CN113804789B - Separation and detection method of compound A with multiple chiral sites and chiral isomer thereof and application of compound A in synthesis process - Google Patents
Separation and detection method of compound A with multiple chiral sites and chiral isomer thereof and application of compound A in synthesis process Download PDFInfo
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Abstract
The invention provides a separation and detection method of a compound A with multiple chiral sites and chiral isomers thereof and application of the compound A in a synthesis process of the compound A; detecting by liquid chromatography; the detection conditions of the liquid chromatography comprise: the chromatographic column is a xylonite polysaccharide coating type chiral chromatographic column or a chemical bonding type chiral chromatographic column; the mobile phase comprises a first solvent and a second solvent according to the volume ratio of (90-97): (3-10), wherein the first solvent is n-hexane, and the second solvent is isopropanol or ethanol. The method comprises the steps of selecting a xylonite polysaccharide coating type chiral chromatographic column or a chemical bonding type chiral chromatographic column, and selecting proper mobile phase components and proportion; the method realizes effective separation and quantitative detection of the compound A with multiple chiral sites and the chiral isomer thereof under the same chromatographic condition, can also accurately detect the content and the chiral purity of the compound A, and can also detect the specific structure and the content of the chiral isomer thereof.
Description
Technical Field
The invention relates to the field of drug synthesis, in particular to a separation and detection method of a compound A with multiple chiral sites and chiral isomers thereof and application of the compound A in a synthesis process of the compound A.
Background
In the field of medicine, tert-butyl (S) -2- ((1R, 2R) -3- ((R) -4-benzyl-2-oxazolidinone-3-yl) -1-hydroxy-2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate is used as an intermediate to prepare a high-activity anti-cancer medicament which can be used for treating cancers such as breast cancer.
The structure of tert-butyl (S) -2- ((1R, 2R) -3- ((R) -4-benzyl-2-oxazolidinone-3-yl) -1-hydroxy-2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate is compound A (Compound A) described above, which is synthesized from N-BOC-L-prolinaldehyde and (R) -4-benzyl-3-propionyl-2-oxazolidinone, but during this synthesis reaction one or more of the other seven compounds represented by the structures described above, compound B (Compound B), compound C (Compound C), compound D (Compound D), compound E (Compound E), compound F (Compound F), compound G (Compound G), and compound H (Compound H), are easily produced due to improper control of conditions or impure chirality of the starting materials.
The compound A serving as an important medical intermediate can continue to react to generate a more complex compound later, if the chirality is not controlled in the synthesis process of the compound A, the product quality is greatly influenced later, and the subsequent separation and purification are more troublesome and more loss is caused. Therefore, strict chiral control of the synthesized product is particularly important.
At present, whether a synthetic product contains chiral isomers or not and which chiral isomer the synthetic product contains can be judged only by combining a plurality of forward chromatographs or combining the forward chromatographs and a plurality of analysis methods, the process is complicated and troublesome, and accurate quantification cannot be realized.
Disclosure of Invention
The invention aims to provide a separation and detection method for quickly, accurately and reliably separating and detecting the compound A with multiple chiral sites and the chiral isomer thereof, which can accurately detect the content and the chiral purity of the compound A and simultaneously detect the specific structure and the content of the chiral isomer, and application of the separation and detection method in a synthesis process of the compound A.
In order to achieve the above purpose, the invention provides the following technical scheme:
a separation and detection method of compound A with multiple chiral sites and chiral isomers thereof adopts liquid chromatography for detection;
the compound A is tert-butyl (S) -2- ((1R, 2R) -3- ((R) -4-benzyl-2-oxazolidone-3-yl) -1-hydroxy-2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate;
the detection conditions of the liquid chromatography comprise: the chromatographic column is a polysaccharide coating type chiral chromatographic column or a chemical bonding type chiral chromatographic column; the mobile phase comprises a first solvent and a second solvent according to the volume ratio of (90-97): (3-10), wherein the first solvent is n-hexane, and the second solvent is isopropanol or ethanol.
