CN108663448B - A kind of detection method of related substances in compound amino acid injection - Google Patents
A kind of detection method of related substances in compound amino acid injection Download PDFInfo
- Publication number
- CN108663448B CN108663448B CN201810457668.7A CN201810457668A CN108663448B CN 108663448 B CN108663448 B CN 108663448B CN 201810457668 A CN201810457668 A CN 201810457668A CN 108663448 B CN108663448 B CN 108663448B
- Authority
- CN
- China
- Prior art keywords
- amino acid
- compound amino
- acid injection
- solution
- kynurenine
- 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
- -1 compound amino acid Chemical class 0.000 title claims abstract description 94
- 238000002347 injection Methods 0.000 title claims abstract description 90
- 239000007924 injection Substances 0.000 title claims abstract description 90
- 239000000126 substance Substances 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title abstract description 13
- LDCYZAJDBXYCGN-VIFPVBQESA-N 5-hydroxy-L-tryptophan Chemical compound C1=C(O)C=C2C(C[C@H](N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-VIFPVBQESA-N 0.000 claims abstract description 67
- LDCYZAJDBXYCGN-UHFFFAOYSA-N oxitriptan Natural products C1=C(O)C=C2C(CC(N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-UHFFFAOYSA-N 0.000 claims abstract description 66
- 229940000681 5-hydroxytryptophan Drugs 0.000 claims abstract description 65
- YGPSJZOEDVAXAB-UHFFFAOYSA-N (R)-Kynurenine Natural products OC(=O)C(N)CC(=O)C1=CC=CC=C1N YGPSJZOEDVAXAB-UHFFFAOYSA-N 0.000 claims abstract description 63
- YGPSJZOEDVAXAB-QMMMGPOBSA-N L-kynurenine Chemical compound OC(=O)[C@@H](N)CC(=O)C1=CC=CC=C1N YGPSJZOEDVAXAB-QMMMGPOBSA-N 0.000 claims abstract description 60
- 238000000034 method Methods 0.000 claims abstract description 57
- PPXBTTCMHNWJPE-UHFFFAOYSA-N 2,2,3,3,4,4,4-heptafluorobutanoic acid;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)C(F)(F)C(F)(F)F PPXBTTCMHNWJPE-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000004190 ion pair chromatography Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 93
- 239000012085 test solution Substances 0.000 claims description 43
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 39
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 39
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 20
- 239000013558 reference substance Substances 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 8
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 239000012088 reference solution Substances 0.000 claims description 5
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- LZDNBBYBDGBADK-KBPBESRZSA-N Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-KBPBESRZSA-N 0.000 claims 3
- 150000001413 amino acids Chemical class 0.000 abstract description 13
- 239000012535 impurity Substances 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 229960004799 tryptophan Drugs 0.000 description 26
- 238000012360 testing method Methods 0.000 description 10
- 108010016626 Dipeptides Proteins 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UXJDSNKBYFJNDO-UHFFFAOYSA-N 9-[4-[2-[2-(4-carbazol-9-ylphenyl)phenyl]phenyl]phenyl]carbazole Chemical compound C12=CC=CC=C2C2=CC=CC=C2N1C(C=C1)=CC=C1C1=CC=CC=C1C1=CC=CC=C1C1=CC=C(N2C3=CC=CC=C3C3=CC=CC=C32)C=C1 UXJDSNKBYFJNDO-UHFFFAOYSA-N 0.000 description 1
- 241001397104 Dima Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 206010025476 Malabsorption Diseases 0.000 description 1
- 208000004155 Malabsorption Syndromes Diseases 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000692 cap cell Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- YPJUNDFVDDCYIH-UHFFFAOYSA-N perfluorobutyric acid Chemical compound OC(=O)C(F)(F)C(F)(F)C(F)(F)F YPJUNDFVDDCYIH-UHFFFAOYSA-N 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 235000021075 protein intake Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
Landscapes
- 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)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供一种复方氨基酸注射液中有关物质的检测方法，该方法以5‑羟基色氨酸和L‑犬尿氨酸为指针性杂质，通过三氟乙酸‑七氟丁酸离子对色谱方法优化了氨基酸注射液中所含有的各种氨基酸和有关物质的分离和基线稳定性，检测灵敏度高，准确性和重现性好，能够满足复方氨基酸注射液中有关物质控制的要求。
The invention provides a method for detecting related substances in compound amino acid injection. The method uses 5-hydroxytryptophan and L-kynurenine as index impurities, and adopts a trifluoroacetic acid-heptafluorobutyric acid ion pair chromatography method. The separation and baseline stability of various amino acids and related substances contained in amino acid injection are optimized, with high detection sensitivity, accuracy and reproducibility, which can meet the requirements of related substances control in compound amino acid injection.
Description
Technical Field
The invention relates to a method for detecting related substances in an injection, in particular to a method for detecting related substances in a compound amino acid injection.
Background
Amino acids are the basic units that make up human proteins, and are also the starting materials for the body to synthesize antibodies, hormones, enzymes and other tissues. The compound amino acid injection is generally used for patients who have insufficient protein intake, malabsorption and the like and have amino acid which can not meet the metabolic needs of organisms, and is also used for improving the nutritional status of the patients after the operation. However, the pharmacopoeia of all countries does not contain a method for detecting related substances in compound amino acid injection. Although pharmacopoeia collects detection methods of related substances in amino acid raw materials, the related substances are residual process impurities in the production process of amino acid, are mostly endogenous amino acids and are covered by other amino acid raw materials in a compound preparation formula, so that the detection requirements of the related substances in the compound amino acid injection cannot be met.
