Method for determining residual solvents in amifostine by gas chromatography

Abstract

The present invention belongs to the field of analysis and detection, and specifically relates to a method for simultaneously detecting residual organic solvents of N,N-dimethylformamide and sulfolane in amifostine by gas chromatography, which includes sample pretreatment and gas chromatograph detection method, and the steps are as follows: take amifostine, first add water to completely dissolve it, then add an organic solvent to filter after amifostine is precipitated, and take the filtrate and directly enter the gas chromatograph according to the gas chromatography detection conditions. The sample pretreatment method of the present invention can not only completely dissolve the DMF and sulfolane wrapped in the amifostine sample, but also remove the high-boiling point amifostine and directly enter the gas chromatograph, avoiding the contamination of the gas chromatograph and the detection interference caused by the degradation impurities of amifostine. The gas chromatograph detection method provided by the present invention can simultaneously and accurately detect the content of residual organic solvents of N,N-dimethylformamide and sulfolane in amifostine.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a method for simultaneously detecting N,N-dimethylformamide and sulfolane residual organic solvents in amifostine by gas chromatography. Background Art

[0002] Amifostine is an organosulfide phosphate cell protectant developed by CLINIGEN. After being hydrolyzed and dephosphorylated by alkaline phosphatase in tissues, it is metabolized into active metabolites containing sulfhydryl groups (H2N-(CH2)3-NH-(CH2)2-SH), which scavenge free radicals in tissues and reduce cytotoxicity. In 1995, the first injection was approved for marketing in the United States to reduce the cumulative nephrotoxicity caused by repeated use of cisplatin in advanced ovarian cancer; to reduce the incidence of moderate to severe dry mouth in patients receiving postoperative radiotherapy for head and neck cancer (where the irradiation window includes most of the parotid gland). It was first approved in China in 2001 for the adjuvant treatment of various cancers, reducing the toxicity of the kidneys, bone marrow, heart, ears and nervous system caused by chemotherapy, and its use before radiotherapy can significantly reduce the occurrence of dry mouth and mucositis.

[0003] However, since DMF and sulfolane organic solvents are used in the production process of amifostine, the higher the content of residual solvents, the greater the toxicity to the human body. According to ICH "Q3C (R8): Impurities: Guidelines for Residual Solvents": Since residual solvents cannot help treatment, all residual solvents should be removed as much as possible to meet the quality standards of preparations, production quality management standards (GMP) or other quality requirements. The residual solvent content of the preparation should not be higher than the level supported by safety data. Among them, DMF and sulfolane are both Class II organic solvents, with boiling points of 153°C and 285°C, respectively, and safety limits in drugs are 0.088% and 0.016%, respectively. Therefore, we need to control their residues to ensure the safety of drugs.

[0004] At present, there are many literature reports on methods for detecting residual solvents DMF and sulfolane, such as literature 1 (Tong Wenliang et al. Gas chromatography determination of sulfolane in industrial wastewater [J], Chemical Production and Technology, 2010, 17 (2): 33-35.) using a gas chromatography column HP-1 elastic capillary column (30m × 0.53mm × 1μm), injection port temperature 300℃; FID detector temperature 300℃; column temperature program: 100℃ for 2min, increase to 260℃ at 15℃ / min and maintain for 10min; carrier gas (nitrogen) volume flow 4ml / min; tail gas volume flow 30ml / min; hydrogen volume flow 30ml / min, air volume flow 300ml / min, no split. Preparation of test solution: The test sample was dissolved in saturated sodium chloride solution and extracted with dichloromethane, and the dichloromethane layer was directly injected. Reference 2 (Jiang Caiduan et al. Determination of N,N-dimethylformamide residue in amifostine raw material by gas chromatography [J], Science and Technology Innovation and Application, 2014, 5: 41.) A gas chromatography column RTX-624 (30m×0.53mm×3.0μm) was used; injection port temperature was 150℃; detector temperature was 280℃; column temperature program: 70℃ for 1min, then heated to 100℃ at 8℃ / min, and kept for 5min; finally heated to 200℃ at 30℃ / min; split ratio 2:1; column head pressure: 4.0psi; injection volume 1ml; headspace bottle temperature 80℃; quantitative loop temperature 100℃; transfer tube temperature 110℃, sample equilibration time 30min. Preparation of test solution: After the test sample was dissolved in water, a certain amount of anhydrous sodium sulfate was added and headspace injection was used.

