CN112578044B - Method for detecting Fmoc-AEEA content by high performance liquid chromatography - Google Patents
Method for detecting Fmoc-AEEA content by high performance liquid chromatography Download PDFInfo
- Publication number
- CN112578044B CN112578044B CN202011389963.7A CN202011389963A CN112578044B CN 112578044 B CN112578044 B CN 112578044B CN 202011389963 A CN202011389963 A CN 202011389963A CN 112578044 B CN112578044 B CN 112578044B
- Authority
- CN
- China
- Prior art keywords
- fmoc
- aeea
- mobile phase
- detecting
- content
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a method for detecting Fmoc-AEEA content by using a high performance liquid chromatography, wherein the specific chromatographic conditions are that a chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 210-220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; mobile phase B was 0.1% TFA acetonitrile and mobile phase flow rate was 0.8-1.2 ml/min. The method for measuring the content of the main component Fmoc-AEEA has the characteristics of rapidness and accuracy.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to a method for detecting Fmoc-AEEA content by using a high-performance liquid phase.
Background
[2- [2- (Fmoc-amino) ethoxy ] acetic acid with the chemical structural formula:
For process reasons, the product usually comprises Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA and Fmoc-beta-Ala impurities, and the like, and in the prior art, no detection method is available for simultaneously distinguishing the main component Fmoc-AEEA from the impurities so as to accurately determine the content of the main component Fmoc-AEEA.
Disclosure of Invention
In order to solve the problems, the invention discloses a high-efficiency liquid phase detection method for Fmoc-AEEA content, wherein the impurities comprise Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA and Fmoc-beta-Ala.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting Fmoc-AEEA content by using a high performance liquid chromatography is characterized in that a sample to be detected comprises the following main components of Fmoc-AEEA: Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA, and Fmoc-beta-Ala; detecting the content of the main component Fmoc-AEEA by adopting an external standard method; the specific detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 210-220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; the mobile phase B is 0.1 percent TFA acetonitrile, and the flow rate of the mobile phase is 0.8-1.2 ml/min;
the elution procedure was: within 0-20min, the mobile phase A is reduced from 70% to 0, and the mobile phase B is increased from 30% to 100%; keeping the mobile phase A at 0 and the mobile phase B at 100% within 20-25 min; in 25-27min, the mobile phase A is increased from 0 to 70 percent, and the mobile phase B is decreased from 100 percent to 30 percent; keeping the mobile phase A at 70% and the mobile phase B at 30% within 27-35 min;
(2) drawing a standard curve: taking acetonitrile as a solvent, preparing Fmoc-AEEA standard substance into samples with different concentrations, detecting the standard substance samples with different concentrations according to the analysis method in the step (1), obtaining peak areas corresponding to the peaks of the main product Fmoc-AEEA with different concentrations, and drawing a standard curve;
(3) preparing a sample solution to be detected: dissolving a sample to be detected in acetonitrile by taking the acetonitrile as a solvent;
(4) and (3) detecting the sample solution to be detected obtained in the step (3) according to the analysis method in the step (1) to obtain the peak area of the main product Fmoc-AEEA, and substituting the obtained peak area into the standard curve obtained in the step (2) to obtain the content of the main product Fmoc-AEEA in the sample to be detected.
Further, in the step (3), the preparation concentration of the sample to be detected is 0.8-1.2 mg/mL.
Further, the amount of the sample was 5. mu.L.
Further, the detection wavelength was 220 nm.
Further, the flow rate of the mobile phase was 1 mL/min.
The invention also provides a method for detecting the content of related substances in Fmoc-AEEA by using a high performance liquid chromatography, wherein the related substances comprise Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA and Fmoc-beta-Ala, and the content of each related substance is detected by using a main component self-control method; specific chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 210-220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; the mobile phase B is 0.1 percent TFA acetonitrile, and the flow rate of the mobile phase is 0.8-1.2 ml/min;
the elution procedure was: within 0-20min, the mobile phase A is reduced from 70% to 0, and the mobile phase B is increased from 30% to 100%; keeping the mobile phase A at 0 and the mobile phase B at 100% within 20-25 min; in 25-27min, the mobile phase A is increased from 0 to 70 percent, and the mobile phase B is decreased from 100 percent to 30 percent; within 27-35min, mobile phase A was maintained at 70% and mobile phase B at 30%.
