CN106950306B - Method for determining content of cysteine in compound amino acid injection - Google Patents
Method for determining content of cysteine in compound amino acid injection Download PDFInfo
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Abstract
The invention provides a method for measuring the content of cysteine in compound amino acid injection, which comprises the steps of preparing a cysteine aqueous solution as a reference solution; taking the compound amino acid injection as a test solution; deriving cysteine in the reference substance solution and the test substance solution by adopting N- (1-pyrene) maleimide; and performing high performance liquid chromatography analysis on the derived reference substance solution and the sample solution, and obtaining the content of cysteine in the compound amino acid injection by adopting an external standard method according to liquid chromatogram charts of the derived reference substance solution and the sample solution. The method provided by the invention can realize accurate determination of the cysteine content in the compound amino acid injection, and has the advantages of good specificity, good precision and higher recovery rate.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for determining the content of cysteine in compound amino acid injection.
Background
At present, the compound amino acid injection approved by the country has more than ten varieties, such as 18AA, 18AA-I, 18AA-II, 18AA-IV, 18AA-V and the like. Among the 20 amino acids that synthesize proteins, cysteine is the most unstable and is easily oxidized into cystine and other substances that are insoluble in water. Therefore, whether the content of the cysteine in the compound amino acid injection meets the specification directly reflects the product quality and the process level.
In the prior art, a colorimetric method is reported to be adopted for measuring the content of cysteine, specifically, a color developing agent and cysteine are used for carrying out color development reaction under the condition of a certain pH value, and a UV method is used for detecting; however, the colorimetric method is poor in specificity, and the color developing agent and other amino acids also develop color to different degrees under corresponding pH value conditions, so that the determination result is interfered. In the prior art, cysteine is oxidized into cystine by adopting a strong oxidant, and then the post-column derivation of ninhydrin is adopted to carry out determination by adopting an amino acid analyzer; however, the condition for oxidizing the cysteine by the method is violent and is not easy to control, the sensitivity of an amino acid analyzer is low, the cysteine with higher concentration is required, and the method is not suitable for measuring the content of the cysteine in the compound amino acid injection. In recent years, high performance liquid chromatography has been widely used for amino acid analysis, but derivatization of amino acids is generally required to improve detection sensitivity. In the prior art, the derivatization of cysteine by using iodoacetic acid-o-phthalaldehyde, iodoacetic acid-PICO TAG and N-ethylmaleimide as derivatization agents is reported, but the problems of multiple derivatization steps and interference of other amino acids exist, and the content of cysteine in the compound amino acid injection cannot be accurately determined.
Disclosure of Invention
The invention aims to provide a method for measuring the content of cysteine in compound amino acid injection, which has good specificity, good precision and higher recovery rate.
The invention provides a method for determining the content of cysteine in a compound amino acid injection, which comprises the following steps:
(1) preparing a cysteine water solution as a reference substance solution;
taking the compound amino acid injection as a test solution;
deriving cysteine in the reference substance solution and the test substance solution by adopting N- (1-pyrene) maleimide;
(2) and (2) carrying out high performance liquid chromatography analysis on the derivatized reference solution and the test solution obtained in the step (1), and obtaining the content of cysteine in the compound amino acid injection by adopting an external standard method according to liquid chromatogram of the derivatized reference solution and the test solution.
Preferably, the molar ratio of cysteine to N- (1-pyrene) maleimide upon derivatization in step (1) is 1: (1-3).
Preferably, the N- (1-pyrene) maleimide in the step (1) is used in the form of an N- (1-pyrene) maleimide acetonitrile solution.
Preferably, the mass concentration of the N- (1-pyrene) maleimide acetonitrile solution is 0.15-0.25 mg/mL.
Preferably, the temperature for derivation in the step (1) is 15-25 ℃.
Preferably, the derivation time in the step (1) is 15-25 min.
Preferably, the HPLC analysis in step (2) is performed by using a PDA detector.
Preferably, the detection wavelength of the HPLC analysis in the step (2) is 274 nm.
Preferably, the HPLC analysis in step (2) is performed by using an octadecylsilane bonded silica gel chromatographic column.
Preferably, the column temperature of the HPLC analysis in the step (2) is 30-40 ℃.
The invention provides a method for measuring the content of cysteine in compound amino acid injection, which comprises the steps of preparing a cysteine aqueous solution as a reference solution; taking the compound amino acid injection as a test solution; deriving cysteine in the reference substance solution and the test substance solution by adopting N- (1-pyrene) maleimide; and performing high performance liquid chromatography analysis on the derived reference substance solution and the sample solution, and obtaining the content of cysteine in the compound amino acid injection by adopting an external standard method according to liquid chromatogram charts of the derived reference substance solution and the sample solution. The method provided by the invention can realize accurate determination of the cysteine content in the compound amino acid injection, has good specificity, good precision and higher recovery rate, can indirectly reflect the degradation condition of other amino acids in the compound amino acid injection through the change of the cysteine content, and effectively controls the quality of the compound amino acid injection product.
Drawings
FIG. 1 is a graph of the ultraviolet absorption spectrum of a cysteine derivative;
FIG. 2 is a graph showing an ultraviolet absorption spectrum of N- (1-pyrene) maleimide;
FIG. 3 is a liquid chromatogram of a negative control solution in example 1;
FIG. 4 is a liquid chromatogram of the test solution of example 1;
FIG. 5 is a liquid chromatogram of a negative control solution of example 2;
FIG. 6 is a liquid chromatogram of the test solution of example 2;
FIG. 7 is a liquid chromatogram of a negative control solution of example 3;
FIG. 8 is a liquid chromatogram of the test solution in example 3.
