Method for detecting tribromoacetone related substances by gas chromatography

Abstract

The invention discloses a method for detecting related substances of tribromoacetone by gas chromatography, which belongs to the technical field of chemical detection, the related substances of tribromoacetone are detected by gas chromatography, an instrument is a gas chromatograph, a detector is an FID detector, a capillary column taking 100% methylpolysiloxane (MEGA-1) as a stationary liquid is used as a chromatographic column, carrier gas is N2, the flow velocity is 2.0 mL / min, the split ratio is 50: 1, and the detection time is 30 minutes. Temperature programming: the initial temperature is 80 DEG C, and the temperature is raised to 250 DEG C at the rate of 10 DEG C per minute and maintained for 10 minutes; a sample injection mode is as follows: direct sample injection is performed, the temperature of a sample injection port is 260 DEG C, and the temperature of a detector is 280 DEG C; according to the present invention, the sample introduction volume is 0.2 [mu] l, the related substances in the tribromoacetone can be effectively separated, the content of the related substances can be quantitatively detected, the acetone, the bromoacetone, the 1, 3-dibromoacetone and the 1, 1, 3, 3-tetrabromoacetone sequentially appear peaks, and the method has characteristics of simple operation, strong specificity, good repeatability, good intermediate precision and good durability, and can accurately and rapidly determine the content of the various related substances in the tribromoacetone.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for detecting related substances in tribromoacetone using a gas chromatography method. Background Art

[0002] Tribromoacetone is a key starting material in the synthesis of methotrexate (MTX). Methotrexate, a folic acid analogue, exhibits antiproliferative, anti-inflammatory, and immunomodulatory effects. It is an immunosuppressant widely used clinically to treat a variety of conditions, including malignancies and autoimmune diseases. Since its discovery in the 1940s, it has been used to treat childhood leukemia and psoriasis since the 1950s. It is now a commonly used anti-tumor drug in clinical practice, primarily for acute leukemia, breast cancer, choriocarcinoma, and malignant hydatidiform mole. It is also used in rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, ectopic pregnancy, and ophthalmology. Methotrexate is one of the pharmaceutical industry's greatest success stories, as its discovery of indications diverges significantly from its original purpose, with low and high doses being used for distinct indications. Its main dosage forms include tablets, injections, injections, and oral solutions, and it is included in the national medical insurance catalog.

[0003] Controlling the quality of methotrexate starting materials from the source is crucial. Currently, few methods for detecting tribromoacetone-related substances have been reported. Due to the diverse, unstable, and highly reactive impurities in tribromoacetone, there are no direct methods for simultaneously detecting multiple impurities in tribromoacetone. Therefore, developing a method that can effectively detect multiple impurities in tribromoacetone is crucial, providing a simple and effective method for in-process control and finished product testing. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for detecting the impurity content of related substances (acetone, bromoacetone, 1,3-dibromoacetone, 1,1,3,3-tetrabromoacetone) in tribromoacetone. The present invention adopts the following technical solutions:

[0005] A method for detecting tribromoacetone-related substances by gas chromatography comprises the following steps:

[0006] 1) Instrument and chromatographic conditions: Gas chromatograph; MEGA-1 (50 m × 0.32 mm × 1.0 μm) or a capillary column of similar polarity; FID detector; initial column temperature 70–90°C, then raised at a rate of 8–12°C / min to a final temperature of 250°C; holding time at the final temperature for 8–12 min; nitrogen carrier gas.

[0007] In order to achieve better detection effect, the carrier gas flow rate is 1.5-2.5 ml / min, the split ratio is 50:1; the injection port temperature is 250-270°C, the detector temperature is 270-290°C; the injection method is direct injection; and the injection volume is 0.2 μL.

[0008] 2) Prepare the following solutions:

[0009] a. Preparation of each impurity location solution:

[0010] Acetone positioning solution: Take 1 ml of acetone into the injection vial.

[0011] Bromoacetone positioning solution: Take 20 μl of bromoacetone, place it in a sample vial, add 1 ml of acetone, and shake well.

[0012] 1,3-Dibromoacetone positioning solution: Take 0.1g of 1,3-dibromoacetone, place it in a sample vial, add 1ml of acetone, and shake well.

