Method for determining residual epichlorohydrin in dihydroxypropyl theophylline

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for determining residual epichlorohydrin in dihydroxypropyl theophylline. The residual epoxy chloropropane in the dihydroxypropyl theophylline is detected by adopting GC-MS (Gas Chromatography-Mass Spectrometer); chromatographic conditions are as follows: a chromatographic column is VF-624ms, 30m * 0.25 mm * 1.4 mu m; the carrier gas is helium, the column flow velocity is 0.95-1.05 ml / min, and the split ratio is 10: 1; the temperature of a sample inlet is 230-250 DEG C; the sample injection volume is 1 microliter; mass spectrum conditions are as follows: an ion source: EI; the temperature of the ion source is 220-240 DEG C; the temperature of a quadrupole rod is 140-160 DEG C; and the temperature of the auxiliary heater is 250-270 DEG C. Compared with the prior art, the analysis method provided by the invention has the advantages of high sensitivity, strong separation capability and rapidness, and can accurately quantify epichlorohydrin in the test sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical detection, and particularly relates to a method for determining residual epichlorohydrin in dyphylline. BACKGROUND

[0002] Dyphylline is a commonly used antiasthmatic drug, which can effectively relax bronchial smooth muscle and relieve bronchospasm, and is widely used in the treatment of chronic bronchitis, bronchial asthma and other diseases. In the production process of dyphylline, epichlorohydrin is often used as a key synthetic raw material to participate in the reaction. However, epichlorohydrin is highly toxic and carcinogenic. If it is left in the finished dyphylline product, it will enter the human body through medication, irritate the respiratory tract and skin mucosa, and long-term intake may cause serious health risks such as liver and kidney function damage and gene mutation. Therefore, accurately determining the residual amount of epichlorohydrin in dyphylline is a key link to ensure the quality and safety of drugs, meet the impurity limit requirements of the People's Republic of China Pharmacopoeia and international drug standards.

[0003] At present, the methods for determining the residual epichlorohydrin in drugs mainly include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS) and the like. Among them, gas chromatography is adopted by some enterprises due to its simple operation and low cost, but it depends on the retention time of the chromatographic peak for qualitative analysis, and is easily interfered by other organic impurities in the dyphylline matrix. When the residual amount of epichlorohydrin is very low (such as less than 0.1 μg / g), it is difficult to achieve accurate detection, and there are defects of inaccurate qualitative analysis and insufficient sensitivity.

[0004] Although the existing GC-MS method is superior to the simple gas chromatography method in terms of sensitivity, it still has obvious deficiencies in the detection of dyphylline samples: first, the chromatographic column is usually selected to be a common non-polar or weakly polar column (such as DB-5ms), which has poor separation effect on epichlorohydrin and dyphylline degradation products, and is prone to chromatographic peak overlap, affecting the quantitative accuracy; second, the programmed temperature rate is not reasonably set, and a single low temperature rate (such as 5-10 ℃ / min) is usually used, resulting in a long analysis period (usually more than 30 min), and high-boiling impurities are easily left in the column, affecting subsequent detection; third, the mass spectrometry parameters are not optimized for epichlorohydrin, and the ion source temperature and auxiliary heater temperature are not properly set, resulting in weak response signals of epichlorohydrin characteristic ions (such as m / z 62, 92), and the detection limit is difficult to meet the detection requirements of trace residues in drugs.

[0005] These defects make the existing method unable to efficiently and accurately determine the residual amount of epichlorohydrin in dyphylline, and it is difficult to meet the requirements of drug production quality control and supervision. SUMMARY

[0006] This application provides a method for determining residual epichlorohydrin in dihydroxypropyltheophylline. Compared with the prior art, the analytical method provided in this application has high sensitivity, strong separation ability, and is rapid, and can accurately quantify epichlorohydrin in the test sample.

[0007] This application provides a method for determining residual epichlorohydrin in dihydroxypropyltheophylline, using the following technical solution: A method for determining residual epichlorohydrin in dihydroxypropyltheophylline, using GC-MS to detect residual epichlorohydrin in dihydroxypropyltheophylline; The chromatographic conditions are as follows: Chromatographic column: VF-624ms, 30m × 0.25mm × 1.4μm; Carrier gas: helium; column flow rate: 0.95-1.05 ml / min; split ratio: 10:1. Inlet temperature: 230-250℃; Inlet volume: 1μl; The mass spectrometry conditions are as follows: Ion source: EI; Ion source temperature 220-240℃; Quadrupole temperature 140-160℃; Auxiliary heater 250-270℃.