Detailed Description
The invention provides a method for determining the content of cysteine in a compound amino acid injection, which comprises the following steps:
(1) preparing a cysteine water solution as a reference substance solution;
taking the compound amino acid injection as a test solution;
deriving cysteine in the reference substance solution and the test substance solution by adopting N- (1-pyrene) maleimide;
(2) and (2) carrying out high performance liquid chromatography analysis on the derivatized reference solution and the test solution obtained in the step (1), and obtaining the content of cysteine in the compound amino acid injection by adopting an external standard method according to liquid chromatogram of the derivatized reference solution and the test solution.
The invention prepares cysteine water solution as reference solution. The concentration of the reference solution is not particularly limited in the present invention, and may be a concentration matching cysteine in the test solution, which is well known to those skilled in the art. The method for preparing the solution of the reference substance is not particularly limited in the present invention, and the solution preparation method known to those skilled in the art may be adopted.
The invention uses the compound amino acid injection as a test solution. The invention has no special limitation on the type of the compound amino acid injection, and the compound amino acid injection containing cysteine, which is well known to the technical personnel in the field, can be adopted, and the specific type is 18AA-V, 18AA-VII or 18 AA-IX.
According to the invention, N- (1-pyrene) maleimide is adopted to derivatize cysteine in the reference solution and the test solution. The N- (1-pyrene) maleimide is stable in structure and not easy to degrade, the N- (1-pyrene) maleimide is used as a derivatization agent, cysteine derivatives obtained after derivatization of cysteine have strong absorption in an ultraviolet region, interference of other amino acids in the compound amino acid injection can be shielded, and accurate determination of the cysteine content in the compound amino acid injection is realized. In the present invention, the reaction formula of N- (1-pyrene) maleimide and cysteine is as follows:
in the present invention, the molar ratio of cysteine to N- (1-pyrene) maleimide at the time of derivatization is preferably 1: (1-3), specifically 1: 1. 1: 2 or 1: 3.
in the present invention, the N- (1-pyrene) maleimide is preferably used in the form of an N- (1-pyrene) maleimide acetonitrile solution. In the invention, the mass concentration of the N- (1-pyrene) maleimide acetonitrile solution is preferably 0.15-0.25 mg/mL, more preferably 0.18-0.22 mg/mL, and most preferably 0.20 mg/mL.
In the invention, the pH value of the reaction system when the N- (1-pyrene) maleimide derivatizes the cysteine is preferably 5.6-6.4. When the cysteine is derived, the compound amino acid injection is preferably diluted according to the pH value and the concentration of the total amino acid contained in the compound amino acid injection. Therefore, on one hand, the pH value requirement of a reaction system during derivatization is met, and on the other hand, the overload of a chromatographic column or the adverse effect on high performance liquid chromatography equipment cannot be caused during subsequent high performance liquid chromatography analysis. In the embodiment of the invention, when the cysteine content in the compound amino acid injection 18AA-V, 18AA-VII or 18AA-IX is measured, the compound amino acid injection is diluted by 5 times, and then mixed with N- (1-pyrene) maleimide acetonitrile solution for derivatization reaction.
In the invention, the derivation temperature is preferably 15-25 ℃, more preferably 18-22 ℃, and most preferably 20 ℃; the derivation time is preferably 15-25 min, more preferably 18-22 min, and most preferably 20 min.
After the derivatization is completed, the method carries out high performance liquid chromatography analysis on the obtained derivatized reference substance solution and the sample solution, and obtains the content of cysteine in the compound amino acid injection by adopting an external standard method according to the liquid chromatogram of the derivatized reference substance solution and the sample solution.
The mobile phase and the elution mode adopted in the high performance liquid chromatography are not particularly limited, and the mobile phase and the elution mode which are well known to those skilled in the art can be adopted according to the type of the compound amino acid injection to be detected.
Specifically, in the invention, when the compound amino acid injection 18AA-V is used as a sample, the following gradient elution procedure is preferably adopted for the high performance liquid chromatography:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 18 | 40 | 60 |
| 25 | 10 | 90 |
| 33 | 10 | 90 |
| 34 | 90 | 10 |
| 40 | 90 | 10 |
Wherein the mobile phase A is 0.01mol/L sodium dihydrogen phosphate solution, and the pH value of the mobile phase A is 2.4-3.4; mobile phase B was acetonitrile.
In the invention, the pH value of the mobile phase A is preferably 2.4-3.4, and more preferably 2.8 +/-0.1. The pH value of the mobile phase A is preferably adjusted by phosphoric acid; the mass percentage concentration of the phosphoric acid is preferably 8-12%, and more preferably 10%.
Specifically, in the invention, when the compound amino acid injection 18AA-VII is used as a sample, the following gradient elution procedure is preferably adopted for the high performance liquid chromatography analysis:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
Wherein the mobile phase A is a 0.015mol/L sodium acetate solution, and the pH value of the mobile phase A is 2.8 +/-0.1; mobile phase B was acetonitrile.
The pH value of the mobile phase A is preferably adjusted by phosphoric acid; the mass percentage concentration of the phosphoric acid is preferably 8-12%, and more preferably 10%.
Specifically, in the present invention, when the compound amino acid injection 18AA-IX is used as a sample, the following gradient elution procedure is preferably adopted for the hplc analysis:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
| 30 | 90 | 10 |
Wherein the mobile phase A is 0.01mol/L sodium dihydrogen phosphate solution, and the pH value of the mobile phase A is 2.8 +/-0.1; mobile phase B was acetonitrile.
