CN111366660A - Method for measuring content of dimethyl sulfate in Etegravir - Google Patents
Method for measuring content of dimethyl sulfate in Etegravir Download PDFInfo
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- CN111366660A CN111366660A CN201811600349.3A CN201811600349A CN111366660A CN 111366660 A CN111366660 A CN 111366660A CN 201811600349 A CN201811600349 A CN 201811600349A CN 111366660 A CN111366660 A CN 111366660A
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- VAYGXNSJCAHWJZ-UHFFFAOYSA-N dimethyl sulfate Chemical compound COS(=O)(=O)OC VAYGXNSJCAHWJZ-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 11
- 238000001212 derivatisation Methods 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 239000000523 sample Substances 0.000 claims description 20
- 239000003085 diluting agent Substances 0.000 claims description 14
- 238000001819 mass spectrum Methods 0.000 claims description 13
- 239000002253 acid Substances 0.000 claims description 8
- 238000010586 diagram Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 5
- 239000012488 sample solution Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- BCVXHSPFUWZLGQ-UHFFFAOYSA-N mecn acetonitrile Chemical compound CC#N.CC#N BCVXHSPFUWZLGQ-UHFFFAOYSA-N 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims 6
- XXSSRSVXDNUAQX-QGZVFWFLSA-N 1-(4-fluorophenyl)-5-[(4s)-2-oxo-4-phenyl-1,3-oxazolidin-3-yl]pentane-1,5-dione Chemical compound C1=CC(F)=CC=C1C(=O)CCCC(=O)N1C(=O)OC[C@@H]1C1=CC=CC=C1 XXSSRSVXDNUAQX-QGZVFWFLSA-N 0.000 claims 2
- -1 2-mercaptopyridine acetonitrile Chemical compound 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- OLNTVTPDXPETLC-XPWALMASSA-N ezetimibe Chemical compound N1([C@@H]([C@H](C1=O)CC[C@H](O)C=1C=CC(F)=CC=1)C=1C=CC(O)=CC=1)C1=CC=C(F)C=C1 OLNTVTPDXPETLC-XPWALMASSA-N 0.000 claims 1
- 229960000815 ezetimibe Drugs 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000004454 trace mineral analysis Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 239000012071 phase Substances 0.000 description 12
- 238000005303 weighing Methods 0.000 description 8
- 239000012483 derivatization solution Substances 0.000 description 6
- WHMDPDGBKYUEMW-UHFFFAOYSA-N pyridine-2-thiol Chemical compound SC1=CC=CC=N1 WHMDPDGBKYUEMW-UHFFFAOYSA-N 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229960003586 elvitegravir Drugs 0.000 description 2
- JUZYLCPPVHEVSV-LJQANCHMSA-N elvitegravir Chemical compound COC1=CC=2N([C@H](CO)C(C)C)C=C(C(O)=O)C(=O)C=2C=C1CC1=CC=CC(Cl)=C1F JUZYLCPPVHEVSV-LJQANCHMSA-N 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229940099797 HIV integrase inhibitor Drugs 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003084 hiv integrase inhibitor Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for measuring the content of dimethyl sulfate in Etegravir, which adopts chemical derivatization and combines a high performance liquid chromatography-mass spectrometer to measure the content of the dimethyl sulfate in the Etegravir. 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 measuring the content of dimethyl sulfate in Eviet by adopting chemical derivatization and combining a high performance liquid chromatography-mass spectrometer. The method can well measure the content of trace dimethyl sulfate in the Etegravir, provides guarantee for the healthy and safe medication of patients, and belongs to the technical field of medicines.
Background
Ericivir, chemical name Elvitegravir.
Is an HIV integrase inhibitor that interferes with the replication of the HIV virus by blocking viral integration into the human cell genome, and is useful in the treatment of adult HIV-1 infectors without any known Elvitegravir resistance-related mutations. The structure of the eptigevir is as follows:
the structure of the etifovir containing reaction residual dimethyl sulfate is as follows:
accurate and quantitative determination of the content of dimethyl sulfate in the eptigravir 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, no method for measuring the content of trace dimethyl sulfate in the Etegravir exists, and the content of the dimethyl sulfate in the Etegravir is measured by adopting chemical derivatization and combining with a high performance liquid chromatography-mass spectrometer. 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
The direct quantitative determination of trace dimethyl sulfate has various defects. 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 the derivatization reagent 2-mercaptopyridine can rapidly and quantitatively perform chemical reaction with methoxy groups is utilized, and the derivative formed by the derivatization 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:
the good solubility of the etifovir in Acetonitrile (Acetonitrile) was found by experiment. The present invention will use Acetonitrile (Acetonitriles) 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.