[0005] The DCM solvent used in the prior art document 1 has a low boiling point, high volatility and high toxicity. At the same time, amifostine is insoluble in DCM. The inventors tried to use water as a solvent, adopt the gas chromatography conditions of document 1, and directly inject the sample spiked solution to detect the peaks of DMF and sulfolane. It was found that the peaks of DMF and sulfolane appeared earlier, water had obvious interference with the detection of DMF, and the peak tailing of sulfolane was more serious; the inventors adopted the gas chromatography conditions of document 2 for headspace injection, and found that sulfolane did not appear or its headspace sensitivity did not meet the detection requirements; therefore, the prior art cannot simultaneously and accurately detect the residual solvents of DMF and sulfolane in amifostine, and a method for simultaneously detecting multiple residual solvents in amifostine is urgently needed. Summary of the invention

[0006] The first object of the present invention is to provide a pre-treatment of an amifostine test sample, which comprises the following steps: taking the test sample amifostine and placing it in a volumetric flask, first adding water to completely dissolve it, then adding an organic solvent, filtering after the amifostine is precipitated, and taking the filtrate. The pre-treatment method of the sample of the present invention can make the DMF and sulfolane wrapped in the amifostine sample all dissolve out, and can remove the high-boiling amifostine at the same time, avoid it directly entering the gas chromatograph, damage the instrument, and also avoid the detection interference caused by the degradation impurities of amifostine.

[0007] The second object of the present invention is to provide a method for detecting residual solvents DMF and sulfolane in amifostine by gas chromatography, and the gas phase detection method of the present invention can accurately detect residual solvents DMF and sulfolane in amifostine at the same time. The method is simple to operate, has high sensitivity, good reproducibility, accurate results and good specificity.

[0008] To achieve the above object, the present invention adopts the following technical scheme: take the test sample amifostine, first add water to completely dissolve it, then add an organic solvent to precipitate the amifostine and filter it, take the filtrate and directly enter the gas chromatograph system to record the chromatogram;

[0009] The concentration of the amifostine after dissolving in water in the present invention is 125 mg / ml to 250 mg / ml, preferably 125 mg / ml;

[0010] The organic solvent in the present invention is dimethyl sulfoxide, anhydrous ethanol, ethyl acetate, N-methylpyrrolidone, preferably anhydrous ethanol;

[0011] The volume ratio of water to organic solvent in the present invention is 1:9 to 6:4, preferably 2:8;

[0012] The organic solvent in the present invention is anhydrous ethanol, and the volume ratio of water to anhydrous ethanol is 2:8;

[0013] In the gas chromatography detection conditions of the present invention, the chromatographic column is a gas phase capillary column of 30m×0.53mm×3μm DB-624 or 30m×0.53mm×1μm DB-WAX, preferably 30m×0.53mm×3μm DB-624.

[0014] The temperature rise program in the gas chromatography detection conditions of the present invention is as follows: the initial temperature is 100°C, maintained for 2 minutes, heated to 150°C at a rate of 15°C / min, maintained for 5 minutes, and then heated to 250°C at a rate of 30°C / min, maintained for 5 minutes.

[0015] The detector in the gas chromatography detection conditions of the present invention is: hydrogen flame ionization detector, electron capture detector, preferably hydrogen flame ionization detector.

[0016] The gas chromatography detection conditions in the present invention include an injection port temperature of 300°C and a detector temperature of 300°C.

[0017] The flow rate in the gas chromatography detection condition described in the present invention is: 4.0 ml / min.

[0018] The carrier gas in the gas chromatography detection conditions of the present invention is nitrogen.

[0019] The split mode in the gas chromatography detection conditions of the present invention is splitless or the split ratio is 20:1 to 10:1, preferably splitless.

[0020] The gas chromatography detection conditions in the present invention are as follows: the injection volume is 0.5 to 1 μl, preferably 0.5 μl.

[0021] The present invention also provides a method for determining N,N-dimethylformamide and sulfolane residual solvents in amifostine by gas chromatography, comprising the following steps:

[0022] (1) Test solution: Place amifostine in a volumetric flask and add water to dissolve it so that the concentration of the amifostine solution is 125 mg / ml to 250 mg / ml. Then add an organic solvent to precipitate the amifostine and filter it to obtain the filtrate.