Disclosure of Invention
The invention provides a method for detecting related substances in a compound amino acid injection, which determines the pointer impurities in the compound amino acid injection, has high detection sensitivity, high accuracy and good reproducibility, and can meet the requirements of related substance control in the compound amino acid injection.
The invention relates to a method for detecting related substances in a compound amino acid injection, which comprises the following steps:
(1) preparation of test solution and control solution:
taking the compound amino acid injection as a test solution directly;
preparing at least 3 5-hydroxytryptophan control solutions with different concentrations and at least 3L-kynurenine control solutions with different concentrations;
(2) injecting the 5-hydroxytryptophan reference substance solutions with different concentrations into a high performance liquid chromatograph, recording chromatograms of the 5-hydroxytryptophan reference substance solutions with different concentrations under the wavelength of 280nm, and making a standard curve or a regression equation by taking the concentration of 5-hydroxytryptophan as a horizontal coordinate and taking a peak area corresponding to the concentration as a vertical coordinate; injecting the L-kynurenine reference substance solutions with different concentrations into a high performance liquid chromatograph, recording chromatograms of the L-kynurenine reference substance solutions with different concentrations under the wavelength of 365nm, and making a standard curve or a regression equation by taking the concentration of the L-kynurenine as a horizontal ordinate and taking a peak area corresponding to the concentration as a vertical coordinate;
(3) injecting the test solution into a high performance liquid chromatograph, recording chromatograms of the test solution at 280nm and 365nm, calculating the amount of 5-hydroxytryptophan in the test solution according to a standard curve or a regression equation by using the peak area of 5-hydroxytryptophan in the chromatogram at 280nm of the test solution according to the standard curve or the regression equation and not exceeding 0.2% of the labeled amount of tryptophan in the compound amino acid injection, and calculating the amount of L-kynurenine in the test solution according to the standard curve or the regression equation by using the peak area of L-kynurenine in the chromatogram at 365nm of the test solution and not exceeding 0.2% of the labeled amount of tryptophan in the compound amino acid injection;
wherein, the compound amino acid injection contains tryptophan.
The inventor of the invention finds that tryptophan is the most unstable amino acid in 20 amino acids, and 5-hydroxytryptophan and L-kynurenine are the impurities which appear earliest, generate fastest and have the most amount for compound amino acid injection containing tryptophan, so that 5-hydroxytryptophan and L-kynurenine are determined as the indicative impurities in the compound amino acid injection.
As will be understood by those skilled in the art, various tryptophan-containing compound amino acid injections can be detected by the method of the present invention, including but not limited to: the compound amino acid (15) dipeptide (2) injection, the compound amino acid injection (18AA) (specifications comprise 5% and 12%), the compound amino acid injection (18AA-I), the compound amino acid injection (18AA-II) (specifications comprise 5%, 8.5% and 11.4%), the compound amino acid injection (18AA-III), the compound amino acid injection (18AA-V), the compound amino acid injection (18 AA-VII), the compound amino acid injection (20AA), the compound amino acid injection (15AA), the compound amino acid injection (14AA), the compound amino acid injection (17AA) and the like, preferably, the compound amino acid (15) dipeptide (2) injection.
In a preferred embodiment, in step (1), a control solution containing the test solution is further prepared; in the step (2), further injecting the control solution into a high performance liquid chromatograph, and recording the chromatogram of the control solution under the wavelength of 280 nm; in the step (3), according to a self-comparison method, if other chromatographic peaks are detected in the 280nm wavelength chromatogram of the test solution, the content of a single peak area is not more than 0.5% of the tryptophan labeled amount in the compound amino acid injection through comparison with the peak area of the tryptophan in the comparison solution, and the content of a total peak area is not more than 5% of the tryptophan labeled amount in the compound amino acid injection through comparison with the peak area of the tryptophan in the comparison solution.
It will be appreciated by those skilled in the art that various solvents commonly used in the art can be used to formulate the 5-hydroxytryptophan control solution and the L-kynurenine control solution. Preferably, the solvent of the control solution is dilute hydrochloric acid aqueous solution, and more preferably hydrochloric acid aqueous solution with the concentration of 0.1 mol/L.
It will be appreciated by those skilled in the art that a variety of solvents commonly used in the art can be used to formulate control solutions for the test solutions. Preferably, the solvent of the control solution is dilute aqueous hydrochloric acid, more preferably aqueous hydrochloric acid with a concentration of 0.1 mol/L.
