CN111812259A - Method for detecting alanyl glutamine impurity in compound amino acid injection - Google Patents
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Abstract
The invention belongs to the technical field of pharmaceutical chemistry detection, and particularly relates to a method for detecting alanyl glutamine impurities in a compound amino acid injection. The detection method comprises the following steps: preparing an L-pyroglutamic acid reference solution, preparing a compound amino acid injection (the component of which contains alanyl glutamine) test solution by adopting cation exchange resin, determining the conditions of high performance liquid chromatography, detecting the L-pyroglutamic acid reference solution and the compound amino acid injection test solution by adopting the high performance liquid chromatography, and calculating the impurity content of the alanyl glutamine in the test solution by using a correction factor method. The method can accurately detect the content of alanyl glutamine impurities in a complex sample, does not involve the use of an organic solvent in the experimental process, only uses low-price pyroglutamic acid as a reference substance, and greatly saves the daily detection cost.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry detection, and particularly relates to a method for detecting alanyl glutamine impurities in a compound amino acid injection.
Background
Alanyl glutamic acidAminoamide is a very important dipeptide in parenteral nutrition products, and can be rapidly decomposed into two amino acids which are necessary for human bodies in the human bodies: alanine and glutamic acid, and has no toxic and side effects, and the application of the alanine and the glutamic acid in parenteral nutrition is increasingly paid attention. Alanyl glutamine is degraded under the action of high temperature and other conditions, and for compound products, when a conventional HPLC (high performance liquid chromatography) method is used for detection, complex amino acid components interfere with the detection of four impurities of the alanyl glutamine, namely L-pyroglutamic acid, cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid). Simone[1]The detection of alanyl glutamine impurity in parenteral amino acid injection is carried out by LC-ESI-MS/MS, but the LC-ESI-MS/MS equipment is expensive and general laboratories do not have the condition. Simone[2]The two-dimensional reverse chromatography and HILIC separation mode is adopted by the people, and the IT-MS and the CAD are used as detectors to analyze the alanyl glutamine impurity in the parenteral amino acid injection, the method has expensive equipment and complex operation, and the condition is not provided in common laboratories.
Non-patent documents: [1] simone Schiesel, Michael Lammerhofer, Alexander Leitner, Wolfgang Lindner. journal of Chromatography A,1259(2012)111-120.
[2]Simone Schiesel,Michael Lammerhofer,Wolfgang Lindner.Journal ofChromatography A,1259(2012)100-110.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting alanyl glutamine impurity in compound amino acid injection aiming at the defects of the prior art. The detection method is simple, convenient, rapid and low in cost, the amino acid components in the compound amino acid injection are removed by using the cation exchange resin for adsorption, the subsequent measurement of alanyl glutamine impurities L-pyroglutamic acid, cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) is facilitated, the interference caused by the amino acid components is not needed to be worried about, the diluent and the mobile phase are both aqueous phases in the measurement process, the use of any organic solvent is not involved, the environment is friendly, and the cost is saved.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) preparing an L-pyroglutamic acid reference substance solution;
(2) injecting a proper amount of compound amino acid injection into a chromatographic column filled with cation exchange resin, leaching by using ultrapure water as an eluting solvent, and collecting eluent to obtain a test sample solution;
(3) determining the conditions of the high performance liquid chromatography:
a chromatographic column: the specification of the hydrophilic modified alkyl silane chemically bonded silica gel column is 50-250 mm multiplied by 2.1-4.6 mm, and the particle size of the filler is 1.7-5 mu m;
mobile phase: 5-50 mmol/L, diammonium hydrogen phosphate, pH 1.9-2.1;
flow rate: 0.9-1.1 mL/min;
column temperature: 23-27 ℃;
detection wavelength: 200-220 nm;
sample introduction volume: 5-50 mu L;
(4) detecting the L-pyroglutamic acid reference solution and the compound amino acid injection test solution by adopting a high performance liquid chromatography, and recording corresponding chromatograms;
(5) according to the experiment, the correction factors of the relative retention time and response of three impurities including cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) to L-pyroglutamic acid are calculated, and then the content of alanyl-glutamine impurity in the test solution is calculated according to the peak area of the L-pyroglutamic acid reference substance.
