CN109085255B - Method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and enantiomer thereof by using HPLC method - Google Patents
Method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and enantiomer thereof by using HPLC method Download PDFInfo
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Abstract
The invention discloses a method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and an enantiomer thereof by an HPLC (high performance liquid chromatography) method. The method can simply, accurately and efficiently analyze and prepare the 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole with single optical activity and the enantiomer thereof, thereby realizing the quality control of the 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole.
Description
Technical Field
The invention relates to a chromatographic analysis separation method, in particular to a high performance liquid chromatography for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and enantiomers thereof, belonging to the technical field of chemical raw material preparation.
Background
Urine leucocyte is an important index of conventional urine detection, and the pyrrolyl ester is an important substrate in a reagent band module for determining the urine leucocyte by a drying method, and the determination principle is as follows: the neutrophil cytoplasm in the leucocyte contains esterase which acts on the pyrrole ester in the test paper module to cause the pyrrole ester to generate hydrolysis reaction and further couple with diazonium salt to form purple complex, and the shade of the color of the purple complex is in direct proportion to the number of the neutrophil. The pyrrole esters are specifically classified into (-) -pyrrole esters with chemical names of 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and (+) -pyrrole esters with chemical names of 3- (N-p-toluenesulfonyl-D-alanyloxy) -5-phenylpyrrole, and the structure of the (-) -pyrrole esters is shown as the formula (1):
in formula 1, A: (-) -pyrrolyl ester; b: (+) -pyrrole ester;
the esterase has different stereoselectivity to two enantiomers of the pyrrole ester, the stereoselectivity to the (-) -pyrrole ester is stronger, the selectivity to the (+) -pyrrole ester is weaker than that to the other enantiomer, and therefore, the pyrrole ester used in the urine leukocyte test paper is generally the (-) -pyrrole ester. Therefore, how to analyze and prepare (-) -pyrrolate and enantiomers thereof and control the content of (+) -pyrrolate impurities are very important, and further, the quality of urine leukocyte test paper is ensured. However, no relevant report is found on the analytical separation method of the two enantiomers of the pyrrole ester.
Therefore, how to establish a method for simply, rapidly and efficiently analyzing and separating two enantiomers of pyrrole ester so as to realize the quality control of the pyrrole ester is a technical problem to be solved at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a liquid chromatography method using a chiral stationary phase to analyze and separate two enantiomers of pyrrole ester, thereby realizing quality control and preparation of a single enantiomer.
The invention provides a method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by HPLC method, which adopts a chiral chromatographic column and uses normal phase chromatography for analysis and separation; the chiral chromatographic column is a polysaccharide derivative coating type chiral chromatographic column, and specifically, the surface of silica gel is coated with one of amylose-tris (3, 5-dimethylphenyl carbamate), amylose-tris [ (S) -alpha-methylphenyl carbamate ], cellulose-tris (3, 5-dimethylphenyl carbamate), amylose-tris (5-chloro-2-methylphenyl carbamate) or cellulose-tris (3-chloro-4-methylphenyl carbamate); the mobile phase used by the normal phase chromatography is a mixed solvent of n-hexane-isopropanol or a mixed solvent of n-hexane-ethanol;
the analysis method comprises the following steps:
(1) (+) -pyrrole ester and (-) -pyrrole ester reference substances are respectively dissolved by ethanol and are prepared into reference substance solutions with the concentration of 0.1 mg/mL;
(2) dissolving a (+/-) -pyrrolyl ester test sample with ethanol to prepare a test sample solution with the concentration of 0.1-1 mg/mL; the sample injection amount is 5-10 mu L;
