CN115326949A - Method for separating and detecting L-pyroglutamic acid and enantiomer thereof - Google Patents

Abstract

The invention relates to a method for measuring the content of enantiomer D-pyroglutamic acid in L-pyroglutamic acid by high performance liquid chromatography. The method uses a chromatographic column coated with amylose as a filler, alkane as a mobile phase A, a mixed solution as a mobile phase B, and adopts a gradient elution mode and an external standard method to measure L-pyroglutamic acid and enantiomers thereof. The method provided by the invention can accurately separate and measure the enantiomer in the L-pyroglutamic acid, has strong specificity, high sensitivity, high accuracy and simple operation, and is convenient for effectively controlling the content of the enantiomer in the L-pyroglutamic acid.

Description

Method for separating and detecting L-pyroglutamic acid and enantiomer thereof

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and measuring L-pyroglutamic acid and enantiomers thereof by using a high performance liquid chromatography.

Background

L-Pyroglutamic acid (L-Pyroglutamic acid) is a natural humectant, has been used as a cosmetic raw material and an oxidase inhibitor for a long time, and is also a main raw material of novel immune promoters pidotimod, L-theanine and other medicaments. In recent years, with the improvement of living standard of people, the demand of cosmetics and medicines is increasing, and the market demand of L-pyroglutamic acid is also increasing. The chemical name of the L-pyroglutamic acid is (S) - (-) -2-pyrrolidone-5-carboxylic acid, and the chemical structural formula is as follows:

l-pyroglutamic acid has 1 chiral center in the molecule, and thus one enantiomer, D-pyroglutamic acid, exists. Quality control is required in the production of L-pyroglutamic acid. However, at present, no reference is made to United states Pharmacopeia USP, european Pharmacopeia EP and Chinese Pharmacopeia Ch.P. and no relevant literature reports exist on the separation and determination of L-pyroglutamic acid and its enantiomer by using a gradient elution method. In order to better and more accurately control the content of the enantiomer of the L-pyroglutamic acid and to control the risk of bringing about quality in the subsequent use process to the maximum extent, the inventors developed an analytical method suitable for simply, rapidly and accurately determining the content of the enantiomer of the L-pyroglutamic acid.

Disclosure of Invention

The invention provides a method for separating and detecting L-pyroglutamic acid and enantiomers thereof, which is characterized by comprising the following steps:

a) Taking a linear starch coated filler as a chiral separation chromatographic column;

b) Taking lower alkane as a mobile phase A, and taking the mixed solution as a mobile phase B;

c) The mobile phase adopts a gradient elution method, the flow rate of the mobile phase is 0.5-2.0 mL/min, the detection wavelength is 200-280 nm, and the column temperature is 20-45 ℃;

the chiral separation chromatographic column is CHIRALPAK AD-H, the specification is 250mm multiplied by 4.6mm, and the diameter is 5 mu m;

the mixed solution is a mixed solution of trifluoroacetic acid and an alcohol solution;

the gradient elution method of the mobile phase comprises the following steps:

time (min)	Lower alkane (%)	Mixed solution (%)
			0	100～90	0～10
1	100～90	0～10
			9	90～70	10～30
15	90～70	10～30
			15.1	100～90	0～10
25	100～90	0～10

The structural formula of the L-pyroglutamic acid and the enantiomer D-pyroglutamic acid thereof is shown as follows:

in some embodiments, the lower alkane is selected from one or more of n-hexane, cyclohexane, n-pentane and n-heptane, and the alcohol solution is selected from one or more of methanol, ethanol, isopropanol and n-propanol.

In some embodiments, the lower alkane is n-hexane, and the alcohol solution is a mixed solution of ethanol and isopropanol.

In some embodiments, the alcohol solution has a ratio of isopropanol: ethanol volume ratio of 2:1-1:2, trifluoroacetic acid concentration of 0.01% -0.15% V/V.

In some embodiments, the ratio of isopropyl alcohol: the volume ratio of ethanol is 1:1.

in some embodiments, the trifluoroacetic acid concentration is 0.1% V/V.

