CN113024392A - Deuterated kynurenine and application thereof in analysis of amino acid configuration in polypeptide - Google Patents
Deuterated kynurenine and application thereof in analysis of amino acid configuration in polypeptide Download PDFInfo
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Abstract
The invention provides deuterated kynurenine and application thereof in analysis of amino acid configuration in polypeptide, which comprises a synthetic process of the deuterated kynurenine, confirmation of the structure of the deuterated kynurenine based on high-resolution mass spectrometry and nuclear magnetic technology, and determination of the configuration of the kynurenine in daptomycin by using the deuterated kynurenine as a reference substance through liquid chromatography-high-resolution mass spectrometry. The method determines the deuterated sites and the deuterated numbers of the deuterated kynurenines for the first time, can judge the configuration of the kynurenines in the polypeptide, and is suitable for the quality control of the polypeptide drugs.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry and pharmaceutical analytical chemistry, in particular to deuterated kynurenine and application thereof in analysis of amino acid configuration in polypeptide.
Background
Daptomycin is a cyclic lipopeptide antibiotic and has a strong bactericidal effect on drug-resistant gram-positive bacteria in vivo and in vitro. Daptomycin consists of 10 amino acid residues to form a cyclic peptide part, 3 amino acids to form a linear peptide part, and a terminal amino acid is connected with a decanoyl fatty chain, and the structural formula I is as follows:
the change of amino acid configuration has great influence on the antibacterial activity of daptomycin (Penschig, Wangxisasai, Paphiu, Huchanghua, Liao national institute, pharmaceutical Proc., 2018,53(6):839-844), and a reasonable method must be established for monitoring the amino acid and the configuration thereof in daptomycin to ensure the safety of drugs.
At present, the analysis of the amino acid configuration is to hydrolyze polypeptide in hydrochloric acid by heating and then analyze by liquid chromatography or liquid chromatography mass spectrometry by adopting a chiral derivation method, but the method cannot exclude whether alpha-carbon configuration conversion exists in the heating process. Deuterium-substituted hydrochloric acid heavy water solution is selected as a hydrolysis reagent, hydrogen on alpha-carbon can be converted into deuterium if the alpha-carbon undergoes configuration conversion, and the elution time is changed, so that whether the configuration conversion occurs in the heating process or not can be distinguished.
Kynurenine (Kynurenine, Kyn) in daptomycin is a non-protein amino acid, and the structural formula II is shown as follows. At present, no literature on a preparation method, a deuterated site and application of deuterated kynurenine is reported (Zhang Jiayu, Wazhenwan, Zhang Yu, Han Weina, Haerbin medical university report, 2014,48(3):255 + 259; Murai Y, Wang L, Masuda K, Sakihama Y, Hashidoko Y, Hatanaka Y, Hashimoto M.2013,2013(23),5111 + 5116).
On the basis, the research on the structure of the deuterated kynurenine and the application of the deuterated kynurenine to the amino acid configuration analysis in daptomycin are an important research direction.
Disclosure of Invention
In order to realize the technical purpose, the invention provides deuterated kynurenine and application thereof in analysis of amino acid configuration in polypeptide, which comprises a synthetic process of the deuterated kynurenine, confirmation of the structure of the deuterated kynurenine based on high-resolution mass spectrometry and nuclear magnetic technology, and determination of the configuration of the kynurenine in daptomycin by using the deuterated kynurenine as a reference substance through liquid chromatography-high-resolution mass spectrometry.