(3) setting the proportion of the n-hexane-isopropanol mixed solvent or the n-hexane-ethanol mixed solvent to be 90: 10-0: 100, respectively; setting the flow rate to be 0.4-1.0 mL/min; setting the temperature of a liquid chromatographic column incubator to be 20-40 ℃; the detection wavelength is 210 nm;
(4) precisely absorbing (+) -pyrrole ester and (-) -pyrrole ester reference substances and (+/-) -pyrrole ester test samples to be respectively injected into a liquid chromatographic column, and finishing the analysis of (-) -pyrrole ester and enantiomers thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substances;
the preparation method comprises the following steps:
(1) (+) -pyrrole ester and (-) -pyrrole ester reference substances are respectively dissolved by ethanol and are prepared into reference substance solutions with the concentration of 1 mg/mL;
(2) dissolving a (+/-) -pyrrolyl ester test sample with ethanol to prepare a test sample solution with the concentration of 5-10 mg/mL; the sample injection amount is 1-10 mL;
(3) setting the proportion of the n-hexane-isopropanol mixed solvent or the n-hexane-ethanol mixed solvent to be 90: 10-0: 100, respectively; setting the flow rate to be 5-50 mL/min; the detection wavelength is 210 nm;
(4) respectively injecting (+) -pyrrole ester and (-) -pyrrole ester reference substances and (+/-) -pyrrole ester test samples into a liquid chromatographic column, respectively receiving corresponding main peaks according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substances, concentrating and drying to finish the preparation of the (-) -pyrrole ester and the enantiomer thereof;
further, according to the method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by using the HPLC method, the chiral chromatographic column filler used in the analysis method is silica gel coated with amylose-tris (3, 5-dimethylphenyl carbamate) on the surface, the used mobile phase is n-hexane-isopropanol (70: 30-0: 100) or n-hexane-ethanol (70: 30-0: 100), and the set flow rate is 0.4-1.0 mL/min.
The experimental results show that: the method adopts a chiral chromatographic column with the surface of silica gel coated with amylose-tris (3, 5-dimethylphenyl carbamate), adopts a n-hexane-isopropanol (70:30) mixed solvent or a n-hexane-ethanol (70:30) mixed solvent as a mobile phase, adopts a better sample injection amount and a better wavelength, can ensure the stability of the pyrrole ester, improves the symmetry of chromatographic peaks, and obtains a better separation effect, wherein the separation degree is more than 16.
Further, the method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by using the HPLC method is characterized in that the chiral chromatographic column filler used in the analysis method is silica gel coated with amylose-tris [ (S) -alpha-methylphenyl carbamate ] on the surface, the used mobile phase is n-hexane-isopropanol (75: 25-30: 70) or n-hexane-ethanol (85: 15-40: 60), and the set flow rate is 0.5-1.0 mL/min.
The experimental results show that: the method adopts a chiral chromatographic column with the surface of silica gel coated with amylose-tri [ (S) -alpha-methylphenyl carbamate ], adopts a n-hexane-isopropanol (75:25) mixed solvent or a n-hexane-ethanol (85:15) mixed solvent as a mobile phase, adopts a better sample injection amount and a better wavelength, can ensure the stability of the pyrrole ester, improves the symmetry of chromatographic peaks, and obtains a better separation effect, wherein the separation degree is more than 6.
Further, the method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by using the HPLC method is characterized in that the chiral chromatographic column filler used in the analysis method is silica gel coated with cellulose-tri (3, 5-dimethylphenyl formate) on the surface, the used mobile phase is n-hexane-isopropanol (90: 10-70: 30) or n-hexane-ethanol (90: 10-70: 30), and the set flow rate is 0.8-1.0 mL/min.
The experimental results show that: the method adopts a chiral chromatographic column with a silica gel surface coated with cellulose-tri (3, 5-dimethylphenyl carbamate), adopts a n-hexane-isopropanol (90:10) mixed solvent or a n-hexane-ethanol (90:10) mixed solvent as a mobile phase, adopts a better sample injection amount and a better wavelength, can ensure the stability of the pyrrole ester, improves the symmetry of chromatographic peaks, and obtains a better separation effect, wherein the separation degree is more than 3.
