Quantitative detection method for benzene in eltrombopag ethanolamine bulk drug
By optimizing the carrier gas and solvent using gas chromatography, and combining headspace sampling and a flame ionization detector, the problem of detecting benzene residues in eltrombopag ethanolamine raw material was solved, achieving highly specific and low-cost quantitative detection results.
Patent Information
- Application Number
- CN202511448392.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2026-01-13
AI Technical Summary
Existing technologies cannot effectively and accurately detect benzene residues in eltrombopag ethanolamine raw materials, and the use of helium as a carrier gas is costly and has poor stability, making it difficult to meet the minimum benzene limit requirements in ICH Q3C.
Gas chromatography was used with nitrogen as the carrier gas and dimethyl sulfoxide as the solvent. The quantitative detection of benzene in eltrombopag ethanolamine raw material was performed through headspace injection mode combined with a flame ionization detector. The optimization of the chromatographic column, temperature program and detector was specifically included.
It achieves highly specific, sensitive, and accurate quantitative detection of benzene, meets the minimum requirements of ICH Q3C, ensures product quality and safety, and is easy to operate and has a low cost.
Smart Images
Figure CN121324527A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of detection, specifically relating to a quantitative detection method for benzene in eltrombopag ethanolamine raw material. Background Technology
[0002] Eltrombopag ethanolamine, chemically named 3'-{(2Z)-2-[1-(3,4-dimethylyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-yl]hydrazinoyl}-2'-hydroxy-3-diphenylcarboxylic acid-2-aminoethanol (eltrombopag), has the molecular formula C 25 H 22 N4O4·2(C2H7NO); structural formula: .
[0003] Eltrombopag ethanolamine is a non-peptide thrombopoietin receptor (TPO-R) agonist. It is used to treat chronic immune (idiopathic) thrombocytopenic purpura (ITP) that has not responded well to prior therapy with glucocorticoids, immunoglobulins, etc.
[0004] Benzene is a colorless liquid with an aromatic odor. It is volatile, flammable, and toxic. It can damage human hematopoietic stem cells, reducing their replication and affecting the hematopoietic microenvironment, thus causing hematopoietic dysfunction. Furthermore, benzene can covalently bind to DNA, inhibiting DNA transcription and causing cell proliferation disorders. Benzene can also directly damage DNA, inducing mutations or chromosomal aberrations. The Chinese Pharmacopoeia classifies benzene as a Class 1 solvent, and its use should be avoided in drug production.
[0005] The synthesis of eltrombopag ethanolamine raw material uses reagents that may contain benzene, such as methanol, ethanol, and isopropanol. Alcohol solvents are easily contaminated by benzene, so it is crucial to develop an effective detection method for benzene in drugs.
[0006] There are currently no research reports on the detection of benzene in eltrombopag ethanolamine raw materials. Hang Weiwei, Determination of Benzene Residue in Drug U by Headspace Gas Chromatography [J], Health Must-Read, 2012, 11(3), disclosed a method for determining benzene residue in drugs, but helium is used as the carrier gas, which is more expensive and less stable than nitrogen. According to ICH Q3C, benzene is a Group 1 carcinogen, with a limit of 2 ppm in raw materials. The limit is low. The solubility of eltrombopag ethanolamine raw materials in N,N-dimethylformamide is worse than that in dimethyl sulfoxide, which means that the method cannot be used to determine benzene in eltrombopag ethanolamine raw materials.
[0007] Therefore, it is necessary to develop a detection method with high specificity, sensitivity and accuracy for the quantitative detection of benzene in the preparation process of eltrombopag ethanolamine raw material, so as to ensure product quality and thus solve the product safety problem. Summary of the Invention
[0008] To address the shortcomings of existing technologies, this invention provides a method for the quantitative detection of benzene in eltrombopag ethanolamine raw material. The method of this invention is simple to operate, has good specificity, and high sensitivity and accuracy, which can fully meet the requirements for quantitative determination of benzene in the preparation process of eltrombopag ethanolamine raw material, enabling better control of product quality and ensuring drug safety.