In a first aspect, the invention provides deuterated kynurenine, which has structural formulas (1) and (2)
In a second aspect, the invention provides a preparation method of the deuterated kynurenine, which specifically comprises the following steps:
step S1, heating hydrolysis: dissolving L-kynurenine (3) in a deuterated hydrochloric acid heavy water solution with the concentration of 0.5-20 mg/mL, filling nitrogen, sealing, and heating to obtain a first mixed solution;
step S2, purifying the first mixed solution through liquid chromatography, collecting deuterated kynurenine fraction, and freeze-drying to obtain yellowish powdery deuterated kynurenine;
the specific operating conditions of the liquid chromatography are as follows: an octadecyl bonded silica gel reverse phase chromatographic column is adopted, a mobile phase A is 0.01-1% formic acid water solution, a mobile phase B is acetonitrile or methanol, and the content of the mobile phase A: the flow rate of the mobile phase B is set to be 0.5-2mL/min, the column temperature is 25-40 ℃, the sample injection amount is 10-100 mu L, and the absorption wavelength is 190-280 nm.
Preferably, the molar concentration of the deuterated hydrochloric acid heavy water solution is 4-10 mol/L.
Preferably, the heating temperature is 85-130 ℃, and the heating time is 5-24 h.
In a third aspect, the invention provides a method for detecting a kynurenine configuration in daptomycin, which specifically comprises the following steps:
step a1, chiral Marfey's reagent derivatization:
respectively placing a daptomycin test solution and a reference solution into a liquid-phase sample injection bottle, and performing derivatization reaction on the daptomycin test solution and the reference solution by adopting a chiral Marfey's reagent solution to obtain a derivatized reference solution and a test solution;
step A2: performing high performance liquid chromatography-mass spectrometry on the derivatized reference substance solution and the derivatized test substance solution;
the test solution comprises an L-kynurenine solution, a D-kynurenine solution and an L-deuterated kynurenine solution, wherein the L-deuterated kynurenine solution comprises the L-deuterated kynurenine.
Preferably, the daptomycin has the following structural formula (4), and the amino acid at the 13 th position is L-kynurenine.
Preferably, step a1 further includes the following steps:
step A11, the preparation method of the daptomycin test solution comprises the following steps:
dissolving daptomycin into a heavy water solution of deuterated hydrochloric acid, wherein the concentration of the daptomycin is 0.5-5mg/mL, filling nitrogen, sealing, heating and hydrolyzing to obtain a hydrolyzed solution, cooling the hydrolyzed solution to room temperature, adding a proper amount of 4-8mol/L sodium hydroxide solution, and neutralizing to neutrality to obtain a daptomycin test solution;
step A12, preparation method of the reference solution:
the preparation method of the reference substance solution comprises the following steps: respectively dissolving D-kynurenine, L-kynurenine and the deuterated kynurenine in a heavy water solution of deuterated hydrochloric acid to obtain a reference solution; the concentration of the reference substance solution is 0.3-5 mg/mL;
step A13, a preparation method of the chiral Marfey's reagent solution:
and dissolving the Marfey's reagent in a solvent to obtain the chiral Marfey's reagent solution with the concentration of 0.5-4 mg/mL.
Preferably, in the step A11, the temperature is 100 ℃ and the temperature is 140 ℃, and the time is 5-24 h.
Preferably, in step A13, the Marfey's reagent is one of Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-valinamide (L/D-FDVA), Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-alaninamide (L/D-FDAA), Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-leucinamide (L/D-FDLA) and Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-phenylalanyl (L/D-FDPA).
Preferably, in step a13, the solvent of the Marfey's reagent is dimethyl sulfoxide, acetone or ethanol.
Preferably, step a2 specifically includes: respectively measuring 20-80 mu L, L-kynurenine solution, D-kynurenine solution and 10-60 mu L of deuterated kynurenine solution of daptomycin test sample solution, respectively placing the daptomycin test sample solution, 20-80 mu L of chiral Marfey's reagent solution and 10-50 mu L of 0.01-0.1mol/L triethylamine solution into liquid-phase sample bottles, uniformly mixing, placing the mixture into a water bath at 30-80 ℃ for reaction for 40-100min, taking out, cooling to room temperature, and adding 10-50 mu L of 0.02-0.08mol/L formic acid solution to make the mixture neutral.
