CN111443151A - Method for detecting content of trace cysteine in compound amino acid injection - Google Patents
Method for detecting content of trace cysteine in compound amino acid injection Download PDFInfo
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Abstract
The invention relates to a method for detecting the content of trace cysteine in a compound amino acid injection, which comprises the following steps: preparing a reference solution, preparing a test solution, respectively deriving cysteine in the reference solution and the test solution by adopting an N- (1-pyrenyl) maleimide acetonitrile solution, determining chromatographic conditions, carrying out high performance liquid chromatography analysis on the derived reference solution and the test solution obtained in the step three under the condition of the step four, and obtaining the content of the cysteine in the compound amino acid injection by adopting an external standard method according to liquid chromatograms of the derived reference solution and the test solution. The method provided by the invention can realize accurate determination of the content of the trace cysteine in the compound amino acid injection, and has the advantages of good specificity, good precision, good recovery rate and high sensitivity.
Description
Technical Field
The invention relates to a method for detecting the content of cysteine in compound amino acid injection, in particular to a method for detecting the content of trace cysteine in compound amino acid injection by applying high performance liquid chromatography.
Background
The compound amino acid injection is a parenteral nutrition drug, has various types and different prescriptions, has 18 compound amino acid injections containing cystine, cysteine and acetylcysteine according to incomplete statistics, and researches of the subject group find that the cystine and the acetylcysteine in the compound amino acid injection can be degraded to generate trace cysteine. Therefore, in the compound amino acid injection, the content of the cysteine can reflect the quality of the product.
Cysteine has high polarity, the retention is weak and difficult to separate by using a C18 chromatographic column, and the retention of a polar compound by the C18 chromatographic column is enhanced by adding an ion pair reagent into a mobile phase by an ion pair chromatography, but the problems of unreproducible retention time and short service life of the chromatographic column exist; because the specifications of cystine, cysteine and acetylcysteine in the prescription of the compound amino acid injection are small and the number of coexisting amino acids is large, the common method for analyzing the pre-column or post-column derived amino acid cannot accurately quantify the cysteine due to insufficient sensitivity or poor separation degree of the cysteine and the coexisting amino acid.
In the prior art (CN 106950306 a), it is reported that N- (1-pyrenyl) maleimide is used as a derivatization agent to derivatize cysteine, and high performance liquid chromatography is used to determine the content of cysteine (cysteine contained in the prescription) in the compound amino acid injection, in the technique, an ultraviolet detector is used, and the sensitivity of the method is not sufficient for determining the content of trace cysteine in the compound amino acid injection containing cystine and acetylcysteine, which does not contain cysteine in the prescription.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for detecting the content of trace cysteine in a compound amino acid injection with good specificity, good sensitivity and high derivatization reaction speed.
According to the technical scheme provided by the invention, the method for detecting the content of the trace cysteine in the compound amino acid injection comprises the following steps:
step one, adding water into cysteine to dilute the cysteine into a control solution with the concentration of 0.0639-20.04 mg/L;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution;
thirdly, respectively derivatizing cysteine in the reference substance solution and the sample solution by adopting an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8-2.6 mmol/L, wherein the volume ratio of the reference substance solution and the sample solution to react with the N- (1-pyrenyl) maleimide acetonitrile solution is 1: 3, and adding a hydrochloric acid solution to mix uniformly in a vortex manner after the derivatization is finished;
step four, determining the following chromatographic conditions:
the chromatographic column adopts a 250 × 4.6mm and 5 mu m octadecylsilane chemically bonded silica C18 chromatographic column;
mobile phase: the mobile phase A is 10mM sodium dihydrogen phosphate buffer solution, and the mobile phase B is acetonitrile;
the linear gradient elution conditions were: 0-8 minutes: 60% -56% of mobile phase A and 40% -44% of mobile phase B; 9-17 minutes: 10% mobile phase a, 90% mobile phase B; after 18 minutes: 60% -56% of mobile phase A and 40% -44% of mobile phase B;
setting the flow rate to be 0.9-1.1 m L/min;
the column temperature is set to be 33-37 ℃;
the excitation wavelength was set to 330 nm;
the emission wavelength was set at 376 nm;
the injector temperature was set to 4 ℃;
the injection volume is set to 10 mu L;
and step five, carrying out high performance liquid chromatography analysis on the derivatized reference substance solution and the sample solution obtained in the step three under the condition of the step four, 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 substance solution and the sample solution.
