A method for quantitatively analyzing dimethyl sulfate content by derivatization method

Abstract

The present invention belongs to the technical field of chemical detection, and specifically relates to a method for quantitatively analyzing the content of dimethyl sulfate by derivatization. The method uses sodium phenol as a derivatization reagent to form a derivative anisole with dimethyl sulfate, and the peak area of ​​anisole in a test solution is measured by gas chromatography-mass spectrometry to calculate the content of dimethyl sulfate. Compared with the prior art, the analytical method has the following advantages: (1) the derivative product prepared by the present invention is stable within 11 hours, and the peak areas of the target product are 32224, 33873, 31960, and 31560 at 0, 2, 6, and 11 hours, respectively, with an RSD of 3.14%, a good peak shape, and no significant change in the peak shape of the target product due to programmed temperature increase; (2) the method has high sensitivity. After detection, the quantitative limit of dimethyl sulfate is 0.1057 ng, and the detection limit is 0.0032 ng, which fully meets the determination requirements; (3) the derivatization reagent and dimethyl sulfate can immediately undergo a derivatization reaction, which greatly improves the overall detection efficiency and is simple to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for quantitatively analyzing the content of dimethyl sulfate by a derivatization method. Background Art

[0002] Dimethyl sulfate is mutagenic / genotoxic both in vitro and in vivo. According to ICH M7, the threshold of toxicological concern (TTC) is 1.5 μg / day. Currently, the commonly used analytical methods for detecting dimethyl sulfate include direct detection by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Due to the poor stability of dimethyl sulfate at room temperature, direct analytical detection will produce large errors or the detection process requires a new preparation, resulting in cumbersome experimental operations; and dimethyl sulfate is a commonly used methylation reagent with active chemical properties, so the derivatization method combined with gas chromatography-mass spectrometry is used to determine dimethyl sulfate.

[0003] Regarding the determination method of dimethyl sulfate, the following documents have disclosed relevant information:

[0004] Prior art 1: directly using gas chromatography or gas chromatography-mass spectrometry to determine the content of dimethyl sulfate; Disadvantages: However, dimethyl sulfate has poor stability at room temperature and is difficult to accurately determine. It usually needs to be freshly prepared before use, and the test process is time-consuming and laborious; for samples with lower limit requirements, the detection sensitivity of gas chromatography may not meet the detection requirements; CN 113848279A discloses a method for detecting residual reagent dimethyl sulfate in a drug, and the content of monomethyl sulfate is detected by ion chromatography: 250 mg of the drug pantoprazole sodium to be tested is accurately weighed, placed in a 50 ml volumetric flask, dissolved with 30 mmol / L sodium hydroxide solution and fixed to the scale, and shaken well; after filtering the above solution through a solid phase extraction column, the content of monomethyl sulfate is directly determined by ion chromatography.

[0005] Prior art 2: CN111505182A discloses a method for determining dimethyl sulfate in drugs by derivatization gas chromatography-mass spectrometry, which uses sodium iodide and sodium thiosulfate dissolved in water as a derivatization solution, adds acetonitrile and dimethyl sulfate to react to generate methyl iodide, and then uses gas chromatography-mass spectrometry headspace injection to calculate the dimethyl sulfate content by methyl iodide peak area. The derivatization reaction time of this method is short: 30 minutes, and the derivative product methyl iodide is a volatile substance, which can reduce the interference of non-volatile substances on the detection;

[0006] Shortcomings of the second technology: The derivatization reagent sodium iodide usually requires water as a solvent, and multiple injections will shorten the service life of the ion source of the gas chromatography-mass spectrometer; the derivative product has poor stability, and the derivatization reaction is difficult to control. It is usually necessary to add an internal standard to ensure the accuracy of the detection, and the experimental operation is cumbersome.

[0007] Prior art three: CN 10364545C discloses a method for preparing anisole, comprising the following steps: (1) reacting an excess of 10%-30% dimethyl sulfate with a sodium phenolate solution (prepared by equimolar ratio of 20% (by weight) sodium hydroxide solution and phenol) at 10-30° C. for 1 hour, at 40° C. for half an hour, and at reflux temperature for half an hour; (2) azeotropic distillation is performed to distill out the anisole, and the distilled water is partially returned to the reactor during the distillation process; (3) when the anisole obtained by azeotropic distillation is close to 90% of the theoretical yield, a 20% (by weight) sodium hydroxide solution is added to the reactor, and the reaction is continued for half an hour to one hour to distill out all the anisole generated by the reaction, and then the anisole product is dried and redistilled.