[0023] (2) Reference solution: Take N,N-dimethylformamide and sulfolane solvents respectively and place them in a volumetric flask. Add water to dissolve them and then add anhydrous ethanol to dilute to the mark so that the concentrations of N,N-dimethylformamide and sulfolane solutions are 0.022 mg / ml and 0.004 mg / ml respectively.

[0024] (3) Subject the test solution and the reference solution to gas chromatography, record the chromatograms, and analyze the results using the external standard method.

[0025] Wherein, in step (1) and step (2), the volume ratio of the added water to the anhydrous ethanol is 2:8.

[0026] The beneficial effects of the present invention are:

[0027] (1) The sample pretreatment of the present invention can dissolve all the DMF and sulfolane contained in the amifostine sample and obtain accurate quantitative detection. At the same time, the high-boiling-point amifostine can be removed and directly enter the gas chromatograph to avoid contamination of the gas chromatograph and detection interference caused by the degradation impurities of amifostine.

[0028] (2) The present invention can accurately detect the contents of DMF and sulfolane residual solvents simultaneously, and the detection method has the advantages of high accuracy, high sensitivity, good reproducibility and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 In Example 1 of the present invention, the solvent is water, and the chromatogram is obtained after injecting the test sample spiked solution A into the gas chromatograph;

[0030] Figure 2 In Example 1 of the present invention, the solvent is ethyl acetate, and the chromatogram is obtained after the test sample spiked solution B is injected into the gas chromatograph;

[0031] Figure 3 In Example 1 of the present invention, the solvent is dimethyl sulfoxide, and the chromatogram is obtained after injecting the test sample spiked solution C into the gas chromatograph;

[0032] Figure 4 In Example 1 of the present invention, the solvent is N-methylpyrrolidone, and the chromatogram is obtained after injecting the test sample spiked solution D into the gas chromatograph;

[0033] Figure 5 In Example 1 of the present invention, the solvent is anhydrous ethanol, and the sample spiked solution E is injected into the gas chromatograph to obtain a chromatogram;

[0034] Figure 6 In Example 2 of the present invention, the solvent is water and ethanol in a volume ratio of 6:4, and the chromatogram is obtained after injecting the test sample spiked solution G into the gas chromatograph;

[0035] Figure 7 is the gas chromatogram of the blank solution in Example 3 of the present invention;

[0036] Figure 8 is a gas chromatogram of the test solution in Example 3 of the present invention;

[0037] Fig. 9 is a gas chromatogram of the test sample spiked solution in Example 3 of the present invention;

[0038] Fig.10 The gas chromatography condition parameter split ratio in Example 4 of the present invention is 20:1, and the chromatogram is obtained after the test sample spiked solution E is injected into the gas chromatograph;

[0039] Fig.11 The gas chromatography condition parameter split ratio in Example 4 of the present invention is 10:1, and the chromatogram is obtained after the test sample spiked solution E is injected into the gas chromatograph;

[0040] Fig.12 The gas chromatographic column in Example 5 of the present invention is DB-WAX, and the other detection conditions remain unchanged. The sample spiked solution E is injected into the gas chromatograph to obtain a chromatogram;

[0041] Fig.13The gas chromatography condition parameter detector in Example 6 of the present invention is ECD, and the other detection conditions remain unchanged. The sample spiked solution E is injected into the gas chromatograph to obtain a chromatogram;

[0042] Fig.14 is a gas chromatogram of the blank solution in Comparative Example 1 of the present invention;

[0043] Fig.15 It is a gas chromatogram of the test sample spiked solution in Comparative Example 1 of the present invention;

[0044] Fig.16 It is the gas chromatogram of the test sample spiked solution in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0045] The present invention provides a method for simultaneously detecting residual organic solvents of N,N-dimethylformamide and cyclopentane sulfone in amifostine. Those skilled in the art can refer to the content of this article and appropriately improve the chromatographic condition parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. The test materials used in the present invention are all common commercial products and can all be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0046] In the present invention, the abbreviations involved have the following meanings:

[0047] DMSO means dimethyl sulfoxide;

[0048] DMF stands for N,N-dimethylformamide;

[0049] NMP means N-methylpyrrolidone;

[0050] EAC means ethyl acetate;

[0051] ECD stands for electron capture detector;

[0052] FID stands for flame ionization detector;

[0053] RSD stands for relative standard deviation;

[0054] Example 1

[0055] 1.1 Gas chromatography conditions

[0056] Chromatographic column: DB-624, 30m×0.53mm, 3μm.

