CN114689707A - Detection method of acetylcysteine enantiomer - Google Patents
Detection method of acetylcysteine enantiomer Download PDFInfo
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- CN114689707A CN114689707A CN202011570144.2A CN202011570144A CN114689707A CN 114689707 A CN114689707 A CN 114689707A CN 202011570144 A CN202011570144 A CN 202011570144A CN 114689707 A CN114689707 A CN 114689707A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Abstract
The invention relates to the technical field of analysis and detection, in particular to a detection method of acetylcysteine enantiomer. The detection method of acetylcysteine enantiomer provided by the invention comprises the steps of diluting a sample with a diluent, and then carrying out HPLC detection; the diluent consists of ethanol and isopropanol; the chromatographic conditions for the HPLC assay include: the chromatographic column is a chiral column; the mobile phase consists of normal hexane, isopropanol, ethanol and trifluoroacetic acid, and is eluted isocratically. The method provided by the invention can reduce the pretreatment steps of the sample. The detection result shows that the method of the invention can lead the L-acetylcysteine and the D-acetylcysteine to have better separation degree, and the detection accuracy, sensitivity, repeatability and precision are all good.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a detection method of acetylcysteine enantiomer.
Background
Acetylcysteine (Acetylcysteine) is a mucus dissolving agent and has a strong phlegm dissolving effect. The sulfhydryl contained in the molecule can break the disulfide bond in glycoprotein polypeptide chain in the sputum, thereby reducing the viscosity of the sputum and leading the sputum to be liquefied and easy to expectorate. And DNA fibers in purulent sputum can be broken, so that the sputum can dissolve white sticky sputum and purulent sputum. The acetylcysteine contains a chiral center, and contains 2 configurations of an L configuration and a D configuration, wherein the L configuration is an effective configuration, and the D configuration is controlled as an impurity.
Currently, the pharmaceutical formulations of acetylcysteine on the market include oral preparations, injections, and aerosol inhalants. Among them, there are 3 enterprises selling acetylcysteine injection on the market at home. Acetylcysteine is unstable in aqueous solution, amido bond is easy to break, and N, N are easy to form by oxidation’diacetyl-L-cystine, which is temperature sensitive and susceptible to degradation by cleavage of the sulfhydryl bond at high temperatures. The isomer acetyl-D-cysteine and acetyl-L-cysteine have the same functional group, the physical and chemical properties are close, and the isomer acetyl-D-cysteine and acetyl-L-cysteine are unstable to oxygen and high temperature. Therefore, quality control of acetylcysteine injection, especially detection of isomer, is very important. In the existing detection method, the separation degree of isomer chromatographic peaks is insufficient, and the detection accuracy is low.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for detecting acetylcysteine enantiomers, which can achieve good separation.
The detection method of acetylcysteine enantiomer provided by the invention comprises the steps of diluting a sample with a diluent, and then carrying out HPLC detection;
the diluent consists of ethanol and isopropanol;
the chromatographic conditions for the HPLC assay include:
a chromatographic column: a chiral column;
mobile phase: consists of normal hexane, isopropanol, ethanol and trifluoroacetic acid, and is eluted isocratically.
In the chromatographic conditions provided by the invention, good separation of acetylcysteine and an enantiomer thereof is realized by improving a sample treatment mode and optimizing the chromatographic conditions, derivatization is not needed before detection, heating treatment is avoided, the risk of sample degradation in a pretreatment process is reduced, and the detection process is simplified.
The chromatographic column adopted by the invention is a chiral column, and the filler of the chromatographic column is silica gel with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface. The chromatographic column has good solvent tolerance, for example, the chromatographic column can tolerate water in the acetylcysteine injection, so that a pretreatment mode of the acetylcysteine injection which is more favorable for detection can be selected. In some embodiments, the chromatography column is an CHIRALPAKIG chromatography column. In some embodiments, the size of the column is 4.6X 250mm, 5 μm.
The experiment of the invention shows that the selection of the chromatographic column, the column temperature and the selection of the mobile phase have extremely obvious influence on the sample separation degree. In some embodiments, the column temperature of the chromatography column in the detection method of the invention is 30 ℃. In the detection method, the flow rate of the mobile phase is 1.5 mL/min. The time of elution of the mobile phase is 30-40 min. In some embodiments, the elution time of the mobile phase is 30min or 40 min.
