CN112557573B - Method for measuring AEEA-AEEA content - Google Patents
Method for measuring AEEA-AEEA content Download PDFInfo
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- CN112557573B CN112557573B CN202011620495.XA CN202011620495A CN112557573B CN 112557573 B CN112557573 B CN 112557573B CN 202011620495 A CN202011620495 A CN 202011620495A CN 112557573 B CN112557573 B CN 112557573B
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Abstract
The invention discloses a method for measuring AEEA-AEEA content, which comprises the steps of diluting a sample to be detected, and then detecting the diluted sample in a high performance liquid chromatograph; the chromatographic conditions are as follows: chromatographic column: welch Ultimate HILIC Silica,250 mm. Times.4.6mm 5. Mu.m; mobile phase flow rate: 1.0mL/min; column temperature: 35+/-2 ℃; detection wavelength: 205nm; elution gradient procedure: within 0-10min, mobile phase A is increased from 10% to 20%, mobile phase B is decreased from 90% to 80%, within 10-45min, mobile phase A is increased from 20% to 30%, mobile phase B is decreased from 80% to 70%, within 45-50min, mobile phase A is maintained at 30%, mobile phase B is maintained at 70%, within 50-51min, mobile phase A is decreased from 30% to 10%, mobile phase B is increased from 70% to 90%, within 51-66min, mobile phase A is maintained at 10%, mobile phase B is maintained at 90%; wherein, the mobile phase A is 0.01mol/L ammonium acetate aqueous solution, and the mobile phase B is acetonitrile. The invention can effectively separate the product from each impurity, thereby realizing the content detection of the target product.
Description
Technical Field
The invention belongs to the field of chemical analysis, and particularly relates to a method for measuring AEEA-AEEA content.
Background
AEEa-AEEa, chinese name 17-amino-10-oxo-3,6,12,15-tetraoxa-9-aza-heptadecanoic acid, does not produce various impurities in the process of producing AEEa-AEEa, wherein impurities 2A methyl ester, AEEa, beta-Ala-AEEa (beta-AlA-A), beta-Ala-2A and 3A are several major impurities. The polarity of the product AEEA-AEEA and each impurity is larger, and no AEEA-AEEA and no measuring method for the impurities exist in the prior art so as to detect the content of AEEA-AEEA.
Disclosure of Invention
In order to solve the problems, the invention provides a method for measuring the content of AEEA-AEEA, which is based on a high performance liquid chromatograph and can effectively separate a target product from other impurities so as to accurately measure the content of the target product.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for measuring AEEA-AEEA content includes diluting a sample to be detected, and detecting in a high performance liquid chromatograph; the chromatographic conditions are as follows: chromatographic column: welch Ultimate HILIC Silica,250 mm. Times.4.6mm 5. Mu.m; mobile phase flow rate: 1.0mL/min; column temperature: 35+/-2 ℃; detection wavelength: 205nm; elution gradient procedure: within 0-10min, mobile phase A is increased from 10% to 20%, mobile phase B is decreased from 90% to 80%, within 10-45min, mobile phase A is increased from 20% to 30%, mobile phase B is decreased from 80% to 70%, within 45-50min, mobile phase A is maintained at 30%, mobile phase B is maintained at 70%, within 50-51min, mobile phase A is decreased from 30% to 10%, mobile phase B is increased from 70% to 90%, within 51-66min, mobile phase A is maintained at 10%, mobile phase B is maintained at 90%; wherein, the mobile phase A is 0.01mol/L ammonium acetate aqueous solution, and the mobile phase B is acetonitrile.
Further, the impurities in the sample to be detected include impurity 2A methyl ester, impurity AEEA, impurity beta-Ala-2A and impurity 3A.
Further, the diluent of the sample to be tested is a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 1:1.
Further, the specification of the column was 250mm×4.6mm 5. Mu.m.