Preferably, in the step A2, in the high performance liquid chromatography, a chromatographic column is C8 or C18, a mobile phase A is 0.01-0.2% formic acid aqueous solution, a mobile phase B is acetonitrile or methanol, the column temperature is 25-50 ℃, the flow rate is 1-2mL/min, and gradient elution is adopted.
In a fourth aspect, the present invention provides a method for detecting kynurenine configuration in a polypeptide, which is characterized by specifically comprising the following steps:
step a1, chiral Marfey's reagent derivatization:
respectively placing a polypeptide test solution and a reference solution in a liquid-phase sample injection bottle, and performing derivatization reaction on the daptomycin test solution and the reference solution by adopting a chiral Marfey's reagent solution to obtain a derivatized reference solution and a derivatized test solution;
step A2: performing high performance liquid chromatography-mass spectrometry on the derivatized reference substance solution and the derivatized test substance solution;
the test solution comprises an L-kynurenine solution, a D-kynurenine solution, and an L-deuterated kynurenine solution comprising L-deuterated kynurenine as described in any one of claims 1 to 4.
Compared with the prior art, the invention has the technical effects that:
the method determines the deuterated sites and the deuterated numbers of the L-deuterated kynurenine for the first time, can judge the configuration of the kynurenine in the polypeptide by not generating alpha configuration transformation in the heating hydrolysis process of the L-deuterated kynurenine, and is suitable for the quality control of the polypeptide drugs.
Drawings
FIG. 1 is a mass spectrum of L-deuterated kynurenine;
FIG. 2 is a nuclear magnetic hydrogen spectrum of L-deuterated kynurenine;
FIG. 3 is a mass spectrometric total ion flow diagram of D-kynurenine-D-FDVA derivative (A), L-kynurenine-D-FDVA derivative;
FIG. 4 is a mass spectrum of L-kynurenine-D-FDVA derivative;
FIG. 5 is a total ion flow diagram of L-deuterated kynurenine-D-FDVA derivatives;
FIG. 6 is a mass spectrum of L-deuterated kynurenine-D-FDVA derivative;
FIG. 7 is a total ion flow diagram (A) of daptomycin after hydrolysis to amino acids with deuterated hydrochloric acid, D-FDVA derivatization, and an ion flow diagram (B) obtained by extraction of ion m/z 493.20;
FIG. 8 is a mass spectrum of a daptomycin hydrolyzed L-deuterated kynurenine-D-FDVA derivative.
Detailed Description
The main used reagents are: l-kynurenine (batch No.: CNU8L), daptomycin (batch No.: 933039).
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
Instrument and reagent
The invention mainly uses the following instruments in the research process: agilent model 1290 high performance liquid chromatograph-6550 QTOF-MS (Agilent Technologies, USA).
Example 1
The embodiment provides a preparation method of deuterated kynurenine, which specifically comprises the following steps:
step S1, heating hydrolysis: dissolving L-kynurenine in 6mol/L deuterium hydrochloric acid heavy water solution to obtain a first mixed solution, wherein the concentration is 2mg/mL, the heating temperature is 110 ℃, and the reaction time is 7 hours;
step S2, purifying by liquid chromatography, collecting deuterated kynurenine fraction, and freeze-drying to obtain yellowish powdery deuterated kynurenine;
the specific operating conditions of the liquid chromatogram are as follows: an octadecyl bonded silica gel reverse phase chromatographic column is adopted, a mobile phase A is 0.1% formic acid water solution, a mobile phase B is acetonitrile, and the content of the mobile phase A: the flow rate of mobile phase B was set to 0.8mL/min at 50:50(v/v), the column temperature was 35 ℃, the sample size was 100 μ L, and the absorption wavelength was 254 nm.
The method comprises the following steps:
the structure of the deuterated kynurenine prepared in example 1 was verified by liquid chromatography-mass spectrometry, specifically performed as follows:
dissolving deuterated kynurenine in a 10% acetonitrile solution with the concentration of 1mg/mL, wherein a mobile phase A is 0.1% formic acid water solution, a mobile phase B is acetonitrile, and the content of the mobile phase A: the flow rate of the mobile phase B is set to be 0.8mL/min, the column temperature is 35 ℃, the sample injection amount is 10L, the absorption wavelength is 254nm, and the mass spectrum scanning range is m/z 100-800.
The mass spectrogram is shown in figure 1, the M/z of the excimer ion peak is 213.1234([ M + H ] +), and the ion peak with M/z of 212.1113([ M + H ] +) can be observed, which has theoretical molecular weights 4 and 3 respectively compared with kynurenine (M/z 209.0848, [ M + H ] +), which indicates that four hydrogen and three hydrogen are respectively replaced by deuterium.
The second method comprises the following steps:
the structure of the deuterated kynurenine prepared in example 1 is verified by nuclear magnetic resonance hydrogen spectroscopy, and the specific operation is as follows:
weighing 5mg of deuterated kynurenine, dissolving in deuterated hydrochloric acid solution, and measuring the hydrogen spectrum by a nuclear magnetic resonance spectrometer, wherein the hydrogen spectrum is shown in the attached figure 2: 1H-NMR (500MHz, DCl) with chemical shifts for hydrogen δ 6.05(s,1H), 5.72(s,1H), 2.60(s,1H), 1.96(d, J ═ 5.1Hz, 0.5H). The chemical shift of the 3-hydrogen on the benzene ring is 6.05, the chemical shift of the 5-hydrogen is 5.72, and the H in the 4-and 6-positions on the benzene ring are deuterated. Integration of H at position 7 showed 0.5H, indicating that the deuteration of the two hydrogens on the methylene at position 7 was 75%. Further illustrated are deuterated kynurenines in which 4 hydrogens are deuterated or 3 hydrogens are deuterated. Meanwhile, H (chemical shift 2.60) at position 8 is not deuterated.
From the above information, the transformation process and chemical structure of deuterated kynurenine were deduced as follows:
example 2
This example provides a method for determining the configuration of kynurenine in daptomycin, comprising the following steps:
step a1, chiral Marfey's reagent derivatization:
step A11, preparation of daptomycin test solution: dissolving daptomycin into a heavy water solution of deuterated hydrochloric acid, wherein the concentration of the daptomycin is 1mg/mL, filling nitrogen, sealing, heating and hydrolyzing to obtain a hydrolyzed solution, cooling the hydrolyzed solution to room temperature, adding a proper amount of 4-8mol/L sodium hydroxide solution, and neutralizing to neutrality to obtain a daptomycin test solution;
step a12, preparation of control solution: respectively dissolving D-kynurenine, L-kynurenine and deuterated kynurenine in example 1 in a heavy aqueous solution of deuterated hydrochloric acid to obtain a reference solution; the concentration of the reference substance solution is 0.5 mg/mL;
step A13, a preparation method of the chiral Marfey's reagent solution: dissolving the D-FDVA reagent in a solvent to obtain the chiral Marfey's reagent solution with the concentration of 2 mg/mL;
respectively measuring 50 mu L, L-kynurenine, D-kynurenine and 30 mu L-deuterated kynurenine solution of daptomycin test sample solution, respectively placing the daptomycin test sample solution into a liquid-phase sample injection bottle, adding 50 mu L D-FDVA solution and 30 mu L0.05 mol/L triethylamine solution, uniformly mixing, placing the mixture in a water bath at 40 ℃ for reacting for 60min, taking out the mixture, cooling the mixture to room temperature, and adding 30 mu L0.05 mol/L formic acid solution to enable the mixture to be neutral;
step A2, high performance liquid chromatography-high resolution mass spectrometry:
operating parameters of the high performance liquid chromatography: a conventional C18 reversed phase chromatographic column is adopted, a mobile phase A is 0.1% formic acid water solution, a mobile phase B is acetonitrile, gradient elution is carried out, the composition of the mobile phase is shown in table 1, the detection wavelength is 340nm, the column temperature is 40 ℃, the flow rate is 1.5mL/min, split flow is carried out before mass spectrum detection, and the split flow ratio is 2: 1.
