CN115616133A - Method for detecting cysteine in compound amino acid injection and application thereof - Google Patents
Method for detecting cysteine in compound amino acid injection and application thereof Download PDFInfo
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Abstract
The invention provides a detection method of cysteine in compound amino acid injection, application and application thereof, belonging to the technical field of analytical chemistry. The detection method comprises the following steps: adopting high performance liquid chromatography, wherein the chromatographic conditions comprise: a chromatographic column of octadecylsilane bonded silica gel filler is adopted, mobile phase is used for gradient elution, and an ultraviolet detector is used for detection. The cysteine detection method provided by the invention effectively improves the peak shape and the separation degree of cysteine by screening and optimizing chromatographic conditions such as the mobile phase proportion, pH, flow rate, column temperature, wavelength and the like, and realizes accurate quantitative detection of the substance. The detection method is verified by systematic methodology, has the advantages of strong specificity, good linear relation, good accuracy, strong durability, good stability and the like, and can quantitatively detect the cysteine in the compound amino acid injection, thereby ensuring the effectiveness and safety of clinical medication of the related compound amino acid injection.
Description
Technical Field
The invention relates to the field of analytical chemistry, and particularly relates to a detection method and application of cysteine in compound amino acid injection.
Background
The compound amino acid injection is a common parenteral nutrition injection and has a wide clinical application range. At present, the variety of amino acids in the market is more, for example, compound amino acid injection (18 AA-VII), compound amino acid injection (18 AA-IX) and the like contain cysteine, but the cysteine is easy to be oxidized and degraded, so that the product quality is unstable, the medication safety of patients and the effectiveness of medicines are influenced, and accurate quantitative research on the cysteine content is needed.
According to the existing literature reports, after a sample is derived by using various deriving reagents, quantitative detection is carried out by using various high-precision instruments such as an amino acid analyzer, a high performance liquid chromatograph and the like; however, derivatization by using a derivatization agent is complicated, and a sample after derivatization is unstable and is greatly influenced by derivatization reaction time, concentration and the like, so that a measurement result is unstable. In the prior art, a colorimetric method is also reported for detection, cysteine is subjected to color development reaction by using a color developing agent, and detection is performed by using an ultraviolet-visible spectrophotometry method after reaction.
Disclosure of Invention
The technical problem to be solved by the invention is that the existing detection method for cysteine in compound amino acid injection has many defects, and the invention aims to provide a detection method for cysteine in compound amino acid injection and application thereof, which are convenient to operate and can accurately and quantitatively detect the content of cysteine in compound amino acid injection, thereby ensuring the quality of compound amino acid injection and improving the effectiveness and safety of clinical medication.
The invention is realized by the following technical scheme:
a method for detecting cysteine in compound amino acid injection comprises the following steps: adopting high performance liquid chromatography, wherein the chromatographic conditions comprise: performing gradient elution by using a chromatographic column of octadecylsilane bonded silica gel filler and a mobile phase, detecting by using an ultraviolet detector, wherein the detection wavelength is 195-205 nm, the flow rate of the mobile phase is 0.9-1.1 ml/min, the sample injection amount is 10-30 mu l, and the column temperature is 25-35 ℃;
wherein, the mobile phase comprises a mobile phase A and a mobile phase B, the ratio of the mobile phase A: a mixed solution of methanol and a sodium octane sulfonate buffer salt solution; mobile phase B: an aqueous acetonitrile solution.
Further, in a preferred embodiment of the present invention, the mobile phase a is a mixed solution of methanol and 3 to 6mmol/L sodium octane sulfonate buffer solution; wherein the volume ratio of the sodium octane sulfonate buffer solution to the methanol is 97:3 to 99:1.
further, in a preferred embodiment of the present invention, the pH of the mobile phase a =1.9 to 2.1.
Further, in a preferred embodiment of the present invention, the mobile phase a is adjusted in pH with phosphoric acid.