Preferably, 3-7, more preferably 3-5, and even more preferably 3 control solutions of 5-hydroxytryptophan of different concentrations are prepared. One skilled in the art would be able to select the concentration of 5-hydroxytryptophan in a plurality of control solutions used to create a standard curve or regression equation. Preferably, the minimum concentration is lower than 0.1% of the marked amount of tryptophan in the compound amino acid injection, and the maximum concentration is higher than 0.4% of the marked amount of tryptophan in the compound amino acid injection. For example, for a compound amino acid (15) dipeptide (2) injection, wherein the labeled amount of tryptophan is 0.95g/500ml, the concentrations of 5-hydroxytryptophan in the 3 control solutions used to make the standard curve or regression equation can range from 0.2 to 0.8. mu.g/ml, 1.6 to 2.4. mu.g/ml, and 8 to 10. mu.g/ml, respectively, e.g., the concentrations of 5-hydroxytryptophan in the 3 control solutions can range from about 0.5. mu.g/ml, about 2.0. mu.g/ml, and about 9.0. mu.g/ml, respectively. The linear range of the method of the invention exceeds 0.1-0.4% of the labeled amount of tryptophan.
Preferably, 3 to 7, more preferably 3 to 5, and still more preferably 3 different concentrations of the L-kynurenine control solution are formulated. Those skilled in the art will be able to select the concentration of L-kynurenine in a plurality of control solutions used to make a standard curve or regression equation. Preferably, the minimum concentration is lower than 0.1% of the marked amount of tryptophan in the compound amino acid injection, and the maximum concentration is higher than 0.4% of the marked amount of tryptophan in the compound amino acid injection. For example, for a compound amino acid (15) dipeptide (2) injection, wherein the labeled amount of tryptophan is 0.95g/500ml, the concentrations of L-kynurenine in the 3 control solutions used to make the standard curve or regression equation may range from 0.2 to 0.8. mu.g/ml, 1.6 to 2.4. mu.g/ml, and 8 to 10. mu.g/ml, respectively, e.g., the concentrations of L-kynurenine in the 3 control solutions may range from about 0.5. mu.g/ml, about 2.0. mu.g/ml, and about 9.0. mu.g/ml, respectively. The linear range of the method of the invention exceeds 0.1-0.4% of the labeled amount of tryptophan.
Preferably, the volume percentage of the test solution in the control solution is 1%, that is, the test solution is diluted 100 times with the solvent. If other chromatographic peaks are detected in the 280nm wavelength chromatogram of the test solution, the single peak area is not more than 0.5 times of the peak area of tryptophan in the control solution, and the total peak area is not more than 5 times of the peak area of tryptophan in the control solution.
Preferably, the chromatographic conditions are: adopting trifluoroacetic acid-heptafluorobutyric acid ion pair chromatography; the chromatographic column is a reversed phase C18 chromatographic column, preferably a 4.6mm × 250mm, 3 μm specification, more preferably a reversed phase C18AQ chromatographic column; the detector is an ultraviolet detector.
Preferably, mobile phase a is 5.0mmol/L heptafluorobutyric acid-0.3% trifluoroacetic acid-water, mobile phase B is acetonitrile; preferably, a gradient elution is used, the elution procedure being shown in table 1 below; preferably, the flow rate is 0.5 to 1.0ml/min, more preferably 0.7 to 0.9ml/min, and still more preferably 0.8 ml/min. Preferably, the column temperature is 35-45 deg.C, more preferably 40 deg.C.
TABLE 1 gradient elution procedure
According to the invention, through a large number of researches, 5-hydroxytryptophan and L-kynurenine are determined as indicative impurities in the compound amino acid injection, and related substances in the compound amino acid injection can be detected and controlled by detecting and controlling the 5-hydroxytryptophan and the L-kynurenine in the compound amino acid injection. Particularly, the invention optimizes the separation and baseline stability of various amino acids and related substances contained in the amino acid injection by a trifluoroacetic acid-heptafluorobutyric acid ion pair chromatographic method, and improves the detection sensitivity. Proved by methodology verification, the method disclosed by the invention is sensitive, accurate and good in reproducibility, and can meet the requirements of related substance control of the compound amino acid injection.
Drawings
FIG. 1280 nm wavelength 5-hydroxytryptophan reference chromatogram
FIG. 2365 nm wavelength L-kynurenine reference solution chromatogram
FIG. 3280 nm wavelength chromatogram of test solution
FIG. 45-hydroxytryptophan standard curve chart
FIG. 5 Standard graph of kynurenine
FIG. 6280 nm open-air stirring 1 hr sample solution chromatogram
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.
Example 1 detection of related substances in Compound amino acid injection
1. Instrument for measuring the position of a moving object
Thermo DIONEX UltiMate model 3000 high performance liquid chromatograph (online degasser, double ternary gradient pump, DIONEX UltiMate 3000Diode Array Detector, DIONEX UltiMate 3000Autosampler, DIONEX UltiMate 3000Column unit, Chromeleon 7.2 chromatography workstation) (Thermo fisher, usa); a balance (XP205, METTLER TOLEDO, Switzerland); Milli-Q ultra pure water devices (Integral 10, Merck Millipore, Germany); water phase needle filter (polyethersulfone, batch No. 9G020320, Shanghai' an general scientific instruments Co., Ltd., China).
2. Reagent
Acetonitrile (Merck chemical technology (Shanghai) Co., Ltd., HPLC purity 99.9%, batch No. 1312001 YT); trifluoroacetic acid (99% by Bailingwei Tech Co., Ltd., Beijing, batch No. L490O 140); heptafluorobutyric acid (Beijing Dima technical Co., Ltd., 99.5%, lot number: 7281190); hydrochloric acid: beijing chemical plant, batch No. 20140910, content w/%: 38.0; the water is ultrapure water (greater than 18.2M omega cm).