Compared with the prior art, the invention has the beneficial effects that:
in the present invention, the impurities to be separated include: Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA and Fmoc-beta-Ala, in particular to the separation of Fmoc-beta-Ala and a main product Fmoc-AEEA; in the method, TFA (trifluoroacetic acid) is added into the mobile phase, so that the peak shape can be effectively improved; in order to separate all impurities in a short time, particularly Fmoc-beta-Ala and Fmoc-AEEA, the invention adopts a gradient elution procedure, the total time of the elution procedure is 35min, and all impurities can be effectively separated under the elution procedure. The method for measuring the content of the main component Fmoc-AEEA has the characteristics of rapidness and accuracy.
Drawings
FIG. 1 is a diagram showing the main component peaks;
FIG. 2 is a diagram showing the positions of impurities and main components.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to illustrate only some, but not all, of the embodiments of the present invention. All other embodiments that can be obtained by a person skilled in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.
Example 1
A method for detecting Fmoc-AEEA content by high performance liquid chromatography comprises the following steps of: Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA, and Fmoc-beta-Ala; detecting the content of the main component Fmoc-AEEA by adopting an external standard method; the specific detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; the mobile phase B is 0.1% TFA acetonitrile, and the flow rate of the mobile phase is 1 mL/min; the sample injection amount is 5 mu L;
the elution procedure was:
(2) drawing a standard curve: taking a proper amount of Fmoc-AEEA standard substance, precisely weighing, adding acetonitrile to dissolve and respectively preparing solutions with the concentrations of 0.2, 0.6, 1.0, 1.4, 1.6 and 2.0mg/mL, then carrying out sample removal according to the chromatographic conditions specified in the step (1), and recording a chromatogram; taking the peak area of Fmoc-AEEA as an ordinate y and the concentration of Fmoc-AEEA solution as an abscissa x to obtain a regression equation y of 11032x +527, and R 2 Therefore, Fmoc-AEEA is good in the linear range of 0.2-2.0 mg/mL because of 0.9998;
(3) preparing a sample solution to be detected: dissolving a sample to be detected in acetonitrile by taking the acetonitrile as a solvent, wherein the preparation concentration of the sample to be detected is about 1.0 mg/mL;
(4) and (3) detecting the sample solution to be detected obtained in the step (3) according to the analysis method in the step (1) to obtain the peak area of the main product Fmoc-AEEA, and substituting the obtained peak area into the standard curve obtained in the step (2) to obtain the content of the main product Fmoc-AEEA in the sample to be detected.
Example 2
And (3) repeatability experiment: and (4) taking the same batch of samples to be tested Fmoc-AEEA, repeatedly preparing 6 parts according to the method in the step (3), carrying out content determination according to the method in the step (4), and recording a chromatogram. The results are shown in table 1, and the average content of 6 parts is 99.72% and RSD (n ═ 6) is 0.43% under the condition of the chromatogram, and the results show that the reproducibility of the content measurement method is good.
TABLE 1 results of repeated experiments
Example 3
Accuracy experiment: precisely weighing about 80mg, 100mg and 120mg of Fmoc-AEEA sample to be detected, respectively placing the Fmoc-AEEA sample into a 100mL volumetric flask, respectively adding acetonitrile to dilute the sample to a scale, shaking up the sample to prepare sample solutions with the concentrations of 80%, 100% and 120%, respectively, taking Fmoc-AEEA standard substance to prepare a reference substance solution by the same method, carrying out sample injection according to the chromatographic conditions in the step (1), recording a chromatogram, making a standard curve according to chromatographic data of the reference substance solution, and calculating the measured quantity according to the standard curve, wherein the result is shown in a table 2, under the proposed chromatographic conditions, the recovery rate of Fmoc-AEEA under three concentrations is between 98.2% and 99.7%, and the RSD value is 0.44%, namely the method for detecting the Fmoc-AEEA content has good accuracy.
TABLE 2 accuracy test results
Example 4
In order to determine the content of each impurity, based on the above detection method, the content of the relevant substance is detected by using a principal component self-control method:
(1) specific chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; the mobile phase B is 0.1% TFA acetonitrile, and the flow rate of the mobile phase is 1 mL/min; the sample injection amount is 5 mu L;
the elution procedure was:
(2) preparing a sample solution to be tested: precisely weighing a sample to be detected, adding acetonitrile to dissolve and quantitatively diluting the sample to be detected to prepare a solution containing about 1mg in every 1mL, wherein the solution is used as a sample solution to be detected;
(3) preparation of control solution: precisely measuring 1mL of the sample solution to be detected obtained in the step (2), placing the sample solution into a 100mL measuring flask, diluting the sample solution to a scale with a mobile phase, and shaking up to be used as a reference solution;
(4) and (3) measuring related substances: and (2) injecting according to the chromatographic conditions in the step (1), recording a chromatogram, and determining by using a main component self-contrast method, wherein the peak area of a single impurity is not more than 1.5 times of the main peak area of the contrast solution, and the sum of the peak areas of all impurities is not more than 2 times of the main peak area of the contrast solution.
