CN115144498A - Method for detecting content of enantiomers in acetylcysteine and acetylcysteine particles - Google Patents
Method for detecting content of enantiomers in acetylcysteine and acetylcysteine particles Download PDFInfo
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- 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
- G01N30/26—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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention discloses a method for detecting content of enantiomers in acetylcysteine and acetylcysteine particles, which comprises the steps of directly adopting normal-phase high performance liquid chromatography to detect the content of the enantiomers in a test sample; the conditions of the high performance liquid chromatography are as follows: the chromatographic column is a coating type polysaccharide derivative chiral chromatographic column; the mobile phase is n-hexane-absolute ethyl alcohol-trifluoroacetic acid, and the volume ratio of the n-hexane to the absolute ethyl alcohol to the trifluoroacetic acid is (68-72) to (28-32) to (0.08-0.12); the elution mode is isocratic elution. The method provided by the invention can simply, conveniently, accurately and efficiently detect the content of the enantiomer in acetylcysteine, and has important significance for acetylcysteine quality research and control, clinical curative effect and medication safety.
Description
Technical Field
The invention relates to the technical field of drug analysis and detection, and particularly relates to a method for detecting content of an enantiomer in acetylcysteine and acetylcysteine particles.
Background
Acetylcysteine (N-acetyl-L-cysteine, L-NAC) is an amino acid drug, is a mucolytic agent, is suitable for dyspnea and dyspnea caused by a large amount of phlegm-sticky obstruction, and is also suitable for patients with postoperative dyspnea, acute and chronic bronchitis, bronchiectasis, pneumonia, tuberculosis, emphysema and the like with thick sputum and dyspnea, and is also commonly used for treating idiopathic interstitial lung. In recent years, it has been found that a 3% aqueous solution of the medicine can be used as an eye drop (acimetion) for treating keratitis, and with the research and development of fine cosmetics, recent photomedical and photobiological studies show that N-acetyl-L-cysteine can reduce subcutaneous melanin and eliminate the deposited melanin, and has been increasingly paid attention as a whitening cosmetic.
N-acetyl-L-cysteine is widely applied to the fields of medicines, pesticides, chemical industry and the like, only the conventional physicochemical index of N-acetyl-L-cysteine is usually detected at present, but a byproduct, namely N-acetyl-D-cysteine (D-NAC), which is an enantiomer, is generated in the production process, so that the product quality is influenced. Therefore, the research on the chiral resolution technology of NAC is of great significance to the medicine quality research.
At present, in patents 20100297844.9 and 201811516127.3, a derivatization reagent is adopted to perform derivatization reaction with acetylcysteine and an isomer thereof to obtain a derivatization product of the acetylcysteine and the isomer thereof, and then high performance liquid chromatography is used for separation and detection, so that the pre-column derivatization reaction has the disadvantages of complex operation steps, multiple reaction steps and difficulty in popularization and use. Therefore, a separation and purification method capable of directly, rapidly and accurately measuring the content of the acetylcysteine enantiomer is established, and the method has important guiding significance for medicine production and quality improvement.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting content of an enantiomer in acetylcysteine and acetylcysteine particles, so as to overcome the technical problems that complicated pre-column derivatization operation steps are required in the prior art, and the rapid and accurate determination of the content of the enantiomer of acetylcysteine is not facilitated.
The invention is realized by the following steps:
a method for detecting content of enantiomer in acetylcysteine and acetylcysteine granule comprises directly detecting content of enantiomer in a sample by normal phase high performance liquid chromatography;
the conditions of the high performance liquid chromatography are as follows:
the chromatographic column is a coating type polysaccharide derivative chiral chromatographic column;
the mobile phase is n-hexane-absolute ethyl alcohol-trifluoroacetic acid, and the volume ratio of the n-hexane to the absolute ethyl alcohol to the trifluoroacetic acid is (68-72) to (28-32) to (0.08-0.12);
the elution mode is isocratic elution.
In alternative embodiments, the volume ratio of n-hexane, absolute ethanol, and trifluoroacetic acid is 70.