3. Reference substance
L-kynurenine (Sigma, HPLC purity 98%, batch: BCBP8307V), 5-hydroxy-L-tryptophan (Aldrich, HPLC purity 98%, batch: BCBP 2662V).
4. Reagent
Compound amino acid (15), dipeptide (2), injection (batch No. 14121601, double-crane medicine industry), and tryptophan marked amount of 0.95g/500 ml.
5. Detection of related substances in compound amino acid injection
Taking the compound amino acid injection to be directly used as a test solution. Precisely measuring 1ml of the test solution, placing the test solution into a 100ml measuring flask, diluting the test solution to a scale with 0.1mol/L hydrochloric acid aqueous solution, shaking up, and using the test solution as a control solution. Precisely weighing 5-hydroxytryptophan, and preparing 5-hydroxytryptophan reference solutions with 5-hydroxytryptophan concentrations of 0.5105 μ g/ml, 2.002 μ g/ml and 9.544 μ g/ml with 0.1mol/L hydrochloric acid aqueous solution; l-kynurenine is precisely weighed and prepared into L-kynurenine reference substance solutions with the concentrations of 0.5285 mu g/ml, 2.016 mu g/ml and 9.304 mu g/ml by using 0.1mol/L hydrochloric acid aqueous solution.
Using a Capcell PAK C18AQ S3(4.6mm × 250mm, 3 μm) chromatographic column; mobile phase A: 5.0mmol/L heptafluorobutyric acid-0.3% trifluoroacetic acid-water, mobile phase B: acetonitrile, gradient elution, elution procedure see table 2 below; the flow rate is 0.8 ml/min; the column temperature is 40 ℃; detection wavelength: 280nm and 365 nm; in the 280nm wavelength chromatogram, the number of theoretical plates is not less than 10000, and the separation degree of 5-hydroxytryptophan, L-kynurenine and adjacent chromatographic peak is more than 1.5 according to the calculation of 5-hydroxytryptophan peak.
TABLE 2 gradient elution procedure
Precisely measuring 10 μ L of each of the test solution, the control solution and the two control solutions, respectively injecting into a liquid chromatograph, and recording chromatograms of the test solution, the control solution and the 5-hydroxytryptophan control solution at 280nm wavelength and chromatograms of the test solution and the L-kynurenine control solution at 365nm wavelength. Wherein the chromatogram of 5-hydroxytryptophan control solution with concentration of 2.0 μ g/ml at 280nm wavelength, the chromatogram of L-kynurenine control solution with concentration of 2.0 μ g/ml at 365nm wavelength, and the chromatogram of test solution at 280nm wavelength are shown in figures 1-3.
And (3) taking the peak area of 5-hydroxytryptophan in a 280nm wavelength chromatogram of a 5-hydroxytryptophan reference substance solution with the concentrations of 0.5105 mu g/ml, 2.002 mu g/ml and 9.544 mu g/ml as a vertical coordinate and the concentration of 5-hydroxytryptophan as a horizontal coordinate to prepare a standard curve (shown in figure 4), and determining the amount of 5-hydroxytryptophan contained in the test substance solution according to the standard curve by taking the peak area of 5-hydroxytryptophan in the 280nm wavelength chromatogram of the test substance solution according to the standard curve method. In the compound amino acid (15) dipeptide (2) injection with lot number 14121601 detected in the example, the 5-hydroxytryptophan peak is not detected, i.e. the amount of 5-hydroxytryptophan is not more than 0.2% of the labeled amount of tryptophan in the compound amino acid injection.
Taking the peak area of L-kynurenine in the 365nm wavelength chromatogram of the L-kynurenine reference substance solution with the concentrations of 0.5285 mu g/ml, 2.016 mu g/ml and 9.304 mu g/ml as the ordinate and the concentration of L-kynurenine as the abscissa, making a standard curve (see attached figure 5), and determining the amount of L-kynurenine contained in the test substance solution according to the standard curve by taking the peak area of L-kynurenine in the 365nm wavelength chromatogram of the test substance solution according to the standard curve method. In the compound amino acid (15) dipeptide (2) injection with lot number 14121601 detected in the example, no L-kynurenine peak is detected, i.e. the amount of L-kynurenine is not more than 0.2% of the labeled amount of tryptophan in the compound amino acid injection.
The single peak area of other chromatographic peaks detected in the 280nm wavelength chromatogram of the test solution does not exceed 0.5 time of the peak area of tryptophan in the control solution, and the total peak area does not exceed 5 times of the peak area of tryptophan in the control solution.
Placing the compound amino acid injection of the same batch number at room temperature in an open manner, placing the compound amino acid injection on a magnetic stirrer for stirring for 1h due to full consideration of oxidation instability, detecting related substances contained in the compound amino acid injection according to the same method, detecting a 5-hydroxytryptophan peak of a test solution of the compound amino acid injection at a wavelength of 280nm (shown in figure 6), and calculating the concentration of the compound amino acid injection to be 1.078 mu g/ml according to a standard curve method; l-kynurenine was detected at a wavelength of 365nm and its concentration was 0.5907. mu.g/ml according to the standard curve method.