By using liquid chromatography, a polysaccharide-coated chiral chromatographic column or a chemically bonded chiral chromatographic column of amylose-tris- (3, 5-dimethylphenylcarbamate) or cellulose-tris- (3, 5-dimethylphenylcarbamate) type is selected, and a first solvent (n-hexane) and a second solvent (isopropanol or ethanol) are selected in a volume ratio of (90-97): the mixture of (3-10) as a mobile phase; the method has the advantages that the multi-chiral-site compound A and the chiral isomer thereof can be effectively separated and quantitatively detected under the same chromatographic condition, the content and the chiral purity of the compound A can be accurately detected, the specific structure and the content of the chiral isomer can be detected, the separation detection method only needs 30-60min for detection, and the method is rapid, high in accuracy and simple and convenient to operate.
Preferably, the mobile phase is a mixture of the first solvent and the second solvent in a volume ratio of 93:7, the sample can be dissolved more fully, the chiral isomers can be separated better, and the resolution and the sensitivity are improved.
In some embodiments, the flow rate of the mobile phase is 0.8 to 1.2mL/min; preferably, the flow rate of the mobile phase is 1.0mL/min, whereby the chiral isomers can be well separated at an optimum flow rate, improving the degree of separation and durability.
In some embodiments, the column temperature for the liquid chromatography detection is between 15 ℃ and 30 ℃; preferably, the column temperature of the chromatographic column is 20 ℃, so that the damage to the chromatographic column can be reduced, and the separation degree of the compound A and the chiral isomer thereof can be further improved.
In some embodiments, the detector used for the liquid chromatography detection is a variable wavelength scanning ultraviolet detector with a detection wavelength of 200-220nm; preferably, the detection wavelength is 210nm, the content of the compound A and the chiral isomer thereof can be more accurately detected, the detection limit can reach 0.0003mg/ml, and the quantification limit can reach 0.001mg/ml.
In some embodiments, the elution format used for the liquid chromatography detection is mobile phase isocratic elution; the polysaccharide-coated chiral chromatographic column is a xylonite CHIRALPAK AD-H column or a xylonite OD-H column; the chemical bonding type chiral chromatographic column is a xylonite CHIRALPAK IA column; preferably, the specifications of the xylonite CHIRALPAK AD-H column, the xylonite CHIRALCEL OD-H column and the xylonite CHIRALPAK IA column are 250mm x 4.6mm,5um, so that the compound A and the chiral isomer thereof can be simultaneously detected effectively, the chiral isomer can be well separated, the peak shape is better, the separation degree and sensitivity are improved, the stability, repeatability and accuracy of detection are improved, and the more accurate chiral purity of the compound A and the content result of the chiral isomer are obtained.
In some embodiments, the sample for the liquid chromatography detection is solubilized or diluted by a diluent; preferably, the diluent employs the mobile phase. The concentration of the sample solution is 0.8-1.2mg/ml; the sample volume of the sample solution is 5-10 mu L; preferably, the concentration of the sample solution is 1mg/ml.
By selecting a proper sample injection amount and enabling the response value to meet the measurement requirement, continuous high-concentration sample injection is avoided, and the service life of the chromatographic column is prolonged by adopting a proper sample injection concentration; and under the condition of the optimal injection concentration of 1mg/ml, the peaks of the main component compound A and other chiral isomers are completely separated.
The application also provides application of the separation and detection method of the compound A with multiple chiral sites and the chiral isomer thereof in the synthesis process of the compound A.
The invention has the beneficial effects that:
the method comprises the following steps of selecting and adopting a polysaccharide coating type chiral chromatographic column or a chemical bonding type chiral chromatographic column by adopting a liquid chromatography, and selecting a first solvent (n-hexane) and a second solvent (isopropanol or ethanol) according to a volume ratio of (90-97): the mixture of (3-10) as a mobile phase; the method realizes effective separation and quantitative detection of the compound A with multiple chiral sites and the chiral isomer thereof under the same chromatographic condition, can also accurately detect the content and the chiral purity of the compound A, and can detect the specific structure and the content of the chiral isomer thereof.