[0057] Chromatographic conditions: Temperature program: starting temperature 100°C, maintain for 2 minutes, increase the temperature to 150°C at a rate of 15°C per minute, maintain for 5 minutes, then increase the temperature to 250°C at a rate of 30°C per minute, maintain for 5 minutes; Inlet temperature: 300°C; Detector type: FID; Detector temperature: 300°C; Flow rate: 4.0 ml / min; Carrier gas: nitrogen; Split mode: non-split; Injection volume: 0.5 μl.

[0058] 1.2 Test method: Prepare the test sample spike solution with water, EAC, DMSO, NMP and anhydrous ethanol solvents respectively.

[0059] DMF stock solution: Accurately weigh about 110 mg of DMF, place in a 50 ml volumetric flask, dilute to volume with water, and shake well.

[0060] Sulfolane stock solution: Accurately weigh about 20 mg of sulfolane, place in a 50 ml volumetric flask, dilute to volume with water, and shake well.

[0061] Reference substance stock solution: Accurately measure 1 ml of DMF stock solution and 1 ml of sulfolane stock solution, place them in a 20 ml volumetric flask, dilute to the mark with water, and shake well.

[0062] Test sample spiked solution A (water): Take 0.25 g of amifostine, place it in a 10 ml volumetric flask, add the reference sample stock solution to dilute to the scale, and shake well.

[0063] Test sample spiked solution B (EAC): Take 0.25 g of amifostine and place it in a 10 ml volumetric flask. Add 2 ml of reference sample stock solution. Shake to dissolve, then add EAC to dilute to the mark. Mix well and let stand for about 30 min. Take the supernatant.

[0064] Test sample spiked solution C (DMSO): Weigh 0.25 g of amifostine sample and place it in a 10 ml volumetric flask. Add 2 ml of reference sample stock solution to dissolve it completely. Add DMSO to make up to the mark, mix well, filter after the precipitate is separated, and take the filtrate.

[0065] Test sample spiked solution D (NMP): Weigh 0.25 g of amifostine sample and place it in a 10 ml volumetric flask. Add 2 ml of reference stock solution and dissolve it completely. Then add NMP to make up to the mark, mix well, filter after the precipitate is precipitated, and take the filtrate.

[0066] Test sample spiked solution E (anhydrous ethanol): Weigh 0.25 g of amifostine sample and place it in a 10 ml volumetric flask. Add 2 ml of reference stock solution to dissolve it completely. Add anhydrous ethanol to make up to the mark, mix well, filter after the precipitate is separated, and take the filtrate.

[0067] Take the test sample spiked solutions A, B, C, D, and E respectively, inject and test according to the detection conditions of Example 1, record the chromatogram, and the results are shown in the following table:

[0068] Table 1 Solvent screening results

[0069]

[0070] Example 2

[0071] Test method: Prepare the test sample spike solution according to the volume ratio of water to ethanol of 1:9, 2:8 and 6:4 respectively.

[0072] Test sample spiked solution F (water: ethanol = 1:9): Weigh 0.25 g of amifostine sample and place it in a 10 ml volumetric flask, add 1 ml of reference sample stock solution, dissolve it completely by ultrasonication, add anhydrous ethanol to make up the volume, mix well, filter after the precipitate is precipitated, and take the filtrate.

[0073] Test sample spiked solution G (water: ethanol = 6:4): weigh 0.25 g of amifostine sample, place in a 10 ml volumetric flask, add 2 ml of reference stock solution, and after it is completely dissolved, add 4 ml of water, mix well, add anhydrous ethanol to make up to volume, mix well, filter after the precipitate is precipitated, and take the filtrate.

[0074] Take the test sample spiked solutions F and G and the test sample spiked solution E (water: ethanol = 2:8) prepared in Example 1 respectively, inject and test according to the detection conditions of Example 1, record the chromatogram, and the results are shown in the following table:

[0075] Table 2 Water and ethanol ratio screening results

[0076]

[0077] Example 3

[0078] Blank solution: 80% ethanol in water.

