Detection method of acetylcysteine injection enantiomer

Abstract

The invention relates to the technical field of analysis and detection, and discloses an acetylcysteine injection enantiomer detection method, which comprises the following steps: freeze-drying a sample, diluting with a solvent, and directly carrying out HPLC (High Performance Liquid Chromatography) detection; chromatographic conditions of HPLC detection comprise: a chromatographic column is a chiral column; a mobile phase is composed of normal hexane, isopropanol, ethanol and trifluoroacetic acid, and gradient elution is carried out, by adopting the method provided by the invention for detection, the pretreatment steps of a sample can be reduced, and the elution of unknown impurities in the sample is accelerated and the operation time is shortened by utilizing gradient elution. The detection result shows that the method provided by the invention can enable the L-acetylcysteine and the D-acetylcysteine to have better separation degree, and the specificity, the sensitivity, the repeatability and the accuracy of the detection are good.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting enantiomers of acetylcysteine injection. Background Art

[0002] Acetylcysteine is a mucolytic agent with a strong dissolving effect on both white and thick sputum. The sulfhydryl groups in its molecule can cleave disulfide bonds within glycoprotein polypeptide chains in sputum, thereby reducing sputum viscosity and liquefying it for easier expectoration. It can also cleave DNA fibers in purulent sputum, thus dissolving both white, sticky, and purulent sputum. Acetylcysteine contains a chiral center and exists in two configurations: L- and D-cysteine, respectively. The L-configuration is the active configuration, while the D-configuration should be controlled as an impurity.

[0003] Acetylcysteine has been used as a medication for nearly 60 years (approved for marketing in September 1963). It is the acetylated form of the non-essential amino acid L-cysteine. Currently, acetylcysteine is available in the market in injections, inhalers, tablets, and granules.

[0004] The enantiomers acetyl-D-cysteine and acetyl-L-cysteine share the same functional groups and similar physical and chemical properties. They are also unstable to oxygen and high temperatures. Therefore, the detection of enantiomers is particularly important in the quality control of acetylcysteine injection. Existing detection methods have the following problems:

[0005] Sample pretreatment is complex, and some samples require derivatization. Acetylcysteine injection has a high water content, and most normal-phase chromatography systems are hydrophobic, and most chromatographic columns are not water-resistant. No solutions have been reported in the literature.

[0006] The insufficient separation of the enantiomer peaks results in low accuracy of the test results, which is insufficient to meet the requirements of quality control. Summary of the Invention

[0007] The sample pretreatment in the prior art is complex, and some require derivatization operations; acetylcysteine injection has a high water content, and most normal phase chromatography systems are hydrophobic systems, and most chromatographic columns are not water-resistant. The present invention solves the technical problems by adopting the following technical solutions: the method for detecting enantiomers of acetylcysteine injection of the present invention comprises the following steps: the sample is lyophilized, diluted with a solvent, and directly subjected to HPLC detection;

[0008] The chromatographic conditions of the HPLC detection include:

[0009] Chromatographic column: chiral column;

[0010] Mobile phase: composed of n-hexane, isopropanol, ethanol and trifluoroacetic acid, gradient elution.

[0011] In the chromatographic conditions provided in this application, by changing the sample processing method and optimizing the chromatographic conditions, a good separation of acetylcysteine and its enantiomers is achieved, and there is no need to rotary evaporate or freeze-dry the sample before detection, avoiding water bath heating treatment or freeze-drying that may change the sample, thereby simplifying the detection process.

[0012] Preferably, the freeze dryer is a vacuum freeze dryer, the pre-freezing temperature of the freeze dryer is -45°C, the freezing time is 30 minutes to 1 hour, and the freezing time is 3 to 6 hours.

[0013] Preferably, the freeze dryer has a primary drying parameter of heating to -15--20°C in 30-45 minutes and maintaining the primary drying time for up to 24 hours;

[0014] Raise the temperature from -15 to -20°C to -10 to -5°C and maintain for 6 hours;

[0015] Raise the temperature from -10 to -5°C to 5°C and maintain for 6 hours.