In addition, through the detection of the separation detection method, if a certain chiral isomer of the compound is detected, the condition can be reversely controlled due to the reason, so that the condition is adjusted to comprehensively guarantee the product quality, the cost is effectively reduced, the product yield is improved, and the influence on the subsequent application of the compound A is avoided.
Drawings
To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.
FIG. 1 is a liquid chromatogram of compound A and its chiral isomer orientation of example 1 of the present application;
FIG. 2 is a liquid chromatogram of a quantitative limiting solution for locating compound A and its chiral isomer in example 1 of the present application;
FIG. 3 is a liquid chromatogram of a sample of synthetic Compound A of example 1 of the present application;
FIG. 4 is a liquid chromatogram of compound A and its chiral isomer orientation of example 2 of the present application;
FIG. 5 is a liquid chromatogram of a quantitative limiting solution for locating compound A and its chiral isomer of example 2 of the present application;
FIG. 6 is a liquid chromatogram of a sample of synthetic Compound A of example 2 herein;
FIG. 7 is a liquid chromatogram of compound A and its chiral isomer orientation of example 3 of the present application;
FIG. 8 is a liquid chromatogram of a quantitative limiting solution for localization of Compound A and its chiral isomer of example 3 of the present application;
FIG. 9 is a liquid chromatogram of a sample of synthetic Compound A of example 3 herein;
FIG. 10 is a liquid chromatogram of compound A and its chiral isomer orientation of example 4 of the present application;
FIG. 11 is a liquid chromatogram for the localization of Compound A and its chiral isomer of comparative example 1;
FIG. 12 is a liquid chromatogram for the localization of Compound A and its chiral isomer of comparative example 2;
fig. 13 is a liquid chromatogram for the localization of compound a and its chiral isomer of comparative example 3.
Fig. 14 is a liquid chromatogram for the localization of compound a and its chiral isomer of comparative example 4.
Fig. 15 is a liquid chromatogram for the localization of compound a and its chiral isomer of comparative example 5.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
A sample of the synthetic compound A used in the following example was synthesized from N-BOC-L-prolinaldehyde and (R) -4-benzyl-3-propionyl-2-oxazolidinone.
Example 1
Detection conditions are as follows:
the instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: xylonite CHIRALCEL OD-H (250mm 4.6mm,5 um);
mobile phase: n-hexane: ethanol =93:7;
and (3) an elution mode: isocratic elution;
flow rate: 1.0mL/min
Column temperature: 20 deg.C
Detector wavelength: 210nm
Sample introduction volume: 10 μ L
Diluent agent: n-hexane: ethanol =93:7;
operating time: 30min
The experimental steps are as follows:
(1) Preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
(2) Preparing a reference substance quantitative limit solution: 1ml of each of the 8 reference solutions is dissolved and diluted with a diluent to prepare a limiting solution of 0.001mg/ml.
(3) Preparing a sample solution: taking a synthesized compound A sample of 100mg, precisely weighing, placing in a 100mL volumetric flask, dissolving and diluting to a scale with a diluent, shaking up, and preparing into a sample solution of 1mg/mL.
(4) And (3) placing the reference substance solution prepared in the step (1), the positioning limit solution prepared in the step (2) and the sample solution prepared in the step (3) into a sample injector, balancing, injecting the sample solution into a liquid chromatograph, detecting by adopting the detection conditions, and recording a chromatogram.
The experimental results are as follows:
the liquid chromatogram for positioning the compound A and the 7 chiral isomer compounds B, C, D, E, F, G and H is shown in figure 1, and the corresponding data results are shown in the following table 1.