Further, the solvent used in the preparation of the reference solution in step (1) is pure water.
Further, in the step (2), when the compound amino acid injection is eluted by using cation exchange resin, the amino acid components are completely absorbed and removed by the cation exchange resin, and four impurities of L-pyroglutamic acid, cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) are completely eluted.
Further, the size of the column in step (3) was 4.6mm × 250mm, and the particle size was 5 μm.
Further, the mobile phase in the step (3) is (NH) with the concentration of 10mmol/L4)2HPO4The pH was 2.0.
Further, the flow rate in step (3) was set to 1 mL/min.
Further, the column temperature in step (3) was set to 25 ℃.
Further, the detection wavelength in step (3) is set to 205 nm.
Further, the sample amount in step (3) is set to 25 μ L.
Compared with the prior art, the invention has the following effects and benefits:
(1) according to the detection method, firstly, the amino acid components in the compound amino acid injection are removed by adsorption of the cation exchange resin, then the alanyl glutamine impurities are determined by using the conventional reversed-phase HPLC, and the amino acid components in the compound amino acid injection are removed by adsorption of the cation exchange resin, so that the alanyl glutamine impurities of L-pyroglutamic acid, cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) can be conveniently, accurately and quickly determined by using the conventional HPLC, and interference caused by the amino acid components is not worried.
(2) The method is characterized in that L-pyroglutamic acid which is low in price and easy to obtain is selected as a standard substance, and the relative retention time and the response correction factor of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) relative to the L-pyroglutamic acid are calculated through experiments, so that the content of four impurities in a sample can be accurately detected only by preparing an L-pyroglutamic acid reference solution in daily detection, and the time and the cost are greatly saved.
(3) In the experiment, the diluent and the mobile phase are both water phases, and the use of any organic solvent is not involved, so that the environment is friendly, and the cost is saved.
Drawings
FIG. 1 is an HPLC chromatogram of a control solution of L-pyroglutamic acid of the present invention.
FIG. 2 is an HPLC chromatogram of a test solution of the compound amino acid injection in example 1 of the present invention.
FIG. 3 is an HPLC chromatogram of a test solution of the compound amino acid injection in example 2 of the present invention.
FIG. 4 is an HPLC chromatogram of a test solution of the compound amino acid injection in example 3 of the present invention.
FIG. 5 is an HPLC chromatogram of a test solution of the compound amino acid injection in example 4 of the present invention.
FIG. 6 is an HPLC chromatogram of a test solution of the compound amino acid injection in example 5 of the present invention.
FIG. 7 is an HPLC chromatogram of a test solution of compound amino acid injection in example 6 of the present invention.
FIG. 8 is an HPLC chromatogram of a test solution of the compound amino acid injection in example 7 of the present invention.
Description of reference numerals: 1: l-pyroglutamic acid; 2: cyclo- (L-alanyl-glutamine); 3: L-pyroglutamyl-L-alanine; 4: cyclo- (L-alanyl-glutamic acid).