[0079] Reference substance solution: Accurately measure 2 ml of the reference substance stock solution prepared in Example 1, place it in a 10 ml volumetric flask, dilute to volume with anhydrous ethanol, and shake well.

[0080] Reference substance linear stock solution: Accurately measure 2.0 ml of DMF stock solution and 2.0 ml of sulfolane stock solution, place in a 20 ml volumetric flask, add water to dilute to the scale, and shake well.

[0081] Test solution: Take 0.25g of amifostine, weigh accurately, put it in a 10ml volumetric flask, add 2ml of water, shake to dissolve, add anhydrous ethanol to dilute to the scale, shake well, filter after the precipitate is precipitated, and take the filtrate.

[0082] Test sample spiked solution: Take 0.25 g of amifostine, accurately weigh it, and place it in a 10 ml volumetric flask. Accurately add 2 ml of the reference stock solution prepared in Example 1. After shaking to dissolve, add 5 μl each of methanol, ethanol, and ethyl acetate. Add anhydrous ethanol to dilute to the scale, shake well, filter the precipitate, and obtain the filtrate.

[0083] 3.1 Exclusivity

[0084] Test method: Precisely measure 0.5 μl of blank solution, test solution, test spiked solution and reference solution and inject them into gas chromatograph, record the chromatogram, and calculate the content of DMF and sulfolane residual organic solvents in amifostine by peak area according to the external standard method, which is publicly expressed as:

[0085] Content% = (rU / rS) × (CS / CU) × 100%

[0086] Where: rU is the peak area of ​​DMF or sulfolane in the test solution;

[0087] rS is the peak area of ​​DMF or sulfolane in the reference solution;

[0088] CU is the concentration of the test solution;

[0089] CS is the concentration of DMF or sulfolane in the reference solution.

[0090] Test results: Figure 7 It showed that the blank solution did not interfere with the peaks of DMF and sulfolane; Figure 8 It showed that no residual solvents of DMF and sulfolane were detected in the test solution; Fig. 9 It shows that methanol, ethanol and ethyl acetate in the spiked solution of the test sample do not interfere with the peaks of DMF and sulfolane, wherein the separation degrees between the DMF peak and the adjacent peaks are 22.4 and 72.9, respectively; the separation degrees between the sulfolane peak and the adjacent peaks are 9.3 and 7.7, respectively; indicating that the detection method of the present invention has good specificity.

[0091] 3.2 Repeatability

[0092] Test method: Take 6 injections of reference solution and test solution respectively, perform injection and test according to the above test conditions, and record the chromatogram.

[0093] Table 3 Results of 6 repeated tests of reference solution

[0094] frequency 1 2 3 4 5 6 average value RSD DMF 62.987 61.395 61.266 59.684 61.458 62.681 61.579 1.9％ Sulfolane 20.862 20.410 20.352 20.024 20.172 20.776 20.433 1.6％

[0095] Table 4 Results of 6 repeated tests of the spiked solution of the test sample

[0096] name DMF peak area DMF recovery rate / % Sulfolane peak area Sulfolane recovery rate / % 1 61.454 99.7％ 20.641 100.1％ 2 62.653 101.7％ 20.771 100.7％ 3 63.990 103.8％ 21.494 104.2％ 4 61.817 100.3％ 20.649 100.1％ 5 63.919 103.7％ 21.111 102.4％ 6 62.193 100.9％ 20.700 100.4％ average value / 101.7％ / 101.3％ RSD / 1.7％ / 1.6％

[0097] Test results: As shown in Tables 3 to 4, the RSDs of the recoveries of DMF and sulfolane in the six sample spiked solutions were 1.7% and 1.6%, respectively, and the recoveries of the two residual solvents were both between 80% and 120%, indicating that the detection method of the present invention has good repeatability.

[0098] 3.3 Limit of Quantitation & Limit of Detection

[0099] Test method: Accurately measure 0.2 ml of the reference stock solution, place it in a 10 ml volumetric bottle, add 1.8 ml of ultrapure water, dilute to the mark with anhydrous ethanol, shake well, filter, and take the filtrate. Dilute step by step until the peak heights of DMF and sulfolane are both 10 to 30 times the baseline noise, which is the quantitative limit, and this is used as the quantitative limit solution.

