CN115754096A - A kind of detection method of benzene in amifostine - Google Patents
A kind of detection method of benzene in amifostineDownload PDFInfo
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Abstract
Description
Translated fromChinese技术领域technical field
本发明属于检测领域，具体涉及一种氨磷汀中苯的检测方法。The invention belongs to the detection field, and in particular relates to a method for detecting benzene in amifostine.
背景技术Background technique
氨磷汀(Amifostine)，别名安磷汀，化学名称为2-[(3-氨基丙基)氨基]乙基硫代二氢磷酸酯，是含有一个磷酸基团的小分子化合物，为正常细胞的保护剂，用于化疗不良反应的预防。苯为无色、具有芳香气味的液体，具有易挥发、易燃、有毒的特点，可损伤人体造血干细胞，引起造血干细胞复制减少、影响造血微环境，从而引起造血功能障碍；此外，苯还可与DNA共价结合，抑制DNA的转录作用，从而引起细胞增殖障碍。苯也可直接损伤DNA，诱发突变或染色体畸变。《中国药典》将苯列为1类溶剂，在药物生产过程中应避免使用。在氨磷汀生产过程中已不再使用苯作为溶剂，但会大量使用各种醇类溶剂，醇类溶剂容易被苯污染，所以开发药物中苯的有效的检测方法至关重要。Amifostine, also known as Amifostine, chemical name is 2-[(3-aminopropyl) amino] ethyl thiodihydrogen phosphate, is a small molecular compound containing a phosphate group, and is a Protective agent for the prevention of adverse reactions to chemotherapy. Benzene is a colorless liquid with aromatic odor. It is volatile, flammable, and toxic. It can damage human hematopoietic stem cells, reduce the replication of hematopoietic stem cells, affect the hematopoietic microenvironment, and cause hematopoietic dysfunction; in addition, benzene can also Covalently binds to DNA, inhibits DNA transcription, thereby causing cell proliferation disorders. Benzene can also directly damage DNA, induce mutation or chromosomal aberration. "Chinese Pharmacopoeia" lists benzene as a class 1 solvent, which should be avoided in the pharmaceutical production process. In the production process of amifostine, benzene is no longer used as a solvent, but various alcohol solvents are used in large quantities. Alcohol solvents are easily contaminated by benzene, so it is very important to develop an effective detection method for benzene in drugs.
目前还没有氨磷汀中苯检测的研究报道。杭玮玮，顶空进样气相色谱法测定药物U的苯残留量[J],健康必读，2012,11(3),公开了一种药物中苯残留量的测定方法，由于药物溶剂包括氢氧化钠溶液，条件剧烈，会导致氨磷汀降解，且使用顶空瓶，使顶空瓶易被腐蚀，这些不利因素导致其方法不能准确测定氨磷汀中的苯。There is no research report on the detection of benzene in amifostine. Hang Weiwei, Determination of Benzene Residue in Drug U by Headspace Gas Chromatography [J], Health Must Read, 2012, 11(3), discloses a method for the determination of benzene residue in drug, since drug solvents include Sodium solution, under severe conditions, will lead to the degradation of amifostine, and the use of headspace vials makes the vials easily corroded. These unfavorable factors make the method unable to accurately determine the benzene in amifostine.
发明内容Contents of the invention
为了解决上述问题，本发明提供了一种氨磷汀中苯的检测方法，它包括如下步骤：In order to solve the above problems, the invention provides a kind of detection method of benzene in amifostine, and it comprises the steps:
(1)制备对照品溶液：取苯对照品，加DMA溶液溶解，再加水稀释至每1ml含苯0.01～0.8μg，即得；(1) Preparation of reference substance solution: take benzene reference substance, add DMA solution to dissolve, add water to dilute to 0.01-0.8μg of benzene per 1ml, and obtain it;
(2)制备供试品溶液：取氨磷汀，加水溶解，即得；(2) Preparation of the test solution: take amifostine, add water to dissolve, and get final product;
(3)检测：采用顶空进样气相色谱法检测；气相色谱条件：色谱柱DB-624，升温程序：起始温度为40℃，保持8min，之后以30℃/min的速率升温至180℃，维持5min。(3) Detection: detection by headspace gas chromatography; gas chromatography conditions: chromatographic column DB-624, heating program: the initial temperature is 40°C, keep it for 8min, and then raise the temperature to 180°C at a rate of 30°C/min , maintain 5min.