The pH value of the mobile phase A is preferably adjusted by phosphoric acid; the mass percentage concentration of the phosphoric acid is preferably 8-12%, and more preferably 10%.
In the invention, the flow rate of the mobile phase is preferably 0.8-1.2 mL/min, and more preferably 1.0 mL/min.
In the present invention, the high performance liquid chromatography preferably employs a PDA detector.
In the present invention, the detection wavelength of the high performance liquid chromatography is preferably 274 nm. The invention adopts a PDA detector to detect cysteine derivatives and N- (1-pyrene) maleimide, and obtains ultraviolet absorption spectrograms of the cysteine derivatives and the N- (1-pyrene) maleimide, which are shown in a figure 1 and a figure 2. As can be seen from FIGS. 1 and 2, the UV absorption spectra of the cysteine derivative and N- (1-pyrene) maleimide are similar, and the cysteine derivative and N- (1-pyrene) maleimide have larger absorption peaks at a wavelength of 274nm, and most amino acids do not show peaks at the wavelength, so that the interference of other amino acids is avoided.
In the present invention, the high performance liquid chromatography preferably employs an octadecylsilane bonded silica chromatographic column.
In the invention, the column temperature of the high performance liquid chromatography is preferably 30-40 ℃, more preferably 33-37 ℃ and most preferably 35 ℃.
In the present invention, the amount of the sample to be subjected to the HPLC analysis is preferably 10. mu.L.
And analyzing by the high performance liquid chromatography to obtain liquid chromatograms of the derived reference solution and the test solution, and obtaining the content of the cysteine in the compound amino acid injection by adopting an external standard method. In practical application, the content of the cysteine hydrochloride can be calculated according to the content of the cysteine, so that the content of the cysteine hydrochloride in the compound amino acid injection is determined.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Determining the content of cysteine hydrochloride in the compound amino acid injection 18AA-V
(1) The method for determining the content of cysteine hydrochloride in the compound amino acid injection 18AA-V by taking the compound amino acid 18AA-V injection prepared by Hubei Yisemiday pharmaceutical company Limited as a sample comprises the following steps:
(11) precisely weighing cysteine, mixing with water, and preparing 0.06mg/mL cysteine aqueous solution as a reference solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-V, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
mixing N- (1-pyrene) maleimide with acetonitrile to obtain an N- (1-pyrene) maleimide acetonitrile solution with the mass concentration of 0.2mg/mL, wherein the N- (1-pyrene) maleimide acetonitrile solution is used as a derivatization agent solution;
(12) mixing 0.5mL of reference substance solution and 0.5mL of test substance solution in the step (11) with 1mL of derivatization agent solution respectively, and performing derivatization for 20min at the temperature of 20 ℃;
(13) performing high performance liquid chromatography analysis on the derivatized reference solution and the sample solution obtained in the step (12), and obtaining the content of cysteine in the derivatized sample solution by adopting an external standard method according to liquid chromatogram charts of the derivatized reference solution and the sample solution, so as to obtain the content of cysteine in the compound amino acid injection 18 AA-V; calculating the content of cysteine hydrochloride by the content of cysteine to obtain the content of cysteine hydrochloride in the compound amino acid injection 18 AA-V;
wherein, the conditions of the high performance liquid chromatography analysis are as follows:
gradient elution procedure:
wherein the mobile phase A is 0.01mol/L sodium dihydrogen phosphate solution, and the pH value of the mobile phase A is 2.8 +/-0.1; the mobile phase B is acetonitrile;
the flow rate of the mobile phase is 1.0 mL/min;
a PDA detector is adopted, and the detection wavelength is 274 nm;
octadecylsilane chemically bonded silica chromatographic column with the column temperature of 35 ℃;
the amount of sample was 10. mu.L.
(2) Method specificity test
Preparing a mixed solution without cysteine according to the formula of the compound amino acid injection 18AA-V, and diluting the mixed solution by 5 times with water to be used as a negative control solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-V, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
after the negative control solution and the test solution were derivatized as described in step (12), high performance liquid chromatography was performed as described in step (13), and the results are shown in FIGS. 3 and 4. FIG. 3 is a liquid chromatogram of a negative control solution, in which A is a hetero-peak at 12.153min and 2 is N- (1-pyrene) maleimide; FIG. 4 is a liquid chromatogram of a sample solution, in which 1 is a cysteine derivative and 2 is N- (1-pyrene) maleimide. As can be seen from fig. 3 and 4, the negative control solution had no spectral peak at the retention time of the cysteine derivative, and the hetero-peak at 12.153min was not interfering with the cysteine assay; unreacted N- (1-pyrene) maleimide after derivatization did not interfere with cysteine determination.
The purity of the chromatographic peak of the cysteine derivative in the test solution was measured by a PDA detector, and the results are shown in table 1. As can be seen from Table 1, the chromatographic peak of the cysteine derivative is a single pure chromatographic peak, and the method specificity is good.
TABLE 1 chromatographic peak purity test results for cysteine derivatives in test solutions
(3) Linear test
Preparing a mixed solution without cysteine according to the formula of the compound amino acid injection 18AA-V, and diluting the mixed solution by 5 times with water to be used as a negative control solution;
accurately weighing 74.2mg of cysteine, and mixing with 500mL of the negative control solution to obtain a cysteine standard solution; precisely measuring cysteine standard solutions respectively, and adding water to dilute to obtain cysteine standard solutions with mass concentrations of 10.2 mu g/mL, 30.7 mu g/mL, 61.4 mu g/mL, 71.6 mu g/mL and 102.3 mu g/mL respectively;
after the standard cysteine solutions with different dilution concentrations were derived according to the method described in step (12), high performance liquid chromatography was performed according to the method described in step (13) to obtain cysteine derivativesThe peak area of the cysteine is a vertical coordinate, the concentration of the cysteine is a horizontal coordinate, and a linear equation is made on the peak area to the concentration to obtain a standard curve of the cysteine; the standard curve equation, correlation coefficient and linear range of the cysteine are shown in table 2. As can be seen from Table 2, cysteine was linear well within the range of 10.2 to 102.3. mu.g/mL, and the correlation coefficient R was2Is 0.9995.