[0100] Accurately measure 65μl of the reference stock solution, place it in a 10ml volumetric flask, add 1.9ml of ultrapure water, dilute to scale with anhydrous ethanol, shake well, filter, and take the filtrate. Dilute step by step until the peak height of DMF and sulfolane is 3 to 10 times the baseline noise, which is the detection limit and is used as the detection limit solution.

[0101] The quantitative limit solution and the detection limit solution were tested according to the above-mentioned test method and chromatographic conditions, and the chromatograms were recorded.

[0102] Table 5 Results of quantification limit and detection limit of DMF

[0103]

[0104] Table 6 Results of quantification limit and detection limit of sulfolane

[0105]

[0106] Test results: From Tables 5 to 6, it can be seen that the quantitative limit and detection limit of DMF are 0.0090% and 0.0029% respectively; the quantitative limit and detection limit of sulfolane are 0.0018% and 0.0006% respectively, indicating that the sensitivity of the detection method of the present invention meets the detection requirements.

[0107] 3.4 Linearity:

[0108] Test method: Accurately measure 0.2 ml of the reference linear stock solution, place it in a 10 ml volumetric flask, add 1.8 ml of water, dilute to the mark with anhydrous ethanol, shake well, filter, and take the filtrate. This solution is a 20% linear solution.

[0109] Accurately measure 0.5 ml of the linear stock solution, place it in a 10 ml volumetric flask, add 1.5 ml of water, dilute to the mark with anhydrous ethanol, shake well, filter, and take the filtrate. This solution is a 50% linear solution.

[0110] Accurately measure 1.0 ml of the linear stock solution, place it in a 10 ml volumetric flask, add 1 ml of water, dilute to the mark with anhydrous ethanol, shake well, filter, and take the filtrate. This solution is a 100% linear solution.

[0111] Accurately measure 1.5 ml of the linear stock solution, place it in a 10 ml volumetric flask, add 0.5 ml of water, dilute to the mark with anhydrous ethanol, shake well, filter, and take the filtrate. This solution is a 150% linear solution.

[0112] Accurately measure 2.0 ml of the linear stock solution, place it in a 10 ml volumetric flask, add anhydrous ethanol to dilute to the mark, shake well, filter, and take the filtrate. This solution is a 200% linear solution.

[0113] The quantitative limit solution, 20%, 50%, 100%, 150%, and 200% linear solutions were tested according to the above-mentioned detection method and chromatographic conditions, and the chromatograms were recorded.

[0114] Table 7 Linearity results of DMF and sulfolane

[0115]

[0116]

[0117] Test results: As shown in Table 7, the correlation coefficients of DMF and sulfolane are 0.9997 and 0.9998, respectively, indicating that the detection method of the present invention has a good linear relationship.

[0118] 3.5 Accuracy

[0119] Test method: Weigh 250.06 mg of amifostine accurately, place in a 10 ml volumetric flask, add 0.2 ml of reference stock solution accurately, add 1.8 ml of ultrapure water, and shake to dissolve. Add anhydrous ethanol to dilute to the scale, shake well, filter after precipitation, and take the filtrate. Prepare 3 portions as the limit of quantitation (LOQ) recovery rate solution in the same way.

[0120] Accurately weigh 250.34 mg of amifostine, place in a 10 ml volumetric flask, accurately add 0.5 ml of linear stock solution, add 1.5 ml of ultrapure water, shake to dissolve. Add anhydrous ethanol to dilute to scale, shake well, filter after precipitation, and take the filtrate. Prepare 3 portions as 50% recovery solution in the same way.

[0121] Weigh 249.79 mg of amifostine accurately, place in a 10 ml volumetric flask, add 1.0 ml of linear stock solution accurately, add 1.0 ml of ultrapure water, and shake to dissolve. Add anhydrous ethanol to dilute to the scale, shake well, filter after precipitation, and take the filtrate. Prepare 3 portions as 100% recovery solution in the same way.