Further, in a preferred embodiment of the present invention, the volume ratio of acetonitrile to water in the mobile phase B is 70:30 to 90:10.
further, in a preferred embodiment of the present invention, the gradient elution conditions are:
time: 0min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: 18min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: at 18.5min,40 to 60 percent of mobile phase A and 40 to 60 percent of mobile phase B;
time: 21.5min, 40-60% of mobile phase A and 40-60% of mobile phase B;
time: 22min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: 40min, 95-100% of mobile phase A, and 0-5% of mobile phase B.
Further, in the preferred embodiment of the present invention, the wavelength is 200nm.
Further, in the preferred embodiment of the present invention, the flow rate of the mobile phase is 1.0ml/min.
Further, in a preferred embodiment of the present invention, the column temperature is 30 ℃.
The invention also provides application of the method for detecting cysteine in the compound amino acid injection in detecting cysteine in the compound amino acid injection.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention effectively improves the peak shape and the separation degree of the cysteine by screening and optimizing chromatographic conditions such as the mobile phase proportion, pH, flow velocity, column temperature, wavelength and the like, and realizes accurate quantitative detection of the substance. The detection method is verified by systematic methodology, has the advantages of strong specificity, good linear relation, good accuracy, strong durability, good stability and the like, and can quantitatively detect the cysteine in the compound amino acid injection, thereby ensuring the effectiveness and safety of clinical medication of the related compound amino acid injection.
Drawings
In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and that for those skilled in the art, other related drawings can be obtained from these drawings without inventive effort. In the drawings:
FIG. 1 is a schematic HPLC of a cysteine control solution;
FIG. 2 is a schematic HPLC of a suitable solution of the cysteine system.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and the accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limiting the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The technical scheme of the specific implementation mode of the invention is as follows:
the detection method of cysteine in compound amino acid injection provided by the embodiment comprises the following steps: adopting high performance liquid chromatography, wherein the chromatographic conditions comprise: performing gradient elution with mobile phase by using chromatographic column of octadecylsilane bonded silica gel filler, and detecting with ultraviolet detector at wavelength of 195-205 nm, and further at wavelength of 200nm. The flow rate of the mobile phase is 0.9ml/min to 1.1ml/min, and further, the flow rate of the mobile phase is 1.0ml/min. The amount of the sample is 10. Mu.l to 30. Mu.l, further 15. Mu.l to 25. Mu.l, and further 20. Mu.l. The column temperature is 25 ℃ to 35 ℃, and further, the column temperature is 30 ℃.
Wherein, the mobile phase is composed of a mobile phase A and a mobile phase B. Mobile phase A: a mixed solution of methanol and a buffered saline solution of sodium octane sulfonate. The mobile phase A is a mixed solution of methanol and 3-6 mmol/L sodium octane sulfonate buffer solution, and the volume ratio of the sodium octane sulfonate buffer solution to the methanol is 97:3 to 99:1, further, the volume ratio of the sodium octane sulfonate buffer solution to the methanol is 98:2. the pH of the mobile phase a =1.9 to 2.1, and further, the pH of the mobile phase a =2.0. Mobile phase a was pH adjusted with phosphoric acid.
Mobile phase B: an aqueous acetonitrile solution. The volume ratio of acetonitrile to water in the mobile phase B is 70:30 to 90:10, and further, the volume ratio of acetonitrile to water in the mobile phase B is 80:20.
the conditions of the gradient elution are as follows:
time: 0min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: 18min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: at 18.5min,40 to 60 percent of mobile phase A and 40 to 60 percent of mobile phase B;
time: 21.5min,40 to 60 percent of mobile phase A and 40 to 60 percent of mobile phase B;
time: 22min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: 40min, 95-100% of mobile phase A, and 0-5% of mobile phase B.
