylglutaric anhydride 10.786 10.326 11.008 3 - Isobutylglutaric acid 12.654 11.984 12.763 Unknown impurity 14.567 14.029 14.798

[0077] Table 11 Resolution at Different Detector Temperatures

[0078] Condition 300℃ 290℃ 310℃ Acetonitrile - - - 3 - Isobutylglutaric anhydride 10.786 10.526 11.032 3 - Isobutylglutaric acid 12.654 12.471 12.952 Unknown impurity 14.567 14.396 14.686

[0079] Comparative Example 1

[0080] This comparative example provides a method for detecting related substances of 3 - isobutylglutaric anhydride. The detection method is exactly the same as the gas chromatographic conditions in Example 1, except that the chromatographic column in the chromatographic conditions is a capillary column with 5% diphenyl - 95% dimethyl polysiloxane (or similar polarity) as the stationary liquid

[0081] HP - 5 (30m × 0.53mm × 1.0μm), and other conditions remain unchanged.

[0082] Take the test sample solution under Example 1 for injection detection. The test results prove that the main component peak of 3 - isobutylglutaric anhydride fronts, resulting in a lower accuracy of 3 - isobutylglutaric acid.

[0083] Comparative Example 2

[0084] This comparative example provides a method for detecting related substances of 3 - isobutylglutaric anhydride. The detection method is exactly the same as the gas chromatographic conditions in Example 1, except that the chromatographic column in the chromatographic conditions is a capillary column DB - 1701 (30m × 0.53mm × 1.0μm) with 14% cyanopropylphenyl - 86% dimethyl polysiloxane (or similar polarity) as the stationary liquid, and other conditions remain unchanged.

[0085] Take the test sample solution under Example 1 for injection detection. The test results prove that the main component peak of 3 - isobutylglutaric anhydride tails, and it fails to reach baseline separation from 3 - isobutylglutaric acid, resulting in a lower accuracy of 3 - isobutylglutaric acid.

[0086] Comparative Example 3

[0087] This comparative example provides a method for detecting related substances of 3-isobutylglutaric anhydride. The detection method is exactly the same as the gas chromatography conditions in Example 1, except that the chromatographic column in the chromatographic conditions is a capillary column HP-FFAP (30m×0.53mm×1.0μm) with cross-linked polyethylene glycol (or similar polarity) as the stationary liquid, and other conditions remain unchanged.

[0088] Inject the test solution under the item of Example 1 for detection. The test results prove that the chromatographic peaks of 3-isobutylglutaric anhydride and 3-isobutylglutaric acid are broadened, the column efficiency is reduced, resulting in a decrease in accuracy.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting related substances in 3-isobutylglutaric anhydride, characterized in that: Gas chromatography was used for detection, and the chromatographic conditions of the gas chromatography included: The detector is a hydrogen flame ionization detector; The chromatographic column uses a capillary column with polyethylene glycol 20M as the stationary liquid; Heating program: starting temperature 80℃~82℃, maintain for 4.5min~5.5min, increase the temperature to 90℃~110℃ at a rate of 3℃ / min~7℃ / min, maintain for 0min, then increase the temperature to 290℃~310℃ at a rate of 35℃ / min~45℃ / min, maintain for 2.5min~3.5min. The temperature of the injection port is 240℃~260℃; The temperature of the detector is 290°C to 310°C.

2. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 1, characterized in that: The chromatographic column is Agilent DB-WAX, with a specification of 30m×0.53mm and a filler diameter of 1.0μm.

3. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography method also include: the carrier gas is nitrogen.

4. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 3, characterized in that: The flow rate of the nitrogen gas is 2.8 ml / min to 3.2 ml / min.

5. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography method also include: the injection volume is 0.8 μl to 1.2 μl.

6. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography method also include: a split ratio of 5:1 to 15:

1.

7. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 6, characterized in that: The split ratio is 8:1 to 12:

1.

8. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 1, characterized in that: The following steps are involved: Preparation of test solution: dissolve 3-isobutylglutaric anhydride sample in solvent to obtain test solution; preparation of reference solution: dissolve 3-isobutylglutaric anhydride sample and 3-isobutylglutaric acid reference in solvent to obtain reference solution.

9. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 8, characterized in that: The solvent is acetonitrile.

10. The method for detecting related substances in 3-isobutylglutaric anhydride according to claim 8, characterized in that: The concentration of the test solution is 20 mg / ml to 30 mg / ml; the concentration of the 3-isobutylglutaric anhydride sample in the reference solution is 20 mg / ml to 30 mg / ml, and the concentration of the 3-isobutylglutaric acid reference is 20 μg / ml to 30 μg / ml.

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