CN106814144B - Method for determining and analyzing content of dimethyl sulfate in dimethyl fumarate - Google Patents
Method for determining and analyzing content of dimethyl sulfate in dimethyl fumarate Download PDFInfo
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- CN106814144B CN106814144B CN201510872446.8A CN201510872446A CN106814144B CN 106814144 B CN106814144 B CN 106814144B CN 201510872446 A CN201510872446 A CN 201510872446A CN 106814144 B CN106814144 B CN 106814144B
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Abstract
The invention discloses a method for determining and analyzing the content of dimethyl sulfate in dimethyl fumarate, which comprises the steps of using secondary amine as a derivative reagent to form a derivative with dimethyl sulfate in the dimethyl fumarate, and then determining the content of the derivative by using a high performance liquid chromatography-mass spectrometer to obtain the content of the dimethyl sulfate in the dimethyl fumarate. Compared with the prior art, the analysis method has the characteristics of good specificity, good reproducibility, extremely high sensitivity, suitability for trace analysis and the like.
Description
Technical Field
The invention relates to a method for deriving dimethyl sulfate by secondary amine, and the content of the derivative is measured by a high performance liquid chromatography-mass spectrometer, so that the content of dimethyl sulfate in dimethyl fumarate is calculated. The method can well control the content of trace dimethyl sulfate in the raw material medicine, provides guarantee for the healthy and safe medication of patients, and belongs to the technical field of medicines.
Technical Field
Dimethyl fumarate, chemical name Dimethyl fumarate.
Is a medicine for treating multiple sclerosis. The structure is as follows:
the dimethyl fumarate contains reaction residual dimethyl sulfate and has the following structure:
accurate and quantitative determination of the dimethyl sulfate content in dimethyl fumarate is extremely challenging. Dimethyl sulfate is very polar, does not have sensitive ultraviolet absorbing groups, and is easily hydrolyzed.
Therefore, it is difficult to directly measure trace amounts by gas, liquid chromatography and other analytical means.
At present, only a method for determining the content of the constant dimethyl sulfate in the dimethyl fumarate adopts a classical chromatography, and has the defects of great specificity, sensitivity, method precision and repeatability, and cannot provide a reliable trace analysis result. In order to overcome the defects of the original method, the invention adopts secondary amine as a derivatization reagent to form a stable derivative with dimethyl sulfate. The formed derivative has excellent characteristics of liquid chromatography separation, mass spectrum detection and the like. The analysis method has the characteristics of good specificity, high sensitivity, good reproducibility and the like.
Disclosure of Invention
As described above, direct quantitative determination of traces of dimethyl sulfate has various drawbacks. However, by utilizing the characteristic that dimethyl sulfate carries methoxy group, the characteristics of dimethyl sulfate can be modified to convert the dimethyl sulfate into a molecular structure suitable for quantitative analysis. The property that secondary amine can rapidly and quantitatively perform chemical reaction with methoxy groups is utilized, and the derivative formed by the derivative reagent and dimethyl sulfate has the characteristics of strong stability and suitability for reverse phase liquid chromatography separation. In addition, the sensitivity and specificity of the method are greatly improved by the characteristic that the derivative has sensitive absorption on a mass spectrum detector. The chemical reaction formula of the derivatization is as follows:
derivative structure
It was found by experiment that dimethyl fumarate has very good solubility in acetonitrile. The present invention will use acetonitrile as a diluent. The key point of the analysis method is to strictly control the water content in the diluent. Since the diluent contains an indefinite amount of water, this water will hydrolyze the dimethyl sulfate to produce an indefinite amount of sulfuric acid, resulting in a low measurement result and poor reproducibility. To ensure that the water content in the system is constant and below acceptable experimental error, the present invention uses chromatographic grade acetonitrile.
The derivatization reaction in the acetonitrile solvent of chromatographic grade can accurately quantify the dimethyl sulfate in the dimethyl fumarate.
The invention relates to a method for determining and analyzing the content of dimethyl sulfate in dimethyl fumarate, which comprises the steps of forming a derivative by using secondary amine as a derivative reagent and dimethyl sulfate in the dimethyl fumarate, and determining the content of the derivative by using a high performance liquid chromatography-mass spectrometer so as to obtain the content of the dimethyl sulfate in the dimethyl fumarate.
The above dimethyl sulfate derivatization reaction uses a secondary amine and acetonitrile as the derivatizing agent.
The ion mode is selected by adopting a high performance liquid chromatography-mass spectrometer and negative ions.