进一步地，步骤(1)中，所述苯与DMA溶液的质量体积比为20～60mg：100ml；所述加水稀释至每1ml含苯0.04～0.4μg的溶液。Further, in step (1), the mass volume ratio of the benzene to the DMA solution is 20-60 mg:100 ml; the solution is diluted with water to a solution containing 0.04-0.4 μg of benzene per 1 ml.
进一步地，所述DMA溶液的浓度为25～75％，优选50％。Further, the concentration of the DMA solution is 25-75%, preferably 50%.
进一步地，步骤(2)中，所述氨磷汀与水的质量体积比为0.2～2g：5mL，优选1g：5mL。Further, in step (2), the mass volume ratio of the amifostine to water is 0.2-2g:5mL, preferably 1g:5mL.
进一步地，步骤(3)中，所述色谱柱DB-624规格为30m×0.53mm，3μm。Further, in step (3), the specification of the chromatographic column DB-624 is 30m×0.53mm, 3μm.
进一步地，步骤(3)中，所述气相色谱条件中进样口温度200℃，检测器温度250℃，顶空平衡温度80℃，定量环温度90℃，传输线温度100℃，载气为氮气，流速2.0ml/min，分流比5∶1。Further, in step (3), in the gas chromatography conditions, the inlet temperature is 200°C, the detector temperature is 250°C, the headspace equilibrium temperature is 80°C, the quantitative loop temperature is 90°C, the transfer line temperature is 100°C, and the carrier gas is nitrogen , flow rate 2.0ml/min, split ratio 5:1.
进一步地，所述供试品溶液的色谱图中呈现与对照品溶液的色谱图中色谱峰保留时间一致的色谱峰，确定氨磷汀中含有苯。Further, the chromatogram of the test solution presents a chromatographic peak consistent with the retention time of the chromatographic peak in the chromatogram of the reference solution, confirming that amifostine contains benzene.
更进一步地，所述氨磷汀中苯的含量按外标法以峰面积计算。Furthermore, the content of benzene in the amifostine is calculated by the peak area according to the external standard method.
本发明氨磷汀中苯的检测方法，通过以水作为氨磷汀的溶剂，采用DMA溶解苯，再用水稀释配制苯对照品溶液，在特定的升温程序、进样口温度、检测器温度、流速及顶空参数的色谱条件下，使待检样品中的苯与氨磷汀及杂质完全分离，实现氨磷汀中苯的准确测定。本发明方法准确性和灵敏度高，重复性好，具备实际推广应用价值。The detection method of benzene in amifostine of the present invention, by using water as the solvent of amifostine, adopts DMA to dissolve benzene, then dilute with water to prepare benzene reference substance solution, in specific heating program, inlet temperature, detector temperature, Under the chromatographic conditions of the flow rate and headspace parameters, the benzene in the sample to be tested is completely separated from the amifostine and impurities, and the accurate determination of benzene in the amifostine is realized. The method of the invention has high accuracy and sensitivity, good repeatability, and has practical popularization and application value.
以下通过实施例形式的具体实施方式，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实施例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above-mentioned content of the present invention will be further described in detail through specific implementation in the form of examples below. However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.
附图说明Description of drawings
图1试验组①苯对照品溶液Figure 1 Test group ① Benzene reference substance solution
图2试验组①样品加标溶液Figure 2 Test group ① sample spiked solution
图3试验组②苯对照品溶液Figure 3
图4试验组②样品加标溶液Figure 4
图5试验组③苯对照品溶液Figure 5
图6试验组③样品加标溶液Figure 6
图7试验组④苯对照品溶液Figure 7 Test group ④ Benzene reference substance solution
图8试验组④样品加标溶液Figure 8 Test group ④ sample spiked solution
图9单独进样50％DMF溶液Figure 9 Separate injection of 50% DMF solution
图10单独进样50％DMA溶液Figure 10 Separate injection of 50% DMA solution
图11单独进样50％DMSO溶液Figure 11 Separate injection of 50% DMSO solution
图12按照试验组①配制的苯对照品溶液Fig. 12 is prepared according to the benzene reference substance solution of test group 1.