[0008] In this application, a VF-624ms column (30m × 0.25mm × 1.4μm) was selected. This column has a moderately polar stationary phase (containing 6% cyanopropylphenyl-94% dimethylpolysiloxane), which exhibits a greater difference in retention between the polar compound epichlorohydrin (containing epoxy and chlorine atoms, and is highly polar) and the degradation products of dihydroxypropyltheophylline (mostly weakly polar organic compounds). This allows for baseline separation of the two compounds, completely avoiding peak overlap and fundamentally improving quantitative accuracy. In contrast, existing technologies use ordinary non-polar / weakly polar columns (such as DB-5ms), which cannot effectively separate epichlorohydrin and dihydroxypropyltheophylline degradation products, easily leading to peak overlap and quantitative deviation.

[0009] In this application, specific mass spectrometry parameters are set to enhance the ionization efficiency of epichlorohydrin: ion source temperature 220-240℃: matching the thermal stability of epichlorohydrin (boiling point 116℃), avoiding insufficient ionization due to excessively low temperature, or compound decomposition due to excessively high temperature; quadrupole temperature 140-160℃, auxiliary heater 250-270℃: ensuring ion transport efficiency and reducing ion loss during transport; combined with EI ion source (hard ionization source): it can stably generate characteristic fragment ions of epichlorohydrin (C2H3O⁺ at m / z 62, C3H5OCl⁺ at m / z 92), the response signal intensity is improved by 2-3 times compared with the prior art, and the detection limit can be reduced to below 0.05μg / g, fully meeting the requirements for trace drug residue detection. In existing technologies, parameters such as ion source temperature and auxiliary heater temperature are not optimized for epichlorohydrin, resulting in weak response signals of its characteristic ions (m / z 62, 92) and detection limits that cannot meet the requirements for detecting trace residues (e.g., <0.1 μg / g).

[0010] Furthermore, in terms of carrier gas and flow rate, this application uses helium (purity ≥99.999%) as the carrier gas, with a column flow rate of 0.95-1.05 ml / min, which results in less ionization interference compared to nitrogen carrier gas commonly used in existing technologies. A split ratio of 10:1 avoids injection overload while ensuring sufficient sample volume enters the mass spectrometer, balancing sensitivity and resolution. Injection conditions: an injection port temperature of 230-250℃ and an injection volume of 1 μl ensure instantaneous vaporization of epichlorohydrin, reducing sample adsorption and further reducing detection errors.

[0011] Optionally, the chromatographic conditions include a programmed temperature rise method as follows: starting temperature 35-45℃, hold for 2-3 min; increase temperature at a rate of 10-15℃ / min to 120-140℃, hold for 0 min; then increase temperature at a rate of 55-65℃ / min to 230-250℃, hold for 3-4 min.

[0012] Optionally, the chromatographic conditions include a programmed temperature rise method as follows: starting temperature 40℃, hold for 2 min; increase to 130℃ at a rate of 15℃ / min, hold for 0 min; then increase to 250℃ at a rate of 60℃ / min, hold for 4 min.

[0013] This application designs a three-stage gradient temperature program to specifically optimize separation efficiency and impurity removal: Initially, hold at 40℃ for 2 minutes to ensure sufficient focusing of epichlorohydrin (boiling point 116℃) and low-boiling-point impurities, avoiding peak broadening; Increase at 15℃ / min to 130℃ (no hold): rapidly separate epichlorohydrin from medium-boiling-point impurities, shortening the separation time of key components; Increase at 60℃ / min to 250℃ and hold for 4 minutes: rapidly elute high-boiling-point impurities (such as dihydroxypropyltheophylline residue) at high temperature, avoiding residual contamination within the column. Simultaneously, the total analysis cycle can be compressed to within 15 minutes (more than 50% shorter than existing technologies), balancing efficiency and detection stability. Existing technologies use a single low-rate temperature increase (5-10℃ / min), resulting in analysis cycles exceeding 30 minutes, and high-boiling-point impurities easily accumulate within the column, affecting subsequent detection stability.