The method comprises the following steps:
(1) taking dimethyl fumarate raw material or preparation powder, and preparing a sample derivative solution by taking an acetonitrile solution containing secondary amine as a derivative;
(2) the method adopts a chromatographic column of an ion chromatographic column, and adopts a mobile phase of 0.03 to 0.15 percent formic acid or acetic acid aqueous solution of 5 to 80mM ammonium formate or ammonium acetate and acetonitrile for isocratic elution.
(3) Setting the flow rate of the mobile phase to be 0.3-0.6mL/mL, and controlling the column temperature to be 25-40 ℃.
(4) And (3) taking 5uL of the sample derivative solution obtained in the step (1), injecting a sample, and recording a mass spectrum total ion flow diagram.
(5) And (3) selecting negative ions in an ion mode by adopting a mass spectrum detector, sampling the sample derivative solution in the step (1), and recording a mass spectrogram.
The technical scheme adopted by the invention is as follows:
sample pretreatment:
diluting liquid: chromatographic grade acetonitrile was used.
The derived liquid: an approximately 0.2% solution of the secondary amine was prepared with the diluent.
Standard dimethyl sulfate solution: a standard dimethyl sulfate solution of about 60ug/ml is prepared with diluent.
Standard derived solution: precisely adding 500ul of standard dimethyl sulfate solution into a 2ml HPLC sample bottle, precisely adding 500ul of derivative solution, placing in a constant temperature oven, standing at 50 deg.C for 60min, and injecting.
Sample-derived solution: precisely weighing 20mg of sample in a 2ml HPLC sample bottle, precisely adding 500ul of diluent, precisely adding 500ul of derivative solution, placing in a constant temperature oven, standing at 50 deg.C for 60min, and injecting.
The chromatographic column used in the invention is as follows: an ion chromatography column. The flow rate is 0.3-0.6 mL/min. The column temperature is 25-40 ℃. Mobile phase A: 5mM-80mM ammonium formate or acetate in 0.03% -0.15% aqueous formic or acetic acid, mobile phase B: acetonitrile, gradient as follows:
| A | B | |
| 0min | 10％ | 90％ |
| 10min | 10％ | 90％ |
a mass spectrum detector: negative ion mode, [ M-H ]]-:111
Description of the drawings:
FIG. 1: a mass spectrum bar chart of the secondary amine derivative obtained in example 1 according to the present invention;
FIG. 2 is a drawing: the mass spectrum total ion flow chart of the dimethyl sulfate standard substance derivative obtained in the embodiment 1 of the invention;
FIG. 3: the mass spectrum total ion flow chart of the dimethyl fumarate sample derivative obtained in the embodiment 2 is shown;
FIG. 4 is a drawing: according to the sample adding recovery mass spectrum total ion flow chart of the dimethyl fumarate sample obtained in the embodiment 3, provided by the invention, the total ion flow chart is obtained.
Detailed Description
For better understanding of the technical solutions of the present invention, the following embodiments are further described, but not limited to, the present invention.
Example one
Instruments and conditions:
high performance liquid chromatography mass spectrometer: agilent 1260 definition, MS Detector.
A chromatographic column: ion chromatographic column
Mobile phase: a: 5mM-80mM ammonium formate or ammonium acetate, 0.03% -0.15% formic acid or acetic acid solution B and acetonitrile.
The isocratic degree is as follows: 0-15min, and the organic phase ratio is kept 90%.
Column temperature: 35 ℃ is carried out.
Flow rate: 0.5mL/min
Selecting negative ions: 111
Sample introduction volume: 5 ul.
The experimental steps are as follows:
1) preparing a mobile phase A: 50mM ammonium acetate was accurately weighed out and dissolved in 1000mL of water containing 0.1% acetic acid, and mixed well.
2) Diluent A: acetonitrile
3) Preparing a derivative liquid B: 2mL of diethylamine was placed in a 1000mL volumetric flask and A was added to a constant volume.
4) Standard solution C: precisely weighing about 60mg dimethyl sulfate in a 10ml volumetric flask, adding A to a constant volume, shaking up, precisely sucking 100ul into another 10ml volumetric flask, and adding A to a constant volume to obtain C (the concentration of the standard solution is about 60 ug/ml).
5) Standard solution: precisely taking 500ul C and 500ul B in a 2ml HPLC sample feeding bottle, covering a cover, placing in a 50 ℃ oven for 60min, and feeding. A derivative mass spectrum bar chart is recorded, see typical figure 1. The total ion flow graph of the derivative mass spectrum is recorded, and is shown in a typical figure 2.