Claims (6)
1. The method for detecting Fmoc-AEEA content by using the high performance liquid chromatography is characterized in that a sample to be detected comprises the main component Fmoc-AEEA, and the sample to be detected also comprises the following impurities: Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA, and Fmoc-beta-Ala; detecting the content of the main component Fmoc-AEEA by adopting an external standard method; the specific detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 210-220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; the mobile phase B is 0.1% TFA acetonitrile, and the flow rate of the mobile phase is 0.8-1.2 mL/min;
the elution procedure was: within 0-20min, the mobile phase A is reduced from 70% to 0, and the mobile phase B is increased from 30% to 100%; keeping the mobile phase A at 0 and the mobile phase B at 100% within 20-25 min; in 25-27min, the mobile phase A is increased from 0 to 70 percent, and the mobile phase B is decreased from 100 percent to 30 percent; keeping the mobile phase A at 70% and the mobile phase B at 30% within 27-35 min;
(2) drawing a standard curve: taking acetonitrile as a solvent, preparing Fmoc-AEEA standard substance into samples with different concentrations, detecting the standard substance samples with different concentrations according to the analysis method in the step (1), obtaining peak areas corresponding to the peaks of the main product Fmoc-AEEA with different concentrations, and drawing a standard curve;
(3) preparing a sample solution to be detected: dissolving a sample to be detected in acetonitrile by taking the acetonitrile as a solvent;
(4) and (3) detecting the sample solution to be detected obtained in the step (3) according to the analysis method in the step (1) to obtain the peak area of the main product Fmoc-AEEA, and substituting the obtained peak area into the standard curve obtained in the step (2) to obtain the content of the main product Fmoc-AEEA in the sample to be detected.
2. The method for detecting Fmoc-AEEA content by using high performance liquid chromatography as claimed in claim 1, wherein in step (3), the sample to be detected is prepared at a concentration of 0.8-1.2 mg/mL.
3. The method for detecting Fmoc-AEEA content by high performance liquid chromatography as claimed in claim 2, wherein the sample volume is 5 μ L.
4. The method for detecting Fmoc-AEEA content by using high performance liquid chromatography as claimed in claim 1, wherein the detection wavelength is 220 nm.
5. The method for detecting Fmoc-AEEA content in High Performance Liquid Chromatography (HPLC) as claimed in claim 1, wherein the flow rate of the mobile phase is 1 mL/min.
6. A method for detecting contents of related substances in Fmoc-AEEA by using a high performance liquid chromatography is characterized in that the related substances comprise Fmoc-beta-Ala-AEEA, Fmoc-AEEA-AEEA and Fmoc-beta-Ala, and the contents of the related substances are detected by using a main component self-control method; specific chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX SB-C18, the detection wavelength is 210-220nm, and the column temperature is 25 ℃; mobile phase a was 0.1% TFA water; the mobile phase B is 0.1 percent TFA acetonitrile, and the flow rate of the mobile phase is 0.8-1.2 ml/min;
the elution procedure was: in 0-20min, the mobile phase A is reduced from 70% to 0, and the mobile phase B is increased from 30% to 100%; keeping the mobile phase A at 0 and the mobile phase B at 100% within 20-25 min; in 25-27min, the mobile phase A is increased from 0 to 70 percent, and the mobile phase B is decreased from 100 percent to 30 percent; within 27-35min, mobile phase A was maintained at 70% and mobile phase B at 30%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011389963.7A CN112578044B (en) | 2020-12-02 | 2020-12-02 | Method for detecting Fmoc-AEEA content by high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011389963.7A CN112578044B (en) | 2020-12-02 | 2020-12-02 | Method for detecting Fmoc-AEEA content by high performance liquid chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112578044A CN112578044A (en) | 2021-03-30 |
| CN112578044B true CN112578044B (en) | 2022-09-02 |
Family