The liquid chromatogram of the quantitative limiting solution of the compound A and 7 chiral isomer compounds B, C, D, E, F, G and H is shown in figure 2, and the corresponding data results are shown in the following table 2;
the liquid chromatogram of the sample of synthetic compound a is shown in fig. 3, and the corresponding data results are shown in table 3 below.
TABLE 1 Compound A and its chiral isomer positioning results
| Analyte | Retention time | Degree of separation |
| Compound B | 6.481min | - |
| Compound G | 8.060min | 3.71 |
| Compound C | 9.081min | 2.00 |
| Compound F | 11.251min | 3.62 |
| Compound A | 12.202min | 1.60 |
| Compound E | 14.487min | 2.89 |
| Compound H | 16.060min | 1.70 |
| Compound D | 18.705min | 2.53 |
TABLE 2 quantitative limit results for Compound A and its chiral isomers
| Analyte | Retention time | Signal to noise ratio |
| Compound B | 6.481min | 73.4 |
| Compound G | 8.060min | 59.4 |
| Compound C | 9.081min | 46.6 |
| Compound F | 11.251min | 29.8 |
| Compound A | 12.202min | 37.8 |
| Compound E | 14.487min | 30.5 |
| Compound H | 16.060min | 26.6 |
| Compound D | 18.705min | 23.1 |
Table 3 chiral purity results for samples of synthetic compound a
| Analyte | Retention time | Purity of |
| Compound B | 6.481min | 0.266％ |
| Compound G | 8.060min | |
| Compound C | 9.081min | 0.270％ |
| Compound F | 11.251min | 0.435％ |
| Compound A | 12.202min | 99.029％ |
| Compound E | 14.487min | |
| Compound H | 16.060min | |
| Compound D | 18.705min |
Example 2
Detection conditions are as follows:
the instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: xylonite CHIRALPAK AD-H (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =90:10;
and (3) an elution mode: isocratic elution;
flow rate: 1.0mL/min
Column temperature: 20 deg.C
Detector wavelength: 210nm
Sample introduction volume: 5 μ L
Diluent agent: n-hexane: isopropanol =90:10;
operating time: 40min
The experimental steps are as follows:
(1) Preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
(2) Preparing a reference substance quantitative limit solution: respectively dissolving 1ml of the 8 reference substance solutions with a diluent and diluting to prepare a positioning limit solution of 0.001mg/ml.
(3) Preparing a sample solution: taking a synthesized compound A sample of 100mg, precisely weighing, placing in a 100mL volumetric flask, dissolving and diluting to a scale with a diluent, shaking up, and preparing into a sample solution of 1mg/mL.
(4) And (3) placing the reference substance solution prepared in the step (1), the positioning limit solution prepared in the step (2) and the sample solution prepared in the step (3) into a sample injector, balancing, injecting the sample solution into a liquid chromatograph, detecting by adopting the detection conditions, and recording a chromatogram.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 4, and the corresponding data results are shown in Table 4 below.
The liquid chromatogram of the quantitative limiting solution of the compound A and the 7 chiral isomer compounds B, C, D, E, F, G and H is shown in FIG. 5, and the corresponding data results are shown in the following Table 5;
the liquid chromatogram of the sample of synthetic compound a is shown in fig. 6, and the corresponding data results are shown in table 6 below.