In an alternative embodiment, the coated polysaccharide derivative chiral chromatographic column packing is starch-3, 5-ditolyl carbamate.
In an alternative embodiment, the column is a xylonite CHIRALPA IA, 250mm by 4.6mm,5 μm.
In an alternative embodiment, the flow rate of the mobile phase in the high performance liquid chromatography is 0.8 to 1.2mL/min.
In an alternative embodiment, the flow rate of the mobile phase is 1.0mL/min.
In an alternative embodiment, the detection wavelength of the high performance liquid chromatography is 205 to 215nm.
In an alternative embodiment, the detection wavelength is 210nm.
In an alternative embodiment, the column temperature of the high performance liquid chromatography is 24 to 26 ℃.
In an alternative embodiment, the column temperature of the chromatography column is 25 ℃.
In an alternative embodiment, the sample size of the high performance liquid chromatography is 8 to 12. Mu.L.
In an alternative embodiment, the sample size is 10 μ L.
In an optional embodiment, the detecting step includes injecting the blank solution, the system applicability solution, the control solution and the sample solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the enantiomer in the sample according to an external standard method;
the blank solution is absolute ethyl alcohol;
the system applicability solution is a solution which is prepared by absolute ethyl alcohol and contains 2.5-3.5 mg of acetylcysteine in each 1mL and a solution of 15-25 mug of N-acetyl-D-cysteine reference substance;
the control solution is prepared by preparing with anhydrous ethanol to contain 15 per 1mL 25 μ g of a solution of an N-acetyl-D-cysteine control;
the test solution is prepared by absolute ethyl alcohol and contains 2.5-3.5 mg of acetylcysteine in each 1 mL.
In an alternative embodiment, the above system suitability solution is a solution formulated with absolute ethanol to contain 3.0mg of acetylcysteine per 1mL, and 20 μ g of N-acetyl-D-cysteine control per 1 mL;
in an alternative embodiment, the above-mentioned control solution is a solution prepared by using absolute ethyl alcohol to prepare 20 μ g of N-acetyl-D-cysteine control in every 1 mL;
in an alternative embodiment, the test solution is prepared with absolute ethanol to contain 3.0mg of acetylcysteine per 1 mL.
The invention has the following beneficial effects:
the method directly adopts a high performance liquid chromatography method to detect the content of the acetylcysteine and the enantiomer in the acetylcysteine particles, does not need to perform derivatization on a sample, is simple and convenient to prepare the sample, and has the characteristics of good specificity, high accuracy, strong precision and sensitivity, good repeatability and small error, so that the detection method provided by the invention has important significance for the quality research and control of the acetylcysteine and the preparation thereof, and the curative effect and the medication safety of the medicine.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed 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 for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a chromatogram of a system suitability solution in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. 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 conventional products which are not indicated by manufacturers and are commercially available.
Due to the defects of the separation method of acetylcysteine and the isomer thereof in the prior art: the derivatization reaction is carried out firstly, and then the high performance liquid chromatography is used for separation and detection, the pre-column derivatization reaction has complex operation steps and more reaction steps, and is not easy to popularize and use. In order to improve the defects of the detection method, the inventor discovers through a series of creative works that the content of the enantiomers in acetylcysteine and acetylcysteine particles can be directly detected by adopting a coating type chiral chromatographic column with polysaccharide derivatives instead of derivatization, taking n-hexane-absolute ethyl alcohol-trifluoroacetic acid as a mobile phase and optimizing related high performance liquid chromatography conditions, and the effect of derivatization on a sample is not needed.
Based on the method, the invention provides a method for detecting the content of the enantiomer in acetylcysteine and acetylcysteine particles, which comprises the step of directly adopting normal-phase high performance liquid chromatography to detect the content of the enantiomer in a test sample.
Wherein, the conditions of the high performance liquid chromatography are as follows:
the chromatographic column is a coating type polysaccharide derivative chiral chromatographic column.