具体实施方式Detailed ways
实施例1本发明氨磷汀中苯的定性检测Qualitative detection of benzene in the amifostine of the present invention in embodiment 1
1)制备供试品溶液1) Prepare the test solution
取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入水5ml，立即压盖密封，即得。Take 1.0g of amifostine, accurately weigh it, put it in a 20ml headspace bottle, add 5ml of water accurately, press the cap and seal it immediately, and it is ready.
2)制备对照品溶液2) Preparation of reference substance solution
取苯对照品40mg，精密称定，置100ml量瓶中，加50％DMA溶液溶解并稀释至刻度，作为苯对照品贮备液；Take 40 mg of benzene reference substance, accurately weigh it, put it in a 100ml measuring bottle, add 50% DMA solution to dissolve and dilute to the mark, and use it as the benzene reference substance stock solution;
精密量取苯对照品贮备液，加水稀释制成每1ml中含苯0.4μg的溶液。精密量取5ml，置20ml顶空瓶中，立即压盖密封，即得。Precisely measure the stock solution of benzene reference substance, dilute it with water to make a solution containing 0.4 μg of benzene per 1 ml. Precisely measure 5ml, put it in a 20ml headspace bottle, and immediately press the cap to seal it.
3)分别吸取对照品溶液和供试品溶液注入顶空进样气相色谱仪，色谱条件如下：3) draw reference substance solution and need testing solution respectively and inject headspace sampling gas chromatograph, chromatographic conditions are as follows:
色谱柱DB-624，30m×0.53mm，3μm；Chromatographic column DB-624, 30m×0.53mm, 3μm;
升温程序：起始温度40℃，维持8分钟，以每分钟30℃的速率升温至180℃，维持5分钟；Heating program: start at 40°C, maintain for 8 minutes, raise the temperature to 180°C at a rate of 30°C per minute, and maintain for 5 minutes;
进样口温度200℃；检测器温度250℃；顶空平衡温度：80℃；定量环温度：90℃；传输线温度：100℃；载气为氮气，流速2.0ml/min，分流比5∶1。The inlet temperature is 200°C; the detector temperature is 250°C; the headspace equilibrium temperature: 80°C; the quantitative loop temperature: 90°C; the transfer line temperature: 100°C; the carrier gas is nitrogen, the flow rate is 2.0ml/min, and the split ratio is 5:1 .
4)供试品溶液的色谱图中呈现与对照品溶液的色谱图中色谱峰保留时间一致的色谱峰，确定氨磷汀中含有苯。4) in the chromatogram of the need testing solution, there is a chromatographic peak consistent with the retention time of the chromatographic peak in the chromatogram of the reference substance solution, and it is determined that benzene is contained in the amifostine.
实施例2本发明氨磷汀中苯的定量检测
1)制备供试品溶液1) Prepare the test solution
取氨磷汀2g，精密称定，置20ml顶空瓶中，精密加入水5ml，立即压盖密封，即得。Take 2g of amifostine, accurately weigh it, put it in a 20ml headspace bottle, accurately add 5ml of water, immediately press the cap and seal it, and it is ready.
2)制备系列浓度的对照品溶液2) prepare a series of concentrations of the reference substance solution
取苯对照品40mg，精密称定，置100ml量瓶中，加50％DMA溶液溶解并稀释至刻度，作为苯对照品贮备液；Take 40 mg of benzene reference substance, accurately weigh it, put it in a 100ml measuring bottle, add 50% DMA solution to dissolve and dilute to the mark, and use it as the benzene reference substance stock solution;
精密量取苯对照品贮备液，加水稀释制成每1ml中含苯0.8μg的溶液。精密量取5ml，置20ml顶空瓶中，立即压盖密封，即得。Precisely measure the stock solution of benzene reference substance, dilute it with water to make a solution containing 0.8 μg of benzene per 1 ml. Precisely measure 5ml, put it in a 20ml headspace bottle, and immediately press the cap to seal it.
3)分别吸取系列浓度的对照品溶液和供试品溶液注入顶空进样气相色谱仪，色谱条件如下：3) draw the reference substance solution and the need testing solution of serial concentrations respectively and inject into the headspace sampling gas chromatograph, and the chromatographic conditions are as follows:
色谱柱DB-624，30m×0.53mm，3μm；Chromatographic column DB-624, 30m×0.53mm, 3μm;
升温程序：起始温度40℃，维持8分钟，以每分钟30℃的速率升温至180℃，维持5分钟；Heating program: start at 40°C, maintain for 8 minutes, raise the temperature to 180°C at a rate of 30°C per minute, and maintain for 5 minutes;
进样口温度200℃；检测器温度250℃；顶空平衡温度：80℃；定量环温度：90℃；传输线温度：100℃；载气：N2，流速2.0ml/min，分流比5：1。Inlet temperature: 200°C; detector temperature: 250°C; headspace equilibrium temperature: 80°C; quantitative loop temperature: 90°C; transfer line temperature: 100°C; carrier gas: N2, flow rate: 2.0ml/min, split ratio: 5:1 .