TABLE 4 positioning results of Compound A and its chiral isomers
| Analyte | Retention time | Degree of separation |
| Compound G | 7.937min | - |
| Compound F | 8.336min | 0.71 |
| Compound B | 11.097min | 4.54 |
| Compound E | 16.476min | 8.10 |
| Compound H | 18.872min | 2.80 |
| Compound D | 20.552min | 1.87 |
| Compound C | 23.779min | 3.14 |
| Compound A | 29.881min | 2.09 |
TABLE 5 quantitative limit results for Compound A and its chiral isomers
| Analyte | Retention time | Signal to noise ratio |
| Compound G | 7.937min | 26.7 |
| Compound F | 8.336min | 15.6 |
| Compound B | 11.097min | 25.4 |
| Compound E | 16.476min | 14.7 |
| Compound H | 18.872min | 12.1 |
| Compound D | 20.552min | 11.7 |
| Compound C | 23.779min | 11.7 |
| Compound A | 29.881min | - |
Table 6 chiral purity results for samples of synthetic compound a
Example 3
Detection conditions are as follows:
the instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: xylonite CHIRALPAK OD-H (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =97:3;
and (3) an elution mode: isocratic elution;
flow rate: 1.2mL/min
Column temperature: 30 deg.C
Detector wavelength: 210nm
Sample introduction volume: 10 μ L
Diluent agent: n-hexane: isopropanol =97:3;
operating time: 100min
The experimental steps are as follows:
(1) Preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
(2) Preparing a reference substance quantitative limit solution: respectively dissolving 1ml of the 8 reference substance solutions with a diluent and diluting to prepare a positioning limit solution of 0.001mg/ml.
(3) Preparing a sample solution: taking a synthesized compound A sample of 100mg, precisely weighing, placing in a 100mL volumetric flask, dissolving and diluting to a scale with a diluent, shaking up, and preparing into a sample solution of 1mg/mL.
(4) And (3) placing the reference substance solution prepared in the step (1), the positioning limit solution prepared in the step (2) and the sample solution prepared in the step (3) into a sample injector, balancing, injecting the sample solution into a liquid chromatograph, detecting by adopting the detection conditions, and recording a chromatogram.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 7, and the corresponding data results are shown in Table 7 below.
The liquid chromatogram of the quantitative limiting solution of the compound A and the 7 chiral isomer compounds B, C, D, E, F, G and H is shown in FIG. 8, and the corresponding data results are shown in the following Table 8;
the liquid chromatogram of the sample of synthetic compound a is shown in fig. 9, and the results of the corresponding data are shown in table 9 below.
TABLE 7 positioning results of Compound A and its chiral isomers
| Analyte | Retention time | Degree of separation |
| Compound B | 15.346min | - |
| Compound G | 20.261min | 3.91 |
| Compound C | 27.402min | 4.55 |
| Compound F | 30.976min | 1.85 |
| Compound A | 49.223min | 6.38 |
| Compound E | 56.263min | 1.77 |
| Compound H | 77.369min | 4.28 |
| Compound D | 81.059min | 0.60 |
TABLE 8 quantitative limit results for Compound A and its chiral isomers
Table 9 chiral purity results for samples of synthetic compound a
| Analyte | Retention time | Purity of |
| Compound B | 15.346min | 0.291％ |
| Compound G | 20.261min | 0.081％ |
| Compound C | 27.402min | |
| Compound F | 30.976min | 0.277％ |
| Compound A | 49.223min | 98.816％ |
| Compound E | 56.263min | |
| Compound H | 77.369min | |
| Compound D | 81.059min |
Example 4
Detection conditions are as follows:
the instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: xylonite CHIRALPAK IA (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =90:10;
and (3) an elution mode: isocratic elution;
flow rate: 0.8mL/min
Column temperature: 20 deg.C
Detector wavelength: 220nm
Sample introduction volume: 10 μ L
Diluent agent: n-hexane: isopropanol =90:10;
operating time: 60min
The experimental steps are as follows:
preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 10, and the corresponding data results are shown in Table 10 below.
TABLE 10 Compound A and its chiral isomer localization results
| Analyte | Retention time | Degree of separation |
| Compound G | 12.171min | - |
| Compound F | 12.822min | 0.88 |
| Compound B | 16.562min | 4.53 |
| Compound E | 25.184min | 9.81 |
| Compound H | 28.204min | 2.71 |
| Compound D | 32.263min | 3.26 |
| Compound C | 33.918min | 1.13 |
| Compound A | 45.040min | 2.60 |
Comparative example 1
Detection conditions are as follows:
the instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: xylonite CHIRALPAK OD-H (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =85:15;
and (3) an elution mode: isocratic elution;
flow rate: 1.0mL/min
Column temperature: 20 deg.C
Detector wavelength: 210nm
Sample introduction volume: 10 μ L
Diluent agent: n-hexane: isopropanol =85:15;
operating time: 30min
The experimental steps are as follows:
preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 11, and the corresponding data results are shown in Table 11 below.