[0014] Optionally, the detection limit concentration of epichlorohydrin is 0.39 ppm.

[0015] Optionally, the quantitative line concentration of the epichlorohydrin is 1.18 ppm.

[0016] In summary, this application includes at least one of the following beneficial technical effects: The assay method provided in this application has not been reported in the prior art. The method has been validated for system suitability, specificity, limit of detection and limit of quantitation, linearity and range, solution stability, repeatability, intermediate precision, accuracy, and robustness. All validation results meet the acceptable standard requirements, proving that the method is suitable for the detection of epichlorohydrin residues in dihydroxypropyltheophylline.

[0017] This application employs a dual stock solution design (Stock-A1 / A2), in which completely independent dual stock solutions (A1 / A2) are diluted to the same concentration, which can verify systematic errors in the preparation process and avoid random deviations caused by a single path.

[0018] This application employs a salting-out extraction combined technique for sample pretreatment. After dissolving the sample in sodium chloride solution, ethyl acetate is added for extraction. The salting-out effect enhances the transfer efficiency of epichlorohydrin in the organic phase. Compared to conventional methods, traditional liquid-liquid extraction does not require salting-out, but this method significantly improves the extraction rate of impurities in hydrophilic matrices (such as dihydroxypropyltheophylline).

[0019] In this application, it is explicitly required that two samples of the test sample be prepared in parallel, and the stability of the extraction process is verified through data consistency, which meets the GMP requirements for repeatability of key tests.

[0020] In this application, ethyl acetate is used as the blank diluent throughout the process (instead of water or methanol) because it has better solubility for epichlorohydrin and can effectively suppress sample matrix interference.

[0021] In this application, the preparation of the reference solution employs graded dilution control to ensure the accuracy of ultra-trace detection (ng level) and avoid volume errors caused by direct dilution. This method significantly improves the accuracy and anti-interference ability of epichlorohydrin detection, and is particularly suitable for the quality control of trace genotoxic impurities in active pharmaceutical ingredients. Attached Figure Description

[0022] Figure 1 This is a typical spectrum obtained from the specific detection of epichlorohydrin.

[0023] Figure 2 The spectrum obtained by adding the spiked test solution (Sys-4) in the specific detection of epichlorohydrin.

[0024] Figure 3 The graph is a linearity plot obtained from the linear detection of epichlorohydrin. Detailed Implementation

[0025] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0027] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0029] This application provides a method for determining residual epichlorohydrin in dihydroxytheophylline.

[0030] The determination method specifically includes the following steps: The chromatographic conditions are as follows: Chromatographic column: VF-624ms, 30m × 0.25mm × 1.4μm; Carrier gas: helium, column flow rate 1.0 ml / min, split ratio: 10:1; Programmed temperature rise: Initial temperature 40℃, hold for 2 min; increase temperature at 15℃ / min to 130℃, hold for 0 min; then increase temperature at 60℃ / min to 250℃ and hold for 4 min; Inlet temperature: 240℃; injection volume: 1μl.

[0031] The mass spectrometry conditions are as follows: Ion source: EI; Ion source temperature 220-240℃; Quadrupole temperature 140-160℃; Auxiliary heater 250-270℃.

[0032] The principle of this application is as follows: Dihydroxypropyltheophylline is first dissolved in sodium chloride solution, and the supernatant is collected after pretreatment by standing. The test sample is then determined by GC-MS using the external standard method. This detection method first uses gas chromatography (GC) to separate the target analyte in the test sample; the separated sample is then passed through the ionization source of the mass spectrometry system to be converted into charged ions; these ions are arranged in mass-charge ratio order by the mass analyzer, and the arranged charged ions enter the detector to generate a signal, ultimately forming a spectrum. The result is obtained through calculation using a formula.

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] In the following examples, unless otherwise stated, all reagents used were of analytical grade. The specific sources or preparation methods of some reagents are as follows: (1) The water is Grade I water as specified in GB / T6682.

[0036] (2) Blank solution (diluent): ethyl acetate.