Further, the method comprises the following specific steps:
(1) Drawing a standard curve: preparing AEEA-AEEA standard substances into samples with different concentrations, detecting the samples with the different concentrations by using a high performance liquid chromatograph to obtain peak areas corresponding to peaks of main products AEEA-AEEA with different concentrations, and drawing a standard curve;
(2) Preparing a sample diluent to be detected;
(3) The diluent of the sample to be detected enters a high performance liquid chromatography system for detection, the sample injection amount is 20 mu L, and the peak area of the peak of the main product AEEA-AEEA is obtained;
(4) Substituting the peak area obtained in the step (3) into the standard curve obtained in the step (1) to obtain the concentration of the main product AEEA-AEEA in the sample to be detected, and calculating the content according to the calculated concentration and the actual diluted concentration.
Further, the concentration of the diluent in the step (2) is 30mg/mL.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, the polarity of the product AEEA-AEEA and each impurity is larger, almost no retention exists in a common liquid phase C18 column, and the polarity of the product is similar to that of each impurity and is extremely difficult to separate, the Welch Ultimate HILIC Silica chromatographic column is selected, the chromatographic column is combined, and the elution program and the mobile phase of the invention can effectively separate the product from each impurity, so that the content detection of the target product is realized, wherein the mobile phase A is 0.01mol/L ammonium acetate aqueous solution, on one hand, the peak shape can be improved, the tailing phenomenon can be reduced, and on the other hand, the separation between a main peak and an impurity peak is improved due to the reduction of the tailing phenomenon.
In the invention, the ultraviolet absorption of the product AEEA-AEEA and various impurities is weak, the detection wavelength is set to 205nm, the concentration of a sample to be detected is 30mg/mL, the sample injection amount is 20 mu L, the peak absorption of the impurities is increased, the signal is enhanced, and the impurities are prevented from not coming out of the peaks.
Drawings
FIG. 1 shows peak patterns of AEEA-AEEA, 2A methyl ester, AEEA, beta-Ala-2A and 3A.
Detailed Description
The present invention is further described below in conjunction with embodiments, which are merely some, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
Examples
The chromatographic conditions are as follows:
chromatographic column: welch Ultimate HILIC Silica,250 mm. Times.4.6mm 5. Mu.m;
mobile phase flow rate: 1.0mL/min;
column temperature: 35+/-2 ℃;
sample temperature: 25+/-3 ℃;
detection wavelength: 205nm;
elution gradient procedure:
within 0-10min, mobile phase A is increased from 10% to 20%, mobile phase B is decreased from 90% to 80%, within 10-45min, mobile phase A is increased from 20% to 30%, mobile phase B is decreased from 80% to 70%, within 45-50min, mobile phase A is maintained at 30%, mobile phase B is maintained at 70%, within 50-51min, mobile phase A is decreased from 30% to 10%, mobile phase B is increased from 70% to 90%, within 51-66min, mobile phase A is maintained at 10%, mobile phase B is maintained at 90%; wherein, the mobile phase A is 0.01mol/L ammonium acetate aqueous solution, and the mobile phase B is acetonitrile;
(1) Drawing a standard curve: AEEA-AEEA standards were configured as 27 mg/mL, 28 mg/mL, 29 mg/mL, 30mg/mL, 31 mg/mL, 32mg/mL samples, different concentrations of the standardThe standard sample enters a high performance liquid chromatograph to be detected, chromatographic conditions are as described above, peak areas corresponding to the peaks of main products AEEA-AEEA with different concentrations are obtained, a standard curve is drawn, and a regression equation y=87x+1735 is obtained, R 2 = 0.9996, therefore, the linearity is good in the range of 27-32mg/mL, where y is the peak area and x is the sample concentration;
(2) Preparing a sample diluent to be detected, wherein the concentration of the sample to be detected is about 30mg/mL;
(3) The diluent of the sample to be detected enters a high performance liquid chromatography system for detection, the sample injection amount is 20 mu L, and the peak area of the peak of the main product AEEA-AEEA is obtained;
(4) Substituting the peak area obtained in the step (3) into the standard curve obtained in the step (1) to obtain the concentration of the main product AEEA-AEEA in the sample to be detected, thereby calculating the content of AEEA-AEEA,
AEEa-AEEa content = C Calculation of AEEA-AEEA concentration /C Actual dilution concentration of sample *100%。
In fig. 1, the substances corresponding to the peaks are shown in table 1.