In some embodiments, the method further comprises the following steps:

d) Preparing a test solution: taking a proper amount of L-pyroglutamic acid sample, adding a proper amount of diluent to dissolve and dilute the L-pyroglutamic acid sample to prepare a sample solution containing 5-10 mg of L-pyroglutamic acid per 1mL of the sample solution as a sample solution.

e) Preparation of a reference solution: precisely weighing a proper amount of D-pyroglutamic acid, adding a proper amount of diluent to dissolve the D-pyroglutamic acid, and preparing the D-pyroglutamic acid into a solution with the concentration of 0.5-1.5 percent of that of the test solution as a reference solution.

f) Setting the sample introduction volume to be 5-30 mu L, respectively and precisely measuring equal volumes of a test solution and a reference solution, injecting the solutions into a liquid chromatograph, and recording a chromatogram.

In some embodiments, the detection wavelength is 205nm, the column temperature is 35 ℃, the flow rate of the mobile phase is 0.8mL/min, the diluent is ethanol, and the injection volume is 20 μ L.

At present, no analytical method using gradient elution is used for detecting the content of L-pyroglutamic acid enantiomer. The invention is tested and verified repeatedly, using CHIRALPAK AD-H chiral chromatographic column (250 x 4.6mm,5 μm) as separation chromatographic column, n-hexane as mobile phase A, isopropanol: ethanol =50 (containing 0.1% V/V trifluoroacetic acid) as mobile phase B, and the gradient elution method can perform baseline separation on L-pyroglutamic acid and its enantiomer, and can simply, rapidly and accurately control the content of its enantiomer in L-pyroglutamic acid. The invention adopts CHIRALPAKAD-H as a chiral chromatographic column, takes lower alkane and alcohol solution as a mobile phase, has the Retention Time (RT) of the peak position of L-pyroglutamic acid of about 15.5min, the RT of enantiomer of the L-pyroglutamic acid of about 16.5min, the separation degree of more than 1.5, meets the requirements of Chinese pharmacopoeia, has stable peak position, and realizes the separation and determination of the L-pyroglutamic acid and the enantiomer of the L-pyroglutamic acid.

Drawings

FIG. 1 is a liquid chromatogram of a solution of L-pyroglutamic acid and enantiomers thereof useful in a system under isocratic elution.

FIG. 2 is a liquid chromatography overlay of suitable solutions of L-pyroglutamic acid and its enantiomer systems obtained using different diluents under gradient elution conditions.

FIG. 3 is a liquid chromatogram of a blank solution under gradient elution conditions.

FIG. 4 is a liquid chromatogram of a solution of L-pyroglutamic acid and enantiomers thereof suitable for use in a system under gradient elution conditions.

FIG. 5 is a liquid chromatogram of a sensitive solution (0.05%) of L-pyroglutamic acid under gradient elution conditions.

FIG. 6 is a liquid chromatogram of a test solution of L-pyroglutamic acid under gradient elution conditions.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution of the present invention is described below with reference to specific embodiments.

Example 1: screening of chromatographic conditions

1. Apparatus and conditions

Water acquired Arc high performance liquid chromatography;

agilent 1260Infinity II high performance liquid chromatography;

XES205DU electronic analytical balance (mettleritoduo);

l-pyroglutamic acid was purchased from Hongshan-pharmaceutical industries, inc. of Emei,

l-pyroglutamic acid and its enantiomer control were purchased from Shanghai Allantin Biotechnology Ltd;

n-hexane, isopropanol, ethanol and trifluoroacetic acid are all chromatographically pure;

2. experimental methods and results

Example 1-a

A chromatographic column 1: CHIRALPAK AD-H chiral chromatography column (250 x 4.6mm,5 μm);

and (3) chromatographic column 2: CHIRALPAK IC chiral chromatography column (250 × 4.6mm,5 μm);

a chromatographic column 3: CHIRALPAK IG-3 chiral chromatography column (250 x 4.6mm,5 μm);

detection wavelength: 205nm;

column temperature: 30 to 40 ℃;

sample introduction volume: 20 mu L of the solution;

flow rate: 0.6-1.0 mL/min;

mobile phase A: n-hexane, mobile phase B: isopropanol (0.1% trifluoroacetic acid, V/V),

mobile phase A: mobile phase B = 80;

solution preparation

Diluent agent: ethanol (0.1% trifluoroacetic acid, V/V)