[0037] (3) Sodium chloride solution: Weigh 7.50084g of sodium chloride, add 50ml of water to dissolve, and mix well to obtain the solution.

[0038] (4) Preparation of relevant solutions for reference standards: Stock A-1 (Reference Stock Solution A-1): Accurately weigh 10.14 mg of epichlorohydrin, place it in a 5 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the concentration of the prepared solution is 2.0241 mg / ml.

[0039] Stock A-2 (Reference Stock Solution A-2): Accurately weigh 10.01 mg of epichlorohydrin, place it in a 5 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the concentration of the prepared solution is 1.9982 mg / ml.

[0040] Stock-1 (Reference Stock Solution-1): Accurately transfer 78 μl of Stock A-1 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.7883 μg / ml.

[0041] Stock-2 (Reference Stock Solution-2): Accurately transfer 78 μl of Stock A-2 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.5859 μg / ml.

[0042] Std-1 (Reference Solution-1): Accurately transfer 250 μl of Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 78.9417 ng / ml.

[0043] Std-2 (Reference Solution-2): Accurately transfer 250 μl of Stock-2 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 77.9297 ng / ml.

[0044] (5) Preparation of linear solutions: Lin-30% (Linear-30%) solution: Accurately transfer 30 μl of Stock-1 solution into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 23.6825 ng / ml.

[0045] Lin-50% (Linear-50%) solution: Accurately transfer 50 μl of Stock-1 solution into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 39.4709 ng / ml.

[0046] Lin-100% (Linear-100%) solution: Accurately transfer 250 μl of Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 78.9417 ng / ml.

[0047] Lin-150% (linear-150%) solution: Accurately transfer 150 μl of Stock-1 solution into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 118.4126 ng / ml.

[0048] Lin-200% (linear-200%) solution: Accurately transfer 200 μl of Stock-1 solution into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 157.8835 ng / ml.

[0049] (6) Preparation of detection line solution and limit of quantitation solution: LOQ (Limit of Quantification): Same as Lin-30% (Linear-30%) solution.

[0050] LOD (Limit of Detection Solution): Accurately transfer 10 μl of Stock-1 solution into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 7.8942 ng / ml.

[0051] (7) Preparation of repeatable solutions: Re-Spl 1 (Reproducible Solution-1): Accurately weigh 80.18 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the solution is 78.9417 ng / ml.

[0052] Re-Spl 2 (Reproducible Solution-2): Accurately weigh 80.10 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the solution is 78.9417 ng / ml.

[0053] Re-Spl 3 (Reproducible Solution-3): Accurately weigh 80.24 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the solution is 78.9417 ng / ml.

[0054] Re-Spl 4 (Reproducible Solution-4): Accurately weigh 80.13 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the solution is 78.9417 ng / ml.

[0055] Re-Spl 5 (Reproducible Solution-5): Accurately weigh 80.25 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the solution is 78.9417 ng / ml.

[0056] Re-Spl 6 (Reproducible Solution-6): Accurately weigh 80.17 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the solution is 78.9417 ng / ml.

[0057] (8) Preparation of intermediate precision solution: Re-Stock A-1 (Reference Stock Solution A-1): Accurately weigh 10.16 mg of epichlorohydrin, place it in a 5 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the concentration of the prepared solution is 2.0281 ng / ml.

[0058] Re-Stock A-2 (Reference Stock Solution A-2): Accurately weigh 10.21 mg of epichlorohydrin, place it in a 5 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the concentration of the prepared solution is 2.0381 ng / ml.

[0059] Re-Stock-1 (Reference Stock Solution-1): Accurately transfer 78 μl of Re-Stock A-1 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.8195 ng / ml.

[0060] Re-Stock-2 (Reference Stock Solution-2): Accurately transfer 78 μl of Re-Stock A-2 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.8973 ng / ml.

[0061] Re-Std-1 (Reference Solution-1): Accurately transfer 250 μl of Re-Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 79.0974 ng / ml.

[0062] Re-Std-2 (Reference Solution-2): Accurately transfer 250 μl of Re-Stock-2 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 79.4867 ng / ml.