TABLE 2 standard curve equation, correlation coefficient and Linear Range for cysteine
In Table 2, X represents the mass concentration of cysteine, and Y represents the peak area of the liquid chromatogram of the cysteine derivative.
(4) Standard recovery test
Precisely weighing cysteine, mixing with water, and preparing 0.060mg/mL cysteine aqueous solution as a reference substance solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-V, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
precisely weighing cysteine respectively, mixing the cysteine with the test solution to be tested, preparing three parts of mixed solutions with cysteine mass concentrations of 0.048mg/mL, 0.060mg/mL and 0.072mg/mL respectively (the cysteine concentrations are 80%, 100% and 120% of the measured concentration of 0.060mg/mL respectively) as standard adding recovery test solutions;
and (3) after the reference substance solution and the standard-added recovery test solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), calculating according to an external standard method, and calculating the content of cysteine hydrochloride according to the content of cysteine to obtain the recovery rate of cysteine hydrochloride, wherein the result is shown in table 3. As can be seen from table 3, the recovery rate of cysteine hydrochloride was between 97.51% and 101.78%, the average recovery rate was 100.2%, and RSD (%) was 1.4(n ═ 9), indicating that the measurement results were accurate.
TABLE 3 results of the spiked recovery test
(5) Repeatability test and intermediate precision test
Repeatability test
Taking compound amino acid injection 18AA-V with product batch number of 20150101, prepared by Hubei Yisemitian pharmaceutical Co Ltd, as a sample, and diluting the compound amino acid injection 18AA-V by 5 times with water to be used as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.06mg/mL cysteine aqueous solution as a reference solution;
after the test solution and the reference solution are derived according to the method in the step (12), high performance liquid chromatography analysis is carried out according to the method in the step (13), the content of cysteine hydrochloride is calculated according to an external standard method, the content of cysteine is calculated through the content conversion of cysteine, experiments are carried out for 6 times continuously, the peak area of the cysteine derivative and the labeled content of cysteine hydrochloride are compared for each time, and RSD (%) is calculated, and the result is shown in Table 4.
TABLE 4 results of the repeatability tests
| Repeatability test | Peak area of cysteine derivative | Labeled content of cysteine hydrochloride (%) |
| 1 | 3224110 | 90.16 |
| 2 | 3223125 | 90.14 |
| 3 | 3168773 | 88.62 |
| 4 | 3308234 | 92.52 |
| 5 | 3264078 | 91.28 |
| 6 | 3216250 | 89.95 |
| RSD(％) | 1.46 | 1.46 |
Intermediate precision test
The tester A and the tester B adopt different instruments to perform the following intermediate precision tests at different test time:
taking compound amino acid injection 18AA-V with product batch number of 20150101, prepared by Hubei Yisemitian pharmaceutical Co Ltd, as a sample, and diluting the compound amino acid injection 18AA-V by 5 times with water to be used as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.06mg/mL cysteine aqueous solution as a reference solution;
after the test solution and the reference solution are derived according to the method in the step (12), high performance liquid chromatography analysis is carried out according to the method in the step (13), the content of cysteine hydrochloride is calculated according to an external standard method, the content of cysteine is calculated through the content conversion of cysteine, experiments are carried out for 6 times continuously, the peak area of the cysteine derivative and the labeled content of cysteine hydrochloride are compared for each time, and RSD (%) is calculated, and the result is shown in Table 5.
TABLE 5 results of intermediate precision test
| Intermediate precision test | Peak area of cysteine derivative | Labeled content of cysteine hydrochloride (%) |
| 1 | 3297537 | 90.17 |
| 2 | 3189865 | 87.22 |
| 3 | 3238807 | 88.56 |
| 4 | 3308464 | 90.47 |
| 5 | 3206619 | 87.68 |
| 6 | 3286616 | 89.87 |
| RSD(％) | 1.54 | 1.54 |
The average value of 12 marked content data of the cysteine hydrochloride in the repeatability test and the intermediate precision test is 89.72 percent, and the RSD (%) is 1.7 percent, which shows that the method provided by the invention has good precision.
(6) Stability test
Taking compound amino acid injection 18AA-V of which the product batch number is 20150101 and which is prepared by Hubei Yitianmian pharmaceutical Co., Ltd as a sample, sampling at room temperature (22 ℃) for 0h, 1h, 2h and 4h after opening a rubber plug, diluting the compound amino acid 18AA-V injection by 5 times with water respectively, after deriving according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating RSD (%), wherein the results are shown in Table 6, which indicates that the stability of the sample in 4h after opening the seal is good.
TABLE 6 stability test results
(7) Sample assay
Taking 18AA-V of compound amino acid injection prepared by Hubei semiday pharmaceutical company as a sample (the specification of the sample is 100mL and 500mL respectively), and diluting the 18AA-V of compound amino acid injection by 5 times with water to obtain a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.06mg/mL cysteine aqueous solution as a reference solution;
after the sample solution and the reference solution are derived according to the method in the step (12), high performance liquid chromatography analysis is carried out according to the method in the step (13), the content of cysteine hydrochloride is calculated according to an external standard method, and the result is shown in tables 7 and 8.
TABLE 7 measurement results of samples (specification 100mL)
TABLE 8 measurement results of samples (500 mL standard)
As can be seen from the example 1, the method provided by the invention can realize accurate determination of the cysteine content in the compound amino acid injection 18AA-V, and has the advantages of good specificity, good precision and higher recovery rate. The method provided by the invention can effectively control the quality of the compound amino acid injection 18AA-V product, and simultaneously comprehensively considers factors such as large-scale production, storage and transportation, and indirectly reflects that other amino acids in the compound amino acid injection 18AA-V are not degraded under the condition that the content of cysteine hydrochloride is not lower than 70% of the marked content.
Example 2
Determining the content of cysteine in compound amino acid injection 18AA-VII
(1) The method for determining the content of cysteine in the compound amino acid injection 18AA-VII by taking the compound amino acid 18AA-VII injection prepared by Hubei Yisemiday pharmacy Co Ltd as a sample comprises the following steps:
(11) precisely weighing cysteine, mixing with water, and preparing 0.07mg/mL cysteine aqueous solution as a reference solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-VII, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
mixing N- (1-pyrene) maleimide with acetonitrile to obtain an N- (1-pyrene) maleimide acetonitrile solution with the mass concentration of 0.2mg/mL, wherein the N- (1-pyrene) maleimide acetonitrile solution is used as a derivatization agent solution;
(12) mixing 0.5mL of reference substance solution and 0.5mL of test substance solution in the step (11) with 1mL of derivatization agent solution respectively, and performing derivatization for 20min at the temperature of 20 ℃;
(13) performing high performance liquid chromatography analysis on the derivatized reference solution and the sample solution obtained in the step (12), and obtaining the content of cysteine in the derivatized sample solution by adopting an external standard method according to liquid chromatogram charts of the derivatized reference solution and the sample solution, so as to obtain the content of cysteine in the compound amino acid injection 18 AA-VII;
wherein, the conditions of the high performance liquid chromatography analysis are as follows:
gradient elution procedure:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
Wherein the mobile phase A is a 0.015mol/L sodium acetate solution, and the pH value of the mobile phase A is 2.8 +/-0.1; mobile phase B was acetonitrile.
The flow rate of the mobile phase is 1.0 mL/min;
a PDA detector is adopted, and the detection wavelength is 274 nm;
octadecylsilane chemically bonded silica chromatographic column with the column temperature of 35 ℃;
the amount of sample was 10. mu.L.
(2) Method specificity test
Preparing a mixed solution without cysteine according to the formula of the compound amino acid injection 18AA-VII, and diluting the mixed solution by 5 times with water to be used as a negative control solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-VII, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
after the negative control solution and the test solution were derivatized as described in step (12), high performance liquid chromatography was performed as described in step (13), and the results are shown in FIGS. 5 and 6. FIG. 5 is a liquid chromatogram of a negative control solution, in which 2 is N- (1-pyrene) maleimide; FIG. 6 is a liquid chromatogram of a sample solution, in which 1 is a cysteine derivative and 2 is N- (1-pyrene) maleimide. As can be seen from FIGS. 5 and 6, the negative control solution had no peak at the retention time of the cysteine derivative; unreacted N- (1-pyrene) maleimide after derivatization did not interfere with cysteine determination.
The purity of the chromatographic peak of the cysteine derivative in the test solution was measured by a PDA detector, and the results are shown in Table 9. As can be seen from Table 9, the chromatographic peak of the cysteine derivative was a single pure chromatographic peak, and the method specificity was good.
TABLE 9 chromatographic peak purity test results for cysteine derivatives in test solutions
(3) Linear test
Preparing a mixed solution without cysteine according to the formula of the compound amino acid injection 18AA-VII, and diluting the mixed solution by 5 times with water to be used as a negative control solution;
accurately weighing 100.0mg of cysteine, and mixing with 100mL of the negative control solution to obtain a cysteine standard solution; precisely measuring cysteine standard solutions respectively, and adding water to dilute to obtain cysteine standard solutions with mass concentrations of 10.0 [ mu ] g/mL, 30.0 [ mu ] g/mL, 50.0 [ mu ] g/mL, 70.0 [ mu ] g/mL and 90.0 [ mu ] g/mL respectively;
after the obtained cysteine standard solutions with different dilution concentrations are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and taking the peak area of the cysteine derivative as a vertical coordinate, the concentration of cysteine as a horizontal coordinate, and taking a linear equation of the peak area to the concentration to obtain a standard curve of cysteine; the standard curve equation, correlation coefficient and linear range of the cysteine are shown in Table 10. As can be seen from Table 10, cysteine was linear well within the range of 10.0 to 90.0. mu.g/mL, and the correlation coefficient R was2Is 0.9997.
TABLE 10 standard curve equation, correlation coefficient and Linear Range for cysteine
In Table 10, X represents the mass concentration of cysteine, and Y represents the peak area of the liquid chromatogram of the cysteine derivative.
(4) Standard recovery test
Precisely weighing cysteine, mixing with water, and preparing 0.070mg/mL cysteine aqueous solution as a reference solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-VII, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
precisely weighing cysteine respectively, mixing the cysteine with the test solution to be tested, preparing three parts of mixed solution (the cysteine concentration is 80%, 100% and 120% of the determination concentration of 0.070mg/mL respectively) with the cysteine mass concentration of 0.056mg/mL, 0.070mg/mL and 0.084mg/mL respectively, and taking the three parts as the standard-adding recovery test solution;
and (3) after the reference substance solution and the standard adding recovery test solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating according to an external standard method to obtain the recovery rate of the cysteine, wherein the results are shown in a table 11. As can be seen from table 11, the recovery rate of cysteine was between 99.10% and 100.90%, the average recovery rate was 100.08%, and RSD (%) was 0.63(n ═ 9), indicating that the measurement results were accurate.