[0016] Preferably, the freeze dryer analytical drying parameters are: pre-vacuuming below 10 Pa, raising the temperature to 25° C. to 30° C. in 30 minutes to 1 hour, and continuing drying for 5 to 6 hours.

[0017] Experiments have shown that the composition and proportion of the mobile phase have a significant impact on sample separation.

[0018] Preferably, in the mobile phase, the composition of n-hexane, isopropanol, ethanol and trifluoroacetic acid is selected from one of the following:

[0019] n-hexane, ethanol, and trifluoroacetic acid;

[0020] n-hexane, isopropanol, and trifluoroacetic acid;

[0021] n-Hexane, isopropanol, ethanol.

[0022] Preferably, the mobile phase for gradient elution comprises n-hexane, isopropanol, ethanol and trifluoroacetic acid in a volume ratio of (88-98):(0-2):(0-10):0.1.

[0023] Preferably, in the mobile phase for gradient elution, the volume ratio of n-hexane, ethanol and trifluoroacetic acid is 90:10:0.1.

[0024] Preferably, in the mobile phase for gradient elution, the volume ratio of n-hexane, isopropanol and trifluoroacetic acid is 98:2:0.1.

[0025] Preferably, in the mobile phase for gradient elution, the volume ratio of n-hexane, isopropanol and trifluoroacetic acid is 88:2:10:0.1.

[0026] Preferably, the filler of the chromatographic column is silica gel coated with amylose-tris(3,5-dimethylphenylcarbamate).

[0027] The chiral chromatographic column has excellent separation ability and tolerance. For example, it can tolerate a small amount of residual water in acetylcysteine injection. Therefore, it is more conducive to selecting a pretreatment method for acetylcysteine injection.

[0028] Preferably, the solvent is ethanol.

[0029] Preferably, the sample is acetylcysteine injection, or acetylcysteine injection supplemented with an acetylcysteine enantiomer reference substance;

[0030] The volume ratio of the sample to the solvent is 1.5:48.5.

[0031] Preferably, the sample is an acetylcysteine reference substance and an enantiomer reference substance, and the mass-volume ratio of the sample to the solvent is 3 mg:30 μg:1 ml.

[0032] The chromatographic column is a Chiral ND (2) chromatographic column with dimensions of 4.6 × 250 mm and 5 μm;

[0033] The column temperature of the chromatographic column is 30°C.

[0034] The flow rate of the mobile phase was 1.0 ml / min.

[0035] The detector of the HPLC is a UV detector with a detection wavelength of 210 nm;

[0036] The injection volume for the HPLC test was 10 μl.

[0037] The sample is an acetylcysteine enantiomer reference substance, and the mass-volume ratio of the sample to the solvent is 3 mg:100 ml.

[0038] Preferably, the freeze dryer parameters include: pre-freezing temperature -40 ° C, time 45min, 4h. The primary drying parameters are 40min to -20 ° C, continuous primary drying time to 24h, from -20 ° C to -5 ° C, maintain for 6h; from -20 ° C to 5 ° C, maintain for 6h. The analytical drying parameters are pre-vacuum below 10Pa, 30min to raise the temperature to 25. Continue drying for 5h. The chromatographic conditions include: gradient elution, the volume ratio of n-hexane, ethanol and trifluoroacetic acid is 90:10:0.1; chromatographic column: Chiral ND (2), 4.6×250mm, 5μm; detection wavelength: 210nm; column temperature 30 ° C; flow rate: 1.0ml / min, injection volume 10μl. The mobile phase system time is 40min. Under the chromatographic conditions, the separation degree of acetylcysteine peak and enantiomer peak is greater than 7.0.

[0039] Preferably, the freeze dryer parameters include: pre-freezing temperature -45°C, time 45min, 6h. The primary drying parameters are: heating to -20°C in 45min, maintaining the primary drying time for 24h, heating from -20°C to -5°C, maintaining for 6h; heating from -15 to -20°C to 5°C, maintaining for 6h. The analytical drying parameters are: pre-vacuuming below 10Pa, raising the temperature to 30 in 45min. Continue drying for 6h; the chromatographic conditions include: gradient elution, the volume ratio of n-hexane, isopropanol and trifluoroacetic acid is 98:2:0.1; chromatographic column: ChiralND (2), 4.6×250mm, 5μm; detection wavelength: 210nm; column temperature 30°C; flow rate: 1.0ml / min, injection volume 10μl. The mobile phase system time is 40min. Under the chromatographic conditions, the separation degree of acetylcysteine peak and enantiomer peak is greater than 7.0.