[0063] Re-Spl-1 (Intermediate Precision Solution-1): Accurately weigh 80.06 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Re-Std-1 solution, shake for about 2 min, let stand and take the supernatant. The concentration of epichlorohydrin in the prepared solution is 79.0974 ng / ml.

[0064] Re-Spl-2 (Intermediate Precision Solution-2): Accurately weigh 80.05 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Re-Std-1 solution, shake for about 2 min, let stand and take the supernatant. The concentration of epichlorohydrin in the prepared solution is 79.0974 ng / ml.

[0065] Re-Spl-3 (Intermediate Precision Solution-3): Accurately weigh 80.11 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Re-Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of epichlorohydrin in the prepared solution is 79.0974 ng / ml.

[0066] Re-Spl-4 (Intermediate Precision Solution-4): Accurately weigh 80.06 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Re-Std-1 solution, shake for about 2 min, let stand and take the supernatant. The concentration of epichlorohydrin in the prepared solution is 79.0974 ng / ml.

[0067] Re-Spl-5 (Intermediate Precision Solution-5): Accurately weigh 80.10 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Re-Std-1 solution, shake for about 2 min, let stand and take the supernatant. The concentration of epichlorohydrin in the prepared solution is 79.0974 ng / ml.

[0068] Re-Spl-6 (Intermediate Precision Solution-6): Accurately weigh 80.02 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Re-Std-1 solution, shake for about 2 min, let stand and take the supernatant. The concentration of epichlorohydrin in the prepared solution is 79.0974 ng / ml.

[0069] (9) Accuracy solution preparation: A-Spl-1 (Test Solution-1): Accurately weigh 80.15 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of ethyl acetate, shake for about 2 min, let stand and take the supernatant to obtain the solution.

[0070] A-Spl-2 (Test Solution-2): Accurately weigh 80.07 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of ethyl acetate, shake for about 2 min, let stand and take the supernatant to obtain the solution.

[0071] A-Spl L-1 (50% spiked test solution-1): Accurately weigh 80.21 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-50% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 39.4709 ng / ml.

[0072] A-Spl L-2 (50% spiked test solution-2): Accurately weigh 80.03 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-50% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 39.4709 ng / ml.

[0073] A-Spl L-3 (50% spiked test solution-3): Accurately weigh 80.07 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-50% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 39.4709 ng / ml.

[0074] A-Spl M-1 (100% spiked test solution-1): Accurately weigh 80.18 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-100% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 78.9417 ng / ml.

[0075] A-Spl M-2 (100% spiked test solution-2): Accurately weigh 80.10 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-100% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 78.9417 ng / ml.

[0076] A-Spl M-3 (100% spiked test solution-3): Accurately weigh 80.24 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-100% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 78.9417 ng / ml.

[0077] A-Spl H-1 (150% spiked test solution-1): Accurately weigh 80.26 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-1 50% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 118.4126 ng / ml.

[0078] A-Spl H-2 (150% spiked test solution-2): Accurately weigh 80.20 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-150% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 118.4126 ng / ml.

[0079] A-Spl H-3 (150% spiked test solution-3): Accurately weigh 80.03 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Lin-150% solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 118.4126 ng / ml.

[0080] (10) Solution stability: Solution preparation: Sta-Std (Standard Stability Solution): Accurately transfer 250 μl of Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 78.9417 ng / ml.

[0081] Sta-Spl (Spiked Sample Stability Solution): Accurately weigh 80.18 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for about 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 78.9417 ng / ml.

[0082] (11) Preparation of durable solution: Change the injection port temperature: Rob-Stock A-1 (Durability-Reference Stock Solution A-1): Accurately weigh 10.10 mg of epichlorohydrin, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the concentration of the prepared solution is 2.0162 ng / ml.

[0083] Rob-Stock A-2 (Durability-Reference Stock Solution A-2): Accurately weigh 10.14 mg of epichlorohydrin, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the concentration of the prepared solution is 2.0241 ng / ml.

[0084] Rob-Stock-1 (Durability-Reference Stock Solution-1): Accurately transfer 78 μl of Rob-Stock A-1 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.7261 ng / ml.

[0085] Rob-Stock-2 (Durability-Reference Stock Solution-2): Accurately transfer 78 μl of Rob-Stock A-2 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.7883 ng / ml.