In the invention, the volume ratio of n-hexane, isopropanol, ethanol and trifluoroacetic acid in the mobile phase is (88-92): 6-10): 2: 0.1.
In some embodiments, the volume ratio of n-hexane, isopropanol, ethanol, and trifluoroacetic acid in the mobile phase is 90:8:2: 0.1.
In other embodiments, the mobile phase comprises n-hexane, isopropanol, ethanol, and trifluoroacetic acid in a volume ratio of 88:10:2: 0.1.
In other embodiments, the volume ratio of n-hexane, isopropanol, ethanol, and trifluoroacetic acid in the mobile phase is 92:6:2: 0.1.
In the detection method, the detector of the HPLC is an ultraviolet detector, and the detection wavelength is 205 nm.
In the detection method of the present invention, the sample loading amount for HPLC detection is 10. mu.L.
The volume ratio of ethanol to isopropanol in the diluent is (50-60) to (40-50).
In some embodiments, the volume ratio of ethanol to isopropanol in the diluent is 50: 50.
In other embodiments, the volume ratio of ethanol to isopropanol in the diluent is 60: 40.
In the invention, the sample is acetylcysteine injection or acetylcysteine injection added with acetylcysteine isomer standard; the volume ratio of the sample to the diluent is 1.5: 18.5;
the sample is a standard substance of acetylcysteine isomer, and the mass-volume ratio of the sample to the diluent is 5.6 mg: 250 mL.
In some embodiments, the diluent is ethanol: 60:40 (volume ratio) of isopropanol; the chromatographic conditions include: mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid 90:8:2:0.1 (volume ratio); a chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm; detection wavelength: 205 nm; column temperature: 30 ℃; flow rate: 1.5ml/min, injection volume 10 u L. The time for elution of the mobile phase was 30 min. Under the chromatographic conditions, the peak separation degree of acetylcysteine and acetylcysteine isomers is more than 4.2.
In some embodiments, the diluent is ethanol: 60:40 (volume ratio) of isopropanol; the chromatographic conditions include: mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid 88:10:2:0.1 (volume ratio); a chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm; detection wavelength: 205 nm; column temperature: 30 ℃; flow rate: 1.5ml/min, injection volume 10 u L. The time for elution of the mobile phase was 30 min. Under the chromatographic conditions, the peak separation degree of acetylcysteine and acetylcysteine isomers is more than 3.8.
In some embodiments, the diluent is ethanol: 60:40 (volume ratio) of isopropanol; the chromatographic conditions include: mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid 92:6:2:0.1 (volume ratio); a chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm; detection wavelength: 205 nm; column temperature: 30 ℃; flow rate: 1.5ml/min, injection volume 10 u L. The time for elution of the mobile phase was 40 min. Under the chromatographic conditions, the peak separation degree of acetylcysteine from acetylcysteine isomer was 4.7.
In some embodiments, the diluent is ethanol: isopropanol 50:50 (volume ratio); the chromatographic conditions include: mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid 90:8:2:0.1 (volume ratio); a chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm; detection wavelength: 205 nm; column temperature: 30 ℃; flow rate: 1.5ml/min, injection volume 10 u L. The time for elution of the mobile phase was 30 min. Under the chromatographic conditions, the peak separation degree of acetylcysteine and acetylcysteine isomers is more than 4.4.
In the present invention, acetylcysteine isomers were characterized based on acetylcysteine isomer controls. The acetylcysteine isomer was quantified by an external standard method. The detection proves that the limit of the quantification of the acetylcysteine isomer is 1.6-2.3 mu g/mL.
The detection method of acetylcysteine enantiomer provided by the invention comprises the steps of diluting a sample with a diluent, and then carrying out HPLC detection; the diluent consists of ethanol and isopropanol; the chromatographic conditions for the HPLC assay include: the chromatographic column is a chiral column; the mobile phase consists of normal hexane, isopropanol, ethanol and trifluoroacetic acid, and is eluted isocratically. The method provided by the invention can reduce the pretreatment steps of the sample. The detection result shows that the method of the invention can lead the L-acetylcysteine and the D-acetylcysteine to have better separation degree, and the detection accuracy, sensitivity, repeatability and precision are all good.