[0040] Preferably, the freeze dryer parameters include: pre-freezing temperature -45°C, time 45min, 6h. The primary drying parameters are: heating to -20°C in 45min, maintaining the primary drying time for 24h, heating from -20°C to -5°C, maintaining for 6h; heating from -15 to -20°C to 5°C, maintaining for 6h. The analytical drying parameters are: pre-vacuuming below 10Pa, raising the temperature to 30 in 45min. Continue drying for 6h; the chromatographic conditions include: gradient elution, the volume ratio of n-hexane, isopropanol, ethanol and trifluoroacetic acid is 88:2:10:0.1; chromatographic column: ChiralND (2), 4.6×250mm, 5μm; detection wavelength: 210nm; column temperature 30°C; flow rate: 1.0ml / min, injection volume 10μl. The mobile phase system time is 40min. Under the chromatographic conditions, the separation degree of acetylcysteine peak and enantiomer peak is greater than 7.0.

[0041] This application uses an acetylcysteine enantiomer reference substance for qualitative analysis of enantiomers. The acetylcysteine enantiomers were quantified using a self-reference method. Testing revealed a limit of quantification of 1.2 μg / ml and a limit of detection of 0.43 μg / ml for the acetylcysteine enantiomers.

[0042] The beneficial effects of the present invention are as follows:

[0043] The present invention provides a method for detecting enantiomers of acetylcysteine injection, which comprises lyophilizing the sample, diluting it with a solvent, and directly performing HPLC detection; the solvent is ethanol; and the freeze dryer setting parameters include: pre-freezing parameters, primary drying parameters, and analytical drying parameters. The chromatographic conditions of the HPLC detection include: a chiral column; a mobile phase consisting of n-hexane, isopropanol, ethanol, and trifluoroacetic acid, and gradient elution. Using the method provided by the present invention for detection can reduce sample pretreatment steps, accelerate the elution of unknown impurities in the sample using gradient elution, and reduce run time. The test results show that the method of the present invention can achieve better separation between L-acetylcysteine and D-acetylcysteine, and the detection specificity, sensitivity, repeatability, and accuracy are all good. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a chromatogram of Example 1;

[0045] Figure 2 This is the chromatogram of Example 2;

[0046] Figure 3 This is the chromatogram of Example 3. DETAILED DESCRIPTION

[0047] The present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are provided for purposes of illustration and description and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments are chosen and described to better illustrate the principles of the invention and its practical application, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for specific applications.

[0048] The reagents and consumables used in the present invention are all common commercial products and can be purchased on the market. Among them, the enantiomer reference substance is from YC labs Rsearch Standard & Chemicals. The Chiral ND (2) chromatographic column is from Pheromones. The present invention is further described below in conjunction with the examples.

[0049] In Example 1, a method for detecting enantiomers of acetylcysteine injection comprises the following steps:

[0050] 1. Freeze-drying

[0051] Pre-freeze temperature: -40°C, time: 45 minutes, 4 hours. Primary drying parameters: heating to -20°C in 40 minutes, maintaining the primary drying time for up to 24 hours, heating from -20°C to -5°C for 6 hours, and heating from -20°C to 5°C for 6 hours. Drying parameters: pre-vacuum below 10Pa, temperature raised to 25°C in 30 minutes, and drying continued for 5 hours.

[0052] 2. Prepare solution

[0053] Blank excipient solution: Measure 1.5 ml of acetylcysteine injection blank excipient, place it in a 50 ml volumetric flask, dilute to the scale with solvent ethanol, shake well, and use it as the blank excipient solution.