Further, the method for detecting cysteine in compound amino acid injection provided by the embodiment comprises the following steps: adopting high performance liquid chromatography, wherein the chromatographic conditions comprise: the chromatographic column of octadecylsilane chemically bonded silica filler is adopted, gradient elution is carried out by using a mobile phase, detection is carried out by an ultraviolet detector, the detection wavelength is 200nm, the flow rate of the mobile phase is 1.0ml/min, the sample injection amount is 20 mu l, and the column temperature is 30 ℃.
Wherein, the mobile phase is composed of a mobile phase A and a mobile phase B. A mobile phase A: a mixed solution of methanol and a buffered saline solution of sodium octane sulfonate. The mobile phase A is a mixed solution of methanol and 4mmol/L sodium octane sulfonate buffer solution, and the volume ratio of the sodium octane sulfonate buffer solution to the methanol is 98:2. mobile phase a pH =2.0 (pH adjusted with phosphoric acid).
Mobile phase B: and (3) acetonitrile water, wherein the volume ratio of acetonitrile to water in the mobile phase B is 80:20.
the conditions of the gradient elution are as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% mobile phase A,0% mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
The specific embodiment of the invention is as follows: wherein, the first and the second end of the pipe are connected with each other,
compound amino acid injection (18 AA-VII) provided by Sichuan Kelun pharmaceutical industry Co.
Negative samples: compound amino acid injection (18 AA-VII) (without cysteine) is provided by Sichuan Konlen pharmaceutical Co.
Example 1: specificity experiments
A method for detecting cysteine in compound amino acid injection comprises the following steps:
reference stock solution: accurately weighing appropriate amount of cysteine hydrochloride reference substance, diluting with mobile phase A to scale, and making into solution containing cysteine about 0.35 mg/ml.
Control solution: precisely measuring the cysteine negative sample and the reference stock solution by 1.0ml respectively, placing into the same 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain (0.035 mg/ml).
Test solution: precisely measuring 1.0ml of the sample, placing in a 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain the final product.
Chromatographic conditions are as follows:
a chromatographic column: zishengtang MG IIC 18, 250mm multiplied by 4.6mm,5 μm;
mobile phase A:4mmol/L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98;
mobile phase B: acetonitrile-water (80;
column temperature: 30 ℃;
flow rate: 1.0ml/min;
wavelength: 200nm;
sample introduction amount: 20 mu l of the mixture;
the gradient elution conditions were as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% mobile phase A,0% mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
The detection method comprises the following steps: the reference solution and the test solution are accurately measured and injected into a high performance liquid chromatograph for detection, and the results are shown in table 1.
TABLE 1 cysteine specificity test results
As can be seen from Table 1, the cysteine retention times in the control solutions and the test solutions were substantially the same. In the test solution, the separation degree of cysteine and adjacent peaks is greater than 1.5, which shows that the separation effect is good, and shows that the chromatographic conditions of the invention can effectively separate and detect cysteine.
Example 2 linear experiment:
a method for detecting cysteine in compound amino acid injection comprises the following steps:
control stock solutions: accurately weighing appropriate amount of cysteine hydrochloride reference substance, diluting with mobile phase A to scale, and making into solution containing cysteine about 0.35 mg/ml.
The control stock solutions were prepared into linear solutions of 70.5200. Mu.g/ml, 52.8900. Mu.g/ml, 35.2600. Mu.g/ml, 17.6300. Mu.g/ml, 7.0520. Mu.g/ml, 0.1410. Mu.g/ml, respectively.
Chromatographic conditions are as follows:
and (3) chromatographic column: zishengtang MG IIC 18, 250mm × 4.6mm,5 μm;
mobile phase A:4mmol/L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98;
mobile phase B: acetonitrile-water (80;
column temperature: 30 ℃;
flow rate: 1.0ml/min;
wavelength: 200nm;
sample introduction amount: 20 μ l.