System applicability solution: taking about 65mg of D-pyroglutamic acid reference substance, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of diluent, ultrasonically dissolving, adding diluent to dilute to scale, shaking up, and using as 0.65mg/mL D-pyroglutamic acid reference substance stock solution. Precisely weighing about 65mg of L-pyroglutamic acid reference substance, placing in a 10mL measuring flask, precisely weighing 1mL of D-pyroglutamic acid reference substance storage solution, placing in the same 10mL measuring flask, adding appropriate amount of diluent, ultrasonically dissolving, adding diluent to scale, and shaking. (L-pyroglutamic acid concentration 6.5mg/mL, D-pyroglutamic acid concentration 0.065 mg/mL)

The flow rate, the column temperature, the proportion of the mobile phase and the chromatographic column in the chromatographic conditions are respectively changed, other conditions and parameters are not changed, and the separation condition of the L-pyroglutamic acid and the enantiomer thereof is investigated. The results are shown in Table 1

Table 1: baseline split status after changing test parameters

As can be seen from the results in the table above, using column 1: CHIRALPAK AD-H chiral chromatography column (250 × 4.6mm,5 μm), detection wavelength: 205nm, column temperature: 35 ℃, injection volume: 20 μ L, flow rate: 1.0mL/min, mobile phase a — mobile phase B =88 is the preferred chromatographic condition.

Example 1 b

Method verification was performed on the basis of example 1-a.

1. Solution preparation

Diluent agent: ethanol (0.1% trifluoroacetic acid, V/V)

Control solution: taking about 65mg of D-pyroglutamic acid reference substance, accurately weighing, placing in a 10mL measuring flask, adding a proper amount of diluent to dissolve, adding the diluent to dilute to a scale, and shaking up to obtain 6.5mg/mL D-pyroglutamic acid reference substance stock solution. Precisely measuring 1mL of D-pyroglutamic acid reference stock solution, placing into a 100mL measuring flask, adding diluent to dilute to scale, and shaking up to obtain the final product. (D-pyroglutamic acid concentration 0.065 mg/mL).

System applicability solution: precisely weighing about 65mg of L-pyroglutamic acid reference substance, placing in a 10mL measuring flask, precisely weighing 1mL of D-pyroglutamic acid reference substance storage solution, placing in the same 10mL measuring flask, adding appropriate amount of diluent to dissolve, adding diluent to dilute to scale, and shaking up to obtain the final product. (L-pyroglutamic acid concentration 6.5mg/mL, D-pyroglutamic acid concentration 0.065 mg/mL)

Sensitivity solution: precisely measuring 1mL of the system applicability solution, placing the system applicability solution in a 100mL measuring flask, adding the diluent to dilute to the scale, shaking up, precisely measuring 1mL, placing the system applicability solution in a 20mL measuring flask, adding the diluent to dilute to the scale, and shaking up to obtain the product. (0.05%)

Test solution: taking about 65mg of a sample, precisely weighing, placing in a 10mL measuring flask, adding a proper amount of diluent to dissolve, adding the diluent to dilute to a scale, and shaking up to obtain the test solution. (test article concentration 6.5 mg/mL).

2. Chromatographic conditions

A chromatographic column: CHIRALPAK AD-H chiral chromatography column (250X 4.6mm,5 μm)

Detection wavelength: 205nm

Column temperature: 35 deg.C

Sample injection volume: 20 μ L

Flow rate: 1.0mL/min

Mobile phase A: n-hexane-mobile phase B: isopropanol (0.1% trifluoroacetic acid, V/V) =88

3. Sample application recovery test

According to the 'analysis method verification guide principle' in 'Chinese pharmacopoeia', preparing a solution with 50% limit concentration, 100% limit concentration and 150% limit concentration of D-pyroglutamic acid (the limit is 1% of the concentration of the L-pyroglutamic acid), and calculating the recovery rate of the D-pyroglutamic acid.

The result shows that the sample recovery rate of the D-pyroglutamic acid is between 163.9% and 262.0% (RSD =18.3%, n = 7), and exceeds the regulation of analysis method verification guiding principle in Chinese pharmacopoeia.