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Example 1
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of an L-pyroglutamic acid reference substance, placing the reference substance into a 100mL measuring flask, dissolving with a proper amount of water, diluting to scale to obtain a reference substance storage solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance storage solution into a 250mL measuring flask, diluting to scale with water, shaking up to obtain a reference substance working solution;
(2) weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L of (NH)4)2HPO4The pH value is 2.0;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by adopting HPLC and recording corresponding chromatograms as shown in figures 1-2;
(5) calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. The relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) with respect to L-pyroglutamic acid are respectively: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
Example 2
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of an L-pyroglutamic acid reference substance, placing the L-pyroglutamic acid reference substance in a 100mL measuring flask, dissolving with a proper amount of water and diluting to scale to obtain a reference substance storage solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance storage solution in a 250mL measuring flask, diluting with water to scale, and shaking up to obtain a reference substance working solution;
(2) weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L of (NH)4)2HPO4The pH value is 2.0;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 1.0 mL/min;
column temperature: 23 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by adopting HPLC and recording corresponding chromatograms, as shown in figure 3;
(5) calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. Wherein the relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) relative to L-pyroglutamic acid are respectively: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
Example 3
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of an L-pyroglutamic acid reference substance, placing the L-pyroglutamic acid reference substance in a 100mL measuring flask, dissolving with a proper amount of water and diluting to scale to obtain a reference substance storage solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance storage solution in a 250mL measuring flask, diluting with water to scale, and shaking up to obtain a reference substance working solution;
(2) weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L of (NH)4)2HPO4The pH value is 2.0;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 1.0 mL/min;
column temperature: 27 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by adopting HPLC and recording corresponding chromatograms, as shown in figure 4;
(5) calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. Wherein the relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) with respect to L-pyroglutamic acid are respectively about: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
Example 4
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of an L-pyroglutamic acid reference substance, placing the L-pyroglutamic acid reference substance in a 100mL measuring flask, dissolving with a proper amount of water and diluting to scale to obtain a reference substance storage solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance storage solution in a 250mL measuring flask, diluting with water to scale, and shaking up to obtain a reference substance working solution;
(2) weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L of (NH)4)2HPO4The pH value is 2.0;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 0.9 mL/min;
column temperature: 25 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by using HPLC and recording corresponding chromatograms, as shown in FIG. 5;
(5) calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. Wherein the relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) relative to L-pyroglutamic acid are respectively: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
Example 5
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of L-pyroglutamic acid reference substance, placing the L-pyroglutamic acid reference substance in a 100mL measuring flask, dissolving with appropriate amount of water, diluting to scale to obtain reference substance stock solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance stock solution in a 250mL measuring flask, diluting with water to scale, and shaking to obtain reference substance working solution.
(2) Weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L(NH)4)2HPO4The pH value is 2.0;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 1.1 mL/min;
column temperature: 25 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by using HPLC and recording corresponding chromatograms, as shown in FIG. 6;
(5) calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. Wherein the relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) relative to L-pyroglutamic acid are respectively: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
Example 6
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of an L-pyroglutamic acid reference substance, placing the L-pyroglutamic acid reference substance in a 100mL measuring flask, dissolving with a proper amount of water and diluting to scale to obtain a reference substance storage solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance storage solution in a 250mL measuring flask, diluting with water to scale, and shaking up to obtain a reference substance working solution;
(2) weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L of (NH)4)2HPO4The pH value is 1.9;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by using HPLC and recording corresponding chromatograms, as shown in FIG. 7;