[0054] System suitability solution: Take 3 mg of the enantiomer reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add solvent ethanol to dissolve and dilute to the scale, shake well, and use it as the enantiomer stock solution; take 3 mg of the acetylcysteine reference substance, accurately weigh it, place it in a sample vial, add 1 ml of the enantiomer stock solution to dissolve it, shake well, and use it as the system suitability solution.

[0055] Test solution: Measure 1.5 ml of acetylcysteine injection, place it in a 50 ml volumetric flask, dilute to the scale with solvent ethanol, shake well, and use it as the test solution.

[0056] Spiked test solution: Measure 1.5 ml of acetylcysteine injection and place it in a 50 ml volumetric flask. Accurately add 10 ml of the enantiomer stock solution, dilute to the mark with solvent ethanol, shake well, and use this as the spiked test solution.

[0057] Control solution: Measure 1 ml of the test solution or the spiked test solution, place it in a 100 ml volumetric flask, dilute to the scale with solvent ethanol, shake well, and use it as the control solution.

[0058] Quantitation limit solution: Measure 1 ml of the enantiomer stock solution, place it in a 25 ml volumetric flask, dilute to the mark with solvent ethanol, shake well, and use it as the quantitation limit solution.

[0059] Detection limit solution: Measure 3.5 ml of the quantification limit solution, place it in a 10 ml volumetric flask, dilute it to the scale with solvent ethanol, shake well, and use it as the detection limit solution.

[0060] 3. HPLC detection

[0061] Solvent: ethanol;

[0062] Mobile phase A: n-hexane, ethanol, and trifluoroacetic acid in a volume ratio of 90:10:0.1;

[0063] Mobile phase B: isopropanol;

[0064] Column: Chiral ND(2), 4.6×250 mm, 5 μm;

[0065] Detection wavelength: 210nm;

[0066] Column temperature: 30°C;

[0067] Flow rate: 1.0 ml / min;

[0068] Injection volume: 10 μl;

[0069] gradient:

[0070] t / min 0 18 22 32 32.1 40 A％ 98 98 90 90 98 98 B％ 2 2 10 10 2 2

[0071] 3.1 Chromatogram

[0072] Accurately measure 10 μl of the spiked test solution, inject the sample for detection, and record the chromatogram as shown in the figure. Figure 1 .according to Figure 1 , theoretical plate number 4813, separation degree 7.1.

[0073] 3.2 Exclusivity

[0074] Accurately measure 10 μl of solvent, blank excipient solution, system suitability solution, test solution, and control solution, inject the sample for testing, record the chromatogram, and obtain the results based on the chromatogram:

[0075] (1) No interference from solvents and blank excipients;

[0076] (2) The resolution of the enantiomer peak and the acetylcysteine peak in the system suitability solution was 8.7;

[0077] (3) There are no other impurity peaks in the test solution that interfere with the detection of enantiomers.

[0078] 3.3 Repeatability

[0079] Accurately measure 10 μl of 6 spiked test solution and 6 self-control solution, inject and test, record the chromatogram, and calculate the results based on the chromatogram:

[0080]

[0081] 3.4 Sensitivity

[0082] Accurately measure 10 μl of the detection limit and quantification limit solutions, inject the sample for detection, record the chromatogram, and obtain the results based on the chromatogram:

[0083]

[0084] 3.5 Accuracy

[0085] Accurately measure 10 μl of 6 spiked test solution and 6 self-control solution, inject and test, record the chromatogram, and calculate the results based on the chromatogram:

[0086]

[0087]

[0088] Example 2: A method for detecting enantiomers of acetylcysteine injection comprises the following steps:

[0089] 1. Freeze-drying

[0090] Pre-freeze temperature: -45°C, time: 45 minutes, 6 hours. Primary drying parameters: heating to -20°C over 45 minutes, maintaining the primary drying time for up to 24 hours; heating from -20°C to -5°C for 6 hours; heating from -15 to -20°C to 5°C for 6 hours. Drying parameters: pre-vacuum below 10 Pa, temperature raised to 30°C over 45 minutes, and drying continued for 6 hours.

[0091] 2. Prepare solution (same as in Example 1).