[0008] The third technical inadequacy is as follows: (1) the reactions that may occur in the reaction system include sodium hydroxide + phenol, sodium phenol + dimethyl sulfate, and sodium hydroxide + dimethyl sulfate, which is relatively complicated. If the dimethyl sulfate content is calculated using the generated anisole, the test result will be lower than the actual situation or even a false negative will occur, making it impossible to accurately control the quality of the sample. (2) Its main purpose is constant preparation, and the amount of reactants is large, so the reaction conditions are more complicated, including steps such as reflux and distillation. The reaction temperature needs to be adjusted according to the progress, which is time-consuming. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for quantitatively analyzing the content of dimethyl sulfate by a derivatization method. The present invention uses sodium phenol as a derivatization reagent to react with dimethyl sulfate to generate anisole, and the content of dimethyl sulfate is calculated by detecting the content of anisole (low melting point and volatile); and has the following advantages: the reaction product is relatively stable, not easily affected by programmed temperature rise, and has high sensitivity, which fully meets the determination requirements.

[0010] The present invention provides a method for quantitatively analyzing the content of dimethyl sulfate by a derivatization method, which mainly uses a sodium phenolate solution as a derivatization reagent to derive dimethyl sulfate into anisole, and obtains the content of dimethyl sulfate by detecting the content of anisole;

[0011] Preferably, in the preparation process of the sodium phenolate solution, isopropanol is used as a co-solvent for sodium phenolate; in the experiment of the present invention, the solubility of sodium phenolate in dimethyl sulfoxide is low, and it is found through experiments that the solubility of sodium phenolate in the organic phase is poor, and when methanol, ethanol and acetone are used as co-solvents, the matrix interference is large. Through optimization, it is finally determined to select isopropanol as the co-solvent.

[0012] Preferably, the sodium phenolate solution is prepared by first adding isopropanol to sodium phenolate, then adding dimethyl sulfoxide, dissolving by ultrasonication, and then diluting with dimethyl sulfoxide, wherein the addition ratio of isopropanol to dimethyl sulfoxide before ultrasonication is 1:10.8-13.2.

[0013] Preferably, the addition ratio of the isopropanol to dimethyl sulfoxide is 1:12.

[0014] The reaction system detected by the present invention is dimethyl sulfoxide-isopropanol, which can meet the trace determination of dimethyl sulfate in most organic compounds and has wide applicability.

[0015] Preferably, in the method provided by the present invention, the derivative product anisole is diluted with dimethyl sulfoxide to obtain a test solution, and the peak area of ​​anisole in the test solution is measured by gas chromatography-mass spectrometry to calculate the content of dimethyl sulfate.

[0016] A method for quantitatively analyzing the content of dimethyl sulfate by a derivatization method comprises the following steps:

[0017] (1) Preparation of sodium phenolate solution: Weigh 0.48-0.52 g of sodium phenolate and place it in a 500 ml volumetric flask. First, add 25 ml of isopropanol and then add 270-330 ml of dimethyl sulfoxide. Dissolve it by ultrasonication. Dilute to the mark with dimethyl sulfoxide and shake well to obtain a sodium phenolate solution of 0.96-1.04 mg / ml.

[0018] (2) Preparation of reference substance stock solution: Take 18-22 mg of dimethyl sulfate and place it in a 10 ml volumetric flask. Dilute it to the mark with 0.96-1.04 mg / ml sodium phenolate solution and shake well. Accurately measure 1 ml and place it in a 200 ml volumetric flask. Dilute it to the mark with 0.96-1.04 mg / ml sodium phenolate solution and shake well to obtain the reference substance stock solution.

[0019] (3) Preparation of reference substance solution: Measure 0.95-1.05 ml of reference substance stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 0.96-1.04 mg / ml sodium phenolate solution. Shake well. Accurately measure 5 ml and place it in a 20 ml headspace flask. Seal the flask to obtain the reference substance solution.