Drawings
FIG. 1 shows a chromatogram of example 1;
FIG. 2 shows a chromatogram of example 2;
FIG. 3 shows a chromatogram of example 3;
FIG. 4 shows a chromatogram of example 4.
Detailed Description
The invention provides a detection method of acetylcysteine enantiomer. Those skilled in the art can use the teachings herein to appropriately modify the chromatographic parameters. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. Wherein the acetylcysteine injection is from Sichuan Virginia pharmaceutical research Co., Ltd, and the isomer reference is from QCC. CHIRALPAK IG the chromatographic column is from xylonite. The invention is further illustrated by the following examples:
example 1
1. Preparing solution
Test solution: measuring 1.5ml of acetylcysteine injection, putting the acetylcysteine injection into a 20ml measuring flask, adding a diluent (ethanol-isopropanol-60: 40v/v) to dilute to a scale, and shaking up to obtain a test solution.
Adding a standard test solution: measuring 1.5ml of acetylcysteine injection, placing the acetylcysteine injection into a 20ml measuring flask, precisely adding 2ml of reference substance stock solution, adding a diluent to dilute the solution to a scale, and shaking the solution uniformly to obtain a solution for adding a standard test substance. 6 parts are prepared in parallel.
Control solution: taking an isomer reference substance of 5.6mg, precisely weighing, placing in a 25ml measuring flask, adding a diluent to dissolve and dilute to scale, shaking up to serve as a reference substance stock solution, precisely weighing a reference substance stock solution of 2ml, placing in a 20ml measuring flask, adding the diluent to dilute to scale, shaking up to serve as a reference substance solution.
Quantitative limiting solution: taking an isomer reference substance of 2mg, accurately weighing, placing in a 25ml measuring flask, adding a diluent to dissolve and dilute to a scale, shaking up, accurately weighing 1ml, placing in a 50ml measuring flask, adding the diluent to dilute to the scale, shaking up, and taking as a limit solution for quantification.
2. HPLC detection
Diluent agent: ethanol: isopropyl alcohol 60:40 (vol/vol)
Mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid ═ 90:8:2:0.1 (volume ratio)
A chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm
Detection wavelength: 205nm
Column temperature: 30 deg.C
Flow rate: 1.5ml/min
The injection volume is 10. mu.L.
2.1 chromatogram
Precisely measuring 10 μ l of test solution, introducing sample, and recording chromatogram as shown in FIG. 1. According to FIG. 1, the number of trays is 6342 and the degree of separation is 4.2.
2.2 repeatability
Precisely measuring 6 parts of standard sample solution by 10 mu L, injecting a sample, recording a chromatogram, and calculating the result according to the chromatogram:
| sample (I) | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Recovery rate | 101.7％ | 104.4％ | 100.7％ | 102.3％ | 104.1％ | 104.0％ | 1.5％ |
| Degree of separation | 4.2 | 4.2 | 4.2 | 4.3 | 4.3 | 4.3 | -- |
| Tailing factor | 1.01 | 0.99 | 0.99 | 0.97 | 0.97 | 0.97 | -- |
2.3 precision
Precisely measuring 10 μ l of the reference solution, and continuously introducing sample for 6 times, wherein the peak area RSD of the isomer is 0.69%.
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Peak area | 78007 | 77215 | 77383 | 78594 | 77305 | 77857 | 0.69％ |
2.4 sensitivity
Precisely measuring 10 mul of limiting solution, and injecting.
| Quantitative limit concentration (mu g/ml) | S/N of isomer peaks | Concentration percentage of the sample (%) |
| 2.3 | 14 | 0.015 |
2.5 solution stability
2.5.1 precisely measuring 10 mu L of reference solution, and injecting samples at different time points, wherein the ratio of the isomer peak area to the 0h peak area is 95.0-105.0%.
| Time(h) | 0 | 2.5 | 7.5 |
| Peak area | 78007 | 77857 | 74912 |
| Ratio to 0h (%) | NA | 99.8 | 96.0 |
2.5.2 precisely measuring 10 mu L of the sample solution, and injecting samples at different time points, wherein the ratio of the isomer peak area to the 0h peak area is 95.0-105.0%.
| Time(h) | 0 | 3.5 | 7.5 | 12 |
| Peak area | 79085 | 80165 | 81091 | 80448 |
| Ratio to 0h (%) | NA | 102.8 | 104.0 | 103.1 |
Example 2
1. Preparing a solution (same as example 1)
2. HPLC detection
Diluent (b): ethanol: isopropyl alcohol 60:40 (vol/vol)
Mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid (88: 10:2:0.1 (volume ratio)
A chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm
Detection wavelength: 205nm
Column temperature: 30 deg.C
Flow rate: 1.5ml/min
2.1 chromatogram
Precisely measuring 10 μ l of test solution, introducing sample, and recording chromatogram as shown in FIG. 2. According to FIG. 2, the number of trays is 5857 and the degree of separation is 3.8.