[0092] 3. HPLC detection

[0093] Solvent: ethanol;

[0094] Mobile phase A: n-hexane, isopropanol, and trifluoroacetic acid in a volume ratio of 98:2:0.1;

[0095] Mobile phase B: ethanol;

[0096] Column: Chiral ND(2), 4.6×250 mm, 5 μm;

[0097] Detection wavelength: 210nm;

[0098] Column temperature: 30°C;

[0099] Flow rate: 1.0 ml / min;

[0100] Injection volume: 10 μl;

[0101] gradient:

[0102] t / min 0 18 22 32 32.1 40 A％ 90 90 80 80 90 90 B％ 10 10 20 20 10 10

[0103] 3.1 Chromatogram

[0104] Accurately measure 10 μl of the spiked test solution, inject the sample for detection, and record the chromatogram as shown in the figure. Figure 2 .according to Figure 2 , theoretical plate number 4779, separation degree 7.2.

[0105] 3.2 Exclusivity

[0106] Accurately measure 10 μl of solvent, blank excipient solution, system suitability solution, test solution, and control solution, inject the sample for testing, record the chromatogram, and obtain the results based on the chromatogram:

[0107] (1) No interference from solvents and blank excipients;

[0108] (2) The resolution of the enantiomer peak and the acetylcysteine peak in the system suitability solution was 8.8;

[0109] (3) There are no other impurity peaks in the test solution that interfere with the detection of enantiomers.

[0110] 3.3 Repeatability

[0111] Accurately measure 10 μl of 6 spiked test solution and 6 self-control solution, inject and test, record the chromatogram, and calculate the results based on the chromatogram:

[0112]

[0113] 3.4 Sensitivity

[0114] Accurately measure 10 μl of the detection limit and quantification limit solutions, inject the sample for detection, record the chromatogram, and obtain the results based on the chromatogram:

[0115]

[0116] 3.5 Accuracy

[0117] Accurately measure 10 μl of 6 spiked test solution and 6 self-control solution, inject and test, record the chromatogram, and calculate the results based on the chromatogram:

[0118] sample 1 2 3 4 5 6 RSD (%) Recovery rate (%) 104.3 101.7 100.9 109.0 98.8 100.9 3.6

[0119] Example 3: A method for detecting enantiomers of acetylcysteine injection comprises the following steps:

[0120] 1. Freeze-drying (same as Example 2).

[0121] 2. Prepare solution (same as in Example 1).

[0122] 3. HPLC detection

[0123] Solvent: ethanol;

[0124] Mobile phase A: n-hexane, isopropanol, ethanol and trifluoroacetic acid in a volume ratio of 88:2:10:0.1,

[0125] Mobile phase B: ethanol;

[0126] Column: Chiral ND(2), 4.6×250 mm, 5 μm;

[0127] Detection wavelength: 210nm;

[0128] Column temperature: 30°C;

[0129] Flow rate: 1.0 ml / min;

[0130] Injection volume: 10 μl;

[0131] gradient:

[0132] t / min 0 18 22 32 32.1 40 A％ 100 100 80 80 100 100 B％ 0 0 20 20 0 0

[0133] 3.1 Chromatogram

[0134] Accurately measure 10 μl of the spiked test solution, inject the sample for detection, and record the chromatogram as shown in the figure. Figure 3 .according to Figure 3 , theoretical plate number 5069, separation degree 7.3.

[0135] 3.2 Exclusivity

[0136] Accurately measure 10 μl of solvent, blank excipient solution, system suitability solution, test solution, spiked test solution, and control solution, inject the sample for detection, record the chromatogram, and obtain the results based on the chromatogram:

[0137] (1) No interference from solvents and blank excipients;

[0138] (2) The resolution of the enantiomer peak and the acetylcysteine peak in the system suitability solution was 8.9;

[0139] (3) There are no other impurity peaks in the test solution that interfere with the detection of enantiomers.

