A method for testing benzene in a bulk drug
By optimizing the diluent and heating temperature in gas chromatography and combining it with headspace sampling technology, the problem of insufficient sensitivity in benzene detection in existing technologies has been solved, achieving highly sensitive, rapid, and accurate analysis and detection of benzene in pharmaceutical raw materials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2026-07-24
AI Technical Summary
Existing methods for benzene detection, such as liquid chromatography and ultraviolet absorption spectrophotometry, are cumbersome to operate and lack sufficient sensitivity. Common gas chromatography methods, on the other hand, have low benzene response and poor sensitivity, making it difficult to meet the minimum detection requirements for benzene in pharmaceutical raw materials.
Gas chromatography combined with headspace sampling was used, with a mixed solution of DMSO and water as the diluent. The benzene content in the active pharmaceutical ingredient was detected by gas chromatography. Specific conditions included a heating equilibrium temperature of 80-100℃, a carrier gas nitrogen flow rate of 2 mL/min, an FID detector, and a 6% cyanopropylphenyl-94% dimethylsiloxane column. The temperature program ranged from 40-240℃. The diluent ratio and heating temperature were optimized to improve detection accuracy.
It achieves highly sensitive, rapid, and accurate detection of benzene residues, avoiding contamination of the liner and injection port caused by excessively high sample concentrations. It provides high specificity, accuracy, and repeatability, meeting the requirements for low-limit analysis of benzene in active pharmaceutical ingredients.
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Figure CN122448995A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of active pharmaceutical ingredient (API) analysis technology, specifically to a method for testing benzene in APIs. Background Technology
[0002] Benzene is a colorless, toxic liquid with a distinctive aromatic odor, widely used in the pharmaceutical field and an important raw material for many organic compounds. Benzene is a commonly used solvent in drug development and is also a recognized process impurity found in toluene and acetone. It is listed as a Class I solvent requiring restricted use in both ICH guidelines and the 2020 edition of the Chinese Pharmacopoeia, Part IV. Therefore, if solvents such as acetone and toluene are used in the synthesis of active pharmaceutical ingredients, the risk of introducing benzene must be considered. The limit for benzene is very low, only 2 ppm, thus necessitating an analytical method for detecting benzene residues that is highly sensitive, specific, accurate, and precise.
[0003] The main methods for detecting and analyzing benzene include liquid chromatography (LC), ultraviolet (UV) spectrophotometry, and gas chromatography (GC). LC typically involves purifying and enriching the benzene sample using pretreatment techniques (such as solid-phase extraction), then injecting it into a high-performance liquid chromatograph (HPLC) for separation using a stationary phase and measurement using a detector to obtain benzene content information. UV spectrophotometry measures benzene content by measuring the absorption of ultraviolet light in a benzene solution. Benzene molecules absorb ultraviolet light in the 200-300 nm range; the concentration of benzene in the sample is determined by measuring whether ultraviolet light is absorbed. Gas chromatography is one of the most commonly used methods for benzene detection. It involves evaporating and separating the benzene in the sample, passing it along with a carrier gas through a chromatographic column, and finally detecting it using a detector. This method utilizes the fact that benzene's time and physical properties in the chromatographic column differ from other compounds to achieve its detection and quantification.
[0004] Liquid chromatography and ultraviolet absorption spectrophotometry both suffer from problems such as complicated pretreatment and insufficient sensitivity. Common gas chromatography methods also have issues such as low benzene response and poor sensitivity. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an analytical testing method for residual solvent benzene. The detection method established in this invention is highly sensitive, convenient and quick to operate, accurate and reliable, and has good repeatability. Method validation has demonstrated the specificity, accuracy, repeatability, limit of detection, limit of quantitation, linearity, and range of this method. Furthermore, this invention limits headspace sampling, avoiding contamination of the liner and injection port due to excessively high sample concentrations. This provides technical support for the analytical detection of benzene in pharmaceutical raw materials. To achieve the above-mentioned objectives, the following technical solutions are required:
[0006] A method for testing benzene in a pharmaceutical raw material, wherein the testing method is gas chromatography: the pharmaceutical raw material is dissolved in a diluent to obtain a test solution; the test solution is headspace sampled and detected by gas chromatography to obtain a gas chromatogram; the content of benzene in the pharmaceutical raw material is calculated based on the peak area of benzene in the gas chromatogram.
[0007] Specifically, the diluent is a mixed solution of DMSO and water, wherein the proportion of water is between 10% and 20%.
[0008] Specifically, the headspace sampling heating equilibrium temperature is 80-100℃.
[0009] Specifically, the carrier gas of the gas chromatograph is nitrogen, and the flow rate is 2 mL / min.
[0010] Specifically, the detector of the gas chromatograph is FID.
[0011] Specifically, the stationary phase of the gas chromatograph column is 6% cyanopropylphenyl-94% dimethylsiloxane.
[0012] Specifically, the temperature program of the gas chromatograph is as follows: start column temperature 40℃, maintain for 3 min, increase to 120℃ at a rate of 10℃ / min, then increase to 240℃ at a rate of 30℃ / min, and maintain for 5 min.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] The detection method established in this invention is highly sensitive, convenient and quick to operate, accurate and reliable, and has good repeatability. The specificity, accuracy, repeatability, limit of detection, limit of quantitation, linearity, and range of the method have been verified through method validation. In addition, this invention limits headspace sampling, avoiding the problem of contamination of the liner and injection port due to excessively high sample concentration. It provides technical support for the analysis and detection of benzene in pharmaceutical raw materials. Attached Figure Description
[0015] Figure 1 This is a comparison spectrum of Example 1;
[0016] Figure 2 The detection limit spectrum for Example 1;
[0017] Figure 3 This is the limit of quantitation spectrum for Example 1. Detailed Implementation
[0018] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
[0019] Example 1
[0020] This embodiment provides a test method for detecting residual solvent benzene in a pharmaceutical raw material, as well as a method optimization process.