The gradient elution conditions were as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% mobile phase A,0% mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
The detection method comprises the following steps: respectively and accurately measuring 6 linear solutions with different concentrations, a reference solution and a 0.1410 mug/ml concentration quantitative limit solution, injecting the solutions into a high performance liquid chromatograph for detection, and obtaining results shown in table 2.
Table 2 cysteine linearity test results.
As can be seen from table 2, the correlation coefficient r =1 of the linear equation indicates that the linear relationship is good under the chromatographic conditions of the present invention.
Example 3 accuracy experiment:
a method for detecting cysteine in compound amino acid injection comprises the following steps:
reference stock solution: accurately weighing appropriate amount of cysteine hydrochloride reference substance, diluting with mobile phase A to scale, and making into solution containing cysteine about 0.35 mg/ml.
Control solution: precisely measuring the cysteine negative sample and the reference stock solution by 1.0ml respectively, placing into the same 10ml measuring flask, diluting with the mobile phase A to scale, and shaking uniformly to obtain the product (0.035 mg/ml).
Negative sample solution: precisely measuring 1.0ml of cysteine-deficient negative sample, placing the cysteine-deficient negative sample in a 10ml measuring flask, diluting the cysteine-deficient negative sample to a scale with the mobile phase A, and shaking up to obtain the cysteine-deficient negative sample.
The solution of the test sample without the added standard: precisely measuring 0.5ml of the sample, placing in a 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain the final product.
70% recovery solution: precisely measuring 0.5ml of the sample, placing into a 10ml measuring flask, precisely adding 0.2ml of the stock solution of the reference substance, diluting with the mobile phase A to scale, and shaking.
100% recovery solution: precisely measuring 0.5ml of the sample, placing in a 10ml measuring flask, precisely adding 0.5ml of the reference stock solution, diluting with mobile phase A to scale, and shaking.
130% recovery solution: precisely measuring 0.5ml of the sample, placing in a 10ml measuring flask, precisely adding 0.8ml of the reference stock solution, diluting with the mobile phase A to scale, and shaking.
Chromatographic conditions are as follows:
a chromatographic column: zishengtang MG IIC 18, 250mm × 4.6mm,5 μm;
mobile phase A:4mmol/L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98;
mobile phase B: acetonitrile-water (80;
column temperature: 30 ℃;
flow rate: 1.0ml/min;
wavelength: 200nm;
sample introduction amount: 20 μ l.
The gradient elution conditions were as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% of mobile phase A,0% of mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
The detection method comprises the following steps: the reference solution, the negative sample solution, the solution without the standard sample, the solution with the recovery rate of 70%, the solution with the recovery rate of 100% and the solution with the recovery rate of 130% are respectively and accurately measured and injected into a high performance liquid chromatograph for detection, and the results are shown in table 3.
Table 3 cysteine accuracy review results.
As shown in Table 3, the recovery rates of the 9 samples added with the standard solution were 99.72% -101.94%, the average recovery rate was 101.32%, and the RSD =0.71%. Indicating that cysteine has good accuracy under the chromatographic conditions of the invention.
Example 4 durability test:
a method for detecting cysteine in compound amino acid injection comprises the following steps:
reference stock solution: accurately weighing appropriate amount of cysteine hydrochloride reference substance, diluting with mobile phase A to scale, and making into solution containing cysteine about 0.35 mg/ml.
Control solution: precisely measuring the cysteine negative sample and the reference stock solution by 1.0ml respectively, placing into the same 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain (0.035 mg/ml).
Test solution: precisely measuring 1.0ml of the sample, placing in a 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain the final product.