[0140] 3.3 Repeatability

[0141] Accurately measure 10 μl of 6 spiked test solution and 6 self-control solution, inject and test, record the chromatogram, and calculate the results based on the chromatogram:

[0142]

[0143] 3.4 Sensitivity

[0144] Accurately measure 10 μl of the detection limit and quantification limit solutions, inject the sample for detection, record the chromatogram, and obtain the results based on the chromatogram:

[0145]

[0146] 3.5 Accuracy

[0147] Accurately measure 10 μl of 6 spiked test solution and 6 self-control solution, inject and test, record the chromatogram, and calculate the results based on the chromatogram:

[0148] sample 1 2 3 4 5 6 RSD (%) Recovery rate (%) 107.9 98.53 105.4 104.9 100.7 101.5 3.4

[0149] Comparative Example 1: A method for detecting enantiomers of acetylcysteine injection comprises the following steps:

[0150] 1. Freeze-drying (same as Example 1).

[0151] 2. Solution preparation (same as Example 1).

[0152] 3. HPLC detection

[0153] Solvent: ethanol;

[0154] Mobile phase: n-hexane, ethanol (isopropanol), and trifluoroacetic acid in a volume ratio of 85:15:0.1;

[0155] Column: Chiral ND(2), 4.6×250 mm, 5 μm;

[0156] Detection wavelength: 210nm;

[0157] Column temperature: 30°C;

[0158] Flow rate: 0.9 ml / min;

[0159] Injection volume: 10 μl;

[0160] Running time: 40 minutes.

[0161] 3.1 Chromatogram

[0162] Accurately measure 10 μl of the test solution and spiked test solution, inject the sample, and record the chromatogram. The chromatogram shows that the resolution between the acetylcysteine peak and the enantiomer peak is low, at 1.5, and the spike recovery is less than 80%. Changing the mobile phase ratio also results in poor resolution, and the spike recovery is not within the 80%-120% range, which does not meet the requirements.

[0163] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field and related fields without making creative efforts should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention shall be implemented in accordance with conventional means in the field unless otherwise specified or limited.

Claims

1. A method for detecting enantiomers of acetylcysteine injection, characterized in that: The samples were lyophilized and diluted with solvent and directly subjected to HPLC detection; The chromatographic conditions of the HPLC detection include: Chromatographic column: chiral column; Mobile phase: composed of n-hexane, isopropanol, ethanol and trifluoroacetic acid, gradient elution.

2. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The freeze dryer is a vacuum freeze dryer with a pre-freezing temperature of -45°C, a freezing time of 30 minutes to 1 hour, and a freezing time of 3 to 6 hours.

3. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The freeze dryer's primary drying parameters are to heat the temperature to -15--20°C within 30-45 minutes, and maintain the primary drying time for up to 24 hours. Raise the temperature from -15 to -20°C to -10 to -5°C and maintain for 6 hours; Raise the temperature from -10 to -5°C to 5°C and maintain for 6 hours.

4. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The analytical drying parameters of the freeze dryer are as follows: pre-vacuuming below 10 Pa, raising the temperature to 25° C. to 30° C. within 30 minutes to 1 hour, and continuing drying for 5 to 6 hours.

5. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: In the mobile phase, the composition of n-hexane, isopropanol, ethanol and trifluoroacetic acid is selected from one of the following: n-hexane, ethanol, and trifluoroacetic acid; n-hexane, isopropanol, and trifluoroacetic acid; n-Hexane, isopropanol, ethanol.

6. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The mobile phase for gradient elution comprises n-hexane, isopropanol, ethanol and trifluoroacetic acid in a volume ratio of (88-98):(0-2):(0-10):0.

1.

7. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The filler of the chromatographic column is silica gel with amylose-tris (3,5-dimethylphenylcarbamate) coated on the surface.

8. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The solvent is ethanol.

9. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The sample is acetylcysteine injection, or acetylcysteine injection added with an acetylcysteine enantiomer reference substance; The volume ratio of the sample to the solvent is 1.5:48.

5.

10. The method for detecting enantiomers of acetylcysteine injection according to claim 1, wherein: The samples are acetylcysteine reference substance and enantiomer reference substance, and the mass-volume ratio of the sample to the solvent is 3 mg:30 μg:1 ml.

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