Derivatization in chromatographic grade acetonitrile solvent can accurately quantify dimethyl sulfate in the Etikvass.
The technical scheme adopted by the invention is as follows:
sample pretreatment:
diluting liquid: chromatographic grade acetonitrile.
Derivatization solution: and preparing a derivative solution of the 2-mercaptopyridine by using the diluent.
Dimethyl sulfate derivative standard solution: and precisely preparing 200.0ng/mL dimethyl sulfate solution by using the diluent. Equal volumes of the solution and the derivatization solution are mixed for derivatization, and then sample injection is carried out.
Test solution: accurately weighing 200.0mg of a sample to be tested in a 10mL volumetric flask, dissolving the sample by using a diluent, fixing the volume to a scale, mixing the solution with the same volume with the derivatization solution, performing derivatization, and injecting the sample.
The chromatographic column used in the invention is as follows: octadecyl bonded silica gel chromatographic column. The flow rate is 0.3-1.2 mL/min. The column temperature is 25-45 ℃. Mobile phase A: 0.05% -0.2% volatile acid aqueous solution, mobile phase B: acetonitrile, gradient as follows: 0min to 8.0min, organicThe phase ratio was changed from 2% to 95%. A mass spectrum detector: positive ion mode, [ M + H ]]+：126
Description of the drawings:
FIG. 1: a bar graph of a standard quality spectrum of the dimethyl sulfate derivative obtained according to the first embodiment of the invention;
FIG. 2 is a drawing: according to the mass spectrum total ion flow diagram of the dimethyl sulfate derivative standard substance obtained in the first embodiment of the invention;
FIG. 3: according to the total ion flow diagram of the mass spectrum of the dimethyl sulfate derivative in the Etegravir test sample obtained in the second embodiment of the invention;
FIG. 4 is a drawing: and (4) sampling and recovering the mass spectrum total ion flow graph in the Etegravir supply obtained according to the third embodiment of the invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
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: octadecyl bonded silica gel chromatographic column
Mobile phase A: 0.05% -0.2% volatile acid aqueous solution
Mobile phase B: acetonitrile
The gradient is as follows: 0-8.0 min, the organic phase ratio is changed from 2% to 95%
Column temperature: 35 deg.C
Sample introduction volume: 2 μ L
Flow rate: 1.0mL/min
Positive ion mode [ M + H]+：126
The experimental steps are as follows:
1) preparing a mobile phase A: precisely measure 1.0mL of volatile acid, dissolve in 1000mL of water, and mix well.
2) And (3) diluting liquid B: acetonitrile
3) Derivatization solution C: accurately weighing 100.0mg of 2-mercaptopyridine, dissolving in a 50mL volumetric flask, and adding the diluent B to a constant volume.
4) Standard solution D: precisely weighing 20.0mg of dimethyl sulfate in a 100mL volumetric flask, adding the diluent B to a constant volume, and shaking up; precisely sucking 100 mu L of the solution into another 100mL volumetric flask, and adding the diluent B to a constant volume; precisely sucking the solution and the derivative solution C with equal volume, mixing, and deriving to obtain standard solution D, injecting sample, and recording mass spectrum bar chart of dimethyl sulfate derivative, as shown in figure 1. Recording the total ion flow diagram of the mass spectrum of the dimethyl sulfate derivative standard substance, and referring to the attached figure 2.
Example two
Instruments and conditions:
high performance liquid chromatography mass spectrometer: agilent 1260 definition, MS Detector.
A chromatographic column: octadecyl bonded silica gel chromatographic column
Mobile phase A: 0.05% -0.2% volatile acid aqueous solution
Mobile phase B: acetonitrile
The gradient is as follows: the organic phase ratio is changed from 2% to 95% in 0-8.0 min.
Column temperature: 35 deg.C
Sample introduction volume: 2 μ L
Flow rate: 1.0mL/min
Positive ion mode [ M + H]+：126
The experimental steps are as follows:
1) preparing a mobile phase A: precisely measure 1.0mL of volatile acid, dissolve in 1000mL of water, and mix well.