[0021] The detection conditions for the gas chromatography method are as follows:
[0022]
[0023]
[0024] Headspace sampler:
[0025] Heating temperature 90℃ Quantitative Loop Temperature 100℃ Transmission line temperature 120℃ Sample heating time 30min Injection time 1.0min Cycle time 32min
[0026] Solution preparation:
[0027]
[0028] Method development and optimization process: mainly involving the optimization of heating temperature and diluent. Different diluents and heating temperatures were screened according to general analytical methods, and the benzene peak area was confirmed.
[0029] The test results are as follows:
[0030] Heating temperature Different diluents Peak area (pA) 80℃ 100% DMSO 3.30 90℃ 100% DMSO 4.02 100℃ 100% DMSO 4.85 80℃ <![CDATA[10%H2O+90%DMSO]]> 5.98 90℃ <![CDATA[10%H2O+90%DMSO]]> 7.32 80℃ <![CDATA[20%H2O+80%DMSO]]> 10.59 90℃ <![CDATA[20%H2O+80%DMSO]]> 12.53
[0031] The detection results show that the peak area of benzene is directly proportional to the proportion of water in the diluent and the heating temperature, which is significant for benzene detection. However, when further increasing the water content or raising the heating temperature, factors such as the boiling point of the solvent, the solubility of the sample, and the water resistance of the column must also be considered.
[0032] Conclusion: Under the same analytical conditions, a heating temperature of 90℃ and a diluent containing 20% H2O are the optimal conditions. To demonstrate the applicability of this method to the detection of residual benzene, methodological validation was performed, including validation of specificity, accuracy, repeatability, limit of detection, limit of quantitation, and linearity.
[0033] Prepare the solution according to the above conditions.
[0034] (1) Specificity: Under the above chromatographic conditions, blank solution and reference solution were injected separately, and the chromatograms were recorded. There were no interfering peaks at the elution position of the target solvent peak in the blank solution.
[0035] (2) Accuracy: Benzene limits of 50%, 100%, and 120% were added to the test solution, and three aliquots of each concentration were prepared. The solutions were injected sequentially, chromatograms were recorded, and the benzene content was calculated. The measured values were compared with the theoretical values to calculate the recovery rate. The recoveries of the nine aliquots ranged from 71.03% to 86.78%, with an average recovery rate of 80.7%. The RSD of the nine recoveries was 6.2%, indicating good accuracy.
[0036] (3) Repeatability: Six test sample solutions were prepared with benzene at 100% of the limit concentration. The samples were injected sequentially under the chromatographic conditions described above, and the chromatograms were recorded. The benzene content was calculated. The measured values were compared with the theoretical values to calculate the recovery rate. The results showed that the recovery rates of the six samples ranged from 71.03% to 82.31%, with an average recovery rate of 76.9%. The RSD of the six results was 4.9%, indicating good repeatability.
[0037] (4) Limit of detection: The limit of quantitation solution in the linear solution was diluted by one time and then injected for analysis. The concentration of benzene was 0.02994 mg / L, the relative concentration was 0.00003%, and the signal-to-noise ratio was 9, indicating that this concentration level can be reliably detected.
[0038] (5) Limit of quantitation: The limit of quantitation solution under linear solution was injected 6 times consecutively. The limit of quantitation concentration of benzene was 0.05988 mg / L, the relative concentration was 0.00006%, the signal-to-noise ratio was 9, the signal-to-noise ratio of the 6 injections was between 18 and 24, and the peak area RSD of the 6 injections was 4.8%, which proved that the concentration level could be reliably quantified.
[0039] (6) Linearity: Prepare linear solutions with limits of 30% to 120% according to the table below, measure the peak area of benzene at each concentration, plot the standard curve with the peak area as the ordinate and the concentration as the abscissa, and calculate the correlation coefficient R, the Y-axis intercept and the sum of squared residuals.
[0040] Preparation of linear solutions
[0041]
[0042]
[0043] The linear regression equation is y = 61.113x - 0.561, the Y-intercept is -0.561, the correlation coefficient R is 1.00, and the 25% of the limit concentration response value is 2.915, which meets the acceptable standard.
[0044] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for testing benzene in a pharmaceutical raw material, characterized in that, The test method is gas chromatography: the active pharmaceutical ingredient is dissolved in a diluent to obtain a test solution; the test solution is headspace sampled and gas chromatographically detected to obtain a gas chromatogram; the content of benzene in the active pharmaceutical ingredient is calculated based on the peak area of benzene in the gas chromatogram.
2. The test method according to claim 1, characterized in that, The diluent is a mixed solution of DMSO and water, wherein the proportion of water is 10% to 20%.
3. The test method according to claim 1, characterized in that, The headspace sampling heating equilibrium temperature is 80-100℃.
4. The test method according to claim 1, characterized in that, The carrier gas of the gas chromatograph is nitrogen, and the flow rate is 2 mL / min.
5. The test method according to claim 1, characterized in that, The detector of the gas chromatograph is FID.
6. The test method according to claim 1, characterized in that, The stationary phase of the gas chromatograph column is 6% cyanopropylphenyl-94% dimethylsiloxane.
7. The test method according to claim 1, characterized in that, The temperature program of the gas chromatograph is as follows: start column temperature 40℃, maintain for 3 min, increase to 120℃ at a rate of 10℃ / min, then increase to 240℃ at a rate of 30℃ / min, and maintain for 5 min.