4)供试品溶液的色谱图中呈现与对照品溶液的色谱图中色谱峰保留时间一致的色谱峰，确定氨磷汀中含有苯，苯的含量按外标法以峰面积计算。4) in the chromatogram of need testing solution, present the chromatographic peak consistent with the chromatographic peak retention time in the chromatogram of reference substance solution, confirm that benzene is contained in the amifostine, and the content of benzene is calculated with peak area by external standard method.
以下用试验例的方式来证明本发明的有益效果Prove the beneficial effect of the present invention below with the mode of test example
试验例1氨磷汀中苯的检测方法研究Study on the detection method of benzene in test example 1 amifostine
1、仪器与材料1. Instruments and materials
1.1仪器1.1 Instrument
气相色谱仪8890+7697A安捷伦Gas Chromatograph 8890+7697A Agilent
电子天平MS205DU梅特勒Electronic Balance MS205DU Mettler
电子天平ME204E/02梅特勒Electronic balance ME204E/02 Mettler
1.2、试药1.2. Test drug
苯对照品(批号：L1923053，购买于aladin，纯度：99.52％)，氨磷汀(原料药，四川汇宇制药提供)，N,N-二甲基乙酰胺(DMA，批号：21157607，购买于Scharlau)，二甲基亚砜(DMSO，批号：20010301，购买于Scharlau)，二甲基甲酰胺(DMF，批号：21388213，购买于Scharlau)Benzene reference substance (batch number: L1923053, purchased from aladin, purity: 99.52%), amifostine (raw material, provided by Sichuan Huiyu Pharmaceutical), N,N-dimethylacetamide (DMA, batch number: 21157607, purchased from Scharlau), dimethyl sulfoxide (DMSO, lot number: 20010301, purchased from Scharlau), dimethylformamide (DMF, lot number: 21388213, purchased from Scharlau)
2、方法筛选与结果2. Method screening and results
试验组①Test group ①
色谱条件：色谱柱DB-624，30m×0.53mm，3μmChromatographic conditions: column DB-624, 30m×0.53mm, 3μm
升温程序：起始温度40℃，维持8分钟，以每分钟30℃的速率升温至180℃，维持5分钟。Heating program: initial temperature is 40°C, maintained for 8 minutes, raised to 180°C at a rate of 30°C per minute, maintained for 5 minutes.
进样口温度200℃；检测器温度250℃；顶空平衡温度：80℃；定量环温度：90℃；传输线温度：100℃；载气：N2，流速2.0ml/min，分流比5：1。Inlet temperature: 200°C; detector temperature: 250°C; headspace equilibrium temperature: 80°C; quantitative loop temperature: 90°C; transfer line temperature: 100°C; carrier gas: N2, flow rate: 2.0ml/min, split ratio: 5:1 .
对照品溶液：取苯约40mg，精密称定，置100ml量瓶中，加50％DMA溶液溶解并稀释至刻度，作为苯对照品贮备液。精密量取苯对照品贮备液适量，加水稀释制成每1ml中约含苯0.4μg的溶液。精密量取5ml，置20ml顶空瓶中，立即压盖密封。(STD溶液)Reference substance solution: Take about 40 mg of benzene, accurately weigh it, put it in a 100ml measuring bottle, add 50% DMA solution to dissolve and dilute to the mark, and use it as the benzene reference substance stock solution. Precisely measure an appropriate amount of benzene reference substance stock solution, dilute with water to make a solution containing about 0.4 μg of benzene per 1 ml. Precisely measure 5ml, put it in a 20ml headspace bottle, and immediately press the cap to seal it. (STD solution)
供试品溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入水5ml，立即压盖密封。The test solution: take 1.0g of amifostine, accurately weigh it, put it in a 20ml headspace bottle, add 5ml of water accurately, and immediately press the cap to seal.