2.2 repeatability
Precisely measuring 6 parts of standard sample solution by 10 mu L, feeding a sample, recording a chromatogram, and calculating the result according to the chromatogram:
| sample (I) | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Recovery rate | 100.8 | 101.9 | 103.2 | 101.0 | 102.8 | 102.5 | 0.96％ |
| Degree of separation | 3.8 | 3.8 | 3.8 | 3.8 | 3.9 | 3.9 | -- |
| Tailing factor | 1.16 | 1.16 | 1.16 | 1.17 | 1.17 | 1.17 | -- |
2.3 precision
Precisely measuring 10 μ l of the reference solution, and continuously introducing sample for 6 times, wherein the peak area RSD of the isomer is 1.5%.
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Peak area | 89488 | 88644 | 88634 | 86648 | 87163 | 86244 | 1.5％ |
2.4 sensitivity
Precisely measuring 10 mu l of quantitative limiting solution and injecting.
| Quantitative limit concentration (mu g/ml) | S/N of isomer peaks | Concentration percentage of the sample (%) |
| 1.6 | 16 | 0.011 |
Example 3
1. Prepare solution (same as example 1)
2. HPLC detection
Diluent (b): ethanol: isopropyl alcohol 60:40 (vol/vol)
Mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid 92:6:2:0.1 (vol.)
A chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm
Detection wavelength: 205nm
Column temperature: 30 deg.C
Flow rate: 1.5ml/min
2.1 chromatogram
Precisely measuring 10 μ l of the test solution, introducing sample, and recording chromatogram as shown in FIG. 3. According to FIG. 3, the number of trailing trays is 6633 and the degree of separation is 4.7.
2.2 repeatability
Precisely measuring 6 parts of standard sample solution by 10 mu L, injecting a sample, recording a chromatogram, and calculating the result according to the chromatogram:
| sample (I) | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Recovery rate | 103.6 | 102.1 | 101.2 | 100.5 | 102.8 | 104.3 | 1.4％ |
| Degree of separation | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | -- |
| Tailing factor | 1.16 | 1.16 | 1.16 | 1.17 | 1.17 | 1.17 | -- |
2.3 precision
Precisely measuring 10 μ l of reference solution, and continuously introducing sample for 6 times, wherein the peak area RSD of isomer is 1.3%.
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Peak area | 85120 | 85081 | 83477 | 85005 | 84332 | 82665 | 1.3％ |
2.4 sensitivity
Precisely measuring 10 mul of limiting solution, and injecting.
| Quantitative limit concentration (mu g/ml) | S/N of isomer peaks | Equivalent to the concentration of the samplePercentage (%) |
| 1.7 | 11 | 0.011 |
Example 4
1. Prepare solution (same as example 1)
2. HPLC detection
Diluent agent: ethanol: isopropanol 50:50 (vol/vol)
Mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid ═ 90:8:2:0.1 (volume ratio)
A chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm
Detection wavelength: 205nm
Column temperature: 30 deg.C
Flow rate: 1.5ml/min
2.1 chromatogram
Precisely measuring 10 μ l of the test solution, introducing sample, and recording chromatogram as shown in FIG. 3. According to FIG. 3, the number of trays is 4555 and the degree of separation is 4.5.
2.2 repeatability
Precisely measuring 6 parts of standard sample solution by 10 mu L, injecting a sample, recording a chromatogram, and calculating the result according to the chromatogram:
| sample (I) | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Recovery rate | 102.1 | 100.1 | 99.5 | 103.3 | 102.8 | 101.1 | 1.5％ |
| Degree of separation | 4.5 | 4.5 | 4.5 | 4.5 | 4.5 | 4.4 | -- |
| Tailing factor | 0.95 | 0.95 | 0.95 | 0.95 | 0.96 | 0.96 | -- |
2.3 precision
Precisely measuring 10 μ l of reference solution, and continuously introducing sample for 6 times, wherein the peak area RSD of isomer is 1.3%.