In some embodiments, the packing material of the coated polysaccharide derivative chiral chromatography column is starch-3, 5-ditolyl carbamate. Specifically, the coated polysaccharide derivative chiral chromatographic column may be xylonite CHIRALPA IA with specification of 250mm × 4.6mm,5 μm.
In some embodiments, the mobile phase is n-hexane-anhydrous ethanol-trifluoroacetic acid, and the volume ratio of the n-hexane, the anhydrous ethanol and the trifluoroacetic acid is (68-72): (28-32): (0.08-0.12).
In order to further improve the detection effect, the inventor optimizes the volume ratio of each component of the mobile phase, wherein the volume ratio of n-hexane, absolute ethyl alcohol and trifluoroacetic acid after optimization is 70.
The trifluoroacetic acid is added, so that the pH value of a mobile phase system can be ensured, the stable elution environment is realized, the dissociation of weakly dissociated compounds is inhibited, and the measurement accuracy is improved.
In some embodiments, the elution mode of the present invention is isocratic.
In some embodiments, the flow rate of the mobile phase in high performance liquid chromatography is 0.8 to 1.2mL/min. Specifically, the flow rate of the mobile phase may be 1.0mL/min.
In some embodiments, the detection wavelength of the high performance liquid chromatography is 205 to 215nm. Specifically, the detection wavelength may be 210nm.
In some embodiments, the column temperature of the high performance liquid chromatography is 24 to 26 ℃. Specifically, the column temperature of the above-mentioned column may be 25 ℃.
In some embodiments, the sample size for HPLC is 8-12 μ L. Specifically, the above-mentioned sample amount may be 10 μ L.
After setting the high performance liquid chromatography conditions, respectively injecting a blank solution, a system applicability solution, a reference solution and a test sample solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the enantiomer in the test sample according to an external standard method.
In some embodiments, the blank solution is absolute ethanol.
In some embodiments, the system suitability solution is formulated by: precisely weighing a proper amount of acetylcysteine and N-acetyl-D-cysteine reference substances, and quantitatively diluting with absolute ethyl alcohol to prepare a solution with acetylcysteine of 2.5-3.5 mg and N-acetyl-D-cysteine of 15-25 mu g in each 1 ml.
Specifically, the preparation method of the system applicability solution comprises the following steps: precisely weighing appropriate amount of acetylcysteine and N-acetyl-D-cysteine reference substances, and quantitatively diluting with anhydrous ethanol to obtain solution containing acetylcysteine 3.0mg and N-acetyl-D-cysteine 20 μ g per 1 ml.
The system applicability solution is used for verifying whether the performance of the high performance liquid chromatography system meets the detection requirement.
In some embodiments, the control solution is formulated by: accurately weighing 10mg of N-acetyl-D-cysteine reference substance, and quantitatively diluting with absolute ethyl alcohol to prepare a solution containing 15-25 mu g of N-acetyl-D-cysteine reference substance in each 1 mL.
Specifically, the preparation method of the control solution comprises the following steps: accurately weighing 10mg of an N-acetyl-D-cysteine reference substance, placing the reference substance into a 50mL volumetric flask, adding absolute ethyl alcohol to dissolve and dilute the reference substance to a scale, and shaking up to be used as a reference stock solution. Precisely transferring 5.0mL of the control stock solution into a 50mL volumetric flask, adding absolute ethyl alcohol to dilute to a scale, and shaking up to obtain the product.
In some embodiments, the test solution is prepared by: absolute ethyl alcohol is used for preparing a solution containing 2.5-3.5 mg of acetylcysteine in each 1 mL.
Specifically, the preparation method of the test solution comprises the following steps: a solution containing 3.0mg of acetylcysteine per 1mL of the solution was prepared using absolute ethanol.
The high performance liquid chromatograph used in the high performance liquid chromatography of the present invention may be any high performance liquid chromatograph that is conventional in the art, and is not limited herein.
The features and properties of the present invention are described in further detail below with reference to examples.