TABLE 11 results of the normalized recovery test
(5) Repeatability test and intermediate precision test
Repeatability test
Taking compound amino acid injection 18AA-VII of which the product batch number is 20140601 and which is prepared by Hubei Yisemitian pharmaceutical company Limited as a sample, and diluting the compound amino acid injection 18AA-VII by 5 times by using water to serve as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.07mg/mL cysteine aqueous solution as a reference solution;
and (3) after the test solution and the reference solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), calculating according to an external standard method, continuously performing experiments for 6 times, comparing the peak area of the cysteine derivative and the labeled content of cysteine for each time, and calculating RSD (%), wherein the results are shown in a table 12.
TABLE 12 results of the repeatability tests
| Repeatability test | Peak area of cysteine derivative | Labeled content of cysteine (%) |
| 1 | 3547609 | 90.5 |
| 2 | 3578264 | 91.2 |
| 3 | 3631192 | 92.6 |
| 4 | 3591465 | 91.6 |
| 5 | 3541780 | 90.3 |
| 6 | 3551058 | 90.5 |
| RSD(％) | 0.97 | 0.97 |
Intermediate precision test
The tester A and the tester B adopt different instruments to perform the following intermediate precision tests at different test time:
taking compound amino acid injection 18AA-VII of which the product batch number is 20140601 and which is prepared by Hubei Yisemitian pharmaceutical company Limited as a sample, and diluting the compound amino acid injection 18AA-VII by 5 times by using water to serve as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.07mg/mL cysteine aqueous solution as a reference solution;
and (3) after the test solution and the reference solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), calculating according to an external standard method, continuously performing experiments for 6 times, comparing the peak area of the cysteine derivative and the labeled content of cysteine for each time, and calculating RSD (%), wherein the results are shown in a table 13.
TABLE 13 results of intermediate precision test
| Intermediate precision test | Peak area of cysteine derivative | Labeled content of cysteine (%) |
| 1 | 3541534 | 90.3 |
| 2 | 3545456 | 90.4 |
| 3 | 3576832 | 91.2 |
| 4 | 3470939 | 88.5 |
| 5 | 3537612 | 90.2 |
| 6 | 3557222 | 90.7 |
| RSD(％) | 1.01 | 1.01 |
The average value of 12 marked content data of cysteine in the repeatability test and the intermediate precision test is 90.67 percent, and the RSD (%) is 1.08 percent, which shows that the method provided by the invention has good precision.
(6) Stability test
Taking compound amino acid injection 18AA-VII of product lot number 20140601 prepared by Hubei Yimiantian pharmacy Co., Ltd as a sample, opening a rubber plug at room temperature (22 ℃), sampling at 0h, 1h, 2h, 3h and 4h respectively, diluting the compound amino acid 18AA-VII injection by 5 times with water respectively, after the derivative is obtained according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating RSD (%), wherein the results are shown in Table 14, which indicates that the stability of the sample after the seal is opened is good within 4 h.
TABLE 14 stability test results
(7) Sample assay
Taking three batches of compound amino acid injection 18AA-VII prepared by small test and pilot test of Hubei one and a half day pharmaceutical company Limited as samples, and diluting the compound amino acid injection 18AA-VII by 5 times by using water to serve as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.07mg/mL cysteine aqueous solution as a reference solution;
and (3) after the test solution and the reference solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating according to an external standard method to obtain the labeled content of cysteine, wherein the results are shown in a table 15.
TABLE 15 results of sample measurement
As can be seen from the example 2, the method provided by the invention can realize accurate determination of the cysteine content in the compound amino acid injection 18AA-VII, and has the advantages of good specificity, good precision and higher recovery rate. The method provided by the invention can effectively control the quality of the compound amino acid injection 18AA-VII product, and simultaneously comprehensively considers factors such as large-scale production, storage and transportation, and indirectly reflects that other amino acids in the compound amino acid injection 18AA-V are not degraded under the condition that the cysteine content is not lower than 80% of the marked content.
Example 3
Determining the content of cysteine in compound amino acid injection 18AA-IX
(1) The method for determining the content of cysteine in the compound amino acid injection 18AA-IX by taking the compound amino acid 18AA-IX injection prepared by Hubei Yisemiday pharmacy Co Ltd as a sample comprises the following steps:
(11) precisely weighing cysteine, mixing with water, and preparing 0.05mg/mL cysteine aqueous solution as a reference solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-IX, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
mixing N- (1-pyrene) maleimide with acetonitrile to obtain an N- (1-pyrene) maleimide acetonitrile solution with the mass concentration of 0.2mg/mL, wherein the N- (1-pyrene) maleimide acetonitrile solution is used as a derivatization agent solution;
(12) mixing 0.5mL of reference substance solution and 0.5mL of test substance solution in the step (11) with 1mL of derivatization agent solution respectively, and performing derivatization for 20min at the temperature of 20 ℃;
(13) performing high performance liquid chromatography analysis on the derivatized reference solution and the sample solution obtained in the step (12), and obtaining the content of cysteine in the derivatized sample solution by an external standard method according to liquid chromatogram charts of the derivatized reference solution and the sample solution, so as to obtain the content of cysteine in the compound amino acid injection 18 AA-IX;
wherein, the conditions of the high performance liquid chromatography analysis are as follows:
gradient elution procedure:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
| 30 | 90 | 10 |
Wherein the mobile phase A is 0.01mol/L sodium dihydrogen phosphate solution, and the pH value of the mobile phase A is 2.8 +/-0.1; the mobile phase B is acetonitrile;
the flow rate of the mobile phase is 1.0 mL/min;
a PDA detector is adopted, and the detection wavelength is 274 nm;
octadecylsilane chemically bonded silica chromatographic column with the column temperature of 35 ℃;
the amount of sample was 10. mu.L.