Example 2 methodological validation
The methodology of the detection method of the present invention is studied with reference to appendix of the four parts of the edition of Chinese pharmacopoeia 2015. Wherein the compound amino acid (15) dipeptide (2) injection (batch numbers: 14121601, 14121701, 14121801, from the double-crane pharmaceutical industry)
1. Specificity test
Respectively taking a blank solvent (0.1mol/L hydrochloric acid aqueous solution), a 5-hydroxytryptophan reference solution (the concentration is 40.72 mu g/ml), an L-kynurenine reference solution (the concentration is 60.80 mu g/ml), a test solution, a 5-hydroxytryptophan positioning solution (the concentration is 400 mu g/ml) and a mixed solution of the L-kynurenine positioning solution (the concentration is 400 mu g/ml) and 1:1:1, injecting the mixed solution into a liquid chromatograph under the same chromatographic conditions as example 1, recording a chromatogram under the wavelength of 280nm, and finding that the blank solvent and other amino acids in the test solution have no interference on the measurement of the 5-hydroxytryptophan and the L-kynurenine, thereby indicating that the invention has good specificity.
2. System suitability test
Control solutions containing 5-hydroxytryptophan and L-kynurenine with concentrations of 3.802 μ g/ml and 3.722 μ g/ml respectively are prepared, injected into a liquid chromatograph under the same chromatographic conditions as in example 1, and chromatograms at wavelengths of 280nm and 365nm are recorded, wherein the theoretical plate number of 5-hydroxytryptophan is 456909, the theoretical plate number of L-kynurenine is 323966, and the separation degree of the two is 8.52.
3. Repeatability survey
6 standard sample solutions containing 5-hydroxytryptophan and L-kynurenine with concentrations of 3.802 and 3.722 μ g/ml and 20% of sample solution are prepared in parallel and injected into a liquid chromatograph under the same chromatographic conditions as example 1, wherein the peak areas RSD of 5-hydroxytryptophan (at 280 nm) and L-kynurenine (at 365 nm) are 0.30% and 1.89%.
4. Limit of quantification
Preparing quantitative limiting solutions containing 5-hydroxytryptophan and L-kynurenine with the concentrations of 0.8144 and 1.216mg/ml respectively, continuously diluting and injecting into a liquid chromatograph under the same chromatographic conditions as in example 1 until the signal-to-noise ratio S/N is more than or equal to 10. The quantitative limits of 5-hydroxytryptophan (at 280 nm) and L-kynurenine (at 365 nm) were 0.1629 μ g/ml and 0.2432 μ g/ml, respectively.
5. Detection limit
Preparing quantitative limiting solutions containing 5-hydroxytryptophan and L-kynurenine with the concentrations of 0.8144 and 1.216mg/ml respectively, continuously diluting and injecting into a liquid chromatograph under the same chromatographic conditions as in example 1 until the signal-to-noise ratio S/N is more than or equal to 3. The detection limits of 5-hydroxytryptophan (at 280nm wavelength) and L-kynurenine (at 365nm wavelength) are 0.08144 μ g/ml and 0.1216 μ g/ml respectively.
6. Precision survey
A control solution (the test solution was diluted 100 times with 0.1mol/L hydrochloric acid aqueous solution) and control solutions containing 5-hydroxytryptophan and L-kynurenine at concentrations of 3.802 and 3.722. mu.g/ml were injected into a liquid chromatograph, and the sample injection was repeated 6 times, respectively, under the same chromatographic conditions as in example 1, wherein the peak areas RSD of 5-hydroxytryptophan (at 280nm wavelength) and L-kynurenine (at 365nm wavelength) in the control solution were 0.10% and 0.33%, respectively, and the peak area RSD of tryptophan (at 280nm wavelength) in the control solution was 0.15%.
7. Linear test
Preparing a series of control solutions containing 5-hydroxytryptophan with the concentrations of 814.4, 407.2, 203.6, 40.72, 8.144, 4.072, 1.629, 0.8144, 0.3258 and 0.1629 mu g/ml and L-kynurenine with the concentrations of 1216, 608.0, 304.0, 60.80, 12.16, 6.080, 2.432, 1.216, 0.4864 and 0.2432 mu g/ml, injecting the control solutions into a liquid chromatograph under the same chromatographic conditions as those of example 1, wherein the linear relation between the concentrations of 5-hydroxytryptophan and peak areas is good, and the regression equation is as follows: Y0.2858X-0.0011, correlation coefficient r 1.0000, method linear range: 0.1629-814.4 mu g/ml; the linear relation between the L-kynurenine concentration and the peak area is good, and the regression equation is as follows: Y0.2014X-0.2513, correlation coefficient r 0.9999, method linear range: 0.2432-1216 μ g/ml.
8. Stability of test solution
A sample solution to be tested which was left at room temperature for 0 hour, 3 hours, 7 hours, 14 hours, 16 hours, 20 hours, 21 hours and 24 hours, a control solution containing 5-hydroxytryptophan at concentrations of 0.5105, 2.002 and 9.544. mu.g/ml and a control solution containing L-kynurenin at concentrations of 0.5285, 2.2016 and 9.304. mu.g/ml were taken and injected into a liquid chromatograph under the same chromatographic conditions as in example 1 to measure the peak areas of 5-OH-Trp and L-KY in the sample to be tested. 5-OH-Trp and L-KY are not detected in the test sample placed within 24 hours, and the compound amino acid injection has good stability at room temperature.