TABLE 1 composition of mobile phase during gradient elution
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 68 | 32 |
| 20 | 62 | 38 |
| 25 | 50 | 50 |
| 32 | 45 | 55 |
| 34 | 35 | 65 |
| 42 | 35 | 65 |
| 43 | 68 | 32 |
| 48 | 68 | 32 |
The total ion flow diagrams of the D/L-kynurenine-D-FDVA derivative are shown in figures 3A and 3B respectively, the retention time is 25.4min and 30.2min respectively, the mass-to-charge ratio of the quasi-molecular ion peaks is the same, wherein the M/z of the L-kynurenine-D-FDVA derivative is 489.1746([ M + H ] +), and is shown in figure 4.
The total ion flow diagram of the L-deuterated kynurenine-D-FDVA derivative is shown in figure 5, the retention time of the L-deuterated kynurenine-D-FDVA derivative is respectively 30.1min and is basically consistent with that of the L-kynurenine-D-FDVA derivative, and the L-kynurenine does not have D-kynurenine and does not have alpha-carbon configuration conversion in the heating process.
The excimer peaks of the L-deuterated kynurenine-D-FDVA derivative have M/z of 492.1918 and 493.1984([ M + H ] +), as shown in FIG. 6, which respectively represents that the L-deuterated kynurenine-D-FDVA derivative contains three deuterium atoms and four deuterium atoms. In addition, a trace ion peak m/z 491.1840 was observed, indicating the presence of the L-deuterated kynurenine-D-FDVA derivative containing two deuterium atoms, but at a lower level, not detectable by nuclear magnetism.
The total ion flow diagram of daptomycin after hydrolysis into amino acid by deuterated hydrochloric acid and D-FDVA derivatization is shown in figure 7A, and the ion flow diagram obtained by extracting ions m/z 493.20 is shown in figure 7B, and the peak is found to exist only at 30.1min, and the elution time of the peak is consistent with that of the peak of L-deuterated kynurenine-D-FDVA derivatization, so that the configuration of kynurenine in daptomycin is L-type. The mass spectrum of the deuterated kynurenine-D-FDVA derivative after daptomycin hydrolysis is shown in figure 7, wherein the m/z of an excimer ion peak is 493.1994, which indicates that the derivative contains four deuterium atoms, the m/z 492.1923 of the ion peak indicates that the derivative contains three deuterium atoms, and the m/z 491.1849 indicates that the derivative contains two deuterium atoms in a trace amount.
In conclusion, the invention provides deuterated kynurenine and application thereof in analysis of amino acid configuration in polypeptide, which comprises the steps of synthesizing deuterated kynurenine, confirming the structure based on high-resolution mass spectrum and nuclear magnetic technology, and determining the configuration of the kynurenine in daptomycin by using the deuterated kynurenine as a reference substance through liquid chromatography-high-resolution mass spectrum. The method determines the deuterated sites and the deuterated numbers of the deuterated kynurenines for the first time, can judge the configuration of the kynurenines in the polypeptide, and is suitable for the quality control of the polypeptide drugs.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (13)
1. Deuterated kynurenine is characterized in that the structural formulas (1) and (2) are
2. A method for the preparation of deuterated kynurenine as recited in claim 1, comprising the steps of:
step S1, heating hydrolysis: dissolving L-kynurenine (3) in a deuterated hydrochloric acid heavy water solution with the concentration of 0.5-20 mg/mL, filling nitrogen, sealing, and heating to obtain a first mixed solution;
step S2, purifying the first mixed solution through liquid chromatography, collecting deuterated kynurenine fraction, and freeze-drying to obtain yellowish powdery deuterated kynurenine;
the specific operating conditions of the liquid chromatography are as follows: an octadecyl bonded silica gel reverse phase chromatographic column is adopted, a mobile phase A is 0.01-1% formic acid water solution, a mobile phase B is acetonitrile or methanol, and the content of the mobile phase A: the flow rate of the mobile phase B is set to be 0.5-2mL/min, the column temperature is 25-40 ℃, the sample injection amount is 10-100 mu L, and the absorption wavelength is 190-280 nm.