[0122] Accurately weigh 250.55 mg of amifostine, place in a 10 ml volumetric flask, accurately add 1.5 ml of linear stock solution, add 0.5 ml of ultrapure water, and shake to dissolve. Add anhydrous ethanol to dilute to the scale, shake well, filter after precipitation, and take the filtrate. Prepare 3 portions as 150% recovery rate solution in the same way.

[0123] Carry out detection according to the above detection method and chromatographic conditions respectively, and record the chromatogram.

[0124] Table 8 Recovery results of DMF at different concentrations

[0125]

[0126] Table 9 Recovery results of different concentrations of sulfolane

[0127]

[0128] Table 10 Average recovery results of DMF and sulfolane

[0129]

[0130]

[0131] Test results: As shown in Tables 8 to 10, the average recoveries of DMF and sulfolane are 100.6% and 105.2%, respectively, and their RSDs are 4.3% and 6.1%, respectively, indicating that the sample pretreatment method of the present invention can extract all of DMF and sulfolane into the solution, and the detection method has good accuracy.

[0132] 3.6 Durability

[0133] Experimental method: The flow rate (3.8 ml / min and 4.2 ml / min), starting temperature (95°C, 105°C), detector temperature (295°C, 305°C) and injection port temperature (295°C, 305°C) were changed respectively. The other chromatographic conditions were the same as those in Example 1. The reference solution and the test solution were injected into the liquid chromatograph respectively, and the chromatograms were recorded.

[0134] Table 11 Durability results of DMF and sulfolane

[0135]

[0136] Test results: As shown in Table 11, within a certain range, the flow rate, starting temperature, detector temperature and injection port temperature are changed respectively, and the peak area RSD values ​​of DMF and sulfolane in the reference solution are less than 10%, indicating that the detection method of the present invention has good durability.

[0137] Example 4

[0138] Test method: The test sample spiked solution was tested at a gas chromatography condition parameter split ratio of 20:1 and 10:1. Other instruments, test conditions and test methods were basically the same as those in Example 1.

[0139] Take the test sample spiked solution E, inject and test according to the above detection conditions, record the chromatogram, and the results are shown in the following table:

[0140] Table 12 Split ratio screening results

[0141] Divert Mode Test results Split ratio = 20:1 from Fig.10 It can be seen that DMF and sulfolane did not elute. Split ratio = 10:1 from Fig.11 It can be seen that the peak areas of DMF and sulfolane are very small, and their sensitivity does not meet the requirements.

[0142] Example 5

[0143] Test method: The gas chromatographic column was replaced with DB-WAX (30 m×0.53 mm×1.0 μm), and other instruments, detection conditions and detection methods were basically the same as those in Example 1. Take the test sample spiked solution E, inject and detect according to the above detection conditions, and record the chromatogram.

[0144] Test results: From Fig.12 It can be seen that the peak shapes of DMF and sulfolane are normal, but the baseline at 13-14 minutes is greatly elevated.

[0145] Example 6

[0146] Test method: The gas phase detector was replaced with ECD, and other instruments, detection conditions and detection methods were basically the same as those in Example 1. Take the test sample spiked solution E, inject and detect according to the above detection conditions, and record the chromatogram.

[0147] Test results: From Fig.13 It can be seen that DMF and sulfolane did not produce peaks in the spiked solution of the test sample.

[0148] Example 7

[0149] Test method: The injection volume was changed to 1 μl, and other instruments, detection conditions, detection methods, and solution preparation were basically the same as those in Example 1. 1 μl of the reference solution was injected into the gas chromatograph, and the chromatogram was recorded.

[0150] Test results: DMF peak bifurcated due to excessive injection volume, which exceeded the liner load. Therefore, the injection volume needed to be reduced.

[0151] Example 8

[0152] Test method: The concentration of the test solution was changed, and other instruments, test conditions and test methods were basically the same as in Example 1.

[0153] Test solution: Take 0.5 g of amifostine, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of water, shake to dissolve, add anhydrous ethanol to dilute to the scale, shake well, let the precipitate separate out and filter.

[0154] Test results: The test sample was difficult to filter.

[0155] Comparative Example 1

[0156] Test method: The gas chromatography conditions of reference 1 were used, except that the dichloromethane solvent used in the reference was replaced by water. The blank solution and the test sample spiked solution were detected by direct injection, and the chromatograms were recorded.