Further, according to the method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by using the HPLC method, the chiral chromatographic column filler used in the analysis method is silica gel coated with amylose-tris (5-chloro-2-methylphenyl carbamate) on the surface, the used mobile phase is n-hexane-isopropanol (80: 20-71: 29) or n-hexane-ethanol (80: 20-71: 29), and the set flow rate is 0.8-1.0 mL/min.
The experimental results show that: the method adopts a chiral chromatographic column with the surface of silica gel coated with amylose-tris (5-chloro-2-methylphenyl carbamate), adopts a n-hexane-isopropanol (75:25) mixed solvent or a n-hexane-ethanol (75:25) mixed solvent as a mobile phase, adopts a better sample injection amount and a better wavelength, can ensure the stability of the pyrrole ester, improves the symmetry of chromatographic peaks, and obtains a better separation effect, wherein the separation degree is more than 2.0.
Furthermore, the method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by using the HPLC method is characterized in that the chiral chromatographic column packing used in the analysis method is silica gel coated with cellulose-tris (3 chloro-4-methylphenyl carbamate) on the surface, the used mobile phase is n-hexane-isopropanol (85: 15-73: 27) or n-hexane-ethanol (85: 15-73: 27), and the set flow rate is 0.8-1.0 mL/min.
The experimental results show that: the method adopts a chiral chromatographic column with the surface of silica gel coated with cellulose-tri (3-chloro-4-methylphenyl carbamate), adopts a n-hexane-isopropanol (80:20) mixed solvent or a n-hexane-ethanol (80:20) mixed solvent as a mobile phase, adopts a better sample injection amount and a better wavelength, can ensure the stability of the pyrrole ester, improves the symmetry of chromatographic peaks, and obtains a better separation effect, wherein the separation degree is more than 1.5.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method adopts the liquid chromatography of the chiral stationary phase to analyze and separate the enantiomers of the pyrrole ester, has simple operation, accuracy and high efficiency, and can effectively control the quality of the pyrrole ester;
(2) the method adopts a more preferable chiral chromatographic column, a more preferable mobile phase, a more preferable sample amount and a more preferable wavelength, thereby ensuring the stability of the pyrrole ester, improving the symmetry of chromatographic peaks and having better separation effect.
(3) Compared with other liquid chromatography of chiral stationary phases, the method of the invention uses the polysaccharide derivative coating type chiral chromatographic column, and has higher separation degree and better separation effect.
Therefore, the method overcomes the defects of the prior art, solves the problem of analysis and separation of the pyrrole ester enantiomer, can quickly, accurately and efficiently analyze and separate the pyrrole ester enantiomer, effectively controls the quality of the pyrrole ester enantiomer, and further ensures the quality of the pyrrole ester.
Therefore, the invention develops a method for analyzing and preparing (-) -pyrrole ester and enantiomer thereof by HPLC method.
Drawings
FIG. 1: HPLC profile, chromatographic conditions: a chromatographic column:
AD-H (250 mm. times.4.6 mm,5.0 μm); mobile phase: n-hexane-ethanol (50: 50); column temperature: at 30 ℃.
FIG. 2: HPLC profile, chromatographic conditions: a chromatographic column:
AD-H (250 mm. times.4.6 mm,5.0 μm); mobile phase: n-hexane-isopropanol (0: 100); column temperature: at 20 ℃.
FIG. 3: HPLC profile, chromatographic conditions: a chromatographic column:
AS-H (250 mm. times.4.6 mm,5.0 μm); mobile phase: n-hexane-ethanol (70: 30); column temperature: at 30 ℃.
FIG. 4: HPLC profile, chromatographic conditions: a chromatographic column:
AS-H (250 mm. times.4.6 mm,5.0 μm); mobile phase: n-hexane-isopropanol (50: 50); column temperature: at 30 ℃.