TABLE 11 Compound A and its chiral isomer localization results
( "-" indicates that the peak is the first peak; "0" means that the peak coincides with the preceding peak )
| Analyte | Retention time | Degree of separation |
| Compound B | 4.890min | - |
| Compound G | 6.055min | 4.01 |
| Compound C | 6.572min | 1.46 |
| Compound F | 8.630min | 3.27 |
| Compound A | 8.630 |
0 |
| Compound E | 9.261min | 0.83 |
| Compound H | 10.632min | 1.98 |
| Compound D | 12.593min | 2.56 |
Comparative example 2
Detection conditions are as follows:
the instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: xylonite CHIRALPAK OD-H (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =98:2;
and (3) an elution mode: isocratic elution;
flow rate: 1.0mL/min
Column temperature: 30 deg.C
Detector wavelength: 210nm
Sample introduction volume: 10 μ L
Diluent agent: n-hexane: isopropanol =98:2;
operating time: 120min
The experimental steps are as follows:
preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 12, and the corresponding data results are shown in Table 12 below.
TABLE 12 positioning of Compound A and its chiral isomers
( "-" indicates that the peak is the first peak; "0" means that the peak coincides with the preceding peak )
Comparative example 3
The instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: CHIRALPAK OJ-H (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =90:10;
and (3) an elution mode: isocratic elution;
flow rate: 1.0mL/min
Column temperature: 20 deg.C
Detector wavelength: 210nm
Sample introduction volume: 5 μ L
Diluent agent: n-hexane: isopropanol =90:10;
operating time: 30min
The experimental steps are as follows:
preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
The experimental results are as follows:
the liquid chromatogram for positioning compound a and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, compound H is shown in fig. 12, and the corresponding data results are shown in table 13 below.
TABLE 13 Compound A and its chiral isomer localization results
( "-" indicates that the peak is the first peak; "0" means that the peak coincides with the preceding peak )
| Analyte | Retention time | Degree of separation |
| Compound G | 7.119min | - |
| Compound F | 7.119 |
0 |
| Compound B | 9.644min | 2.14 |
| Compound C | 10.013 |
0 |
| Compound A | 10.821min | 0.49 |
| Compound H | 10.821 |
0 |
| Compound E | 12.194min | 0.79 |
| Compound D | 14.030min | 1.01 |
Comparative example 4
The instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: CHIRALPAK AS-H (150mm 4.6mm, 5um);
mobile phase: n-hexane: isopropanol =98:2;
and (3) an elution mode: isocratic elution;
flow rate: 0.8mL/min
Column temperature: 20 deg.C
Detector wavelength: 220nm
Sample introduction volume: 5 μ L
Diluent agent: n-hexane: isopropanol =98:2;
operating time: 40min
The experimental steps are as follows:
preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 14, and the corresponding data results are shown in Table 14 below.
TABLE 14 positioning of Compound A and its chiral isomers
( "-" indicates that the peak is the first peak; "0" means that the peak coincides with the preceding peak )
| Analyte | Retention time | Degree of separation |
| Compound G | 9.217min | - |
| Compound F | 9.217 |
0 |
| Compound B | 10.794min | 1.51 |
| Compound H | 10.794 |
0 |
| Compound A | 18.870min | 4.56 |
| Compound C | 19.302 |
0 |
| Compound E | 24.178min | 2.04 |
| Compound D | 27.933min | 1.69 |
Comparative example 5
The instrument comprises the following steps: agilent 1100 liquid chromatograph, VWD detector
A chromatographic column: CHIRALPAK IC (250mm 4.6mm,5 um);
mobile phase: n-hexane: isopropanol =80:20;
and (3) an elution mode: isocratic elution;
flow rate: 1.0mL/min
Column temperature: 30 deg.C
Detector wavelength: 220nm
Sample introduction volume: 5 μ L
Diluent agent: n-hexane: isopropanol =80:20;
operating time: 30min
The experimental steps are as follows:
preparing a positioning reference substance solution: respectively weighing standard compound A, compound B, compound C, compound D, compound E, compound F, compound G and compound H reference substances, precisely weighing 100mg, placing in a 100mL volumetric flask, dissolving with diluent, diluting to scale, shaking, and preparing into 1mg/mL solution as reference solution.