Preferably, in the first step, cysteine is diluted with water to a concentration of 20.04 mg/L to be used as a control solution.
Preferably, in the second step, the compound amino acid injection is diluted by 10 times.
Preferably, in step three, the derivatization time is 10min to 30 min.
Preferably, in the third step, the concentration of the hydrochloric acid solution added after the derivatization reaction is 0.2 mol/L, the volume ratio of the hydrochloric acid solution to the reference solution after derivatization is 1: 10, and the volume ratio of the hydrochloric acid solution to the test solution after derivatization is 1: 10.
Preferably, in step four, the column temperature used is 35 ℃.
Preferably, in step four, the flow rate used is 1.0m L/min.
Preferably, in step four, the linear gradient elutes as follows: 0-8 minutes: 58% mobile phase a, 42% mobile phase B; 9-17 minutes: 10% mobile phase a, 90% mobile phase B; after 18 minutes: 58% mobile phase a, 42% mobile phase B.
Preferably, in the fourth step, 1.56g to 1000m L g of sodium dihydrogen phosphate is accurately weighed in water, after dissolution, phosphoric acid is added to adjust the pH value to 2.80-3.20, and the solution is filtered by a 0.45-micron filter membrane to obtain the sodium dihydrogen phosphate buffer solution.
The method provided by the invention can realize accurate determination of the content of the trace cysteine in the compound amino acid injection, and has the advantages of good specificity, good precision, good recovery rate and high sensitivity. Compared with the prior art, the invention has the following advantages:
the fluorescence detector is adopted, so that the sensitivity is higher and the specificity is better; the operation is simple, and the derivatization reaction is completed within 10min at room temperature; after the derivatization reaction is finished, hydrochloric acid is added, and cysteine derivatization products in the reference solution and the test solution have good stability and can be stable for several days at 4 ℃.
Drawings
FIG. 1 is a chromatogram of a control working solution in example 1.
FIG. 2 is a chromatogram of the test solution in example 1.
FIG. 3 is a chromatogram of the test solution in example 2.
FIG. 4 is a chromatogram of the test solution in example 3.
FIG. 5 is a chromatogram of the test solution in example 4.
FIG. 6 is a chromatogram of the test solution in example 5.
FIG. 7 is a chromatogram of the test solution in example 6.
FIG. 8 is a chromatogram of the test solution in example 7.
FIG. 9 is a chromatogram of the test solution in example 8.
FIG. 10 is a chromatogram of the test solution in example 9.
FIG. 11 is a chromatogram of the sample solution in example 10.
Detailed Description
The present invention will be further described with reference to the following specific examples.
The column used in the following examples was an octadecylsilane bonded silica C18 column with product parameters of 5 μm, 250mm × 4.6.6 mm.
Example 1
A method for detecting the content of cysteine in compound amino acid injection (containing cystine in a prescription) comprises the following steps:
step one, adding water into cysteine to dilute the cysteine into a solution with the concentration of 20.040 mg/L to be used as a reference solution;
step two, adding water into the compound amino acid injection for diluting by 10 times to obtain a test solution;
step three, mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 2.6 mmol/L of 150 mu L with the reference substance solution of 50 mu L in the step one and the test substance solution of 50 mu L in the step two respectively for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L for uniform vortex mixing after derivatization is completed;
step four, chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.0m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
And step five, carrying out high performance liquid chromatography analysis on the derivatized reference substance solution and the sample solution obtained in the step three under the condition of the step four, 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 substance solution and the sample solution.
And (3) detection results: the retention time of the reference working solution and the test solution is consistent. The cysteine derivative was separated from the adjacent chromatographic peak by 7.93 in the test sample solution, as shown in Table 1.
TABLE 1
| Sample (I) | Retention time (min) | Degree of separation |
| Reference working solution | 4.450 | 28.163 |
| Test solution | 4.465 | 9.770 |
Example 2
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.0m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 2.
TABLE 2
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.54 | 8.98 |
Example 3
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.0m L/min;
column temperature: 33 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 3.
TABLE 3
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.556 | 8.97 |
Example 4
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.0m L/min;
column temperature: 37 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 4.
TABLE 4
Example 5
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 0.9m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 5.
TABLE 5
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.867 | 9.01 |
Example 6
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.1m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 6.