供试品加标溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入对照品溶液5ml，立即压盖密封。The test product spiked solution: take 1.0g of amifostine, accurately weighed, put it in a 20ml headspace bottle, accurately add 5ml of the reference substance solution, and immediately press the cap to seal.
试验组②Test group②
色谱条件：色谱柱DB-624，30m×0.53mm，3μmChromatographic conditions: column DB-624, 30m×0.53mm, 3μm
升温程序：起始温度40℃，维持8分钟，以每分钟30℃的速率升温至180℃，维持5分钟。Heating program: initial temperature is 40°C, maintained for 8 minutes, raised to 180°C at a rate of 30°C per minute, maintained for 5 minutes.
进样口温度200℃，检测器温度250℃，顶空平衡温度：80℃，定量环温度：90℃，传输线温度：100℃，载气：N2，流速2.0ml/min，分流比5：1。Inlet temperature 200°C,
对照品溶液：取苯约33.1mg，精密称定，置100ml量瓶中，加50％DMSO溶液溶解并稀释至刻度，作为苯对照品贮备液。精密量取苯对照品贮备液适量，加水稀释制成每1ml中约含苯0.4μg的溶液。精密量取5ml，置20ml顶空瓶中，立即压盖密封。(STD溶液)Reference substance solution: take about 33.1 mg of benzene, weigh it accurately, put it in a 100ml measuring bottle, add 50% DMSO solution to dissolve and dilute to the mark, and use it as the benzene reference substance stock solution. Precisely measure an appropriate amount of benzene reference substance stock solution, dilute with water to make a solution containing about 0.4 μg of benzene per 1 ml. Precisely measure 5ml, put it in a 20ml headspace bottle, and immediately press the cap to seal it. (STD solution)
供试品溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入水5ml，立即压盖密封。The test solution: take 1.0g of amifostine, accurately weigh it, put it in a 20ml headspace bottle, add 5ml of water accurately, and immediately press the cap to seal.
供试品加标溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入对照品溶液5ml，立即压盖密封。The test product spiked solution: take 1.0g of amifostine, accurately weighed, put it in a 20ml headspace bottle, accurately add 5ml of the reference substance solution, and immediately press the cap to seal.
试验组③Test group③
色谱条件：色谱柱DB-624，30m×0.53mm，3μmChromatographic conditions: column DB-624, 30m×0.53mm, 3μm
升温程序：起始温度40℃，维持8分钟，以每分钟30℃的速率升温至180℃，维持5分钟。Heating program: initial temperature is 40°C, maintained for 8 minutes, raised to 180°C at a rate of 30°C per minute, maintained for 5 minutes.
进样口温度200℃，检测器温度250℃，顶空平衡温度：80℃，定量环温度：90℃，传输线温度：100℃，载气：N2，流速2.0ml/min，分流比5：1。Inlet temperature 200°C,
对照品溶液：取苯约34.34mg，精密称定，置100ml量瓶中，加50％DMF溶液溶解并稀释至刻度，作为苯对照品贮备液。精密量取苯对照品贮备液适量，加水稀释制成每1ml中约含苯0.4μg的溶液。精密量取5ml，置20ml顶空瓶中，立即压盖密封。(STD溶液)Reference substance solution: take about 34.34 mg of benzene, weigh it accurately, put it in a 100ml measuring bottle, add 50% DMF solution to dissolve and dilute to the mark, and use it as the benzene reference substance stock solution. Precisely measure an appropriate amount of benzene reference substance stock solution, dilute with water to make a solution containing about 0.4 μg of benzene per 1 ml. Precisely measure 5ml, put it in a 20ml headspace bottle, and immediately press the cap to seal it. (STD solution)
供试品溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入水5ml，立即压盖密封。The test solution: take 1.0g of amifostine, accurately weigh it, put it in a 20ml headspace bottle, add 5ml of water accurately, and immediately press the cap to seal.
供试品加标溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入对照品溶液5ml，立即压盖密封。The test product spiked solution: take 1.0g of amifostine, accurately weighed, put it in a 20ml headspace bottle, accurately add 5ml of the reference substance solution, and immediately press the cap to seal.