Materials: N-acetyl-D-cysteine reference substances (provided by Shanghai Danish Biotechnology Co., ltd.), acetylcysteine (provided by Wuhan Hongyuan Co., ltd.), and acetylcysteine granules (provided by Zhejiang Jinhua Kangenbei biopharmaceutical Co., ltd.).
Reagent: absolute ethanol (ex shanghai seudo chemical), n-hexane (ex Merck), trifluoroacetic acid (ex TCI).
Example 1
The embodiment is a verification of the specificity of the detection method of the invention, and the specific steps are as follows:
(1) Setting the conditions of high performance liquid chromatography:
a chromatographic column: xylonite CHIRALPAK IA, with specification of 250mm × 4.6mm,5 μm.
Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid, wherein the volume ratio of n-hexane, anhydrous ethanol and trifluoroacetic acid is 70.
Flow rate: 1.0mL/min, detection wavelength: 210nm, column temperature: at 25 ℃.
Isocratic elution was used.
(2) Preparing a solution:
the blank solution is absolute ethyl alcohol.
The preparation method of the system applicability solution comprises the following steps: precisely weighing appropriate amount of acetylcysteine and N-acetyl-D-cysteine reference substances, and quantitatively diluting with anhydrous ethanol to obtain solution containing acetylcysteine 3.0mg and N-acetyl-D-cysteine 20 μ g per 1 ml.
The preparation method of the control solution comprises the following steps: accurately weighing 10mg of an N-acetyl-D-cysteine reference substance, placing the reference substance into a 50mL volumetric flask, adding absolute ethyl alcohol to dissolve and dilute the reference substance to a scale, and shaking up to be used as a reference stock solution. Precisely transferring 5.0mL of the control stock solution into a 50mL volumetric flask, adding absolute ethyl alcohol to dilute to a scale, and shaking up to obtain the product.
(3) Determination and analysis: and respectively injecting 10 mu l of blank solution and 10 mu l of system applicability solution into a high performance liquid chromatograph, and recording the chromatogram.
Acceptance criteria: the blank solvent has no interference to detection, and the separation degree of acetylcysteine and N-acetyl-D-cysteine is more than 1.5.
The verification results are shown in fig. 1. As can be seen from FIG. 1, the blank solvent has no interference to the detection of the enantiomer, and the separation degree of acetylcysteine and N-acetyl-D-cysteine is 3.52, which meets the verification requirement of the specificity of the method.
Example 2
The embodiment is a verification of the sampling precision of the detection method: in the same experiment, the control solution was injected 6 times in duplicate, wherein the conditions of high performance liquid chromatography were the same as in example 1.
Acceptance criteria: the peak area RSD of the N-acetyl-D-cysteine is less than or equal to 5 percent after the control solution is repeatedly injected for 6 times.
The verification result is shown in Table 1, RSD of the reference solution in the area of 6 repeated peak injections is less than or equal to 5%, and the requirement of the method for verifying the repeatability is met.
TABLE 1 summary of repeated feeding of N-acetyl-D-cysteine control solutions
Example 3
The present embodiment is a verification of the linearity and range of the detection method. The method comprises the following specific steps:
taking an N-acetyl-D-cysteine reference substance as a material, respectively preparing linear solutions with the concentration ranges from the quantitative limit concentration to 2.0%, wherein the concentrations are respectively as follows: 1.5. Mu.g/mL, 3. Mu.g/mL, 6. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL, 45. Mu.g/mL, 60. Mu.g/mL; the limit percentages are respectively: 0.05%, 0.1%, 0.2%, 0.67%, 1.0%, 1.5%, 2.0%. The conditions of the high performance liquid chromatography were the same as in example 1.
Acceptance criteria: linear regression coefficient R in this range 2 Between 0.990 and 1.000.
The results are shown in Table 2, and the coefficient of regression R of N-acetyl-D-cysteine in this concentration range 2 In the range of 0.990 to 1.000And the verification requirements of the linearity and the range of the method are met.