TABLE 6
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.214 | 8.97 |
Example 7
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 60 percent of the mobile phase A and 40 percent of the mobile phase B are subjected to linear gradient elution for 0 to 8 minutes, 10 percent of the mobile phase A and 90 percent of the mobile phase B are subjected to 9 to 17 minutes, and after 18 minutes, 60 percent of the mobile phase A and 40 percent of the mobile phase B are subjected to linear gradient elution;
the flow rate is 1.0m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 7.
TABLE 7
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 5.034 | 8.46 |
Example 8
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A, namely 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.0, and the solution is filtered by a 0.45-micron filter membrane), a mobile phase B, namely acetonitrile, is subjected to linear gradient elution under the conditions that 56% of the mobile phase A and 44% of the mobile phase B are subjected to linear gradient elution for 0 to 8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are subjected to 9 to 17 minutes, and 56% of the mobile phase A and 44% of the mobile phase B are subjected to 18 minutes later;
the flow rate is 1.0m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 8.
TABLE 8
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.144 | 7.94 |
Example 9
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 2.8, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.0m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 9.
TABLE 9
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.370 | 8.53 |
Example 10
A method for detecting the content of cysteine in compound amino acid injection (containing acetylcysteine in a prescription) by using high performance liquid chromatography comprises the following steps:
step one, adding water into compound amino acid injection (added with cysteine with the concentration of 64 mg/L) to dilute by 10 times to obtain a test solution;
step two, respectively mixing an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8 mmol/L of 150 mu L with the sample solution of 50 mu L of the step one for derivatization, and adding a 0.2 mol/L hydrochloric acid solution of 20 mu L after derivatization is finished, and uniformly mixing in a vortex manner;
step three, the chromatographic conditions are as follows:
chromatographic column (250 × 4.6mm,5 μm) octadecylsilane chemically bonded silica C18 chromatographic column;
a mobile phase A which is 10mM sodium dihydrogen phosphate buffer solution (1.56 g to 1000m L water is accurately weighed, after the sodium dihydrogen phosphate is dissolved, phosphoric acid is added to adjust the pH value to 3.2, and the solution is filtered by a 0.45 mu m filter membrane), a mobile phase B which is acetonitrile is subjected to linear gradient elution, wherein the linear gradient elution conditions are that 58% of the mobile phase A and 42% of the mobile phase B are carried out in 0-8 minutes, 10% of the mobile phase A and 90% of the mobile phase B are carried out in 9-17 minutes, and 58% of the mobile phase A and 42% of the mobile phase B are carried out after 18 minutes;
the flow rate is 1.0m L/min;
column temperature: 35 ℃;
excitation wavelength: 330nm
Emission wavelength: 376nm
Sample injector temperature: 4 deg.C
Sample injection volume of 10 mu L
Step four, performing high performance liquid chromatography analysis on the derivatized sample solution obtained in the step two under the conditions in the step three, and recording the chromatographic peak position of the cysteine derivative product and the separation degree of the adjacent chromatographic peak as shown in table 10.
| Sample (I) | Retention time (min) | Degree of separation |
| Test solution | 4.422 | 8.82 |
Linearity of the method of the invention
The linear relationship was measured according to the chromatographic conditions of example 2, and control solutions were prepared at concentrations of 0.0639 μ g/m L, 0.6386 μ g/m L, 1.5965 μ g/m L, 3.1930 μ g/m L, 6.3860 μ g/m L, 9.5790 μ g/m L, 12.7720 μ g/m L, respectively, and linear regression was performed using the peak areas and the corresponding concentrations, and the results showed that the cysteine concentrations in the control solutions were between 0.0639mg/l and 12.7720mg/l, and when the cysteine concentrations in the test samples were 0.6390mg/l to 127.7200mg/l, the regression was performed using the peak areas and the corresponding concentrations, and the equation y was 105.4809x +2.0419, and the correlation coefficient r was 0.9999.
Accuracy and precision of the method of the invention
The accuracy and precision of the method of the present invention were tested according to the chromatographic conditions of example 1 by adding cysteine to each of the samples not containing cysteine, designing 3 different concentrations, preparing 3 test solutions for each concentration, and evaluating the results of the measurements of 9 samples, as shown in Table 10.
Description of the drawings: the N-acetylcysteine reference substance contains a trace amount of cysteine, and the N-acetylcysteine reference substance is added in the cysteine blank test sample, so that the trace amount of cysteine can be introduced, and the cysteine should be deducted when the recovery rate is calculated.