9. Accuracy of
2.0mL of 9 portions of the sample (lot No. 14121701) were precisely measured, and each of the 9 portions was placed in a 10mL volumetric flask, and 3 portions of each of control samples (5-hydroxytryptophan, L-kynurenine concentrations 19.92 and 19.82 μ g/mL) were precisely measured and added to the flask, and the solutions were diluted to the scale with 0.1mol/L HCl to prepare solutions having mass concentrations of about 1.90 μ g/mL, 3.80 μ g/mL and 7.60 μ g/mL, and the solutions were injected into a liquid chromatograph under the same chromatographic conditions as in example 1, wherein the average recovery rate of 5-hydroxytryptophan was 98.86%, the average recovery rate of RSD was 0.78% (n-9), the average recovery rate of L-kynurenine was 104.8%, and the average recovery rate of RSD was 1.78% (n-9).
10. Durability test
Preparing reference substance solutions containing 5-hydroxytryptophan and L-kynurenine with concentrations of 3.802 and 3.722 mu g/ml respectively, taking a test sample with a batch number of 14121701, filtering, injecting into a liquid chromatograph respectively, and observing the influence of the peak areas of 5-hydroxytryptophan and L-kynurenine in the reference substance solution and the influence of the peak areas of 5-hydroxytryptophan and L-kynurenine in the test sample solution under the conditions of flow rate +/-0.1 ml/min and column temperature +/-5 ℃ respectively under the chromatographic conditions.
The results show that the detection conditions change within a certain range (flow rate +/-0.1 ml/min, column temperature +/-5 ℃) and have little influence on the determination of the 5-hydroxytryptophan and the L-kynurenine.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (27)
1. A method for detecting related substances in compound amino acid injection is characterized by comprising the following steps:
(1) preparation of test solution and control solution:
taking the compound amino acid injection as a test solution directly;
preparing at least 3 5-hydroxytryptophan control solutions with different concentrations and at least 3L-kynurenine control solutions with different concentrations;
(2) injecting the 5-hydroxytryptophan reference substance solutions with different concentrations into a high performance liquid chromatograph, recording chromatograms of the 5-hydroxytryptophan reference substance solutions with different concentrations under the wavelength of 280nm, and making a standard curve or a regression equation by taking the concentration of 5-hydroxytryptophan as a horizontal coordinate and taking a peak area corresponding to the concentration as a vertical coordinate; injecting the L-kynurenine reference substance solutions with different concentrations into a high performance liquid chromatograph, recording chromatograms of the L-kynurenine reference substance solutions with different concentrations under the wavelength of 365nm, and making a standard curve or a regression equation by taking the concentration of the L-kynurenine as a horizontal ordinate and taking a peak area corresponding to the concentration as a vertical coordinate;
(3) injecting the test solution into a high performance liquid chromatograph, recording chromatograms of the test solution at 280nm and 365nm, calculating the amount of 5-hydroxytryptophan in the test solution according to a standard curve or a regression equation by using the peak area of 5-hydroxytryptophan in the chromatogram at 280nm of the test solution according to the standard curve or the regression equation and not exceeding 0.2% of the labeled amount of tryptophan in the compound amino acid injection, and calculating the amount of L-kynurenine in the test solution according to the standard curve or the regression equation by using the peak area of L-kynurenine in the chromatogram at 365nm of the test solution and not exceeding 0.2% of the labeled amount of tryptophan in the compound amino acid injection;
wherein, the compound amino acid injection contains tryptophan;
wherein, trifluoroacetic acid-heptafluorobutyric acid ion pair chromatography is adopted, the mobile phase A is 5.0mmol/L heptafluorobutyric acid-0.3 percent trifluoroacetic acid-water, the mobile phase B is acetonitrile, gradient elution is adopted, and the elution procedure is shown in the following table:
wherein the chromatographic column is a reversed phase C18 chromatographic column; the flow rate is 0.5-1.0 ml/min; the column temperature is 35-45 ℃.
2. The method according to claim 1, wherein the compound amino acid injection is compound amino acid 15 dipeptide 2 injection, compound amino acid injection 18AA-I, compound amino acid injection 18AA-II, compound amino acid injection 18AA-III, compound amino acid injection 18AA-V, compound amino acid injection 18 AA-VII, compound amino acid injection 20AA, compound amino acid injection 15AA, compound amino acid injection 14AA, compound amino acid injection 17 AA.
3. The method of claim 2, wherein the compound amino acid injection is a compound amino acid 15-dipeptide 2 injection.
4. The method of claim 2, wherein the compound amino acid injection 18AA is 5% or 12% in size.
5. The method of claim 2, wherein the compound amino acid injection 18AA-II is 5%, 8.5% or 11.4% in size.
6. The method according to claim 1, wherein in the step (1), a control solution containing the test solution is further prepared; in the step (2), further injecting the control solution into a high performance liquid chromatograph, and recording the chromatogram of the control solution under the wavelength of 280 nm; in the step (3), according to a self-comparison method, if other chromatographic peaks are detected in the 280nm wavelength chromatogram of the test solution, the content of a single peak area is not more than 0.5% of the tryptophan labeled amount in the compound amino acid injection through comparison with the peak area of the tryptophan in the comparison solution, and the content of a total peak area is not more than 5% of the tryptophan labeled amount in the compound amino acid injection through comparison with the peak area of the tryptophan in the comparison solution.