TABLE 1
The method is subjected to methodological verification:
(1) Accuracy experiment: three parts of AEEA-AEEA standard products about 270mg, 300mg and 320mg are precisely weighed and respectively placed in 10mL volumetric flasks, diluent is respectively added to the scales, shaking is carried out uniformly, sample injection is carried out according to the chromatographic conditions, peak areas are recorded, concentration is calculated according to the regression equation, and measured values are obtained according to the measured value = concentration x 10mL, and the result is shown in Table 2, so that the invention has good accuracy.
TABLE 2
(2) Repeatability: precisely weighing about 300mg of AEEA-AEEA standard substance, placing the AEEA-AEEA standard substance in a 10mL volumetric flask, adding a diluent to a scale, shaking uniformly, taking six samples as samples to be tested, carrying out sample injection according to the chromatographic conditions, measuring the six samples to be tested, recording peak areas, calculating the concentration according to the regression equation, and obtaining a measured value according to the measured value = concentration x 10mL, wherein the result is shown in Table 3, so that the invention has good repeatability.
TABLE 3 Table 3
(3) Intermediate precision: six different operators respectively sample six samples to be tested in repeatability verification on six different dates by using six different instruments, record peak areas, calculate concentration according to the regression equation, obtain measured values according to the measured value = concentration x 10mL, and the result is shown in table 4, so that the invention has good intermediate precision.
TABLE 4 Table 4
Claims (6)
1. A method for measuring AEEA-AEEA content is characterized in that a sample to be detected is diluted and then enters a high performance liquid chromatograph for detection; the sample to be detected is a product AEEA-AEEA; the chromatographic conditions are as follows: chromatographic column: welch Ultimate HILIC Silica; mobile phase flow rate: 1.0mL/min; column temperature: 35+/-2 ℃; detection wavelength: 205nm; elution gradient procedure: within 0-10min, mobile phase A is increased from 10% to 20%, mobile phase B is decreased from 90% to 80%, within 10-45min, mobile phase A is increased from 20% to 30%, mobile phase B is decreased from 80% to 70%, within 45-50min, mobile phase A is maintained at 30%, mobile phase B is maintained at 70%, within 50-51min, mobile phase A is decreased from 30% to 10%, mobile phase B is increased from 70% to 90%, within 51-66min, mobile phase A is maintained at 10%, mobile phase B is maintained at 90%; wherein, the mobile phase A is 0.01mol/L ammonium acetate aqueous solution, and the mobile phase B is acetonitrile;
the impurities in the sample to be detected comprise impurity 2A methyl ester, impurity AEEA, impurity beta-Ala-2A and impurity 3A.
2. The method for determining AEEA-AEEA content according to claim 1, wherein the diluent of the sample to be tested is a mixed solvent of acetonitrile and water.
3. A method of determining AEEa-AEEa content according to claim 2, characterized in that the volume ratio of acetonitrile to water is 1:1.
4. A method of determining AEEa-AEEa content according to claim 1, characterized in that the specification of the chromatographic column is 250mm x 4.6mm 5 μm.
5. The method for determining AEEa-AEEa content according to claim 1, characterized by the specific steps of:
(1) Drawing a standard curve: preparing AEEA-AEEA standard substances into standard substance samples with different concentrations, detecting the standard substance samples with different concentrations by a high performance liquid chromatograph to obtain peak areas corresponding to the peaks of the AEEA-AEEA with different concentrations, and drawing a standard curve;
(2) Preparing a sample diluent to be detected;
(3) The diluent of the sample to be detected enters a high performance liquid chromatography system for detection, the sample injection amount is 20 mu L, and the peak area of the AEEA-AEEA peak is obtained;
(4) Substituting the peak area obtained in the step (3) into the standard curve obtained in the step (1) to obtain the concentration of AEEA-AEEA in the sample to be detected, and calculating the content according to the calculated concentration and the actual diluted concentration.
6. The method according to claim 5, wherein the concentration of the diluent in the step (2) is 30mg/mL.
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