FIG. 5: HPLC profile, chromatographic conditions: a chromatographic column:
OD-H (250X 4.6mm, 5.0. mu.m); mobile phase: n-hexane-ethanol (75: 25); column temperature: at 30 ℃.
FIG. 6: HPLC profile, chromatographic conditions: a chromatographic column:
OD-H (250X 4.6mm, 5.0. mu.m); mobile phase: n-hexane-isopropanol (75: 25); column temperature: 40 deg.C
FIG. 7: HPLC profile, chromatographic conditions: a chromatographic column:
amylose-2 (250X 4.6mm,5.0 μm); mobile phase: n-hexane-isopropanol (80: 20); column temperature: 40 deg.C
FIG. 8: HPLC profile, chromatographic conditions: a chromatographic column:
cellulose-2 (250X 4.6mm,5.0 μm); mobile phase: n-hexane-isopropanol (80: 20); column temperature: 40 deg.C
Detailed Description
The invention will be further explained by the description of embodiments with reference to the drawings, but the scope of protection of the invention is not limited to the embodiments.
Example 1: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (70:30)
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 17.0, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 2: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (0:100)
Column temperature: 20 deg.C
Flow rate: 0.4mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 6.8, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 3: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (50:50)
Column temperature: 30 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 11.7, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 1.
Example 4: analytical separation of (-) -pyrrolate and (+) -pyrrolate
A chromatographic column:
AD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (70:30)
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 16.1, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 5: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (0:100)
Column temperature: 20 deg.C
Flow rate: 0.4mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 4.3, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 2.
Example 6: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (50:50)
Column temperature: 20 deg.C
Flow rate: 0.4mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 7.3, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 7: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (85:15)
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 6.1, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 8: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (40:60)
Column temperature: 20 deg.C
Flow rate: 0.5mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.7, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 9: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (70:30)
Column temperature: 30 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of test sample mother liquor, placing the test sample mother liquor in a 10mL volumetric flask, dissolving the test sample mother liquor in ethanol, diluting the test sample mother liquor to a scale, and shaking the test sample mother liquor uniformly to obtain a test sample solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 3.6, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 3.
Example 10: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (75:25)
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 6.3, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 11: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (30:70)
Column temperature: 20 deg.C
Flow rate: 0.5mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.8, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 12: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (50:50)
Column temperature: 30 deg.C
Flow rate: 0.5mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 3.9, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 4.
Example 13: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (90:10)
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 3.4, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 14: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (70:30)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.6, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 15: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (75:25)
Column temperature: 30 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.8, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 5
Example 16: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (90:10)
Column temperature: 40 deg.C
Flow rate: 1.0mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 3.0, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 17: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (70:30)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.6, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 18: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (75:25)
Column temperature: 40 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.9, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 6
Example 19: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (80:20)
Column temperature: 40 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 2.1, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 7
Example 20: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (71:29)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 2.2, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 21: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (75:25)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 2.0, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 22: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (80:20)
Column temperature: 40 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 2.3, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 23: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (71:29)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 2.1, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 24: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (75:25)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 2.0, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 25: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Cellulose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (85:15)
Column temperature: 40 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 10 μ L
Sample concentration: 0.1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely sucking 1mL of a test product mother solution, placing the test product mother solution in a 10mL volumetric flask, dissolving the test product mother solution in ethanol, diluting the solution to a scale, and shaking up to obtain a test product solution. Respectively and precisely sucking 10 mu L of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution respectively, entering a liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and an enantiomer thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.6, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated. See figure 8
Example 26: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Cellulose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (73:27)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.7, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 27: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
ellulose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-isopropanol (80:20)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.7, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 28: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Cellulose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (85:15)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.7, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 29: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Cellulose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (73:27)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.5, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 30: analytical separation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Cellulose-2(250×4.6mm,5.0μm)
mobile phase: n-hexane-ethanol (80:20)
Column temperature: 20 deg.C
Flow rate: 0.8mL/min
Sample introduction volume: 5 μ L
Sample concentration: 1mg/mL
Detection wavelength: 210nm
Experimental procedure
Respectively and precisely measuring 1mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. The (+/-) -pyrrole ester is precisely weighed to be 10mg, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to the scale, and shaken uniformly to be used as a mother solution of a test sample. Precisely absorbing 5 mu L of each mother liquor of (+) -pyrrole ester, (-) -pyrrole ester reference substance and (±) -pyrrole ester test sample into liquid chromatogram, and finishing the analysis of (-) -pyrrole ester and enantiomer thereof according to the retention time of (+) -pyrrole ester and (-) -pyrrole ester reference substance.