Examples 1 to c

An attempt was made to use a gradient elution method based on example 1-b;

a chromatographic column: CHIRALPAK AD-H chiral chromatography column (250 x 4.6mm,5 μm);

detection wavelength: 205nm;

column temperature: 35 ℃;

sample introduction volume: 20 mu L of the solution;

flow rate: 0.8mL/min;

mobile phase A: n-hexane,

mobile phase B1: isopropanol (0.1% trifluoroacetic acid, V/V),

mobile phase B2: isopropanol-ethanol =50 (0.1% trifluoroacetic acid, V/V);

solution preparation

Diluent 1: ethanol (0.1% trifluoroacetic acid, V/V)

Diluent 2: ethanol

System applicability solution: taking about 65mg of D-pyroglutamic acid reference substance, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of diluent to dissolve, adding diluent to dilute to scale, shaking up, and using as 0.65mg/mL D-pyroglutamic acid reference substance stock solution. Precisely weighing about 65mg of L-pyroglutamic acid reference substance, placing in a 10mL measuring flask, precisely weighing 1mL of D-pyroglutamic acid reference substance storage solution, placing in the same 10mL measuring flask, adding appropriate amount of diluent to dissolve, adding diluent to dilute to scale, and shaking up to obtain the final product. (L-pyroglutamic acid concentration 6.5mg/mL, D-pyroglutamic acid concentration 0.065 mg/mL)

The diluent is diluent 2, other conditions and parameters are unchanged, and the separation condition of L-pyroglutamic acid and the enantiomer thereof by different gradient elution is investigated.

The results are shown in Table 2:

table 2: baseline split status after changing test parameters

In summary, the optimal chromatographic conditions are as follows:

and (3) chromatographic column: CHIRALPAK AD-H chiral chromatography column (250 x 4.6mm,5 μm);

detection wavelength: 205nm;

column temperature: 35 ℃;

sample introduction volume: 20 mu L of the solution;

flow rate: 0.8mL/min;

mobile phase A: n-hexane,

mobile phase B: isopropanol-ethanol =50 (0.1% trifluoroacetic acid, V/V),

the gradient elution was as follows:

the results of screening experiments performed with varying diluent types with reference to the preferred gradient elution conditions described above are shown in FIG. 2.

As can be seen by comparing the system suitability solution chromatogram obtained by using the diluent 2, a large unknown single impurity appears in the system suitability solution chromatogram obtained by using the diluent 1.

The preferred diluent is ethanol.

Example 2 validation of analytical methods

1. Apparatus and conditions

Water acquired Arc high performance liquid chromatography;

XES205DU electronic analytical balance (mettleritoduo);

l-pyroglutamic acid was obtained from Hongshan pharmaceutical industry, inc. of Emei mountain, and L-pyroglutamic acid and its enantiomer control were obtained from Allatin Biotechnology, inc. of Shanghai;

the normal hexane, the isopropanol, the ethanol and the trifluoroacetic acid are all chromatographically pure;

2. experimental methods and results

1. Solution preparation

Diluent agent: ethanol

Control solution: taking about 65mg of D-pyroglutamic acid reference substance, precisely weighing, placing in a 10mL measuring flask, adding appropriate amount of diluent to dissolve, adding diluent to dilute to scale, shaking up, and using as 6.5mg/mL D-pyroglutamic acid reference substance stock solution. Precisely measuring 1mL of D-pyroglutamic acid reference stock solution, placing into a 100mL measuring flask, adding diluent to dilute to scale, and shaking up to obtain the final product. (D-pyroglutamic acid concentration 0.065 mg/mL).

System applicability solution: taking about 65mg of D-pyroglutamic acid reference substance, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of diluent, ultrasonically dissolving, adding diluent to dilute to scale, shaking up, and using as 0.65mg/mL D-pyroglutamic acid reference substance stock solution. Precisely weighing about 65mg of L-pyroglutamic acid reference substance, placing in a 10mL measuring flask, precisely weighing 1mL of D-pyroglutamic acid reference substance storage solution, placing in the same 10mL measuring flask, adding appropriate amount of diluent, ultrasonically dissolving, adding diluent to scale, and shaking. (L-pyroglutamic acid concentration 6.5mg/mL, D-pyroglutamic acid concentration 0.065 mg/mL).