TABLE 2 summary of acetylcysteine isomer linearity and Range evaluation results
Example 4
The present embodiment is a verification of the accuracy of the detection method. The method comprises the following specific steps:
taking an N-acetyl-D-cysteine reference substance and acetylcysteine as materials, respectively preparing 0.1%, 1% and 2% of standard sample adding solutions, preparing 3 parts of standard sample adding solutions with concentrations of 0.1% and 2% in parallel, preparing 6 parts of standard sample adding solutions with concentrations of 1% in parallel, and detecting isomers of the standard sample adding solutions, wherein the conditions of the high performance liquid chromatography are the same as those of example 1.
Acceptance criteria: the recovery rate of the solution of the standard sample is between 90 and 110 percent.
The experimental results are shown in table 3, and the recovery rates of the 12 test sample solutions are all between 90% and 110%, so that the verification requirement of the method accuracy is met.
TABLE 3 summary of acetylcysteine isomer method accuracy evaluation results
Example 5
This example is a verification of the reproducibility of the detection method: a1% standard-added test solution was prepared by Analyzer # 1, the preparation method and detection method were the same as in example 4, and the experiment was repeated 6 times.
Acceptance criteria: the isomer recovery rate RSD of the added sample solution is less than or equal to 10 percent.
The experimental results are shown in Table 4, and the RSD of the detection result obtained by independently repeating the experiment 6 times by the same experimenter is less than or equal to 10 percent, so that the method meets the verification requirement of the repeatability of the method.
TABLE 4 summary of the repeatability verification results of the acetylcysteine isomer testing method
Example 6
This example is a verification of the reproducibility of the detection method: the sample isomer 1% of the sample was added and tested by the analyst 2 on different instruments on different working days according to the experimental method of example 5, and the experiment was repeated 6 times.
Acceptance criteria: the recovery rate RSD of the isomer in the solution in the added sample is less than or equal to 15 percent.
The experimental results are shown in table 5, different experimenters repeat the experiment for 12 times on different instruments on different working days, the RSD of the detection result is less than or equal to 15%, and the requirement of the verification of the intermediate precision of the method is met.
TABLE 5 summary of intermediate precision verification results of acetylcysteine isomer detection methods
Example 7
This example is the verification of the quantitation limit and detection limit of the detection method. The method comprises the following specific steps:
taking an N-acetyl-D-cysteine reference substance as a material, gradually diluting with absolute ethyl alcohol, measuring 10 mu L, injecting into a high performance liquid chromatograph, recording a chromatogram, and determining the concentration as the limit of quantitation and the limit of detection when the signal-to-noise ratio of a target peak is close to 10 and 3.
Acceptance criteria: the signal-to-noise ratio of the quantitative limit solution is close to 10, and the RSD of the peak area of 6 repeated tests is less than or equal to 10 percent; the detection limit solution signal-to-noise ratio is close to 3.
The experimental result is shown in Table 6, the signal-to-noise ratio of the N-acetyl-D-cysteine control solution is close to 10, and the peak area RSD is less than or equal to 10%; the signal-to-noise ratio of the detection limit solution is close to 3. The verification requirements of the method quantification limit and the detection limit are met.
TABLE 6 summary of the quantitative limits and detection limit evaluation results of the acetylcysteine isomer detection method
Example 8
In this example, the content of the enantiomers in acetylcysteine and acetylcysteine particles is detected, and the steps are as follows:
the preparation method of the test solution comprises the following steps: acetylcysteine and acetylcysteine particles are precisely weighed and prepared into a solution containing 3.0mg of acetylcysteine per 1mL of absolute ethyl alcohol. The test solution was sampled and tested according to the method and conditions of example 1. The results are shown in Table 7.
TABLE 7 summary of acetylcysteine and acetylcysteine particle isomer measurements
| Name (R) | Isomer content (%) |
| Acetylcysteine (API) | Not detected out |
| Acetylcysteine granule (self-grinding) | Not detected out |
| Acetylcysteine granules (original grinding) | Not detected out |
Comparative example 1
The difference from example 1 is the change of column type: the column was CHIRALPAK ZWIX (+), 0.4 cm. Times.15 cm. Times.3 μm, and the other conditions were the same as in example 1.