[0020] (4) Preparation of blank group: Accurately measure 5 ml of 0.96-1.04 mg / ml sodium phenolate solution, place it in a 20 ml headspace bottle, and seal it.

[0021] (5) Preparation of linear solution: Accurately measure 0.1 ml, 0.3 ml, 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml of the reference substance stock solution (2) and place them in 10 ml volumetric flasks respectively. Dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, and accurately measure 5 ml of each solution and place them in 20 ml headspace bottles. Seal the bottle to obtain the linear solution.

[0022] (6) Preparation of detection limit solution: Accurately measure 0.1 ml of the reference substance stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, accurately measure 3 ml of the solution and place it in a 10 ml volumetric flask, dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, accurately measure 5 ml and place it in a 20 ml headspace bottle, seal, and the detection limit solution is obtained.

[0023] (7) Preparation of stability solution: Accurately measure 5 ml of the reference substance stock solution (2) and place it in a 50-ml volumetric flask. Dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, accurately measure 5 ml and place it in a 20-ml headspace bottle. Seal the bottle and take 4 aliquots. Inject the samples into the headspace at 0 h, 2 h, 6 h, and 11 h to examine the stability of the solution.

[0024] (8) Determination of derivative products: Weigh 19.5-20.5 mg of anisole and place it in a 10-ml volumetric flask. Dilute to the mark with dimethyl sulfoxide. Shake well. Accurately measure 0.1 ml and place it in a 100-ml volumetric flask. Dilute to the mark with dimethyl sulfoxide. Shake well. Accurately measure 5 ml as the sample to be tested (test solution) and place it in a 20-ml headspace bottle and seal it.

[0025] (9) Detection: Measure the headspace of the sample solution in the vial using a gas chromatography-mass spectrometer to obtain the peak area and calculate the content of dimethyl sulfate.

[0026] The chemical reaction formula derived from the above is as follows:

[0027]

[0028] That is, 2C6H5NaO+C2H6O4S→2C7H8O;

[0029] Preferably, during the detection process (9), the program starts at 38-42°C and maintains for 7-8.5 min, then heats up to 58-62°C at a rate of 4.5-5.5°C / min and maintains for 1-1.5 min, then heats up to 230-250°C at a rate of 38-42°C / min and maintains for 1-2 min.

[0030] Preferably, during the detection process (9), the program starts at 40°C, maintains for 8 minutes, then increases the temperature to 60°C at a rate of 5°C / min and maintains for 1 minute, and then increases the temperature to 240°C at a rate of 40°C / min and maintains for 1 minute.

[0031] Preferably, during the detection process, the chromatographic conditions are: HP-5ms UI chromatographic column, column length 30m, inner diameter 0.25mm, liquid film thickness 0.25μm; flow rate 1.0ml / min; split ratio 20:1; injection port temperature 220°C.

[0032] Preferably, during the detection process, when the solution to be tested is placed in the headspace sampler of the gas chromatography-mass spectrometer for equilibrium, the equilibrium temperature is 100° C. and the equilibrium time is 30 min.

[0033] The beneficial effects of the present invention are:

[0034] (1) The derivative product prepared by the present invention is stable within 11 hours. After testing, the peak areas of the test solution at 0, 2, 6, and 11 hours are 32224, 33873, 31960, and 31560, respectively, and the RSD is 3.14%; the peak shape of the target is good, and the peak shape of the target does not change significantly due to the programmed temperature increase;

[0035] (2) It has high sensitivity. After testing, the quantitative limit of dimethyl sulfate is 0.1057 ng and the detection limit is 0.0032 ng, which fully meets the determination requirements;

[0036] (3) It overcomes the shortcomings of multiple injections in the prior art, can better maintain the quality of the instrument and extend its life; at the same time, since the derivative product of the present invention has good stability, it is not necessary to add an internal standard to ensure the accuracy of the detection, making the test operation simpler;

[0037] (4) The derivatization reagent and dimethyl sulfate can immediately undergo a derivatization reaction, which greatly improves the overall detection efficiency and is simple to operate;