Chromatographic conditions are as follows:
a chromatographic column: zishengtang MG IIC 18, 250mm multiplied by 4.6mm,5 μm;
mobile phase A:4mmol/L sodium octane sulfonate buffer (pH adjusted with phosphoric acid) and methanol;
and (3) mobile phase B: acetonitrile-water (80;
sample injection amount: 20 mu l of the mixture;
the gradient elution conditions were as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% of mobile phase A,0% of mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
Cysteine durability was examined by varying the column temperature, flow rate, wavelength and pH, set as in table 4 below:
TABLE 4 durability Condition parameters
| Condition | Column temperature deg.C | Flow rate ml/min | Wavelength nm | pH value | Mobile phase a ratio |
| Example 4-1a | 25 | 1.0 | 200 | 2.0 | 98：2 |
| Example 4-1b | 35 | 1.0 | 200 | 2.0 | 98：2 |
| Example 4- |
30 | 0.9 | 200 | 2.0 | 98：2 |
| Examples 4 to |
30 | 1.1 | 200 | 2.0 | 98：2 |
| Examples 4 to |
30 | 1.0 | 195 | 2.0 | 98：2 |
| Examples 4 to |
30 | 1.0 | 205 | 2.0 | 98：2 |
| Examples 4 to |
30 | 1.0 | 200 | 1.9 | 98：2 |
| Examples 4 to |
30 | 1.0 | 200 | 2.1 | 98：2 |
| Examples 4 to |
30 | 1.0 | 200 | 2.0 | 97：3 |
| Examples 4 to |
30 | 1.0 | 200 | 2.0 | 99：1 |
The detection method comprises the following steps: the durability condition parameters are set according to the table 4, the sample solution is accurately measured and detected in the high performance liquid chromatograph, and the result is shown in the table 5.
TABLE 5 cysteine durability test results
As can be seen from table 5, after fine adjustment of the chromatographic conditions, the cysteine content of the test sample was 89.64% to 92.32%, the average content was 90.68%, and RSD =0.93%. Indicating that the cysteine process is robust under the chromatographic conditions of the present invention.
Example 5 stability experiment:
a method for detecting cysteine in compound amino acid injection comprises the following steps:
reference stock solution: accurately weighing appropriate amount of cysteine hydrochloride reference substance, diluting with mobile phase A to scale, and making into solution containing cysteine about 0.35 mg/ml.
Control solution: precisely measuring the cysteine negative sample and the reference stock solution by 1.0ml respectively, placing into the same 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain (0.035 mg/ml).
Test solution: precisely measuring 1.0ml of a sample, placing into a 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain the final product.
Chromatographic conditions are as follows:
a chromatographic column: zishengtang MG IIC 18, 250mm × 4.6mm,5 μm;
mobile phase A:4mmol/L sodium octane sulfonate buffer (pH adjusted to 2.0 with phosphoric acid): methanol = (98;
mobile phase B: acetonitrile-water (80;
column temperature: 30 ℃;
flow rate: 1.0ml/min;
wavelength: 200nm;
sample introduction amount: 20 mu l of the mixture;
the gradient elution conditions were as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% mobile phase A,0% mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
The detection method comprises the following steps: the reference solution and the sample solution are accurately measured and detected in a high performance liquid chromatograph, and the results are shown in table 6.
TABLE 6 cysteine stability test results
| Point in time | Peak area of control | Sample content |
| 0h | 394.587 | 93.60％ |
| 2h | 396.184 | 93.57％ |
| 4h | 395.631 | 93.62％ |
| 8h | 395.923 | 93.65％ |
| 12h | 394.107 | 94.06％ |
| 16h | 395.106 | 93.57％ |
| 24h | 395.209 | 93.81％ |
| RSD | 0.19％ | 0.19％ |
As can be seen from Table 6, the area of the control peak and the RSD content of the sample are within 1% within 24 h. Indicating that cysteine is stable well under the chromatographic conditions of the present invention.
Comparative example 1 screening test for chromatographic conditions
A method for detecting cysteine in compound amino acid injection comprises the following steps:
reference stock solution: accurately weighing appropriate amount of cysteine hydrochloride reference substance, diluting with mobile phase A to scale, and making into solution containing cysteine about 0.35 mg/ml.