试验组④Test group④
色谱条件：色谱柱DB-624，30m×0.53mm，3μm；Chromatographic conditions: column DB-624, 30m×0.53mm, 3μm;
升温程序：起始温度40℃，维持8分钟，以每分钟30℃的速率升温至180℃，维持5分钟。Heating program: initial temperature is 40°C, maintained for 8 minutes, raised to 180°C at a rate of 30°C per minute, maintained for 5 minutes.
进样口温度200℃，检测器温度250℃，顶空平衡温度：80℃，定量环温度：90℃，传输线温度：100℃，载气：N2，流速2.0ml/min，分流比5：1。Inlet temperature 200°C,
对照品溶液：取苯约40mg，精密称定，置100ml量瓶中，加20％DMA溶液溶解并稀释至刻度，制成每1ml中约含苯0.4mg的溶液，作为苯对照品贮备液。精密量取苯对照品贮备液适量，加水稀释制成每1ml中约含苯0.4μg的溶液。精密量取5ml，置20ml顶空瓶中，立即压盖密封。(STD溶液)Reference substance solution: Take about 40mg of benzene, weigh it accurately, put it in a 100ml measuring bottle, add 20% DMA solution to dissolve and dilute to the mark, and make a solution containing about 0.4mg of benzene per 1ml, as the benzene reference substance stock solution. Precisely measure an appropriate amount of benzene reference substance stock solution, dilute with water to make a solution containing about 0.4 μg of benzene per 1 ml. Precisely measure 5ml, put it in a 20ml headspace bottle, and immediately press the cap to seal it. (STD solution)
供试品溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入水5ml，立即压盖密封。The test solution: take 1.0g of amifostine, accurately weigh it, put it in a 20ml headspace bottle, add 5ml of water accurately, and immediately press the cap to seal.
供试品加标溶液：取氨磷汀1.0g，精密称定，置20ml顶空瓶中，精密加入对照品溶液5ml，立即压盖密封。The test product spiked solution: take 1.0g of amifostine, accurately weighed, put it in a 20ml headspace bottle, accurately add 5ml of the reference substance solution, and immediately press the cap to seal.
使用顶空进样气相色谱仪，按照试验组①、试验组②、试验组③、试验组④的方法进行实验，结果见表1和图1～8。Using a headspace sampling gas chromatograph, the experiment was carried out according to the methods of test group ①,
表1不同样品溶剂对检测的影响结果Table 1 The effect of different sample solvents on detection
从结果可见：分别采用50％DMA、50％DMSO、50％DMF为稀释剂，苯回收率均能达到80％～120％之间，但使用50％DMSO为稀释剂，如图4所示，供试品加标溶液中在保留时间约8.6min左右出现了一个很大的色谱峰，推测可能为氨磷汀与DMSO反应产生的杂质。As can be seen from the results: using 50% DMA, 50% DMSO, and 50% DMF as the diluent respectively, the recovery rate of benzene can reach between 80% and 120%, but using 50% DMSO as the diluent, as shown in Figure 4, A large chromatographic peak appeared in the spiked solution of the test product at a retention time of about 8.6 minutes, which may be an impurity produced by the reaction between amifostine and DMSO.
为验证对照品溶剂对检测结果准确性的影响，进一步单独进样50％DMF、50％DMA、50％DMSO以及试验组①配制的苯对照品溶液，按照前述色谱条件检测，结果见图9～12。从结果可见：50％DMSO和50％DMF，在苯出峰处(保留时间约为11.92min)均会出现苯的干扰峰，会干扰苯的检测，50％DMA中无干扰峰。In order to verify the influence of the solvent of the reference substance on the accuracy of the test results, 50% DMF, 50% DMA, 50% DMSO and the benzene reference solution prepared by the test group ① were further injected separately, and tested according to the aforementioned chromatographic conditions. The results are shown in Figure 9- 12. It can be seen from the results: 50% DMSO and 50% DMF, there will be interference peaks of benzene at the benzene peak (retention time is about 11.92min), which will interfere with the detection of benzene, and there will be no interference peaks in 50% DMA.
综合考虑回收率和干扰峰影响，最终确定试验组①的方法适宜于氨磷汀中的苯的检测，故将试验组①的方法用于方法学考察。Considering the recovery rate and the influence of interference peaks, the method of test group ① was finally determined to be suitable for the detection of benzene in amifostine, so the method of test group ① was used for methodological investigation.