As can be seen from Table 10, the cysteine concentration in the test sample was in the range of 3.1740mg/l to 9.7360mg/l, the average recovery rate was 100.2%, and the RSD was 1.3%.
Quantitative limits of the method of the invention
The limit of quantitation of the method of the present invention was tested under the chromatographic conditions of example 1, and the results showed that the limit of quantitation of the detection method of the present invention was 1.8870 mg/L for the cysteine concentration in the test sample under the condition that the signal-to-noise ratio was not less than 10.
Claims (9)
1. A method for detecting the content of trace cysteine in compound amino acid injection is characterized by comprising the following steps:
step one, adding water into cysteine to dilute the cysteine into a control solution with the concentration of 0.0639-20.04 mg/L;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution;
thirdly, respectively derivatizing cysteine in the reference substance solution and the sample solution by adopting an N- (1-pyrenyl) maleimide acetonitrile solution with the concentration of 0.8-2.6 mmol/L, wherein the volume ratio of the reference substance solution and the sample solution to react with the N- (1-pyrenyl) maleimide acetonitrile solution is 1: 3, and adding a hydrochloric acid solution to mix uniformly in a vortex manner after the derivatization is finished;
step four, determining the following chromatographic conditions:
the chromatographic column adopts a 250 × 4.6mm and 5mm octadecylsilane chemically bonded silica C18 chromatographic column;
mobile phase: the mobile phase A is 10mM sodium dihydrogen phosphate buffer solution, and the mobile phase B is acetonitrile;
the linear gradient elution conditions were: 0-8 minutes: 60% -56% of mobile phase A and 40% -44% of mobile phase B; 9-17 minutes: 10% mobile phase a, 90% mobile phase B; after 18 minutes: 60% -56% of mobile phase A and 40% -44% of mobile phase B;
setting the flow rate to be 0.9-1.1 m L/min;
the column temperature is set to be 33-37 ℃;
the excitation wavelength was set to 330 nm;
the emission wavelength was set at 376 nm;
the injector temperature was set to 4 ℃;
the injection volume is set to 10 mu L;
and step five, carrying out high performance liquid chromatography analysis on the derivatized reference substance solution and the sample solution obtained in the step three under the condition of the step four, 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 substance solution and the sample solution.
2. The method for detecting the content of the micro cysteine in the compound amino acid injection as claimed in claim 1, wherein in the first step, the cysteine is diluted by adding water to be 20.04 mg/L and then is used as a reference solution.
3. The method for detecting the content of the micro cysteine in the compound amino acid injection according to claim 1, which is characterized in that: in the second step, the compound amino acid injection is diluted by 10 times.
4. The method for detecting the content of the micro cysteine in the compound amino acid injection according to claim 1, which is characterized in that: in the third step, the derivation time is 10 min-30 min.
5. The method for detecting the content of the micro cysteine in the compound amino acid injection as claimed in claim 1, wherein in the third step, the concentration of the hydrochloric acid solution added after the derivatization reaction is 0.2 mol/L, the volume ratio of the hydrochloric acid solution to the reference solution after the derivatization is 1: 10, and the volume ratio of the hydrochloric acid solution to the test solution after the derivatization is 1: 10.
6. The method for detecting the content of the micro cysteine in the compound amino acid injection according to claim 1, which is characterized in that: in the fourth step, the column temperature used was 35 ℃.
7. The method for detecting the content of the micro cysteine in the compound amino acid injection as claimed in claim 1, wherein the flow rate used in the fourth step is 1.0m L/min.
8. The method for detecting the content of the micro cysteine in the compound amino acid injection according to claim 1, which is characterized in that: in the fourth step, the linear gradient elution conditions are as follows: 0-8 minutes: 58% mobile phase a, 42% mobile phase B; 9-17 minutes: 10% mobile phase a, 90% mobile phase B; after 18 minutes: 58% mobile phase a, 42% mobile phase B.
9. The method for detecting the content of the micro cysteine in the compound amino acid injection as claimed in claim 1, wherein in the fourth step, 1.56g to 1000m L g of sodium dihydrogen phosphate is accurately weighed, dissolved, added with phosphoric acid to adjust the pH value to 2.80 to 3.20, and filtered by a 0.45 μm filter membrane to obtain the sodium dihydrogen phosphate buffer solution.
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| CN115856160A (en) * | 2023-02-28 | 2023-03-28 | 长沙晶易医药科技股份有限公司 | Method for determining content of related substances in compound tranexamic acid tablet |
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