3. The method of claim 2, wherein the deuterium oxide-heavy aqueous solution has a molar concentration of 4 to 10 mol/L.
4. The method of claim 2, wherein the heating temperature is 85 ℃ to 130 ℃ and the heating time is 5h to 24 h.
5. A method for detecting kynurenine configuration in daptomycin is characterized by comprising the following steps:
step a1, chiral Marfey's reagent derivatization:
respectively placing a daptomycin test solution and a reference solution into a liquid-phase sample injection bottle, and performing derivatization reaction on the daptomycin test solution and the reference solution by adopting a chiral Marfey's reagent solution to obtain a derivatized reference solution and a test solution;
step A2: performing high performance liquid chromatography-mass spectrometry on the derivatized reference substance solution and the derivatized test substance solution;
the test solution comprises an L-kynurenine solution, a D-kynurenine solution, and a deuterated kynurenine solution, the deuterated kynurenine solution comprising deuterated kynurenine as defined in any one of claims 1 to 4.
6. The method for detecting according to claim 5, wherein daptomycin has the formula (4) below, and the amino acid at position 13 is L-kynurenine.
7. The detecting method according to claim 5, wherein the step A1 further comprises the steps of:
step A11, the preparation method of the daptomycin test solution comprises the following steps:
dissolving daptomycin into a heavy water solution of deuterated hydrochloric acid, wherein the concentration of the daptomycin is 0.5-5mg/mL, filling nitrogen, sealing, heating and hydrolyzing to obtain a hydrolyzed solution, cooling the hydrolyzed solution to room temperature, adding a proper amount of 4-8mol/L sodium hydroxide solution, and neutralizing to neutrality to obtain a daptomycin test solution;
step A12, preparation method of the reference solution:
respectively dissolving D-kynurenine, L-kynurenine and the deuterated kynurenine in a heavy water solution of deuterated hydrochloric acid to obtain a reference solution; the concentration of the reference substance solution is 0.3-5 mg/mL;
step A13, a preparation method of the chiral Marfey's reagent solution:
and dissolving the Marfey's reagent in a solvent to obtain the chiral Marfey's reagent solution with the concentration of 0.5-4 mg/mL.
8. The detection method according to claim 7, wherein in the step A11, the temperature is 100 ℃ and the time is 140 ℃ and the time is 5-24 h.
9. The detection method according to claim 7, wherein in the step A13, the Marfey's reagent is one of Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-valinamide, Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-alaninamide, Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-leucinamide, and Nalpha- (5-fluoro-2, 4-dinitrophenyl) -L/D-phenylalanyl.
10. The assay of claim 7 wherein, in step A13, the solvent of the Marfey's reagent is dimethyl sulfoxide, acetone or ethanol.
11. The detection method according to claim 5, wherein the step A2 specifically includes: respectively measuring 20-80 mu L, L-kynurenine solution, D-kynurenine solution and 10-60 mu L of deuterated kynurenine solution of daptomycin test sample solution, respectively placing the daptomycin test sample solution, 20-80 mu L of chiral Marfey's reagent solution and 10-50 mu L of 0.01-0.1mol/L triethylamine solution into liquid-phase sample bottles, uniformly mixing, placing the mixture into a water bath at 30-80 ℃ for reaction for 40-100min, taking out, cooling to room temperature, and adding 10-50 mu L of 0.02-0.08mol/L formic acid solution to make the mixture neutral.
12. The detection method according to claim 5, wherein in the step A2, in the high performance liquid chromatography, the chromatographic column is C8 or C18, the mobile phase A is 0.01 to 0.2 percent formic acid aqueous solution, the mobile phase B is acetonitrile or methanol, the column temperature is 25 to 50 ℃, the flow rate is 1 to 2mL/min, and gradient elution is adopted.