[0009] To achieve the above-mentioned objectives of this invention, the specific technical solution adopted by this invention is as follows: A quantitative detection method for benzene in eltrombopag ethanolamine raw material includes the following steps: injecting the test solution and the reference solution into a gas chromatograph via headspace injection, recording the chromatograms, and performing quantitative analysis of the chromatograms using the external standard method; The chromatographic detection conditions are as follows: Chromatographic column: A capillary column with 6% cyanopropylphenyl-94% dimethyl polysiloxane as the stationary phase or a capillary column with equivalent performance. The column temperature is programmed: the initial temperature is 55℃-65℃, maintained for 3 minutes, then increased to 150℃ at a rate of 5℃ per minute, and then increased to 240℃ at a rate of 30℃ per minute, and maintained for 5 minutes. The headspace flask was equilibrated at 90℃-100℃ for 20 minutes.
[0010] Preferably, the preparation process of the test solution includes: taking eltrombopag ethanolamine raw material, dissolving it in dimethyl sulfoxide, and thus obtaining the solution.
[0011] More preferably, the mass-to-volume ratio of eltrombopag ethanolamine raw material to dimethyl sulfoxide is 0.8-1.2 g: 5 mL.
[0012] Preferably, the preparation process of the reference solution includes: taking benzene reference standard and diluting it with dimethyl sulfoxide to a concentration of 0.0001 mg to 0.0004 mg benzene per 1 mL.
[0013] Preferably, the chromatographic column is a DB-624 or a capillary column with equivalent performance; the column has dimensions of 30m × 0.32mm and 3μm.
[0014] Preferably, the detection conditions are: an initial temperature of 55-65℃, more preferably 60℃.
[0015] Preferably, the detection conditions are: the temperature of the imported sample is 195-205℃, and more preferably 200℃.
[0016] Preferably, the detector is a hydrogen flame ionization detector.
[0017] Preferably, the detector temperature is 295-305℃, and more preferably 300℃.
[0018] Preferably, the detection conditions are: column flow rate of 1.9-2.1 mL / min and split ratio of 5:1.
[0019] Preferably, the detection conditions are: quantitative loop temperature 95-105℃, pipeline temperature 105-115℃, and more preferably quantitative loop temperature 100℃ and pipeline temperature 110℃.
[0020] This invention also relates to the application of the above-mentioned quantitative detection method in detecting the benzene content in eltrombopag ethanolamine raw materials or preparations.
[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention employs gas chromatography headspace sampling mode, using nitrogen as the carrier gas and dimethyl sulfoxide as the solvent for analysis and detection. This method is highly specific, sensitive, and accurate, simple to operate, and low in cost. It can accurately quantify the benzene content in eltrombopag ethanolamine raw material during preparation and is suitable for routine detection of benzene content in eltrombopag ethanolamine raw material. This method provides assurance for product quality. Attached Figure Description
[0022] Figure 1 This is a gas chromatogram of a blank solution; Figure 2 This is the gas chromatogram of the reference solution; Figure 3 This is the gas chromatogram of the test sample solution; Figure 4 This is a linear regression plot of benzene. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0024] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.
[0025] Example 1 (1) Chromatographic conditions: Instrument: Gas chromatograph (Thermo trace 1300), detector is flame ionization detector (FID); Column: DB-624, 30m × 0.32mm, 3μm; Column temperature: The initial temperature is 60℃, maintained for 3 minutes, then increased to 150℃ at a rate of 5℃ per minute, and then increased to 240℃ at a rate of 30℃ per minute, and maintained for 5 minutes. The injection port temperature is 200℃; the detector temperature is 300℃. The headspace flask was equilibrated at 95°C for 20 minutes. Air flow rate: 350 mL / min; Hydrogen flow rate: 35 mL / min; Column flow rate: 2.0 mL / min; Metering loop temperature: 100℃; Pipeline temperature: 110℃; Blank solvent: water.