Example two
Instruments and conditions:
high performance liquid chromatograph: agilent 1260 definition, MS Detector.
A chromatographic column: ion chromatographic column
Mobile phase: a: 5mM-80mM ammonium formate or ammonium acetate, 0.03% -0.15% formic acid or acetic acid solution B and acetonitrile.
The isocratic degree is as follows: 0-15min, and the organic phase ratio is kept 90%.
Column temperature: 35 ℃ is carried out.
Flow rate: 0.5mL/min
Selecting negative ions: 111
Sample introduction volume: 5 ul.
The experimental steps are as follows:
1) preparing a mobile phase A: 50mM ammonium acetate was accurately weighed out and dissolved in 1000mL of water containing 0.1% acetic acid, and mixed well.
2) Diluent A: acetonitrile
3) Preparing a derivative liquid B: 2mL of diethylamine was placed in a 1000mL volumetric flask and A was added to a constant volume.
4) Sample solution D about 20.0mg of dimethyl fumarate sample was precisely weighed, 500ul A and 500ul B were precisely weighed into a 2ml HPLC sample introduction bottle, the cap was closed, and the bottle was placed in an oven at 50 ℃ for 60min for sample introduction. The total ion flow graph of the derivative mass spectrum is recorded, and is shown in a typical figure 3.
EXAMPLE III
Instruments and conditions:
high performance liquid chromatograph: agilent 1260 definition, MS Detector.
A chromatographic column: ion chromatographic column
Mobile phase: a: 5mM-80mM ammonium formate or ammonium acetate, 0.03% -0.15% formic acid or acetic acid solution B and acetonitrile.
The isocratic degree is as follows: 0-15min, and the organic phase ratio is kept 90%.
Column temperature: 35 ℃ is carried out.
Flow rate: 0.5mL/min
Selecting negative ions: 111
Sample introduction volume: 5 ul.
The experimental steps are as follows:
1) preparing a mobile phase A: 50mM ammonium acetate was accurately weighed out and dissolved in 1000mL of water containing 0.1% acetic acid, and mixed well.
2) Diluent A: acetonitrile
3) Preparing a derivative liquid B: 2mL of diethylamine was placed in a 1000mL volumetric flask and A was added to a constant volume.
4) Standard solution C: precisely weighing about 60mg dimethyl sulfate in a 10ml volumetric flask, adding A to a constant volume, shaking up, precisely sucking 100ul into another 10ml volumetric flask, and adding A to a constant volume to obtain C (the concentration of the standard solution is about 60 ug/ml).
5) Sample solution E about 20.0mg of dimethyl fumarate sample was precisely weighed, 500ul C and 500ul B were precisely weighed into a 2ml HPLC sample introduction bottle, the cap was closed, and the bottle was placed in an oven at 50 ℃ for 60min for sample introduction. The total ion flow graph of the derivative mass spectrum is recorded, and is shown in a typical figure 4.
Claims (2)
1. A method for measuring and analyzing the content of dimethyl sulfate in dimethyl fumarate is characterized by comprising the following steps: using secondary amine as a derivatization reagent to form a derivative with dimethyl sulfate in dimethyl fumarate, and then measuring the content of the derivative by using a high performance liquid chromatography-mass spectrometer, thereby obtaining the content of dimethyl sulfate in dimethyl fumarate; selecting negative ions in an ion mode, wherein the derivatization agent is dimethylamine or diethylamine acetonitrile solution, and a product in the derivatization reaction is monomethyl sulfate;
the determination analysis method comprises the following steps:
(1) taking dimethyl fumarate raw material or preparation powder, and preparing a sample derivative solution by taking an acetonitrile solution containing secondary amine as a derivative;
(2) adopting ion chromatographic column, and making the mobile phase be 0.03% -0.15% formic acid or acetic acid aqueous solution of 5mM-80mM ammonium formate or ammonium acetate and acetonitrile, and making isocratic elution, in which the isocratic is 0-15min, and the organic phase is retained for 90%.
(3) Setting the flow rate of the mobile phase to be 0.3-0.6mL/mL, and controlling the column temperature to be 25-40 ℃;
(4) taking 5uL of the sample derivative solution obtained in the step (1), carrying out sample introduction, and recording a mass spectrum total ion flow diagram;
(5) and (3) selecting negative ions in an ion mode by adopting a mass spectrum detector, sampling the sample derivative solution in the step (1), and recording a mass spectrum ion flow diagram.
2. The assay of claim 1, wherein the percentage by volume of secondary amine to acetonitrile as derivatizing agent is in the range of 0.1% to 0.3%.
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