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Peak area | 75508 | 77653 | 77088 | 76357 | 75894 | 75139 | 1.3％ |
2.4 sensitivity
Precisely measuring 10 mul of limiting solution, and injecting.
| Quantitative limit concentration (mu g/ml) | S/N of isomer peaks | Concentration percentage of the sample (%) |
| 2.1 | 13 | 0.014 |
Comparative example 1
1. Reference is made to the "determination of isomers in inhaled acetylcysteine solution by HPLC" in the literature for solutions prepared and methods for HPLC detection.
Diluent agent: methanol
Mobile phase: n-hexane: ethanol: trifluoroacetic acid (v/v) 90:10:0.1 (vol/v)
A chromatographic column: CHIRALPAK IC, 4.6X 250mm, 5 μm
Detection wavelength: 215nm
Column temperature: 25 deg.C
Flow rate: 1.5ml/min
The injection volume is 10. mu.L.
2.1 chromatogram
Precisely measuring 10 μ l of sample solution, introducing sample, tailing factor of 1.02, number of column plates of 8357, resolution of 2.0, and recovery rate of added standard of 88.6%.
The method reported in the literature has low separation degree and insufficient accuracy.
Comparative example 2
1. Prepare solution (same as example 1)
2. HPLC detection
Diluent (b): ethanol
Mobile phase: n-hexane: isopropyl alcohol: ethanol: trifluoroacetic acid ═ 90:6:4:0.1 (vol.)
And (3) chromatographic column: CHIRALPAK IG, 4.6X 250mm, 5 μm
Detection wavelength: 210nm
Column temperature: 25 deg.C
Flow rate: 1.0ml/min
The injection volume is 10. mu.L.
2.1 chromatogram
Precisely measuring 10 μ l of sample solution, feeding sample, tailing factor of 0.79, number of plates of 2037, and degree of separation of 3.1. The composition of a mobile phase and a diluent are changed, and the peak shape difference of an isomer peak and the number of tower plates are low, so that the detection requirements are not met.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The detection method of acetylcysteine enantiomer is characterized in that a sample is diluted by a diluent and then is subjected to HPLC detection;
the diluent consists of ethanol and isopropanol;
the chromatographic conditions for the HPLC assay include:
a chromatographic column: a chiral column;
mobile phase: consists of normal hexane, isopropanol, ethanol and trifluoroacetic acid, and is eluted isocratically.
2. The detection method according to claim 1, wherein the packing material of the chromatography column is silica gel having amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded to the surface thereof.
3. The method of claim 2, wherein the chromatography column is CHIRALPAKIG chromatography column with a size of 4.6 x 250mm, 5 μm.
4. The detection method according to any one of claims 1 to 3, wherein the column temperature of the chromatography column is 30 ℃.
5. The detection method according to claim 1, wherein in the mobile phase, the volume ratio of n-hexane, isopropanol, ethanol and trifluoroacetic acid is (88-92): (6-10): 2: 0.1.
6. the detection method according to claim 1 or 5, wherein the flow rate of the mobile phase is 1.5 mL/min.
7. The detection method according to claim 1, wherein the detector of HPLC is an ultraviolet detector, and the detection wavelength is 205 nm.
8. The method of claim 1, wherein the amount of sample is 10 μ L for HPLC assay.
9. The detection method according to claim 1, wherein the volume ratio of ethanol to isopropanol in the diluent is (50-60): (40-50).
10. The detection method according to claim 1 or 9,
the sample is acetylcysteine injection or acetylcysteine injection added with acetylcysteine isomer standard; the volume ratio of the sample to the diluent is 1.5: 18.5;
the sample is a standard substance of acetylcysteine isomer, and the mass-volume ratio of the sample to the diluent is 5.6 mg: 250 mL.
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| US20200277258A1 (en) * | 2017-09-20 | 2020-09-03 | Nacuity Pharmaceuticals, Inc. | Method for Preparation of N-Acetyl Cysteine Amide and Derivatives Thereof |
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| CN101968470A (en) * | 2010-09-30 | 2011-02-09 | 湖北新生源生物工程股份有限公司 | Method for separating and measuring acetylcysteine enantiomers |
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