The experimental results are as follows: the separation degree of two enantiomers of (-) -pyrrole ester and (+) -pyrrole ester is 1.5, and the (-) -pyrrole ester and the enantiomer thereof can be completely separated.
Example 31: preparation and isolation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AD-H(250×30mm,10.0μm)
mobile phase: n-hexane-isopropanol (0:100)
Flow rate: 20.0mL/min
Sample introduction volume: 10mL
Sample concentration: 10mg/mL
Detection wavelength: 210nm
The preparation method comprises the following steps: respectively and precisely measuring 10mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. Precisely weighing 100mg of (+/-) -pyrrole ester, placing the (+/-) -pyrrole ester in a 10mL volumetric flask, dissolving the (+/-) -pyrrole ester in ethanol, diluting the dissolved (+/-) -pyrrole ester to a scale mark, and shaking the mixture uniformly to serve as a test solution. Respectively sucking 10mL of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution into a liquid chromatogram, respectively receiving corresponding main peaks according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance, concentrating and drying to obtain 37mg of (-) -pyrrole ester and 42mg of (+) -pyrrole ester.
Example 32: preparation and isolation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
AS-H(250×10mm,10.0μm)
mobile phase: n-hexane-ethanol (70:30)
Flow rate: 5.0mL/min
Sample introduction volume: 1mL of
Sample concentration: 5mg/mL
Detection wavelength: 210nm
The preparation method comprises the following steps: respectively and precisely measuring 10mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. 50mg of (+/-) -pyrrole ester is precisely weighed, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to a scale mark, and shaken uniformly to be used as a test solution. Respectively sucking 1mL of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution into a liquid chromatogram, respectively receiving corresponding main peaks according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance, concentrating and drying to obtain 2.1mg of (-) -pyrrole ester and 2.5mg of (+) -pyrrole ester.
Example 33: preparation and isolation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
OD-H(250×20mm,10.0μm)
mobile phase: n-hexane-ethanol (90:10)
Flow rate: 19.0mL/min
Sample introduction volume: 1mL of
Sample concentration: 10mg/mL
Detection wavelength: 210nm
The preparation method comprises the following steps: respectively and precisely measuring 10mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. Precisely weighing 100mg of (+/-) -pyrrole ester, placing the (+/-) -pyrrole ester in a 10mL volumetric flask, dissolving the (+/-) -pyrrole ester in ethanol, diluting the dissolved (+/-) -pyrrole ester to a scale mark, and shaking the mixture uniformly to serve as a test solution. Respectively sucking 1mL of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution into a liquid chromatogram, respectively receiving corresponding main peaks according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance, concentrating and drying to obtain 4.2mg of (-) -pyrrole ester and 3.9mg of (+) -pyrrole ester.
Example 34: preparation and isolation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Amylose-2(250×30mm,10.0μm)
mobile phase: n-hexane-isopropanol (75:25)
Flow rate: 50.0mL/min
Sample introduction volume: 10mL
Sample concentration: 10mg/mL
Detection wavelength: 210nm
The preparation method comprises the following steps: respectively and precisely measuring 10mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. Precisely weighing 100mg of (+/-) -pyrrole ester, placing the (+/-) -pyrrole ester in a 10mL volumetric flask, dissolving the (+/-) -pyrrole ester in ethanol, diluting the dissolved (+/-) -pyrrole ester to a scale mark, and shaking the mixture uniformly to serve as a test solution. Respectively sucking 10mL of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample solution into a liquid chromatogram, respectively receiving corresponding main peaks according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substance, concentrating and drying to obtain 39mg of (-) -pyrrole ester and 35mg of (+) -pyrrole ester.