[0038] (5) The reaction system detected by the present invention is dimethyl sulfoxide-isopropanol, which can meet the trace determination of dimethyl sulfate in most organic compounds and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0040] Figure 1 is the derivative blank group chromatogram;

[0041] Figure 2 It is the chromatogram of the reference solution;

[0042] Figure 3 It is the chromatogram of anisole solution;

[0043] Figure 4 It is a linear graph;

[0044] Figure 5 This is the chromatogram of anisole when methanol is used as a cosolvent;

[0045] Figure 6 This is the chromatogram of anisole when ethanol is used as a cosolvent;

[0046] Figure 7This is the chromatogram of anisole when acetone is used as a cosolvent;

[0047] Figure 8 This is the chromatogram of anisole when isopropanol is used as a cosolvent;

[0048] Fig. 9 This is the chromatogram of anisole when only dimethyl sulfoxide is used as the solvent. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0050] Example 1

[0051] A method for quantitatively analyzing the content of dimethyl sulfate by a derivatization method comprises the following steps:

[0052] (1) Preparation of derivatization reagent (sodium phenolate solution);

[0053] (2) taking dimethyl sulfate as a raw material and preparing anisole with sodium phenolate solution;

[0054] (3) Dilute anisole with dimethyl sulfoxide to prepare a test solution, perform headspace injection, and record a spectrum.

[0055] The chromatographic conditions and mass spectrometry conditions of the gas chromatography-mass spectrometry detection process are as follows:

[0056]

[0057] Method Validation of Chromatographic Methods:

[0058] 1. Exclusivity

[0059] (1) Preparation of derivatization reagent (sodium phenolate solution): Weigh 0.5 g of sodium phenolate and place it in a 500 ml volumetric flask. First, add 25 ml of isopropanol and then add 300 ml of dimethyl sulfoxide. Dissolve by ultrasonication. Dilute to scale with dimethyl sulfoxide and shake well to obtain a 1 mg / ml sodium phenolate solution.

[0060] (2) Preparation of reference substance stock solution: accurately weigh 20 mg of dimethyl sulfate reference substance, place in a 10 ml volumetric flask, dilute to scale with 1 mg / ml sodium phenolate solution, shake well, accurately measure 1 ml, place in a 200 ml volumetric flask, dilute to scale with 1 mg / ml sodium phenolate solution, shake well to obtain reference substance stock solution;

[0061] (3) Blank group: accurately measure 5 ml of 1 mg / ml sodium phenolate solution, place it in a 20 ml headspace bottle, seal it, and inject it into the headspace to obtain spectrum 1;

[0062] (4) Determination of reference substance solution: accurately measure 1 ml of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to scale with 1 mg / ml sodium phenolate solution, shake well, accurately measure 5 ml and place it in a 20 ml headspace bottle, seal, and perform headspace injection to obtain Spectrum 2;

[0063] At this time, the concentration of the reference solution is 1ug / ml, and the required sodium phenolate concentration is 1 / 126×2×116.02=1.84ug / ml;

[0064] The concentration at the highest point of linearity is about 2ug / ml, and the required sodium phenolate concentration is 2 / 126×2×116.02=3.68ug / ml;

[0065] The concentration of sodium phenolate in the sodium phenolate solution actually used is 1 mg / ml, which is much higher than the theoretical concentration, ensuring complete derivatization;

[0066] (5) Determination of derivative products: Take 20 mg of anisole, accurately weigh it, put it in a 10 ml volumetric flask, dilute it to the mark with dimethyl sulfoxide, shake it well, accurately measure 0.1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with dimethyl sulfoxide, shake it well, accurately measure 5 ml, put it in a 20 ml headspace bottle, seal it, and inject it into the headspace to obtain chromatogram 3;

[0067] Detection conclusion: It can be seen from the spectrum that the blank does not interfere with the determination and the specificity of the detection method is good.

[0068] 2. Linear relationship and range investigation

[0069] Accurately measure 0.1 ml, 0.3 ml, 0.5 ml, 1.0 ml, 1.5 ml and 2.0 ml of the reference substance stock solution prepared in 1(1) above, and place them in 10 ml volumetric flasks respectively. Dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, and accurately measure 5 ml respectively, and place them in 20 ml headspace vials. The vials are sealed and injected into the headspace to obtain chromatogram 4. The linearity and range results are shown in the following table.