3、方法学考察3. Methodological investigation
3.1系统适用性3.1 System suitability
50％DMA:取N,N-二甲基乙酰胺150ml,加水150ml，摇匀。50% DMA: Take 150ml of N,N-dimethylacetamide, add 150ml of water, and shake well.
对照品贮备：取苯35.96mg，置100ml量瓶中，用50％DMA稀释至刻度,摇匀。Reference substance storage: Take 35.96mg of benzene, put it in a 100ml measuring bottle, dilute to the mark with 50% DMA, and shake well.
对照品原液：精密量取对照品贮备1ml，置100ml量瓶中，用水稀释至刻度,摇匀。Reference substance stock solution: Accurately measure 1ml of the reference substance in reserve, put it in a 100ml measuring bottle, dilute with water to the mark, and shake well.
对照品溶液std：精密量取对照品原液10ml，置100ml量瓶中，用水稀释至刻度,摇匀。精密量取5ml,置20ml顶空瓶中,密封,平行6份。Reference substance solution std: Accurately measure 10ml of the reference substance stock solution, put it in a 100ml measuring bottle, dilute with water to the mark, and shake well. Precisely measure 5ml, put it in a 20ml headspace bottle, seal it, and make 6 copies in parallel.
取对照品溶液，按照试验组的色谱条件进样分析，结果见表2。The reference substance solution was taken and analyzed according to the chromatographic conditions of the test group, and the results are shown in Table 2.
表2系统适用性结果Table 2 System Suitability Results
从表2可见：平行6份对照品溶液主峰面积RSD小于10％，说明进样精密度良好。It can be seen from Table 2 that the RSD of the main peak area of 6 parallel reference substance solutions is less than 10%, indicating that the injection precision is good.
3.2、重复性3.2. Repeatability
sample&std 1：取氨磷汀AF1122001V 1.0010g，置20ml顶空瓶中，加对照品溶液5ml，密封。sample&std 1: Take 1.0010g of amifostine AF1122001V, put it in a 20ml headspace bottle, add 5ml of reference solution, and seal it.
sample&std 2：取氨磷汀AF1122001V 1.0017g，置20ml顶空瓶中，加对照品溶液5ml，密封。sample&std 2: Take 1.0017g of amifostine AF1122001V, put it in a 20ml headspace bottle, add 5ml of reference solution, and seal it.
sample&std 3：取氨磷汀AF1122001V 1.0019g，置20ml顶空瓶中，加对照品溶液5ml，密封。sample&std 3: Take 1.0019g of amifostine AF1122001V, put it in a 20ml headspace bottle, add 5ml of reference solution, and seal it.
sample&std 4：取氨磷汀AF1122001V 1.0020g，置20ml顶空瓶中，加对照品溶液5ml，密封。sample&std 4: Take 1.0020g of amifostine AF1122001V, put it in a 20ml headspace bottle, add 5ml of reference solution, and seal it.
sample&std 5：取氨磷汀AF1122001V 1.0034g，置20ml顶空瓶中，加对照品溶液5ml，密封。sample&std 5: Take 1.0034g of amifostine AF1122001V, put it in a 20ml headspace bottle, add 5ml of reference solution, and seal it.
sample&std 6：取氨磷汀AF1122001V 1.0040g，置20ml顶空瓶中，加对照品溶液5ml，密封。sample&std 6: Take 1.0040g of amifostine AF1122001V, put it in a 20ml headspace bottle, add 5ml of reference solution, and seal it.
取以上溶液，按照试验组的色谱条件进样分析，结果见表3Take the above solution, inject and analyze according to the chromatographic conditions of the test group, the results are shown in Table 3
表3重复性结果Table 3 Repeatability Results
从表3可见：于供试品中加入限度浓度的苯，平行6份的回收率在80％～120％，RSD小于10％，重复性良好。It can be seen from Table 3: when benzene at a limit concentration is added to the test product, the recovery rate of 6 parallel copies is 80% to 120%, the RSD is less than 10%, and the repeatability is good.
3.3定量限和检测限3.3 Quantitation limit and detection limit
对照品贮备液：精密称取苯42.64mg，置100ml量瓶中，用50％DMA稀释至刻度,摇匀。Reference substance stock solution: Accurately weigh 42.64mg of benzene, put it in a 100ml measuring bottle, dilute to the mark with 50% DMA, and shake well.