[0026] (2) Solution preparation: Reference solution: Accurately weigh benzene and dilute with dimethyl sulfoxide to prepare a solution containing 0.0004 mg benzene per mL. Accurately measure 2 mL, place it in a headspace bottle, and seal. Perform two replicates.
[0027] Test solution: Accurately weigh 400 mg of eltrombopag ethanolamine raw material (batch number RD-EB10-21002, manufacturer: Guangdong Raffles Pharmaceutical Technology Co., Ltd.), place it in a top-capped empty bottle, accurately add 2 mL of dimethyl sulfoxide, and seal. Perform two parallel tests.
[0028] (3) Determination: Accurately measure 1.0 mL of the reference solution and the test solution, inject them into the gas chromatograph, and record the chromatograms.
[0029] Calculation formula: ; Where: At is the peak area of each residual solvent in the chromatogram of the test sample solution; As represents the peak area of each residual solvent in the chromatogram of the reference solution; Cs represents the concentration of each residual solvent in the reference solution, in mg / mL; Ct represents the concentration of the test solution, in mg / mL.
[0030] Judgment criteria: Calculated by peak area using the external standard method, the residual amount of benzene shall not exceed 0.0002%.
[0031] Test results are shown Figures 1 to 3 , Figure 1 This is a gas chromatogram of a blank solution. Figure 2 This is the gas chromatogram of the reference solution. Figure 3 This is the gas chromatogram of the test sample solution. The results show that benzene was not detected.
[0032] Methodological investigation 1. Specificity test Prepare one part each of blank solution, reference solution, test solution, and spiked solution of test solution, inject them, and record the chromatogram.
[0033] Blank solution: Accurately measure 2 mL of dimethyl sulfoxide, place it in a 20 mL headspace vial, cap it, and seal it.
[0034] Reference solution: Accurately weigh 20 mg of benzene reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and shake well to obtain the reference stock solution. Accurately transfer 1.0 mL of the reference stock solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well. Accurately measure 1.0 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well. Accurately transfer 2.0 mL of the reference solution into a 20 mL headspace vial, cap, and seal to obtain the reference solution.
[0035] Test solution: Accurately weigh 400 mg of the test sample and place it in a 20 mL headspace vial. Accurately transfer 2 mL of dimethyl sulfoxide into the same headspace vial, cap, and seal.
[0036] Spiked solution for test sample: Accurately weigh 400 mg of test sample and place it in a 20 mL headspace vial. Accurately transfer 2 mL of reference solution to the same headspace vial, cap, and seal.
[0037] The results are shown in Table 1. The blank solution showed no interfering peak at the benzene peak, and the test solution showed no other interfering peak at the benzene peak. The elution times of benzene in the reference solution and the spiked test solution were consistent. The retention time of benzene in the spiked test solution and the reference solution differed by 0.26%, indicating good method specificity.
[0038]
[0039] 2. Limit of Detection and Limit of Quantification The reference solution from the "Specificity Test" section was serially diluted and injected, and the chromatogram was recorded. The limit of quantitation (LOQ) was set at a signal-to-noise ratio (S / N) ≥ 10 for benzene peaks, and the result was 0.5 ppm. The limit of detection (LOD) was set at a S / N ≥ 3 for benzene peaks, and the result was 0.048 ppm. The LOQ solution was injected three times consecutively, the peak area was recorded, and the peak area RSD was calculated, resulting in a value of 1.36%. This method can effectively determine benzene, with extremely low LOD and LOD. Specific results are shown in Tables 2 and 3.