7. The method of claim 1, wherein the solvent used to prepare the control solution is dilute aqueous hydrochloric acid.
8. The method according to claim 7, wherein the solvent for preparing the control solution is an aqueous hydrochloric acid solution having a concentration of 0.1 mol/L.
9. The method of claim 6, wherein the solvent of the control solution is dilute aqueous hydrochloric acid.
10. The method according to claim 9, wherein the solvent of the control solution is an aqueous hydrochloric acid solution having a concentration of 0.1 mol/L.
11. The method of claim 1, wherein 3-7 control solutions of 5-hydroxytryptophan of different concentrations are prepared.
12. The method of claim 11, wherein 3-5 control solutions of 5-hydroxytryptophan of different concentrations are prepared.
13. The method of claim 1, wherein the plurality of control solutions used to make the standard curve or regression equation have a minimum concentration of 5-hydroxytryptophan that is less than 0.1% and a maximum concentration that is greater than 0.4% of the amount of tryptophan in the compound amino acid injection.
14. The method as claimed in claim 13, wherein the compound amino acid injection is compound amino acid 15 dipeptide 2 injection, the labeled amount of tryptophan is 0.95g/500ml, and the concentration ranges of 5-hydroxytryptophan in 3 reference solutions used for preparing standard curves or regression equations are 0.2-0.8 μ g/ml, 1.6-2.4 μ g/ml and 8-10 μ g/ml, respectively.
15. The method of claim 14, wherein the concentration of 5-hydroxytryptophan in the 3 control solutions is 0.5 μ g/ml, 2.0 μ g/ml, and 9.0 μ g/ml, respectively.
16. The method of claim 1, wherein 3 to 7 different concentrations of L-kynurenine control solutions are formulated.
17. The method of claim 16, wherein 3 to 5 different concentrations of L-kynurenine control solutions are formulated.
18. The method of claim 1, wherein the plurality of control solutions used to prepare the standard curve or regression equation have a minimum concentration of L-kynurenine less than 0.1% and a maximum concentration of L-kynurenine greater than 0.4% of the amount of tryptophan in the compound amino acid injection.
19. The method as claimed in claim 18, wherein the compound amino acid injection is compound amino acid 15 dipeptide 2 injection, the labeled amount of tryptophan is 0.95g/500ml, and the concentration ranges of L-kynurenine in 3 control solutions used for preparing standard curves or regression equations are 0.2-0.8 μ g/ml, 1.6-2.4 μ g/ml and 8-10 μ g/ml, respectively.
20. The method of claim 19, wherein the concentration of L-kynurenine in the 3 control solutions is 0.5 μ g/ml, 2.0 μ g/ml, 9.0 μ g/ml, respectively.
21. The method of claim 1, wherein the test solution is 1% by volume of the control solution, i.e., the test solution is diluted 100-fold with the solvent; if other chromatographic peaks are detected in the 280nm wavelength chromatogram of the test solution, the single peak area is not more than 0.5 times of the peak area of tryptophan in the control solution, and the total peak area is not more than 5 times of the peak area of tryptophan in the control solution.
22. The method of claim 1, wherein the chromatography column is 4.6mm x 250mm, 3 μm in size.
23. The method of claim 22, wherein the chromatography column is a reverse phase C18AQ chromatography column.
24. The method of claim 1, wherein the detector is an ultraviolet detector.
25. The method of claim 1, wherein the flow rate is 0.7-0.9 ml/min.
26. The method of claim 25, wherein the flow rate is 0.8 ml/min.
27. The method of claim 1, wherein the column temperature is 40 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810457668.7A CN108663448B (en) | 2018-05-14 | 2018-05-14 | A kind of detection method of related substances in compound amino acid injection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810457668.7A CN108663448B (en) | 2018-05-14 | 2018-05-14 | A kind of detection method of related substances in compound amino acid injection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108663448A CN108663448A (en) | 2018-10-16 |
| CN108663448B true CN108663448B (en) | 2021-07-13 |
Family
ID=63778387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810457668.7A Active CN108663448B (en) | 2018-05-14 | 2018-05-14 | A kind of detection method of related substances in compound amino acid injection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108663448B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406684B (en) * | 2018-12-26 | 2021-11-12 | 北京哈三联科技有限责任公司 | Detection method for determining content of impurity B, C, D in tryptophan |
| CN112578031A (en) * | 2019-09-30 | 2021-03-30 | 天津药业研究院股份有限公司 | Method for detecting tryptophan impurities and application |
| CN112697934A (en) * | 2020-12-10 | 2021-04-23 | 武汉久安药业有限公司 | Method for detecting content of pyroglutamic acid in compound amino acid injection |
| CN112858556B (en) * | 2021-01-14 | 2022-11-22 | 费森尤斯卡比华瑞制药有限公司 | Method for detecting tryptophan impurities in compound amino acid solution |
| CN112946099B (en) * | 2021-01-25 | 2022-12-13 | 石家庄四药有限公司 | Method for detecting related substances in amino acid glucose injection |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101776662A (en) * | 2010-01-15 | 2010-07-14 | 中南大学湘雅二医院 | Method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method |