13. A method for detecting kynurenine configuration in polypeptide is characterized by comprising the following steps:
step a1, chiral Marfey's reagent derivatization:
respectively placing a polypeptide test solution and a reference solution in a liquid-phase sample injection bottle, and performing derivatization reaction on the daptomycin test solution and the reference solution by adopting a chiral Marfey's reagent solution to obtain a derivatized reference solution and a derivatized test solution;
step A2: performing high performance liquid chromatography-mass spectrometry on the derivatized reference substance solution and the derivatized test substance solution;
the test solution comprises an L-kynurenine solution, a D-kynurenine solution and a deuterated kynurenine solution, the deuterated kynurenine solution comprising the deuterated kynurenine as defined in any one of claims 1 to 4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114280179A (en) * | 2021-12-22 | 2022-04-05 | 北京美福润医药科技股份有限公司 | Pretreatment of exenatide and method for detecting isomer in His amino acid eluate obtained by pretreatment |
| CN116177498A (en) * | 2022-12-12 | 2023-05-30 | 江南大学 | Chiral selenium nano-film with light response, preparation method thereof and application thereof in detection of L-kynurenine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0989865A (en) * | 1995-07-20 | 1997-04-04 | Kazuhiro Imai | Method for analyzing d/l amino acid sequence of peptide, protein |
| US20060120961A1 (en) * | 2004-10-29 | 2006-06-08 | Target Discovery, Inc. | Glycan analysis using deuterated glucose |
| JP2018100906A (en) * | 2016-12-20 | 2018-06-28 | 花王株式会社 | Method for separation of chiral amino acid |
| CN110297044A (en) * | 2018-03-21 | 2019-10-01 | 中国医学科学院药物研究所 | A method of identification amino acid and peptides absolute configuration and optical purity |
| CN111505142A (en) * | 2020-04-26 | 2020-08-07 | 上海市食品药品检验所 | Polymyxin B amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method |
-
2021
- 2021-02-09 CN CN202110176709.7A patent/CN113024392B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0989865A (en) * | 1995-07-20 | 1997-04-04 | Kazuhiro Imai | Method for analyzing d/l amino acid sequence of peptide, protein |
| US20060120961A1 (en) * | 2004-10-29 | 2006-06-08 | Target Discovery, Inc. | Glycan analysis using deuterated glucose |
| JP2018100906A (en) * | 2016-12-20 | 2018-06-28 | 花王株式会社 | Method for separation of chiral amino acid |
| CN110297044A (en) * | 2018-03-21 | 2019-10-01 | 中国医学科学院药物研究所 | A method of identification amino acid and peptides absolute configuration and optical purity |
| CN111505142A (en) * | 2020-04-26 | 2020-08-07 | 上海市食品药品检验所 | Polymyxin B amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method |
Non-Patent Citations (2)
| Title |
|---|
| 刘浩等: "手性衍生化-HPLC法测定达托霉素中8种手性氨基酸的D，L-构型", 《中国抗生素杂志》 * |
| 刘浩等: "适用于硫酸多黏菌素B中氨基酸D-/L-构型测定的LC-MS分析技术", 《科技成果 》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114280179A (en) * | 2021-12-22 | 2022-04-05 | 北京美福润医药科技股份有限公司 | Pretreatment of exenatide and method for detecting isomer in His amino acid eluate obtained by pretreatment |
| CN114280179B (en) * | 2021-12-22 | 2024-03-15 | 北京美福润医药科技股份有限公司 | Pretreatment of exenatide and detection method of isomer in His amino acid eluent obtained by pretreatment |
| CN116177498A (en) * | 2022-12-12 | 2023-05-30 | 江南大学 | Chiral selenium nano-film with light response, preparation method thereof and application thereof in detection of L-kynurenine |
| CN116177498B (en) * | 2022-12-12 | 2023-11-24 | 江南大学 | Chiral selenium nano-film with light response, preparation method thereof and application thereof in detection of L-kynurenine |
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