The experimental results are as follows:
the liquid chromatogram for positioning compound A and its 7 chiral isomers, compound B, compound C, compound D, compound E, compound F, compound G, and compound H is shown in FIG. 15, and the corresponding data results are shown in Table 15 below.
TABLE 15 positioning of Compound A and its chiral isomers
( "-" indicates that the peak is the first peak; "0" means that the peak coincides with the preceding peak )
| Compound G | 5.382min | - |
| Compound F | 6.242min | 3.16 |
| Compound B | 6.421min | 0.59 |
| Compound E | 6.792min | 1.20 |
| Compound A | 8.004min | 2.49 |
| Compound C | 8.004 |
0 |
| Compound D | 8.786min | 1.40 |
| Compound H | 9.188min | 0.83 |
And (4) conclusion:
the experimental results of the above examples 1-4 show that, by using the chromatographic separation detection method of the present application, compound a and 7 chiral isomers thereof can be well separated under the same chromatographic condition, and in addition, the liquid chromatographic separation detection method can also be used for qualitative and quantitative analysis, the limit of quantitation of compound a and chiral isomers thereof can reach 0.001mg/ml, which chiral isomers are contained in the synthesized compound a sample, and the forward purity of compound a and the content of impurities of each chiral isomer can be directly obtained. Wherein, the embodiment 1 is the optimal condition, under the chromatographic condition, the peaks and the front of the peaks are completely separated, the separation degree is more than 1.5, the analysis time is effectively shortened, and the time and the mobile phase are saved.
The experimental result of the comparative example 1 shows that the chiral chromatographic column of the type cellulose-tri- (3, 5-dimethylphenyl carbamate) is used, but the content of isopropanol in the mobile phase is too high, the mobile phase is not suitable, so that the separation of the peaks of chiral isomers is incomplete, two chiral isomer peaks are completely coincided, the separation with the compound A is not complete, and the qualitative and quantitative detection cannot be carried out, thereby indicating that the method of the comparative example 1 is not suitable for the chiral purity detection and the chiral isomer detection of the compound A.
The experimental result of the comparative example 2 shows that the chiral chromatographic column of the type cellulose-tris- (3, 5-dimethylphenylcarbamate) is used, but the content of n-hexane in the mobile phase is too high, the mobile phase is not suitable, so that the separation between the peaks of the chiral isomers is incomplete under the method, the two chiral isomer peaks are completely overlapped, the analysis time is too long, the qualitative and quantitative detection cannot be carried out, and the method of the comparative example 2 is not suitable for the chiral purity detection and the chiral isomer detection of the compound A.
The experimental result of the comparative example 3 shows that, as the chiral chromatographic column of amylose-tri- (3, 5-dimethylphenyl carbamate) or cellulose-tri- (3, 5-dimethylphenyl carbamate) is not used, other conditions are met, the spectrum shows that the peaks of a plurality of chiral isomers have overlapping wrapping phenomenon under the method, the separation from the compound A is incomplete, and the qualitative and quantitative detection cannot be carried out, so that the method of the comparative example 3 is not suitable for the chiral purity detection and the chiral isomer detection of the compound A.