[0040]
[0041]
[0042] 3. Precision Repeatability and intermediate precision experiments were conducted at different times by two analysts using chromatographic columns from the same manufacturer but with different serial numbers. Following the methods described in the "Specificity Test" section, one blank solution, two reference solutions, and six spiked solutions of the test sample were prepared, and each was injected once, with chromatograms recorded. In the repeatability experiment, the RSD of benzene content in the six spiked solutions of the test sample was 1.7%. In the intermediate precision experiment, the RSD of benzene content in the six spiked solutions of the test sample was 0.6%, and the RSD of the 12 results was 7.5%, indicating good method precision. Specific data are shown in Table 4.
[0043]
[0044] 4. Linear Prepare one aliquot of a linear solution at a LOQ-150% concentration, inject each solution once, and record the chromatogram. Perform linear regression with concentration on the x-axis and peak area on the y-axis. The linear equation is as follows: y =0.4294 x +0.0004; the ratio of y-intercept to peak area at 100% concentration was 0.2%; the correlation coefficient r = 1.00; linearity was good in the range of 0.1028 μg / mL (LOQ) to 0.6166 μg / mL (150%). Results are shown in Table 5 and... Figure 4 .
[0045] Blank solution: Same as under "Specificity test".
[0046] Linear stock solution: Accurately weigh 40 mg of benzene reference standard into a 100 mL volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and shake well. Accurately transfer 1.0 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to obtain the solution.
[0047] LOQ linear solution: Prepare according to the method for preparing the LOQ solution under the "Detection Limit and Quantification Limit" section. Accurately transfer 2.0 mL into a 20 mL headspace vial, cap, and seal.
[0048] 50% linear solution: Accurately transfer 1.0 mL of the linear stock solution to a 20 mL volumetric flask, add dimethyl sulfoxide to the mark, and shake well. Accurately transfer 2.0 mL to a 20 mL headspace vial, cap, and seal.
[0049] 80% linear solution: Accurately transfer 2.0 mL of the linear stock solution to a 25 mL volumetric flask, add dimethyl sulfoxide to the mark, and shake well. Accurately transfer 2.0 mL to a 20 mL headspace vial, cap, and seal.
[0050] 100% Linear Solution: Accurately transfer 2.0 mL of the linear stock solution to a 20 mL volumetric flask, add dimethyl sulfoxide to the mark, and mix well. Accurately transfer 2.0 mL to a 20 mL headspace vial, cap, and seal.
[0051] 120% linear solution: Accurately transfer 3.0 mL of the linear stock solution to a 25 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 2.0 mL to a 20 mL headspace vial, cap, and seal.
[0052] 150% linear solution: Accurately transfer 3.0 mL of the linear stock solution to a 20 mL volumetric flask, add dimethyl sulfoxide to the mark, and shake well. Accurately transfer 2.0 mL to a 20 mL headspace vial, cap, and seal.
[0053]
[0054] 5. Accuracy Three aliquots of accuracy solutions at LOQ-150% concentration were prepared, and each was injected once. Chromatograms were recorded. The recoveries of benzene in the LOQ accuracy solutions ranged from 101.5% to 103.7%, with an RSD of 1.3%. The recoveries of benzene in the 50%–150% accuracy solutions ranged from 102.6% to 106.0%, with an RSD of 1.2% for the nine accuracy recoveries. The method showed good accuracy, and the results are shown in Table 6.
[0055] Prepare one blank solution, two reference solutions, and two test solution according to the methods under "Specificity Test". The preparation methods for other accuracy solutions are as follows: LOQ accuracy solution: Quantitatively dilute according to the concentration of the quantitation limit solution under the "Detection Limit and Quantitation Limit" section. Prepare 3 parallel portions.
[0056] 50% Accuracy Solution: Accurately weigh 20 mg of benzene reference standard into a 20 mL volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 1.0 mL of the above solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 1.0 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well to obtain the accuracy stock solution. Accurately weigh 400 mg of the test sample into a 20 mL headspace vial, accurately transfer 2.0 mL of the above accuracy stock solution into the same headspace vial, cap, and seal. Prepare three parallel aliquots.