[0086] Rob-Std-1 (Durability-Reference Solution-1): Accurately transfer 250 μl of Rob-Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 78.6303 ng / ml.

[0087] Rob-Std-2 (Durability-Reference Solution-2): Accurately transfer 250 μl of Rob-Stock-2 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 78.9417 ng / ml.

[0088] Rob-Spl (Durability-Spiked Test Solution): Accurately weigh 80.15 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Rob-Std-1 solution, shake for about 2 min, let stand and take the supernatant. The concentration of the prepared solution is 78.6303 ng / ml.

[0089] Change the column flow rate: Rob-Stock A-1 (Durability-Reference Stock Solution A-1): Accurately weigh 10.23 mg of epichlorohydrin, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the concentration of the prepared solution is 2.0421 ng / ml.

[0090] Rob-Stock A-2 (Durability-Reference Stock Solution A-2): Accurately weigh 9.98 mg of epichlorohydrin, place it in a 5 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the concentration of the prepared solution is 1.9922 ng / ml.

[0091] Rob-Stock-1 (Durability-Reference Stock Solution-1): Accurately transfer 78 μl of Rob-Stock A-1 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.9285 ng / ml.

[0092] Rob-Stock-2 (Durability-Reference Stock Solution-2): Accurately transfer 78 μl of Rob-Stock A-2 solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 15.5392 ng / ml.

[0093] Rob-Std-1 (Durability-Reference Solution-1): Accurately transfer 250 μl of Rob-Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 79.6424 ng / ml.

[0094] Rob-Std-2 (Durability-Reference Solution-2): Accurately transfer 250 μl of Rob-Stock-2 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 77.6961 ng / ml.

[0095] Rob-Spl (Durability-Spiked Test Solution): Accurately weigh 80.07 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Rob-Std-1 solution, shake for about 2 min, let stand and collect the supernatant. The concentration of the prepared solution is 79.6424 ng / ml.

[0096] The present application will be further described in detail below with reference to the embodiments and test results.

[0097] Example 1

[0098] This embodiment provides a method for determining residual epichlorohydrin in dihydroxytheophylline.

[0099] Specifically, the following steps are included: (1) Preparation and storage of the sample: Accurately weigh 80 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, add 4 ml of ethyl acetate, shake for about 2 min, let stand and take the supernatant. Store at room temperature.

[0100] (2) The chromatographic conditions are as follows: Chromatographic column: VF-624ms, 30m × 0.25mm × 1.4μm; Carrier gas: helium, column flow rate 1.0 ml / min, split ratio: 10:1; Programmed temperature rise: Initial temperature 40℃, hold for 2 min; increase temperature at 15℃ / min to 130℃, hold for 0 min; then increase temperature at 60℃ / min to 250℃ and hold for 4 min; Inlet temperature: 240℃; Inlet volume: 1μl; (3) The mass spectrometry conditions are as follows: Ion source: EI; Ion source temperature 230℃; Quadrupole temperature 150℃; Auxiliary heater 260℃.

[0101] (4) Calculation method: Sample content calculation: X (ppm) = (As × C) R ) / (A R ×Cs).

[0102] Where, —As is the peak area of ​​the target impurity in the sample; —AR is the average peak area of ​​the target impurity from 6 injections in Std-1; —C R -Cs represents the concentration of the target impurity in Std-1 (ng / ml); -Cs represents the sample concentration (mg / ml).

[0103] Performance testing The limit for residual epichlorohydrin in dihydroxypropyltheophylline is ≤0.0002 mg / ml.

[0104] II. Verification Project The following items were analyzed using the analysis method provided in Example 1.

[0105] (1) System precision Experimental method: Inject Std-1 solution into 6 syringes (injection volume 1 μl / injection), and Std-2 solution into 1 syringe (injection volume 1 μl / injection). Record the mass spectrum and peak area. The required RSD of the 6-injection Std-1 solution is ≤10%, and the recovery rate of Std-2 solution relative to Std-1 solution is 90.0%~110.0%.

[0106] The experimental results are shown in Table 1.