Sensitivity solution: precisely measuring 1mL of the system applicability solution, placing the system applicability solution in a 100mL measuring flask, adding the diluent to dilute to the scale, shaking up, precisely measuring 1mL, placing the system applicability solution in a 20mL measuring flask, adding the diluent to dilute to the scale, and shaking up to obtain the product. (0.05%).

Test solution: taking about 65mg of L-pyroglutamic acid sample, precisely weighing, placing in a 10mL measuring flask, adding appropriate amount of diluent to dissolve, adding diluent to dilute to scale, and shaking up to obtain the final product. (L-pyroglutamic acid concentration 6.5 mg/mL).

2. Chromatographic conditions

A chromatographic column: CHIRALPAK AD-H chiral chromatography column (250 x 4.6mm,5 μm);

mobile phase A: n-hexane

Mobile phase B: isopropyl alcohol: ethanol =50 (V/V, containing 0.1% trifluoroacetic acid)

Elution gradient:

column temperature: 35 ℃;

flow rate: 0.8mL/min;

sample introduction amount: 20 mu L of the solution;

3. system suitability survey

The diluent, the system applicability solution and the sample solution are respectively sampled for measurement, and the result shows that the L-pyroglutamic acid (RT is about 15.5 min) and the D-pyroglutamic acid (RT is about 16.5 min) are sequentially subjected to peak discharge, the baselines of the L-pyroglutamic acid and the D-pyroglutamic acid are completely separated, the separation degree is 1.52, and the blank does not interfere with the measurement. The blank solution chromatogram is shown in FIG. 3, and the system suitability chromatogram is shown in FIG. 4.

4. Line survey

Accurately weighing appropriate amount of D-pyroglutamic acid reference substance, adding diluent, dissolving, diluting, shaking, preparing into solution with series concentration, injecting into liquid chromatograph, and performing linear regression to obtain regression equation of D-pyroglutamic acid. The results show that the linear relationship is good in the concentration range of 0.0065-0.26mg/mL (namely 10% -400% of the limit concentration), and R2=0.9997.

5. Sensitivity investigation

A reference sample of L-pyroglutamic acid (65 mg) is precisely weighed, dissolved by adding a diluent and uniformly shaken to be diluted into a solution (namely 0.05%) of the L-pyroglutamic acid (0.00325 mg/mL). The result shows that the chromatographic peak signal-to-noise ratio of the L-pyroglutamic acid is 25.5 under the concentration condition, and the sensitivity is high. The chromatogram of the sensitive solution is shown in FIG. 5.

6. Accuracy test

The recovery rate of D-pyroglutamic acid was calculated by preparing solutions of D-pyroglutamic acid with 50% limiting concentration accuracy, 100% limiting concentration accuracy and 150% limiting concentration accuracy (limiting 1% of the concentration of L-pyroglutamic acid) according to a conventional method. The results showed that the recovery of D-pyroglutamic acid was between 96.1% and 102.9% (RSD =2.8%, n = 6). The method is good in accuracy.

7. Investigation of solution stability

Standing the above control solution at room temperature, sampling at 0h, 0.5h, 1.0h, 12.5h and 19.5h for determination, and recording chromatogram. The result shows that the percentage of the peak area of the D-pyroglutamic acid reference substance solution to the peak area of 0h at different time points is between 99.8 and 104.3 percent, and the requirement is met.

And (4) taking the test solution to be placed at room temperature, injecting samples for measurement for 0 hour and 15 hours respectively, and recording a chromatogram. The result shows that the ratio of the peak area of D-pyroglutamic acid to the peak area of 0h in the test solution after being placed for 15h is 94.8%, which meets the requirement.

8. Repeatability survey

And respectively preparing a diluent, a system applicability solution, a reference substance solution, a test sample solution and a 100% limit concentration standard test sample solution, carrying out sample injection measurement, and recording a chromatogram. The results showed that the sample recovery of D-pyroglutamic acid was between 97.8% and 101.4% (RSD =1.3%, n = 6), and the average recovery was 99.5%. The method is proved to have good repeatability.