[0070] Table 1 Linearity and range results:

[0071]

[0072] 3. Limit of quantification and detection limit

[0073] (1) Accurately measure 0.1 ml of the reference substance stock solution prepared in step 1(1) above and place it in a 10 ml volumetric flask. Dilute to the mark with 1 mg / ml sodium phenolate solution and shake well. Prepare 6 portions in parallel and accurately measure 5 ml of each portion and place them in 20 ml headspace bottles. Seal the bottles to obtain the quantitation limit solution.

[0074] Accurately measure 0.1 ml of the reference substance stock solution prepared in 1(1) above, place it in a 10 ml volumetric flask, dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, accurately measure 3 ml of the solution, place it in a 10 ml volumetric flask, dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, accurately measure 5 ml, place it in a 20 ml headspace bottle, seal, and the detection limit solution is obtained.

[0075] The results of the limit of quantification and the limit of detection are shown in the table below:

[0076] LOQ-1 to LOQ-6 are the limits of quantification, and LOD is the limit of detection;

[0077] Table 2 Test results of limit of quantitation and limit of detection

[0078]

[0079] 4. Investigation of solution stability

[0080] Preparation and testing of stability solution: Accurately measure 5 ml of the reference substance stock solution prepared in 1(1) above and place it in a 50 ml volumetric flask. Dilute to the mark with 1 mg / ml sodium phenolate solution, shake well, accurately measure 5 ml and place it in a 20 ml headspace bottle. Seal the bottle and take 4 portions. After standing at room temperature for 0 h, 2 h, 6 h, and 11 h, perform headspace sampling to examine the stability of the solution.

[0081] The solution stability results are shown in the table below:

[0082] Table 3 Solution stability results

[0083]

[0084] From the perspective of the stability of the peak area, the derivatization reaction has been fully reacted, and the stability of the peak area is not affected after being placed at room temperature; at the same time, since the derivative product of the present invention has good stability, it is not necessary to add an internal standard to ensure the accuracy of the detection, making the test operation simpler and more efficient;

[0085] The derivatization reagent and dimethyl sulfate can immediately undergo a derivatization reaction, which greatly improves the overall detection efficiency and is simple to operate.

Claims

1. A method for quantitatively analyzing the content of dimethyl sulfate by a derivatization method, characterized in that: Using a derivatization reagent to convert dimethyl sulfate into a derivative anisole, diluting the derivative anisole with dimethyl sulfoxide to obtain a test solution, and using a gas chromatography-mass spectrometer to measure the peak area of ​​anisole in the test solution to calculate the content of dimethyl sulfate; Wherein, the derivatization reagent is a sodium phenolate solution, and isopropanol is used as a cosolvent when preparing the derivatization reagent; The preparation method of sodium phenolate solution is as follows: first add isopropanol to sodium phenolate, then add dimethyl sulfoxide, dissolve by ultrasonication, and dilute with dimethyl sulfoxide to obtain the solution. Before ultrasonication, the volume ratio of isopropanol to dimethyl sulfoxide is 1:10.8-13.

2. During the detection by gas chromatography-mass spectrometry, the program starts at 38-42°C, keeps for 7-8.5 minutes, then heats up to 58-62°C at a rate of 4.5-5.5°C / min and keeps for 1-1.5 minutes, then heats up to 230-250°C at a rate of 38-42°C / min and keeps for 1-2 minutes. Chromatographic conditions of the detection process: HP-5ms UI column, column length 30 m, inner diameter 0.25 mm, liquid film thickness 0.25 μm; flow rate 1.0 mL / min; split ratio 20:1; injection port temperature 220°C; During the detection, the solution to be tested was placed in the headspace sampler of the gas chromatography-mass spectrometer for equilibrium, with an equilibrium temperature of 100°C and an equilibrium time of 30 min.

2. The method according to claim 1, characterized in that The derivatization reagent is a 0.96-1.04 mg / mL sodium phenolate solution.

3. The method according to claim 1, characterized in that Before ultrasonic treatment, the volume ratio of isopropanol to dimethyl sulfoxide was 1:12.

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