Control solution: precisely measuring the cysteine negative sample and the reference stock solution by 1.0ml respectively, placing into the same 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain (0.035 mg/ml).
Test solution: precisely measuring 1.0ml of the sample, placing in a 10ml measuring flask, diluting with the mobile phase A to scale, and shaking up to obtain the final product.
Chromatographic conditions are as follows:
a chromatographic column: zishengtang MG IIC 18, 250mm × 4.6mm,5 μm;
mobile phase A:4mmol/L sodium octane sulfonate buffer (pH adjusted with phosphoric acid) and methanol;
mobile phase B: acetonitrile-water (80;
sample introduction amount: 20 mu l of the mixture;
the gradient elution conditions were as follows:
time: 0min,100% of mobile phase A and 0% of mobile phase B;
time: 18min,100% mobile phase a,0% mobile phase B;
time: 18.5min,50% mobile phase a,50% mobile phase B;
time: 21.5min,50% mobile phase A,50% mobile phase B;
time: 22min,100% mobile phase A,0% mobile phase B;
time: 40min,100% mobile phase a,0% mobile phase B.
A control example of chromatographic conditions for setting column temperature, flow rate, wavelength and pH, set as follows in table 7:
TABLE 7 reference example parameters Condition parameters
| Condition | Column temperature deg.C | Flow rate ml/min | Wavelength nm | pH value | Mobile phase a ratio |
| Comparative example 1- |
20 | 1.0 | 200 | 2.0 | 98：2 |
| Comparative examples 1 to |
40 | 1.0 | 200 | 2.0 | 98：2 |
| Comparative examples 1 to |
30 | 0.8 | 200 | 2.0 | 98：2 |
| Comparative examples 1 to |
30 | 1.2 | 200 | 2.0 | 98：2 |
| Comparative examples 1 to |
30 | 1.0 | 190 | 2.0 | 98：2 |
| Comparative examples 1 to |
30 | 1.0 | 210 | 2.0 | 98：2 |
| Comparative examples 1 to |
30 | 1.0 | 200 | 1.8 | 98：2 |
| Comparative examples 1 to |
30 | 1.0 | 200 | 2.2 | 98：2 |
| Comparative examples 1 to |
30 | 1.0 | 200 | 2.0 | 96：4 |
The detection method comprises the following steps: the condition parameters of the reference example are set according to the table 7, the sample solution is accurately measured and detected in the high performance liquid chromatograph, and the result is shown in the table 8.
TABLE 8 cysteine control examination results
In the column temperature examination, the column temperature of 20 ℃ and 40 ℃ has normal content results, and the retention time is influenced by changing the column temperature, but the separation degree is not greatly influenced. And the normal condition shows that the content result at 30 ℃ is normal. The general column temperature and column temperature durability screening results of the investigation instrument are comprehensively combined, so that the column temperature is selected to be in the range of 25-35 ℃.
In the flow rate investigation, the flow rate of 0.8ml/min and the content of 1.2ml/min are normal, the retention time of the target peak of the flow rate of 0.8ml/min is prolonged, but the separation effect is better. The flow rate 1.2ml/min retention time is advanced, there is a risk of poor resolution, while under normal conditions 1.0ml/min content results are normal. Comprehensively combines the screening result of examining the durability of the flow rate, so the flow rate is selected to be 0.9ml/min to 1.1ml/min.
In the wavelength investigation, the content results of the wavelength of 190nm and the wavelength of 210nm are normal, and the 190nm response is higher than that of 210nm, but the wavelength has no great influence on the actual detection. Normal conditions the 200nm content results are normal. The comprehensive combination of the results of wavelength durability screening results shows that the selected wavelength range is 195nm-205 nm.