2) And (3) diluting liquid B: acetonitrile
3) Derivatization solution C: accurately weighing 100.0mg of 2-mercaptopyridine, dissolving in a 50mL volumetric flask, and adding the diluent B to a constant volume.
4) Test article solution E: precisely weighing 200.0mg of the Etegravir sample, and placing the Etegravir sample in a 10mL volumetric flask; respectively and precisely sucking the solution and the derivative solution C with equal volumes, mixing, and deriving to obtain a test sample solution E, and injecting. Recording the total ion flow diagram of the mass spectrum of the dimethyl sulfate derivative in the Etegravir sample, wherein the typical diagram is shown in figure 3.
EXAMPLE III
Instruments and conditions:
high performance liquid chromatography mass spectrometer: agilent 1260 definition, MS Detector.
A chromatographic column: octadecyl bonded silica gel chromatographic column
Mobile phase A: 0.05% -0.2% volatile acid aqueous solution
Mobile phase B: acetonitrile
The gradient is as follows: the organic phase ratio is changed from 2% to 95% in 0-8.0 min.
Column temperature: 35 deg.C
Sample introduction volume: 2 μ L
Flow rate: 1.0mL/min
Positive ion mode [ M + H]+：126
The experimental steps are as follows:
1) preparing a mobile phase A: precisely measure 1.0mL of volatile acid, dissolve in 1000mL of water, and mix well.
2) And (3) diluting liquid B: acetonitrile
3) Derivatization solution C: accurately weighing 100.0mg of 2-mercaptopyridine, dissolving in a 50mL volumetric flask, and adding the diluent B to a constant volume.
4) Standard solution D: precisely weighing 20.0mg of dimethyl sulfate in a 100mL volumetric flask, adding the diluent B to a constant volume, and shaking up; precisely suck 100 μ L of the solution into another 100mL volumetric flask, and add the diluent B to the volume.
5) Sample application and recovery solution F: accurately weighing 200.0mg of the Etegravir sample, placing the sample in a 10mL volumetric flask, dissolving the sample with a standard solution D, fixing the volume to a scale, and uniformly mixing; respectively and precisely sucking the solution and the derivative solution C with equal volumes, mixing, performing derivatization, and injecting a sample. And recording the total ion flow diagram of the sample adding and recovering mass spectrum in the Etigevir supply, and referring to an attached figure 4.
Claims (3)
1. A method for measuring the content of dimethyl sulfate in the ezetimibe is characterized in that chemical derivatization is adopted and an efficient liquid chromatography-mass spectrometer is combined to measure the content of trace dimethyl sulfate in the ezetimibe intermediate.
2. The method of claim 1, wherein the positive ion is selected for ion mode using chemical derivatization in combination with hplc-ms.
3. The method according to claim 1, characterized in that the method comprises the following steps:
(1) taking an ezetimibe intermediate, and preparing a sample solution by taking Acetonitrile (Acetonitrile) as a diluent;
(2) performing gradient elution by using octadecyl bonded silica gel chromatographic column and mobile phase of 0.05-0.2% volatile acid water solution and acetonitrile.
(3) Setting the flow rate of the mobile phase to be 0.3-1.2mL/min, and controlling the column temperature to be 25-45 ℃.
(4) And (3) taking 2 mu L of the sample solution obtained in the step (1), injecting a sample, and recording a mass spectrum total ion flow diagram.
(5) And (3) selecting positive ions in an ion mode by adopting a mass spectrum detector, sampling the sample solution in the step (1), and recording a mass spectrum ion flow diagram.
(6) 2-mercaptopyridine acetonitrile solution is adopted as a derivatization reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811600349.3A CN111366660A (en) | 2018-12-26 | 2018-12-26 | Method for measuring content of dimethyl sulfate in Etegravir |
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| CN201811600349.3A CN111366660A (en) | 2018-12-26 | 2018-12-26 | Method for measuring content of dimethyl sulfate in Etegravir |
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| CN111366660A true CN111366660A (en) | 2020-07-03 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113218719A (en) * | 2021-05-12 | 2021-08-06 | 广东省职业病防治院(广东省职业卫生检测中心) | Dimethyl sulfate air sampling method |
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2018
- 2018-12-26 CN CN201811600349.3A patent/CN111366660A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113218719A (en) * | 2021-05-12 | 2021-08-06 | 广东省职业病防治院(广东省职业卫生检测中心) | Dimethyl sulfate air sampling method |
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Application publication date: 20200703 |