(2) Method specificity test
Preparing a mixed solution without cysteine according to the formula of the compound amino acid injection 18AA-IX, and diluting the mixed solution by 5 times with water to be used as a negative control solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-IX, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
after the negative control solution and the test solution were derivatized as described in step (12), high performance liquid chromatography was performed as described in step (13), and the results are shown in FIGS. 7 and 8. FIG. 7 is a liquid chromatogram of a negative control solution, in which 2 is N- (1-pyrene) maleimide; FIG. 8 is a liquid chromatogram of a sample solution, in which 1 is a cysteine derivative and 2 is N- (1-pyrene) maleimide. As can be seen from FIGS. 7 and 8, the negative control solution had no peak at the retention time of the cysteine derivative; unreacted N- (1-pyrene) maleimide after derivatization did not interfere with cysteine determination.
The purity of the chromatographic peak of the cysteine derivative in the test sample solution was measured by a PDA detector, and the results are shown in Table 16. As can be seen from Table 16, the chromatographic peak of the cysteine derivative was a single pure chromatographic peak, and the method specificity was good.
TABLE 16 chromatographic peak purity test results for cysteine derivatives in test solutions
(3) Linear test
Preparing a mixed solution without cysteine according to the formula of the compound amino acid injection 18AA-IX, and diluting the mixed solution by 5 times with water to be used as a negative control solution;
accurately weighing 97.6mg of cysteine, and mixing with 100mL of the negative control solution to obtain a cysteine standard solution; precisely measuring cysteine standard solutions respectively, and adding water to dilute to obtain cysteine standard solutions with mass concentrations of 9.8 mu g/mL, 19.7 mu g/mL, 39.4 mu g/mL, 59.0 mu g/mL and 78.7 mu g/mL respectively;
after the obtained cysteine standard solutions with different dilution concentrations are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and taking the peak area of the cysteine derivative as a vertical coordinate, the concentration of cysteine as a horizontal coordinate, and taking a linear equation of the peak area to the concentration to obtain a standard curve of cysteine; the standard curve equation, correlation coefficient and linear range of the cysteine are shown in Table 17. As can be seen from Table 17, cysteine was linear well in the range of 9.8 to 78.7. mu.g/mL, and the correlation coefficient R2Is 0.9998.
TABLE 17 standard curve equation, correlation coefficient and Linear Range for cysteine
In Table 2, X represents the mass concentration of cysteine, and Y represents the peak area of the liquid chromatogram of the cysteine derivative.
(4) Standard recovery test
Precisely weighing cysteine, mixing with water, and preparing 0.05mg/mL cysteine aqueous solution as a reference solution;
preparing the compound amino acid injection according to the formula of the compound amino acid injection 18AA-IX, and diluting the compound amino acid injection by 5 times with water to be used as a test solution;
precisely weighing cysteine respectively, mixing the cysteine with the test solution to be tested, preparing three parts of mixed solution with cysteine mass concentrations of 0.04mg/mL, 0.05mg/mL and 0.06mg/mL respectively (the cysteine concentrations are 80%, 100% and 120% of the determination concentration of 0.05mg/mL respectively), and taking the three parts as the standard-adding recovery test solution;
and (3) after the reference substance solution and the standard adding recovery test solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating according to an external standard method to obtain the recovery rate of the cysteine, wherein the results are shown in a table 18. As can be seen from table 18, the recovery rate of cysteine was 97.76% to 100.74%, the average recovery rate was 99.28%, and RSD (%) was 1.06(n ═ 9), indicating that the measurement results were accurate.
TABLE 18 results of the normalized recovery test
(5) Repeatability test and intermediate precision test
Repeatability test
Taking compound amino acid injection 18AA-IX of product batch number 20140601 prepared by Hubei Yisemitian pharmaceutical Co Ltd as a sample, and diluting the compound amino acid injection 18AA-IX by 5 times by using water to serve as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.05mg/mL cysteine aqueous solution as a reference solution;
after the test solution and the reference solution are derived according to the method in the step (12), high performance liquid chromatography analysis is carried out according to the method in the step (13), the calculation is carried out according to an external standard method, the experiment is carried out for 6 times continuously, the peak area of the cysteine derivative and the labeled content of cysteine are compared for each time, and RSD (%) is calculated, and the result is shown in a table 19.
TABLE 19 results of the repeatability tests
Intermediate precision test
The tester A and the tester B adopt different instruments to perform the following intermediate precision tests at different test time:
taking compound amino acid injection 18AA-IX of product batch number 20140601 prepared by Hubei Yisemitian pharmaceutical Co Ltd as a sample, and diluting the compound amino acid injection 18AA-IX by 5 times by using water to serve as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.05mg/mL cysteine aqueous solution as a reference solution;
after the test solution and the reference solution are derived according to the method in the step (12), high performance liquid chromatography analysis is carried out according to the method in the step (13), the calculation is carried out according to an external standard method, the experiment is carried out for 6 times continuously, the peak area of the cysteine derivative and the labeled content of cysteine are compared for each time, and RSD (%) is calculated, and the result is shown in a table 20.