In the pH examination of the mobile phase, the results of the contents of the pH value 1.8 and the pH value 2.2 of the mobile phase are normal, the actual peak generation and separation effect can be influenced by the larger or the lower of different pH values, the risk of poor separation degree exists, and the content result of the pH value 2.0 is normal under the normal condition. Comprehensively combines the results of screening for examining the durability of the pH value of the mobile phase, so that the pH value is selected to be in the range of 1.9-2.1.
In the mobile phase a ratio examination, the mobile phase a ratio 96: the 4 content results are normal, but the peak time is advanced, and the separation effect is not as good as the normal condition. And normal condition 98:2, the separation degree and the content result are normal. Comprehensively combines and examines the screening result of the durability of the proportion of the mobile phase A, so that the proportion range of the sodium octane sulfonate buffer solution to the methanol in the mobile phase A is 97-99.
The invention effectively improves the peak shape and the separation degree of the cysteine by screening and optimizing chromatographic conditions such as the mobile phase proportion, pH, flow velocity, column temperature, wavelength and the like, and realizes accurate quantitative detection of the substance. The detection method is verified by systematic methodology, has the advantages of strong specificity, good linear relation, good accuracy, strong durability, good stability and the like, and can quantitatively detect the cysteine in the compound amino acid injection, thereby ensuring the effectiveness and safety of clinical medication of the related compound amino acid injection.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for detecting cysteine in compound amino acid injection is characterized by comprising the following steps: adopting high performance liquid chromatography, wherein the chromatographic conditions comprise: performing gradient elution by using a chromatographic column of octadecylsilane bonded silica gel filler and a mobile phase, detecting by using an ultraviolet detector, wherein the detection wavelength is 195-205 nm, the flow rate of the mobile phase is 0.9-1.1 ml/min, the sample injection amount is 10-30 mu l, and the column temperature is 25-35 ℃;
wherein, the mobile phase comprises a mobile phase A and a mobile phase B, and the ratio of the mobile phase A: a mixed solution of methanol and a sodium octane sulfonate buffer salt solution; mobile phase B: aqueous acetonitrile.
2. The method for detecting cysteine in compound amino acid injection according to claim 1, wherein the mobile phase A is a mixed solution of methanol and 3-6 mmol/L sodium octane sulfonate buffer solution; wherein the volume ratio of the sodium octane sulfonate buffer solution to the methanol is 97:3 to 99:1.
3. the method for detecting cysteine in compound amino acid injection according to claim 1, wherein the pH of the mobile phase A is = 1.9-2.1.
4. The method for detecting cysteine in compound amino acid injection according to claim 3, wherein the pH of the mobile phase A is adjusted by phosphoric acid.
5. The method for detecting cysteine in compound amino acid injection according to claim 1, wherein the volume ratio of acetonitrile to water in the mobile phase B is 70:30 to 90:10.
6. the method for detecting cysteine in compound amino acid injection according to claim 1, wherein the gradient elution conditions are as follows:
time: 0min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: 18min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: at 18.5min,40 to 60 percent of mobile phase A and 40 to 60 percent of mobile phase B;
time: 21.5min, 40-60% of mobile phase A and 40-60% of mobile phase B;
time: 22min, 95-100% of mobile phase A and 0-5% of mobile phase B;
time: 40min, 95-100% of mobile phase A, and 0-5% of mobile phase B.
7. The method for detecting cysteine in compound amino acid injection according to claim 1, wherein the wavelength is 200nm.
8. The method for detecting cysteine in compound amino acid injection according to claim 1, wherein the flow rate of the mobile phase is 1.0ml/min.
9. The method for detecting cysteine in compound amino acid injection according to claim 1, wherein the column temperature is 30 ℃.
10. The use of the method of any one of claims 1-9 for detecting cysteine in a compound amino acid injection.
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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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| 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 |
| CN115856160B (en) * | 2023-02-28 | 2023-06-06 | 长沙晶易医药科技股份有限公司 | Method for measuring content of related substances in compound tranexamic acid tablet |
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