9. Durability examination

Respectively preparing a diluent, a system applicability solution, a reference substance solution, a test sample solution and a 100% limit concentration standard test sample solution, properly adjusting the flow rate (+ -0.1 mL/min) and the column temperature (+ -3 ℃) for sample injection measurement, and recording the chromatogram. The result shows that the blank solvent has no interference to the detection of the D-pyroglutamic acid under all conditions; in the sensitive solution under each condition, the signal-to-noise ratio of the L-pyroglutamic acid peak is between 25.5 and 72.7; under each condition, the recovery rate of the D-pyroglutamic acid in the standard sample solution with the 100% limit concentration is between 89.6% and 99.5% (RSD =4.1%, n = 5), and the average recovery rate is 95.4%; the amount of D-pyroglutamic acid detected in the test solution was between 0.06% and 0.08% (n = 5). Indicating that the durability of the method is good.

10. Determination of D-pyroglutamic acid content in raw materials

Taking a proper amount of a test sample, preparing a D-pyroglutamic acid reference solution and an L-pyroglutamic acid test sample solution according to the method, respectively injecting samples for determination, and recording a chromatogram map. If the sample solution has impurity peaks with retention time same as that of the reference solution, calculating the content of D-pyroglutamic acid by peak area according to an external standard method. The chromatogram of the test solution is shown in FIG. 6. The contents of D-pyroglutamic acid in the three batches of samples were 0.09%, and 0.10%, respectively.

The above description is only an example of the present invention, and is not intended to limit the scope of the present invention. The method has the advantages of good separation degree, high sensitivity, good durability, accurate quantification and simple and convenient operation.

Claims (8)

1. A method for separating and measuring D-pyroglutamic acid enantiomer in L-pyroglutamic acid by high performance liquid chromatography is characterized in that:

a) Taking a linear starch coated filler as a chiral separation chromatographic column;

b) Taking lower alkane as a mobile phase A, and taking the mixed solution as a mobile phase B;

c) The mobile phase adopts a gradient elution method, the flow rate of the mobile phase is 0.5-2.0 mL/min, the detection wavelength is 200-280 nm, and the column temperature is 20-45 ℃;

the chiral separation chromatographic column is CHIRALPAK AD-H, the specification is 250mm multiplied by 4.6mm, and the diameter is 5 mu m;

the mixed solution is a mixed solution of trifluoroacetic acid and an alcohol solution;

the gradient elution method of the mobile phase comprises the following steps:

time (min) Lower alkane (%) Mixed solution (%) 0 100～90 0～10 1 100～90 0～10 9 90～70 10～30 15 90～70 10～30 15.1 100～90 0～10 25 100～90 0～10

The structural formula of the L-pyroglutamic acid and the enantiomer D-pyroglutamic acid thereof is shown as follows:

2. the method of claim 1, wherein the lower alkane is selected from one or more of n-hexane, cyclohexane, n-pentane and n-heptane, and the alcohol solution is selected from one or more of methanol, ethanol, isopropanol and n-propanol.

3. The method of claim 2, wherein the lower alkane is n-hexane, and the alcohol solution is a mixed solution of ethanol and isopropanol.

4. The method according to any one of claims 1 to 3, wherein the alcohol solution has a molar ratio of isopropanol: ethanol volume ratio of 2:1-1:2, trifluoroacetic acid concentration of 0.01% -0.15% V/V.

5. The process of claim 4, wherein the ratio of isopropyl alcohol: the volume ratio of ethanol is 1:1.

6. the process according to claim 4, wherein the trifluoroacetic acid is at a concentration of 0.1% V/V.

7. The method of claim 1, further comprising the steps of:

d) Preparing a test solution: taking a proper amount of L-pyroglutamic acid sample, adding a proper amount of diluent to dissolve and dilute the L-pyroglutamic acid sample to prepare a sample solution containing 5-10 mg of L-pyroglutamic acid per 1mL of the sample solution as a sample solution.

e) Preparing a reference substance solution: precisely weighing a proper amount of D-pyroglutamic acid, adding a proper amount of diluent for dissolution, and preparing the diluent into a solution with the concentration of 0.5-1.5 percent of the concentration of the test solution as a reference solution.

f) Setting the sample introduction volume to be 5-30 mu L, respectively and precisely measuring equal volumes of a test solution and a reference solution, injecting the solutions into a liquid chromatograph, and recording a chromatogram.

8. The method according to claim 7, wherein the detection wavelength is 205nm, the column temperature is 35 ℃, the flow rate of the mobile phase is 0.8mL/min, the diluent is ethanol, and the injection volume is 20 μ L.

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