[0107] Table 1 System precision - Standard solution test results

[0108] As shown in Table 1, the peak area RSD of epichlorohydrin in Std-1 solution was less than 10.0% for six consecutive injections; the recovery rate of epichlorohydrin in Std-2 solution was in the range of 90.0% to 110.0% compared with Std-1 solution, which met the requirements.

[0109] (2) Exclusivity Solution preparation: Sys-1 (blank solution, diluent): ethyl acetate solution.

[0110] Sys-2 (reference solution): Accurately transfer 250 μl of Stock-1 solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well. The concentration of the prepared solution is 78.9417 mg / ml.

[0111] Sys-3 (test solution): Accurately weigh 80.15 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, add 4 ml of ethyl acetate, shake for 2 min, let stand and take the supernatant to obtain the solution.

[0112] Sys-4 (spiked test solution): Accurately weigh 80.18 mg of dihydroxypropyltheophylline, dissolve it in 2 ml of sodium chloride solution, then add 4 ml of Std-1 solution, shake for 2 min, let stand and take the supernatant to obtain the solution. The concentration of the prepared solution is 78.9417 mg / ml.

[0113] Test method: Inject one sample each of the blank solution, reference solution, test solution, and spiked test solution (injection volume: 1 μl / injection). The blank solution should be free of interference, and the epichlorohydrin peak in the spiked test solution and reference solution should not interfere with each other. The experimental results are shown in Table 2. The target impurities were located using retention time, and the overlay plot is shown below. Figure 1 As shown, the detection spectrum of Sys-4 is as follows: Figure 2 As shown.

[0114] Table 2 Specificity test results

[0115] From Table 2 and Figures 1-2 It can be seen that neither the blank solution (Sys-1) nor the test solution (Sys-3) interfered with the determination of the target peak, and the specificity was good, meeting the requirements; epichlorohydrin eluted at approximately 6 minutes.

[0116] (3) Linear Experimental method: Take each linear solution and inject it once (injection volume is 1 μl / injection). Plot a linear graph with the concentration of epichlorohydrin on the x-axis and the peak area on the y-axis, and calculate the linear correlation coefficient. The R-value is required to be... 2 Value ≥ 0.999. The test results are shown in Table 3 and... Figure 3 As shown.

[0117] Table 3 Results of linear experiments

[0118] From Table 3 and Figure 3 It can be seen that epichlorohydrin exhibits good linearity in the range of 23.6825–157.8835 ng / kg, and R0 2 The value is 0.999, which meets the requirements.

[0119] (4) Limit of quantitation, limit of detection Experimental method: The limit of detection (LOD) solution was injected into 3 injections (1 μl / injection), and the limit of quantitation (LOQ) solution was injected into 6 injections (1 μl / injection). The LOD / LOQ ratio was required to be greater than 3:1; the LOQ ratio was required to be greater than 10:1; and the peak area RSD of the 6 injections was required to be ≤10%. The results are shown in Table 4.

[0120] Table 4. Results of the Limit of Quantitation (LOQ) and Limit of Detection (LOD) tests.

[0121] As shown in Table 4, the detection limit concentration is 7.8942 ng / ml (equivalent to a sample concentration of 0.39 ppm), with a signal-to-noise ratio (S / N) greater than 3:1; the quantitation limit concentration is 23.6825 ng / ml (equivalent to a sample concentration of 1.18 ppm), with a S / N greater than 10:1; and the peak area RSD of the 6 needles is 4.7%, which meets the requirements.

[0122] (5) Repeatability Experimental method: Reproducible solutions were injected separately. The RSD of epichlorohydrin content in the six reproducible solutions was required to be ≤10%. The experimental results are shown in Table 5.

[0123] Table 5 Repeatability Test Results

[0124] As shown in Table 5, the RSD of epichlorohydrin content in the 6 repeatable solutions was 0.7%, which meets the requirements.

[0125] (6) Intermediate precision Experimental method: The solution was prepared again by different personnel on different dates. The intermediate precision solution was taken and injected once (injection volume was 1 μl / injection). The mass spectrum and peak area were recorded. The RSD value of the target impurity content in 12 precision samples was required to be ≤10%. The experimental results are shown in Table 6.

[0126] Table 6 Results of intermediate precision test

[0127] As shown in Table 6, when the solution was re-prepared by different personnel on different dates, the RSD of epichlorohydrin content in the 12 precision samples was 1.7%, which meets the requirements.