对照品原液：精密量取对照品贮备液1ml，置10ml量瓶中，用水稀释至刻度，摇匀。Reference substance stock solution: Accurately measure 1ml of the reference substance stock solution, put it in a 10ml measuring bottle, dilute with water to the mark, and shake well.
对照品溶液STD：精密量取对照品原液1ml，置100ml量瓶中，用水稀释至刻度,摇匀。精密量取5ml,置20ml顶空瓶中,密封。Reference substance solution STD: Accurately measure 1ml of the reference substance stock solution, put it in a 100ml measuring bottle, dilute with water to the mark, and shake well. Accurately measure 5ml, put it in a 20ml headspace bottle, and seal it.
定量限(LOQ)溶液：精密量取对照品溶液5.0ml，置50ml量瓶中，加水稀释至刻度，混匀。Limit of quantification (LOQ) solution: Accurately measure 5.0ml of the reference substance solution, put it in a 50ml measuring bottle, add water to dilute to the mark, and mix well.
检测限(LOD)溶液：精密量取对照品溶液1.7ml，置50ml量瓶中，加水稀释至刻度，摇匀。Limit of detection (LOD) solution: Accurately measure 1.7ml of the reference solution, put it in a 50ml measuring bottle, add water to dilute to the mark, and shake well.
定量限溶液LOQ-1：称取氨磷汀1.0024g至顶空瓶中，精密加入LOQ溶液5.0ml，密封。Limit of quantitation solution LOQ-1: Weigh 1.0024g of amifostine into a headspace vial, add 5.0ml of LOQ solution precisely, and seal it.
定量限溶液LOQ-2：称取氨磷汀1.0007g至顶空瓶中，精密加入LOQ溶液5.0ml，密封。Quantitation limit solution LOQ-2: Weigh 1.0007g of amifostine into a headspace vial, add 5.0ml of LOQ solution precisely, and seal it.
定量限溶液LOQ-3：称取氨磷汀1.0003g至顶空瓶中，精密加入LOQ溶液5.0ml，密封。Limit of quantitation solution LOQ-3: Weigh 1.0003g of amifostine into a headspace vial, add 5.0ml of LOQ solution precisely, and seal it.
定量限溶液LOQ-4：称取氨磷汀1.0014g至顶空瓶中，精密加入LOQ溶液5.0ml，密封。Limit of quantitation solution LOQ-4: Weigh 1.0014g of amifostine into a headspace vial, add 5.0ml of LOQ solution precisely, and seal it.
定量限溶液LOQ-5：称取氨磷汀1.0021g至顶空瓶中，精密加入LOQ溶液5.0ml，密封。Limit of quantitation solution LOQ-5: Weigh 1.0021g of amifostine into a headspace vial, add 5.0ml of LOQ solution precisely, and seal it.
定量限溶液LOQ-6：称取氨磷汀0.9996g至顶空瓶中，精密加入LOQ溶液5.0ml，密封。Limit of quantitation solution LOQ-6: Weigh 0.9996g of amifostine into a headspace vial, add 5.0ml of LOQ solution precisely, and seal it.
检测限溶液LOD：称取氨磷汀0.9998g至顶空瓶中，精密加入LOD溶液5.0ml，密封。Limit of detection solution LOD: Weigh 0.9998g of amifostine into a headspace vial, add 5.0ml of LOD solution precisely, and seal it.
取以上溶液，按照试验组的色谱条件进样分析，定量限和检测限结果分别见表4和表5。The above solution was taken and analyzed according to the chromatographic conditions of the test group. The quantification limit and detection limit results are shown in Table 4 and Table 5, respectively.
表4定量限结果Table 4 quantitation limit result
表5检测限结果Table 5 Detection limit results
从表4和表5可见：平行6份的定量限溶液峰面积RSD小于10％，检测限溶液信噪比为8.8，精密度良好。It can be seen from Table 4 and Table 5 that the peak area RSD of the limit of quantitation solution of 6 parallel samples is less than 10%, the signal-to-noise ratio of the limit of detection solution is 8.8, and the precision is good.
综上，本发明方法使氨磷汀待检样品中的苯与氨磷汀及杂质完全分离，准确性和灵敏度高，重复性好，实现了氨磷汀中苯的准确测定。In summary, the method of the present invention completely separates benzene from amifostine and impurities in the amifostine sample to be tested, has high accuracy, sensitivity and good repeatability, and realizes the accurate determination of benzene in amifostine.
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