The experimental result of the comparative example 4 shows that the chiral chromatographic column of amylose-tri- (3, 5-dimethylphenyl carbamate) or cellulose-tri- (3, 5-dimethylphenyl carbamate) is not used, and the n-hexane content in the mobile phase is too high, so that the mobile phase is not proper, the peaks of a plurality of chiral isomers are overlapped and wrapped, the separation from the compound A is not complete, the peak wrapping phenomenon exists, the qualitative and quantitative detection cannot be carried out, and the method of the comparative example 4 is not suitable for the chiral purity detection and the chiral isomer detection of the compound A.
The experimental results of comparative example 5 above show that, since a chiral column of amylose-tris- (3, 5-dimethylphenylcarbamate) or cellulose-tris- (3, 5-dimethylphenylcarbamate) type is not used and the content of isopropanol in the mobile phase is excessively high, the mobile phase is not suitable, resulting in incomplete separation between the peaks of chiral isomers under the method and incomplete separation from compound a, which cannot be detected qualitatively and quantitatively, indicating that the method of comparative example 5 is not suitable for chiral purity detection and chiral isomer detection of compound a.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Claims (12)
1. A separation detection method of compound A with multiple chiral sites and chiral isomers thereof is characterized in that liquid chromatography is adopted for detection;
the chemical structural formula of the compound A with multiple chiral sites and the chiral isomer thereof is as follows:
the compound A is tert-butyl (S) -2- ((1R, 2R) -3- ((R) -4-benzyl-2-oxazolidone-3-yl) -1-hydroxy-2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate;
the detection conditions of the liquid chromatography comprise: the chromatographic column is a polysaccharide coating type chiral chromatographic column or a chemical bonding type chiral chromatographic column; the mobile phase comprises a first solvent and a second solvent according to the volume ratio of (90-97): (3-10), the first solvent is n-hexane, and the second solvent is isopropanol or ethanol;
the polysaccharide coating type chiral chromatographic column is a CHIRALPAK AD-H column or a CHIRALCEL OD-H column, and the chemical bonding type chiral chromatographic column is a CHIRALPAK IA column.
2. The method for separating and detecting the compound A with multiple chiral sites and the chiral isomer thereof according to claim 1, wherein the mobile phase comprises a first solvent and a second solvent according to a volume ratio of 93:7 in a mixture.
3. The method for separating and detecting the compound A and the chiral isomer thereof according to claim 1, wherein the flow rate of the mobile phase is 0.8-1.2mL/min.
4. The method for separating and detecting compound A and chiral isomers thereof according to claim 3, wherein the flow rate of the mobile phase is 1.0mL/min.
5. The method for separating and detecting the compound A with the multiple chiral sites and the chiral isomer thereof according to claim 1, wherein the temperature of a chromatographic column for detecting the compound A with the liquid chromatography is 15-30 ℃.
6. The method for separating and detecting the compound A with multiple chiral sites and the chiral isomer thereof according to claim 5, wherein the column temperature of the chromatographic column is 20 ℃.
7. The method for separating and detecting compound A and chiral isomers thereof according to claim 1, wherein the detector for the liquid chromatography detection is an ultraviolet detector, and the detection wavelength is 200-220nm.
8. The method for separating and detecting compound A and its chiral isomer according to claim 7, wherein the detection wavelength is 210nm.
9. The method for separating and detecting compound A and its chiral isomer according to claim 1, wherein the specifications of CHIRALPAK AD-H column, CHIRALCEL OD-H column and CHIRALPAK IA column are 250mm x 4.6mm,5um.
10. The method for separating and detecting compound A and chiral isomers thereof according to claim 1, wherein a sample for the detection by the liquid chromatography is dissolved or diluted by a diluent;
the diluent employs the mobile phase.
11. The method for separating and detecting compound a and its chiral isomer according to any of claims 1-10, wherein the concentration of the sample solution used for the detection by liquid chromatography is 0.8-1.2mg/ml;
the sample amount of the sample solution is 5-10 mu L.
12. The use of the separation and detection method of compound a with multiple chiral sites and its chiral isomers according to any one of claims 1 to 11 in the synthesis of compound a.
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