[0128] (7) Accuracy (spiking recovery rate) Experimental method: Nine aliquots of accuracy solutions with three different concentrations (50%, 100%, and 150%) were injected into each syringe (injection volume was 1 μl / injection); the content of propylene oxide was calculated by peak area using the external standard method, and the recovery rate should be in the range of 80-120% and RSD ≤ 10%, and the blank recovery rate was also calculated; the experimental results are shown in Tables 7 and 8.

[0129] Table 7 Results of the Recovery Rate-Blank Recovery Rate Test

[0130] Table 8 Recovery Rate - Results of Spiking Recovery Test

[0131] As shown in Tables 7 and 8, the spiked recoveries of the nine accuracy solutions with three different concentrations (50%, 100%, and 150%) ranged from 96.3% to 107.2%, and the RSD of the recoveries of the nine accuracy solutions was 4.5%, which met the requirements.

[0132] (8) Solution stability Test method: Take 1 μl of Sta-Std (reference stability solution) and Sta-Spl (spiked test sample stability solution) and test at 0h, 6h, 12h and 13h respectively. The ratio of the peak area of ​​the target impurity to the peak area of ​​the target impurity at 0h should be within the range of 80.0% to 120.0%. The results are shown in Tables 9 and 10.

[0133] Table 9. Solution Stability - Sta-Std (Reference Stability Solution) Test Results

[0134] Table 10 Solution Stability - Sta-Spl (Spiked Sample Stability Solution) Test Results

[0135] As shown in Tables 9 and 10, after being placed at room temperature for 13 hours, the peak area ratios of the target impurities at each time point relative to 0 h for both the reference solution and the spiked test solution were between 80.0% and 120.0%, indicating good solution stability.

[0136] (9) Durability Experimental method: The injection port temperature was changed from 240℃ to 230℃ and 250℃; the column flow rate was changed from 1.0 ml / min to 0.95 ml / min and 1.05 ml / min, while other conditions remained unchanged. Two injections of each robust solution were performed (injection volume was 1 μl / injection), and the mass spectra and peak areas were recorded. The target impurity content RSD was required to be ≤10%. The results are shown in Table 11.

[0137] Table 11 Durability Results

[0138] As shown in Table 11, the system suitability meets the requirements under all conditions by changing the injection port temperature and column flow rate; the RSD of epichlorohydrin content measured under all conditions by changing the injection port temperature and column flow rate is 1.8%, which meets the requirements.

[0139] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for determining residual epichlorohydrin in dihydroxypropyltheophylline, characterized in that, GC-MS was used to detect residual epichlorohydrin in dihydroxypropyltheophylline; The chromatographic conditions are as follows: Chromatographic column: VF-624ms, 30m × 0.25mm × 1.4μm; Carrier gas: helium; column flow rate: 0.95-1.05 ml / min; split ratio: 10:

1. Inlet temperature: 230-250℃; Inlet volume: 1μl; The mass spectrometry conditions are as follows: Ion source: EI; Ion source temperature 220-240℃; Quadrupole temperature 140-160℃; Auxiliary heater 250-270℃.

2. The analytical method according to claim 1, characterized in that, The chromatographic conditions are as follows: initial temperature 35-45℃, hold for 2-3 min; increase to 120-140℃ at a rate of 10-15℃ / min, hold for 0 min; then increase to 230-250℃ at a rate of 55-65℃ / min, hold for 3-4 min.

3. The analytical method according to claim 1, characterized in that, The chromatographic conditions are as follows: initial temperature 40℃, hold for 2 min; increase to 130℃ at a rate of 15℃ / min, hold for 0 min; then increase to 250℃ at a rate of 60℃ / min, hold for 4 min.

4. The analytical method according to claim 1, characterized in that, The pretreatment method for the test solution is to dissolve the sample in sodium chloride solution and then extract it with ethyl acetate.

5. The analytical method according to claim 1, characterized in that, The detection limit for epichlorohydrin is 0.39 ppm.

6. The analytical method according to claim 1, characterized in that, The quantitative line concentration of the epichlorohydrin is 1.18 ppm.

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