step four, calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. Wherein the relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) relative to L-pyroglutamic acid are respectively: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
Example 7
A method for detecting alanyl glutamine impurity in compound amino acid injection comprises the following steps:
(1) accurately weighing 56mg of an L-pyroglutamic acid reference substance, placing the L-pyroglutamic acid reference substance in a 100mL measuring flask, dissolving with a proper amount of water and diluting to scale to obtain a reference substance storage solution, accurately transferring 1mL of the L-pyroglutamic acid reference substance storage solution in a 250mL measuring flask, diluting with water to scale, and shaking up to obtain a reference substance working solution;
(2) weighing 3.5g of Dowex 50w X8 resin into a 50mL beaker, soaking the resin in water for 1h, carefully filling the resin into a glass chromatographic column, leaching the glass chromatographic column with 30mL of water, precisely absorbing 2mL of sample into the chromatographic column, leaching the sample with water, and collecting eluent into a 100mL volumetric flask to obtain a sample solution;
(3) determining the conditions of the high performance liquid chromatography:
mobile phase: 10mmol/L of (NH)4)2HPO4The pH value is 2.1;
a chromatographic column: a hydrophilic modified alkyl silane chemical bonding silica gel column with the grain diameter of 5 mu m and the specification of 4.6 multiplied by 250 mm;
detection wavelength: 205 nm;
flow rate: 1.0 mL/min;
column temperature: 25 ℃;
sample introduction volume: 25 mu L of the solution;
(4) analyzing the L-pyroglutamic acid reference substance working solution and the compound amino acid test solution by using HPLC and recording corresponding chromatograms, as shown in FIG. 8;
(5) calculating the content of each impurity according to the peak areas of the impurities in the reference substance and the test substance, wherein the calculation formula is as follows:
in the formula
Cs: the content (mg/mL) of impurities to be detected in the sample;
cst: the content (mg/mL) of L-pyroglutamic acid in the working solution of the reference substance;
as: the peak area of the impurity to be detected in the test sample;
ast: the area average value of the chromatographic peak of the L-pyroglutamic acid in the reference substance working solution;
d: sample dilution factor, 50;
f: correction factor relative to L-pyroglutamic acid. Wherein the relative retention times of cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine, cyclo- (L-alanyl-glutamic acid) relative to L-pyroglutamic acid are respectively: 1.29, 1.44, 1.92; the relative correction factors are: 0.39, 0.60, 0.39.
The foregoing is illustrative of the preferred embodiments of the present invention only and is not to be construed as limiting the claims. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (9)
1. A method for detecting alanyl glutamine impurities in compound amino acid injection is characterized by comprising the following steps:
(1) preparing an L-pyroglutamic acid reference substance solution;
(2) injecting a proper amount of compound amino acid injection into a chromatographic column filled with cation exchange resin, leaching by using ultrapure water as an eluting solvent, and collecting eluent to obtain a test sample solution;
(3) determining the conditions of the high performance liquid chromatography:
a chromatographic column: the specification of the hydrophilic modified alkyl silane chemically bonded silica gel column is 50-250 mm multiplied by 2.1-4.6 mm, and the particle size of the filler is 1.7-5 mu m;
mobile phase: 5-50 mmol/L, diammonium hydrogen phosphate, pH 1.9-2.1;
flow rate: 0.9-1.1 mL/min;
column temperature: 23-27 ℃;
detection wavelength: 200-220 nm;
sample introduction volume: 5-50 mu L;
(4) detecting the L-pyroglutamic acid reference solution and the compound amino acid injection test solution by adopting a high performance liquid chromatography, and recording corresponding chromatograms;
(5) according to the experiment, the correction factors of the relative retention time and response of three impurities including cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) to L-pyroglutamic acid are calculated, and then the content of alanyl-glutamine impurity in the test solution is calculated according to the peak area of the L-pyroglutamic acid reference substance.
2. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the solvent used in the step (1) of preparing the reference solution is pure water.
3. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the compound amino acid injection in step (2) is eluted with cation exchange resin to ensure that all amino acid components are adsorbed and removed by cation resin, and all four impurities of L-pyroglutamic acid, cyclo- (L-alanyl-glutamine), L-pyroglutamyl-L-alanine and cyclo- (L-alanyl-glutamic acid) are eluted.
4. The method for detecting alanyl glutamine impurities in compound amino acid injection according to claim 1, wherein the specification of the chromatographic column in step (3) is 4.6mm x 250mm, and the particle size is 5 μm.
5. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the mobile phase in the step (3) is (NH) with a concentration of 10mmol/L4)2HPO4The pH was 2.0.
6. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the flow rate in step (3) is set to 1 mL/min.
7. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the column temperature in step (3) is set to 25 ℃.
8. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the detection wavelength in step (3) is set to 205 nm.
9. The method for detecting alanyl-glutamine impurities in compound amino acid injection according to claim 1, wherein the sample volume in step (3) is set to 25 μ L.
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