Example 35: preparation and isolation of (-) -pyrrolate and (+) -pyrrolate
Apparatus and conditions
A chromatographic column:
Cellulose-2(250×30mm,10.0μm)
mobile phase: n-hexane-isopropanol (80:20)
Flow rate: 35.0mL/min
Sample introduction volume: 10mL
Sample concentration: 5mg/mL
Detection wavelength: 210nm
The preparation method comprises the following steps: respectively and precisely measuring 10mg of (+) -pyrrolate and (-) -pyrrolate, placing the (+) -pyrrolate and the (-) -pyrrolate in a 10mL volumetric flask, dissolving the (+) -pyrrolate and the (-) -pyrrolate in ethanol, diluting the (+) -pyrrolate and the (-) -pyrrolate to a scale, and shaking up the (+) -pyrrolate and the (-) -pyrrolate to serve as reference substance solutions. 50mg of (+/-) -pyrrole ester is precisely weighed, placed in a 10mL volumetric flask, dissolved in ethanol and diluted to a scale mark, and shaken uniformly to be used as a test solution. Respectively sucking 10mL of mother liquor of (+) -pyrrole ester, a (-) -pyrrole ester reference substance and a (+/-) -pyrrole ester test sample into a liquid chromatogram, respectively receiving corresponding main peaks according to the retention time of the (+) -pyrrole ester reference substance and the (-) -pyrrole ester reference substance, concentrating and drying to prepare 17mg of (-) -pyrrole ester and 24mg of (+) -pyrrole ester.
Claims (6)
1. A method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and its enantiomer by HPLC method is characterized in that a chiral chromatographic column is adopted and normal phase chromatography is used for analysis and separation; the chiral chromatographic column filler is a polysaccharide derivative coating type chiral chromatographic column, and specifically, the surface of silica gel is coated with one of amylose-tris (3, 5-dimethylphenyl carbamate), amylose-tris [ (S) -alpha-methylphenyl carbamate ], cellulose-tris (3, 5-dimethylphenyl carbamate), amylose-tris (5-chloro-2-methylphenyl carbamate) or cellulose-tris (3-chloro-4-methylphenyl carbamate); the mobile phase used by the normal phase chromatography is a mixed solvent of n-hexane-isopropanol or a mixed solvent of n-hexane-ethanol; the 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole is abbreviated as (-) -pyrrolate, and the enantiomer 3- (N-p-toluenesulfonyl-D-alanyloxy) -5-phenylpyrrole is abbreviated as (+) -pyrrolate;
the analysis method comprises the following steps:
(1) (+) -pyrrole ester and (-) -pyrrole ester reference substances are respectively dissolved by ethanol and are prepared into reference substance solutions with the concentration of 0.1 mg/mL;
(2) dissolving a (+/-) -pyrrole ester test sample with ethanol to prepare a test sample solution with the concentration of 0.1-1 mg/mL, wherein the sample amount is 5-10 mu L;
(3) setting the proportion of the n-hexane-isopropanol mixed solvent or the n-hexane-ethanol mixed solvent to be 90: 10-0: 100, respectively; setting the flow rate to be 0.4-1.0 mL/min; setting the temperature of a liquid chromatographic column incubator to be 20-40 ℃; the detection wavelength is 210 nm;
(4) precisely absorbing (+) -pyrrole ester and (-) -pyrrole ester reference substances and (+/-) -pyrrole ester test sample solutions, respectively injecting the (+) -pyrrole ester and (-) -pyrrole ester reference substances into a liquid chromatographic column, and finishing the analysis of (-) -pyrrole ester and enantiomers thereof according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substances;
the preparation method comprises the following steps:
(1) (+) -pyrrole ester and (-) -pyrrole ester reference substances are respectively dissolved by ethanol and are prepared into reference substance solutions with the concentration of 1 mg/mL;
(2) dissolving a (+/-) -pyrrolyl ester test sample with ethanol to prepare a test sample solution with the concentration of 5-10 mg/mL; the sample injection amount is 1-10 mL;
(3) setting the proportion of the n-hexane-isopropanol mixed solvent or the n-hexane-ethanol mixed solvent to be 90: 10-0: 100, respectively; setting the flow rate to be 5-50 mL/min; the detection wavelength is 210 nm;
(4) and injecting (+) -pyrrole ester and (-) -pyrrole ester reference substances and the (+/-) -pyrrole ester test solution into a liquid chromatographic column respectively, receiving corresponding main peaks respectively according to the retention time of the (+) -pyrrole ester and the (-) -pyrrole ester reference substances, concentrating and drying to finish the preparation of the (-) -pyrrole ester and the enantiomer thereof.
2. The method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and the enantiomer thereof according to claim 1, wherein the chiral chromatographic column packing used in the analysis method is silica gel coated with amylose-tris (3, 5-dimethylphenylcarbamate) on the surface, the mobile phase used is N-hexane-isopropanol or N-hexane-ethanol, the ratio of the N-hexane-isopropanol mixed solvent is 70: 30-0: 100, the ratio of the N-hexane-ethanol mixed solvent is 70: 30-0: 100, and the set flow rate is 0.4-1.0 mL/min.
3. The method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and its enantiomer by HPLC method according to claim 1, wherein the chiral chromatographic column packing used in the analysis method is silica gel coated with amylose-tris [ (S) - α -methylphenyl carbamate ] on the surface, the mobile phase used is N-hexane-isopropanol or N-hexane-ethanol, the ratio of N-hexane-isopropanol mixed solvent is 75: 25-30: 70, the ratio of N-hexane-ethanol mixed solvent is 85: 15-40: 60, and the set flow rate is 0.5-1.0 mL/min.
4. The method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and the enantiomer thereof according to claim 1, wherein the chiral chromatographic column packing used in the analysis method is silica gel coated with cellulose-tris (3, 5-dimethylphenyl formate) on the surface, the mobile phase used is N-hexane-isopropanol or N-hexane-ethanol, the ratio of the N-hexane-isopropanol mixed solvent is 90: 10-70: 30, the ratio of the N-hexane-ethanol mixed solvent is 90: 10-70: 30, and the set flow rate is 0.8-1.0 mL/min.
5. The method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and the enantiomer thereof according to claim 1, wherein the chiral chromatographic column packing used in the analysis method is silica gel coated with amylose-tris (5-chloro-2-methylphenyl carbamate), the mobile phase used is N-hexane-isopropanol or N-hexane-ethanol, the ratio of the N-hexane-isopropanol mixed solvent is 80: 20-71: 29, the ratio of the N-hexane-ethanol mixed solvent is 80: 20-71: 29, and the set flow rate is 0.8-1.0 mL/min.
6. The method for analyzing and preparing 3- (N-p-toluenesulfonyl-L-alanyloxy) -5-phenylpyrrole and its enantiomer by HPLC method according to claim 1, wherein the chiral chromatographic column packing used in the analysis method is silica gel coated with cellulose-tris (3 chloro-4-methylphenyl carbamate), the mobile phase used is N-hexane-isopropanol or N-hexane-ethanol, the ratio of N-hexane-isopropanol mixed solvent is 85: 15-73: 27, the ratio of N-hexane-ethanol mixed solvent is 85: 15-73: 27, and the set flow rate is 0.8-1.0 mL/min.
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