| CN104391053A (en) * | 2014-10-30 | 2015-03-04 | 广东新峰药业股份有限公司 | PR-HPLC (phase reversing-high performance liquid chromatography) determination method for simultaneously determining contents of five amino acids in earthworm injection |
| CN107102152A (en) * | 2017-05-17 | 2017-08-29 | 中国医学科学院基础医学研究所 | The protein marker of urine myocardial infarction and its purposes in diagnosis and prognosis |
| CN107907603A (en) * | 2017-10-31 | 2018-04-13 | 华仁药业股份有限公司 | A kind of measure assay method of the amino acids parenteral solution tryptophan in relation to material |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2813580A1 (en) * | 2006-10-13 | 2014-12-17 | Metabolon, Inc. | Biomarkers related to metabolic age and methods using the same |
-
2018
- 2018-05-14 CN CN201810457668.7A patent/CN108663448B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101776662A (en) * | 2010-01-15 | 2010-07-14 | 中南大学湘雅二医院 | Method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method |
| CN104391053A (en) * | 2014-10-30 | 2015-03-04 | 广东新峰药业股份有限公司 | PR-HPLC (phase reversing-high performance liquid chromatography) determination method for simultaneously determining contents of five amino acids in earthworm injection |
| CN107102152A (en) * | 2017-05-17 | 2017-08-29 | 中国医学科学院基础医学研究所 | The protein marker of urine myocardial infarction and its purposes in diagnosis and prognosis |
| CN107907603A (en) * | 2017-10-31 | 2018-04-13 | 华仁药业股份有限公司 | A kind of measure assay method of the amino acids parenteral solution tryptophan in relation to material |
Non-Patent Citations (5)
| Title |
|---|
| Altered tryptophan metabolism in human meningioma;Noble Kumar Talari 等;《Journal of Neuro-Oncology volume》;20160729;第130卷(第1期);第69-77页 * |
| Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry;Lysiane Boulet 等;《Journal of Chromatography B》;20170404;第1054卷;第36-43页 * |
| 比值导数荧光光谱法同时测定色氨酸和5-羟基色氨酸;魏永锋 等;《分析试验室》;20060930;第25卷(第9期);第75-77页 * |
| 高效液相色谱-紫外、荧光双检测器同时测定人尿中犬尿氨酸和犬尿喹啉酸;赵建幸 等;《检验医学》;20090930;第24卷(第9期);第659-662页 * |
| 高效液相色谱-蒸发光散射检测法直接检测20种未衍生基本氨基酸;王玉红 等;《色谱》;20110930;第29卷(第9期);第908-911页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108663448A (en) | 2018-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108663448B (en) | A kind of detection method of related substances in compound amino acid injection | |
| CN107907603B (en) | Method for measuring tryptophan related substances in amino acid injection | |
| CN107367562A (en) | The analyzing detecting method of sulfuric acid Polymyxin B sulfate and application | |
| CN107991415B (en) | Simultaneous separation and determination of pyroglutamic acid and methionine sulfoxide impurities in compound amino acid injection 18AA by liquid chromatography | |
| CN106124667B (en) | A kind of methods of the separation determination Xi Gelieting in relation to substance | |
| CN112763622B (en) | Method for determining plamavir through liquid chromatography | |
| CN102841169B (en) | Method for measuring calcium levofolinate-related substances by using high performance liquid chromatography gradient method | |
| CN115856156B (en) | A method for determining enantiomers of polypeptide protected amino acids | |
| CN117192014A (en) | Detection method of eszopiclone isomer | |
| CN114689737B (en) | Analysis method of S-o-chlorophenylglycine methyl tartrate related substances | |
| CN113820409B (en) | Method for detecting related substances in mother nucleus of moxifloxacin | |
| CN112697934A (en) | Method for detecting content of pyroglutamic acid in compound amino acid injection | |
| CN112394112B (en) | Method for detecting content of hydroxychloroquine oxynitride impurities in hydroxychloroquine sulfate | |
| CN113567607A (en) | Method for detecting content of penicillin G acylase in cefaclor | |
| CN115015457A (en) | Method for analyzing bromoacetic acid impurities in serapager by high performance liquid chromatography | |
| CN108037221B (en) | Method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography | |
| CN117571877B (en) | Method for establishing high-efficiency liquid-phase characteristic spectrum of throat disease pill | |
| CN107748217B (en) | A Method for Establishing Shuangyu Granule GC Quantitative Fingerprint and Its Application | |
| CN108072709B (en) | Method for the determination of enantiomer content in the bulk drug of troxagliptin succinate | |
| CN115112811B (en) | Protein standard substance determination method based on pre-column derivatization-ultra-high performance liquid chromatography | |
| CN113777204B (en) | Detection method of p-hydroxyacetophenone related substances | |
| CN111380978A (en) | Method for simultaneously determining contents of coenzymes NADP and FAD in medicine | |
| CN119861169A (en) | High performance liquid chromatography detection method for 4-aminophenol-2, 6-dicarboxylic acid in mesalazine bulk drug | |
| CN112526013B (en) | Method for detecting concentration of related substances in ibuprofen medicament by using ultra-high liquid chromatography | |
| CN114200067B (en) | High performance liquid chromatography analysis method for 6-bromo-3-hydroxy pyrazine-2-carboxamide and impurities |
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 |