As a result: the isomer and the main peak are sequentially peaked, the separation degree of the isomer and the main peak is 1.40, and the tailing factors are respectively 2.11 and 1.93; the blank at the main peak has an interference peak.
Comparative example 2
The difference from the embodiment 1 is that the composition of the mobile phase is changed, wherein the mobile phase is n-hexane: anhydrous ethanol (70.
As a result: the main peak and the isomer peak have tail type, and the tail factors of the isomer and the main peak are respectively 1.80,1.27.
Comparative example 3
The difference from example 1 is that the flow rate was changed to 0.6mL/min, and the other conditions were the same as example 1.
As a result: the blank interferes with the main peak.
Comparative example 4
The difference from example 1 is that the wavelength was changed to 220nm, and the other conditions were the same as example 1.
As a result: the isomer detection sensitivity is not high, and the limit of quantification can only be 0.15%.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting content of enantiomers in acetylcysteine and acetylcysteine particles is characterized in that the method comprises the steps of directly adopting normal-phase high performance liquid chromatography to detect the content of the enantiomers in a test sample;
the conditions of the high performance liquid chromatography are as follows:
the chromatographic column is a coating type polysaccharide derivative chiral chromatographic column;
the mobile phase is n-hexane-absolute ethyl alcohol-trifluoroacetic acid, and the volume ratio of the n-hexane to the absolute ethyl alcohol to the trifluoroacetic acid is (68-72) to (28-32) to (0.08-0.12);
the elution mode is isocratic elution.
2. The method according to claim 1, wherein the volume ratio of n-hexane, absolute ethanol and trifluoroacetic acid is 70.
3. The method according to claim 1, wherein the coated polysaccharide derivative chiral chromatography column packing material is starch 3, 5-ditolyl carbamate.
4. The method of claim 3, wherein the chromatography column is xylonite CHIRALPA IA, with a size of 250mm x 4.6mm,5 μm.
5. The method according to claim 4, wherein the flow rate of the mobile phase in the high performance liquid chromatography is 0.8-1.2 mL/min;
preferably, the flow rate of the mobile phase is 1.0mL/min.
6. The method according to claim 5, wherein the detection wavelength of the high performance liquid chromatography is 205 to 215nm;
preferably, the detection wavelength is 210nm.
7. The method of claim 6, wherein the column temperature of the high performance liquid chromatography is 24-26 ℃;
preferably, the column temperature of the chromatography column is 25 ℃.
8. The method according to claim 7, wherein the sample volume of the high performance liquid chromatography is 8-12 μ L;
preferably, the sample size is 10 μ L.
9. The method of claim 8, wherein the detecting step comprises injecting the blank solution, the system applicability solution, the control solution and the sample solution into the high performance liquid chromatograph, recording the chromatogram, and calculating the content of the enantiomer in the sample according to an external standard method;
the blank solution is absolute ethyl alcohol;
the system applicability solution is a solution which is prepared by absolute ethyl alcohol and contains 2.5-3.5 mg of acetylcysteine in each 1mL, and a solution of 15-25 mu g of N-acetyl-D-cysteine reference substance;
the reference substance solution is prepared by absolute ethyl alcohol, and each 1mL of the reference substance solution contains 15-25 mug of N-acetyl-D-cysteine;
the test solution is prepared by absolute ethyl alcohol and contains 2.5-3.5 mg of acetylcysteine in each 1 mL.
10. The method of claim 9, wherein the system suitability solution is a solution formulated with absolute ethanol as 3.0mg acetylcysteine per 1mL solution, 20 μ g N-acetyl-D-cysteine control;
preferably, the control solution is a solution prepared by using absolute ethyl alcohol to prepare 20 micrograms of N-acetyl-D-cysteine control substance in every 1 mL;
preferably, the test solution is a solution prepared by using absolute ethyl alcohol to contain 3.0mg of acetylcysteine in every 1 mL.
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