TABLE 20 results of intermediate precision test
| Intermediate precision test | Peak area of cysteine derivative | Labeled content of cysteine (%) |
| 1 | 2012490 | 87.24 |
| 2 | 2025097 | 87.79 |
| 3 | 2145202 | 93.00 |
| 4 | 2111130 | 91.52 |
| 5 | 2111368 | 91.53 |
| 6 | 2113104 | 91.61 |
| RSD(％) | 2.59 | 2.59 |
The average value of 12 marked content data of cysteine in the repeatability test and the intermediate precision test is 90.32%, and RSD (%) is 1.81%, which shows that the method provided by the invention has good precision.
(6) Stability test
Taking compound amino acid injection 18AA-IX of product lot number 20140601 prepared by Hubei Yimiantian pharmaceutical Co., Ltd as a sample, opening a rubber plug at room temperature (22 ℃), sampling at 0min, 80min, 160min, 240min and 320min respectively, diluting the compound amino acid 18AA-IX injection by 5 times with water respectively, after the derivative is obtained according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating RSD (%), wherein the results are shown in Table 21, which indicates that the stability of the sample after the seal is opened is good within 5 h.
TABLE 21 stability test results
(7) Sample assay
Taking three batches of compound amino acid injection 18AA-IX prepared by small test and pilot test of Hubei one and a half day pharmaceutical company Limited as samples, and diluting the compound amino acid injection 18AA-IX by 5 times by using water to serve as a test solution;
precisely weighing cysteine, mixing with water, and preparing 0.05mg/mL cysteine aqueous solution as a reference solution;
and (3) after the test solution and the reference solution are derived according to the method in the step (12), performing high performance liquid chromatography analysis according to the method in the step (13), and calculating according to an external standard method to obtain the labeled content of cysteine, wherein the result is shown in a table 22.
TABLE 22 results of sample measurement
As can be seen from the example 3, the method provided by the invention can realize the accurate determination of the cysteine content in the compound amino acid injection 18AA-IX, and has the advantages of good specificity, good precision and higher recovery rate. The method provided by the invention can effectively control the quality of the compound amino acid injection 18AA-IX products, and simultaneously comprehensively considers factors such as large-scale production, storage and transportation, and indirectly reflects that other amino acids in the compound amino acid injection 18AA-IX are not degraded under the condition that the cysteine content is not lower than 80% of the marked content.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A method for determining the content of cysteine in compound amino acid injection comprises the following steps:
(1) preparing a cysteine water solution as a reference substance solution;
taking the compound amino acid injection as a test solution;
deriving cysteine in the reference substance solution and the test substance solution by adopting N- (1-pyrene) maleimide; the mol ratio of cysteine to N- (1-pyrene) maleimide during derivatization is 1: 1-3, wherein the derivatization temperature is 15-25 ℃, and the derivatization time is 15-25 min; the N- (1-pyrene) maleimide is used in the form of an N- (1-pyrene) maleimide acetonitrile solution, and the mass concentration of the N- (1-pyrene) maleimide acetonitrile solution is 0.15-0.25 mg/mL;
(2) performing high performance liquid chromatography analysis on the derivatized reference solution and the test solution obtained in the step (1), and obtaining the content of cysteine in the compound amino acid injection by adopting an external standard method according to liquid chromatogram of the derivatized reference solution and the test solution; the high performance liquid chromatography adopts a PDA detector, and the detection wavelength is 274 nm; the high performance liquid chromatography adopts an octadecylsilane chemically bonded silica chromatographic column, and the column temperature is 30-40 ℃;
when the compound amino acid injection 18AA-V is used as a test solution, performing high performance liquid chromatography on a sodium dihydrogen phosphate solution with a mobile phase A of 0.01mol/L, wherein the pH value of the mobile phase A is 2.4-3.4, the mobile phase B is acetonitrile, adjusting the pH value of the mobile phase A by using phosphoric acid with the mass percentage concentration of 8-12%, and performing a gradient elution procedure:
0-18 min, mobile phase A: 90% → 40%;
18-25 min, mobile phase A: 40% → 10%;
25-33 min, mobile phase A: 10% → 10%;
33-34 min, mobile phase A: 10% → 90%;
34-40 min, mobile phase A: 90% → 90%;
when the compound amino acid injection 18AA-VII is used as a test solution, performing high performance liquid chromatography on a sodium acetate solution with a mobile phase A of 0.015mol/L, wherein the pH value of the mobile phase A is 2.8 +/-0.1, the mobile phase B is acetonitrile, adjusting the pH value of the mobile phase A by using phosphoric acid with the mass percentage concentration of 8-12%, and performing gradient elution:
0-8 min, mobile phase A: 90% → 40%;
8-18 min, mobile phase A: 40% → 10%;
18-24 min, mobile phase A: 10% → 10%;
24-25 min, mobile phase A: 10% → 90%;
when the compound amino acid injection 18AA-IX is used as a test solution, performing high performance liquid chromatography on a sodium dihydrogen phosphate solution with a mobile phase A of 0.01mol/L, wherein the pH value of the mobile phase A is 2.8 +/-0.1, the mobile phase B is acetonitrile, adjusting the pH value of the mobile phase A by using phosphoric acid with the mass percentage concentration of 8-12%, and performing gradient elution:
0-8 min, mobile phase A: 90% → 40%;
8-18 min, mobile phase A: 40% → 10%;
18-24 min, mobile phase A: 10% → 10%;
24-25 min, mobile phase A: 10% → 90%;
25-30 min, mobile phase A: 90% → 90%.
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