[0157] Test results: From Figures 14 to 15 It can be seen that the elution time of DMF is too early, and the blank solution water has obvious interference with it; at the same time, the elution time of sulfolane is also early and the tailing is serious, indicating that this method is not suitable for the simultaneous detection of DMF and sulfolane solvents in amifostine.

[0158] Comparative Example 2

[0159] Test method: Take the test sample spiked solution, perform sampling and detection according to the gas chromatography conditions of Reference 2, and record the chromatogram.

[0160] Test results: From Fig.16 It can be seen that sulfolane did not appear.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining residual solvents of N,N-dimethylformamide and sulfolane in amifostine by gas chromatography, characterized in that: Take amifostine, first add water to completely dissolve it, then add an organic solvent to precipitate the amifostine and filter it, take the filtrate and directly enter the gas chromatograph according to the gas chromatography conditions, and record the chromatogram; The organic solvent is anhydrous ethanol, and the volume ratio of water to anhydrous ethanol is 2:8; The gas chromatography conditions are as follows: the chromatographic column is a gas phase capillary column of 30m×0.53mm×3μm DB-624 or 30m×0.53mm×1μm DB-WAX; Heating program: starting temperature 100°C, maintain for 2 min, increase to 150°C at a rate of 15°C / min, maintain for 5 min, then increase to 250°C at a rate of 30°C / min, maintain for 5 min; Detector: hydrogen flame ionization detector, electron capture detector; Inlet temperature: 300°C; Detector temperature: 300°C; Flow rate: 4.0ml / min; Carrier gas: nitrogen; Split mode: no split or split ratio of 20:1 to 10:1; Injection volume: 0.5~1μl.

2. The method according to claim 1, characterized in that: The concentration of the amifostine after being dissolved in water is 125 mg / ml to 250 mg / ml.

3. The method according to claim 1, characterized in that: The concentration of the amifostine after being dissolved in water is 125 mg / ml.

4. The method according to claim 1, characterized in that: The chromatographic column is a 30m×0.53mm×3μm DB-624 gas phase capillary column.

5. The method according to claim 1, characterized in that The detector is a hydrogen flame ionization detector.

6. The method according to claim 1, characterized in that The diversion mode is non-diversion.

7. The method according to claim 1, characterized in that The injection volume was 0.5 μl.

8. A method for determining N,N-dimethylformamide and sulfolane residual solvents in amifostine by gas chromatography, characterized in that: The following steps are involved: (1) Test solution: Place amifostine in a volumetric flask, add water to dissolve it so that the concentration of the amifostine solution is 125 mg / ml to 250 mg / ml, then add an organic solvent to precipitate the amifostine, filter it, and take the filtrate to obtain the test solution; (2) Reference solution: Take N,N-dimethylformamide and sulfolane solvents respectively and place them in a volumetric flask. Dissolve them in water and then add anhydrous ethanol to dilute to the mark so that the concentrations of N,N-dimethylformamide and sulfolane solutions are 0.022 mg / ml and 0.004 mg / ml respectively. (3) Subject the test solution and the reference solution to gas chromatography, record the chromatograms, and analyze the results using the external standard method; The organic solvent is anhydrous ethanol; In the steps (1) and (2), the volume ratio of the added water to the anhydrous ethanol is 2:8; The gas chromatography conditions are as follows: the chromatographic column is a gas phase capillary column of 30m×0.53mm×3μm DB-624 or 30m×0.53mm×1μm DB-WAX; Heating program: starting temperature 100°C, maintain for 2 min, increase to 150°C at a rate of 15°C / min, maintain for 5 min, then increase to 250°C at a rate of 30°C / min, maintain for 5 min; Detector: hydrogen flame ionization detector, electron capture detector; Inlet temperature: 300°C; Detector temperature: 300°C; Flow rate: 4.0ml / min; Carrier gas: nitrogen; Split mode: no split or split ratio of 20:1 to 10:1; Injection volume: 0.5~1μl.

9. The method according to claim 8, characterized in that The chromatographic column is a 30m×0.53mm×3μm DB-624 gas phase capillary column.

10. The method according to claim 8, characterized in that The detector is a hydrogen flame ionization detector.

11. The method according to claim 8, characterized in that The diversion mode is non-diversion.

12. The method according to claim 8, characterized in that The injection volume was 0.5 μl.

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