[0013] 1,1,3,3-Tetrabromoacetone positioning solution: Take 0.1g of 1,1,3,3-tetrabromoacetone, place it in a sample vial, add 1ml of acetone solution, and shake well.

[0014] b. Preparation of test solution: Take an appropriate amount of tribromoacetone, dissolve it in warm water, shake well, and re-prepare it before use.

[0015] Inject samples according to the following sequence:

[0016]

[0017] The relevant substances in the tribromoacetone test solution are calculated using the area normalization method.

[0018] Preferably, the chromatographic conditions selected for the gas chromatography analysis of the present invention are as follows:

[0019] Using FID detector;

[0020] Chromatographic column: MEGA-1 (50m×0.32mm×1.0μm) or capillary column with similar polarity;

[0021] Temperature program: start at 80°C, increase the temperature to 250°C at 10°C / min, and maintain for 10 min. Carrier gas: nitrogen, flow rate: 2.0 mL / min, split ratio: 50:1;

[0022] Inlet temperature: 260°C; Detector (FID) temperature: 280°C;

[0023] Injection method: direct injection; injection volume: 0.2μl;

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention can effectively separate related substances in tribromoacetone. A capillary column with common filler (100% methyl polysiloxane) can be used to separate multiple related substances, such as acetone, bromoacetone, 1,3-dibromoacetone, 1,1,3,3-tetrabromoacetone, etc. Since impurities in the above-mentioned analytes are unstable, highly active, and easily converted into each other, the detection method is unstable. The present invention can eliminate the influence of instability by selecting appropriate chromatographic conditions and preparing and injecting the solution immediately after melting. The method is simple to operate, has strong specificity, and is good in repeatability, intermediate precision, and durability. The sample can be directly injected after dissolution, which is convenient to operate and highly accurate, thereby ensuring that the quality of tribromoacetone is controllable. The present invention provides a stable and reliable detection method for detecting related substances of tribromoacetone. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Overlay chromatograms for each positioning solution;

[0027] Figure 2 is the chromatogram of the test solution; DETAILED DESCRIPTION

[0028] The present invention will further supplement the above content with reference to the embodiments (attached drawings), but it should not be understood that the scope of the present invention is limited to the following examples.

[0029] The instruments and chromatographic conditions used in the following examples are as follows: the gas chromatograph is an Agilent 7890A, an FID detector is used, the initial column temperature is 70-90°C, then the temperature is increased at a rate of 8-12°C / min to a final column temperature of 250°C, and the final column temperature is maintained for 8-12 min; the carrier gas is nitrogen, preferably, the carrier gas flow rate is 1.5-2.5 ml / min, and the split ratio is 50:1; the injection port temperature is 250-270°C, and the detector temperature is 270-290°C; the injection method is direct injection; and the injection volume is 0.2 μL.

[0030] Example 1, specificity investigation, such as Figure 1~Figure 2 As shown:

[0031] Accurately measure 0.2 μL of the test solution and each impurity-locating solution, inject them into the gas chromatograph, and record the chromatogram. Under these chromatographic conditions, the main peak in the test solution does not interfere with the detection of known impurities; the minimum resolution in the test solution is 2.3 (greater than 1.5), indicating that this method meets the specificity requirements for the detection of this product.

[0032] Example 2, precision test results:

[0033] (1) Repeatability study

[0034] Accurately measure 6 portions of the test solution, inject 0.2 μL of sample, and inject into the gas chromatograph, recording the chromatogram. The maximum RSD of the test results for 1,3-dibromoacetone, tribromoacetone, 1,1,3,3-tetrabromoacetone, other single impurities, and total impurities is 0.2%, indicating that the repeatability of this method is good. The repeatability test results are as follows:

[0035]

[0036] Note: N / D means not detected

[0037] (2) Intermediate precision study

[0038] Experimenters A and B each prepared six test sample solutions and tested them on different instruments. A 0.2 μL injection volume was injected into the gas chromatograph, and the chromatograms were recorded. The RSDs for 1,3-dibromoacetone, tribromoacetone, 1,1,3,3-tetrabromoacetone, other single impurities, and total impurities were 4.5% at most, exceeding 5%. Therefore, the intermediate precision of this method is good. The intermediate precision test results are as follows:

[0039]

[0040] Example 3, durability investigation:

[0041] Maintaining the basic chromatographic conditions unchanged, the flow rate (±0.2 ml / min), column temperature (±5°C), injection port temperature (±10°C), and detector temperature (±10°C) were adjusted, and the chromatographic column (different brands, models, or batches with the same filler physical and chemical properties) were replaced to examine the durability of the chromatographic conditions. Accurately measure 0.2 μL of the test solution, inject it into the gas chromatograph, and record the chromatogram. The maximum RSD of the durability test results for 1,3-dibromoacetone, tribromoacetone, 1,1,3,3-tetrabromoacetone, other single impurities, and total impurities was 3.7%, not exceeding 5%, and the minimum resolution was 2.2, indicating good durability. The specific test results are as follows:

[0042]

[0043] Example 3, multiple batches of sample detection:

[0044] The validated analytical method was used to test the relevant substances in 5 batches of tribromoacetone test samples. The specific test results are as follows:

[0045]

[0046] The above description is only a preferred embodiment of the present invention. It should be pointed out that those skilled in the art should understand that several improvements can be made without departing from the principles of the present invention. These improvements should also be regarded as the scope of protection of the present invention and should not be regarded as specific limitations on the present invention.

Claims

1. A method for detecting tribromoacetone-related substances by gas chromatography, characterized in that: The following steps are involved: 1) Equipment selection: Use a gas chromatograph with an FID detector and a MEGA-1 or capillary column with similar polarity; 2) Preparation of each impurity localization solution: Acetone positioning solution: Take 1ml of acetone into the injection vial; Bromoacetone positioning solution: Take 20 μl of bromoacetone, place it in a sample vial, add 1 ml of acetone, and shake well; 1,3-Dibromoacetone positioning solution: Take 0.1g of 1,3-dibromoacetone, place it in a sample vial, add 1ml of acetone, and shake well; 1,1,3,3-Tetrabromoacetone positioning solution: Take 0.1g of 1,1,3,3-tetrabromoacetone, place it in a sample vial, add 1ml of acetone solution, and shake well; 3) Preparation of test solution: Dissolve an appropriate amount of tribromoacetone in warm water and shake well. 4) Determination of related substances: Accurately measure 0.2µl of each impurity location solution and test sample solution, inject them into the gas chromatograph, record the chromatogram, and calculate the detection results of each related substance by the area normalization method.

2. The method for detecting tribromoacetone related substances by gas chromatography according to claim 1, wherein: A gas chromatograph and an FID detector are used, and the chromatographic column in the gas chromatography detection is a MEGA-1 capillary column.

3. The method for detecting tribromoacetone related substances by gas chromatography according to claim 1, wherein: The column temperature change process in the gas phase detection is as follows: the initial column temperature is 70-90°C, then the temperature is increased at a rate of 8-12°C / min to a final column temperature of 250°C, and the final column temperature is maintained for 8-12 minutes.

4. The method for detecting tribromoacetone related substances by gas chromatography according to claim 1, wherein: In the gas chromatography detection, the diluent used in the specificity investigation is acetone.

5. The method for detecting tribromoacetone related substances by gas chromatography according to claim 1, wherein: In the gas chromatography detection, the injection port temperature is 250-270° C., the FID detector temperature is 270-290° C., and the carrier gas is nitrogen.

6. The method for detecting tribromoacetone-related substances by gas chromatography according to claim 1, wherein: The gas chromatography test was performed by injecting the sample directly after dissolving the sample, with an injection volume of 0.2 μL.

7. The method for detecting tribromoacetone-related substances by gas chromatography according to claim 1, wherein: The carrier gas flow rate is 1.5-2.5 ml / min, and the split ratio is 50:

1.

8. The method for detecting tribromoacetone-related substances by gas chromatography according to claim 1, wherein: The substances measured in the substance measuring step include at least one of acetone, bromoacetone, 1,3-dibromoacetone, and 1,1,3,3-tetrabromoacetone, or a combination of two or more thereof.

Images