[0057] 100% Accuracy Solution: Accurately weigh 40 mg of benzene reference standard into a 20 mL volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 1.0 mL of the above solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 1.0 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well to obtain the accuracy stock solution. Accurately weigh 400 mg of the test sample into a 20 mL headspace vial, accurately transfer 2.0 mL of the above accuracy stock solution into the same headspace vial, cap, and seal. Prepare three parallel aliquots.
[0058] 150% Accuracy Solution: Accurately weigh 60 mg of benzene reference standard into a 20 mL volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 1.0 mL of the above solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well. Accurately transfer 1.0 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and mix well to obtain the accuracy stock solution. Accurately weigh 400 mg of the test sample into a 20 mL headspace vial, accurately transfer 2.0 mL of the above accuracy stock solution into the same headspace vial, cap, and seal. Prepare three parallel aliquots.
[0059]
[0060] 6. Durability Following the solution preparation method under the "Specificity Test" section, prepare one blank solution, two reference solutions, and two spiked solutions of the test sample. Inject these solutions and record the chromatograms. Under the conditions of an oven temperature of 90℃-100℃, an initial temperature program of 55℃-65℃, and a flow rate of 1.8mL / min-2.0mL / min, the ratio of the benzene content in the spiked solution of the test sample to the result under normal chromatographic conditions was between 0.97 and 1.09, indicating good method robustness. The results are shown in Table 7.
[0061]
[0062] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.
Claims
1. A method for quantitative detection of benzene in eltrombopag ethanolamine raw material, characterized in that, Includes the following steps: The test solution and the reference solution were injected into the gas chromatograph via headspace injection, and the chromatograms were recorded. The external standard method was used to perform quantitative analysis of the chromatograms. The chromatographic detection conditions are as follows: Chromatographic column: A capillary column with 6% cyanopropylphenyl-94% dimethyl polysiloxane as the stationary phase or a capillary column with equivalent performance. The column temperature is programmed: the initial temperature is 55℃-65℃, maintained for 3 minutes, then increased to 150℃ at a rate of 5℃ per minute, and then increased to 240℃ at a rate of 30℃ per minute, and maintained for 5 minutes. The headspace flask was equilibrated at 90℃-100℃ for 20 minutes.
2. The quantitative detection method according to claim 1, characterized in that, The preparation process of the test solution includes: taking eltrombopag ethanolamine raw material, adding dimethyl sulfoxide to dissolve it, and thus obtaining the solution.
3. The quantitative detection method according to claim 1, characterized in that, The preparation process of the reference solution includes: taking benzene reference standard, diluting it with dimethyl sulfoxide to a concentration of 0.0001 mg-0.0004 mg benzene per 1 mL.
4. The quantitative detection method according to claim 1, characterized in that, The chromatographic column is a DB-624 or a capillary column with equivalent performance; the column dimensions are 30m × 0.32mm and 3μm.
5. The quantitative detection method according to claim 1, characterized in that, The detection conditions are as follows: the initial temperature is 55-65℃.
6. The quantitative detection method according to claim 1, characterized in that, The testing conditions are as follows: the temperature of the imported sample is 195-205℃.
7. The quantitative detection method according to claim 1, characterized in that, The detector is a hydrogen flame ionization detector with a detector temperature of 295-305℃.
8. The quantitative detection method according to claim 1, characterized in that, The detection conditions were as follows: column flow rate of 1.9-2.1 mL / min and split ratio of 5:
1.
9. The quantitative detection method according to any one of claims 1-8, characterized in that, The detection conditions are as follows: quantitative loop temperature 95-105℃, pipeline temperature 105-115℃.
10. The application of the quantitative detection method according to any one of claims 1-9 in detecting the benzene content in eltrombopag ethanolamine raw material or preparation.