ID=75126739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011389963.7A Active CN112578044B (en) | 2020-12-02 | 2020-12-02 | Method for detecting Fmoc-AEEA content by high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112578044B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2010011900A (en) * | 2010-10-29 | 2012-04-30 | Bioderpac S A De C V | Method for quantifying amino acids using high-performance liquid chromatography (hplc) from lactic fermentation. |
| CN106198816A (en) * | 2015-05-07 | 2016-12-07 | 华中科技大学 | A kind of kilnitamin content assaying method |
| CN109115899A (en) * | 2018-06-19 | 2019-01-01 | 南京肽业生物科技有限公司 | A kind of analysis method of Fmoc amino acid |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10690635B2 (en) * | 2016-03-23 | 2020-06-23 | Bachem Holding Ag | Purification of glucagon-like peptide 1 analogs |
-
2020
- 2020-12-02 CN CN202011389963.7A patent/CN112578044B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2010011900A (en) * | 2010-10-29 | 2012-04-30 | Bioderpac S A De C V | Method for quantifying amino acids using high-performance liquid chromatography (hplc) from lactic fermentation. |
| CN106198816A (en) * | 2015-05-07 | 2016-12-07 | 华中科技大学 | A kind of kilnitamin content assaying method |
| CN109115899A (en) * | 2018-06-19 | 2019-01-01 | 南京肽业生物科技有限公司 | A kind of analysis method of Fmoc amino acid |
Non-Patent Citations (2)
| Title |
|---|
| Synthesis and In Vitro Analysis of Atrial Natriuretic Peptide-Albumin Conjugates;Roger Leger等;《Bioorganic&Medicinal Chemistry Letters》;20031231;全文 * |
| 长效多肤药物化学修饰的研究进展;姜和等;《重庆理工大学学报(自然科学)》;20120430;第26卷(第4期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112578044A (en) | 2021-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021022876A1 (en) | Method for determining halogenated acid content in chloral hydrate or preparation thereof | |
| CN110031573B (en) | Method for measuring vitamin D content by two-dimensional column switching high performance liquid chromatography | |
| CN113295805B (en) | Method for detecting hydrazine hydrate in medicine | |
| CN109856270A (en) | A method of with 7 index components in hplc simultaneous determination canopy powder granule | |
| CN112578044B (en) | Method for detecting Fmoc-AEEA content by high performance liquid chromatography | |
| CN111965273A (en) | HPLC (high Performance liquid chromatography) method for detecting genotoxic impurities in candesartan cilexetil | |
| CN106814144B (en) | Method for determining and analyzing content of dimethyl sulfate in dimethyl fumarate | |
| CN109507354B (en) | Method for determining content of K powder in human hair by flash evaporation-gas chromatography-mass spectrometry | |
| CN107991415B (en) | Method for simultaneously separating and measuring pyroglutamic acid and methionine sulfoxide impurities in compound amino acid injection 18AA by liquid chromatography | |
| CN115266998A (en) | Method for detecting content of sodium ions in sodium salt molecules | |
| CN113358771B (en) | Method for detecting organic impurities in 2-vinylpyridine | |
| CN108037221B (en) | Method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography | |
| CN109030668B (en) | High performance liquid phase analysis method of gadoxetic acid disodium intermediate | |
| CN108572223B (en) | Method for determining activity inducing substance in polypeptide | |
| CN111443150A (en) | Method for detecting content of acetylcysteine and acetyltyrosine in compound amino acid injection | |
| CN110412164A (en) | A kind of detection method of the related substance of mexiletine hydrochloride | |
| CN114839300B (en) | Method for measuring 6 chemical components in fingered citron by adopting one-measurement-multiple-evaluation method | |
| CN115327005B (en) | Method for detecting clopidogrel oxide related substances | |
| CN112903861B (en) | Method for detecting residual solvent in dextral rabeprazole sodium bulk drug | |
| CN116465990B (en) | Detection method for impurities of 1, 5-hexanediol, 1, 6-hexanediol and 3-methyl-1, 5-pentanediol in atracurium besylate and cis atracurium besylate bulk drug | |
| CN112557573B (en) | Method for measuring AEEA-AEEA content | |
| CN116930368B (en) | Detection method of settop alcohol isomer | |
| CN114660213B (en) | Component content determination method of compound reserpine hydrochlorothiazide tablet | |
| CN115453022B (en) | Method for rapidly judging formaldehyde content in white spirit | |
| CN107091895B (en) | Method for separating and measuring related substances in riociguat raw material medicine by adopting HPLC (high performance liquid chromatography) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |