CN112557520B - Method for detecting TGR-1-corresponding isomer in TGR-1 - Google Patents
Method for detecting TGR-1-corresponding isomer in TGR-1 Download PDFInfo
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- CN112557520B CN112557520B CN201910908350.0A CN201910908350A CN112557520B CN 112557520 B CN112557520 B CN 112557520B CN 201910908350 A CN201910908350 A CN 201910908350A CN 112557520 B CN112557520 B CN 112557520B
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Abstract
The invention provides a novel method 1. TGR-1-enantiomer detection method for detecting isomers in ticagrelor intermediates by adopting a high performance liquid phase separation mode, which is characterized in that: after the isomer reference substance is derived before the solution column, the analysis and detection are carried out by adopting a high-efficiency liquid phase separation mode, and the method has the advantages of good stability, good repeatability and simple operation, can accurately detect the isomer, and is beneficial to the quality control of medicines.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and relates to a method for detecting TGR-1-corresponding isomer in a key starting material TGR-1 of a medicine ticagrelor of a platelet inhibitor.
Background
Ticagrelor is an oral platelet inhibitor drug, and the key starting material TGR-1 synthesis route is as follows:
wherein TGR-1:2- [ [ (3 ar,4s,6r,6 as) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopenta-1, 3-dioxan-4-yl ] oxy ] -ethanol;
TGR-1-enantiomer: 2- [ [ (3 as,4r,6s,6 ar) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopenta-1, 3-dioxan-4-yl ] oxy ] -ethanol.
TGR-1 and TGR-1-enantiomer have the structural formula:
the corresponding isomer of TGR-1 is a byproduct in the reaction, has low content and no ultraviolet absorption, has similar structure and retention time as TGR-1, has similar chromatographic behavior, is difficult to separate in a chromatographic column, is particularly difficult to detect by a conventional HPLC (ultraviolet absorption), and causes a great obstacle in practical detection. In order to effectively control the quality of the product and strictly control the impurity content, we have found a set of analysis methods suitable for detecting TGR-1-corresponding isomer in TGR-1. The method is quick, simple and convenient, and has good stability and specificity; in a certain concentration range, the linear relation between the concentration and the peak area is good; the recovery rate test RSD is less than 10.0%; the minimum detection amount (S/n=3) was 0.003 μg/ml (0.013%); the detection results are consistent in chromatographic column temperature, flow rate, mobile phase proportion and different instruments or chromatographic columns, and the method has good durability.
Disclosure of Invention
The invention aims to provide a method for detecting TGR-1-corresponding isomer in TGR-1, which has good stability and reproducibility and is used for detecting the TGR-1-corresponding isomer.
The technical scheme adopted by the invention for solving the technical problems is as follows: after the TGR-1 and TGR-1-corresponding isomers are subjected to pre-column derivatization treatment, the analysis and detection are carried out by adopting a high performance liquid phase separation mode, and the specific steps are as follows:
1) Derivatization treatment
Taking TGR-1 solution and phthalic dicarboxaldehyde test solution according to the weight ratio of 1:1 molar ratio, and standing at room temperature for 5 minutes to obtain TGR-1 derivative solution.
Taking TGR-1 corresponding isomer solution and phthalic aldehyde test solution according to the ratio of 1:1 molar ratio, and standing at room temperature for 5 minutes to obtain a TGR-1 corresponding isomer derivatization solution.
Reaction mechanism: the method comprises the steps of carrying out 1:1 nucleophilic addition reaction on an aldehyde compound (phthalic dicarboxaldehyde) containing carbonyl and a primary amine compound (TGR-1), wherein a nucleophilic reagent is an amine compound, a nitrogen atom with a lone electron pair in the compound structure attacks a carbon atom with positive charge on a carbonyl group, the nucleophilic addition reaction is completed, an intermediate alpha-hydroxylamine compound is formed, and then Schiff base is formed through further dehydration.
TGR-1 reacts with phthalic aldehyde as follows:
TGR-1 itself has no ultraviolet absorption and cannot be effectively detected by high performance liquid phase separation; when TGR-1-corresponding isomer reacts with the derivatization reagent, schiff base is generated, ultraviolet absorption is strong, and TGR-1-corresponding isomer and TGR-1 are also the corresponding isomers after derivatization, so that high-efficiency liquid phase separation can be adopted for detection.
Derivatizing reagent: 0.8mg/ml of phthalic dicarboxaldehyde test solution;
solvent: methanol;
TGR-1 solution: taking about 10mg of TGR-1, precisely weighing, placing into a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain TGR-1 solution;
TGR-1-enantiomer solution: taking about 10mg of TGR-1-corresponding isomer, precisely weighing, placing into a 10ml measuring flask, adding a solvent for dissolution and dilution to a scale, shaking up, precisely weighing 1ml, placing into a 100ml measuring flask, diluting to the scale with the solvent, shaking up, precisely weighing 1ml, placing into a 10ml measuring flask, diluting to the scale with the solvent, shaking up, and taking as TGR-1-corresponding isomer solution;
TGR-1 derivatization solution: precisely measuring and mixing proper amounts of TGR-1 solution and phthalic aldehyde test solution, and standing at room temperature for 5 minutes to obtain TGR-1 derivatization solution;
TGR-1-enantiomer derivatization solution: precisely measuring a proper amount of TGR-1-enantiomer solution and phthalic aldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain a TGR-1-enantiomer derivatization solution;
blank solvent: precisely measuring a proper amount of each of the solvent and the phthalic aldehyde test solution, uniformly mixing, and standing for 5 minutes at room temperature to serve as a blank solvent;
2) Chromatographic separation
Chromatographic column: chiral chromatographic column (filler is alpha 1-acid glycoprotein, 4.0mm x 100mm,5 μm);
mobile phase: isocratic elution with water-acetonitrile (85:15) or water-acetonitrile (90:10);
flow rate of mobile phase: 0.8-1.2 mL/min;
column temperature: 20-25 ℃;
detection wavelength: 220nm;
detection time: 15min;
3) And (3) analysis and detection: and (3) injecting 20 mu L of the TGR-1 derivatization solution, the TGR-1-enantiomer derivatization solution and the blank solvent in the step (1) into a liquid chromatograph to finish detection of the TGR-1-enantiomer.
Further, we disclose the chromatographic column: chiral chromatography column, AGP 4.0mm x 100mm,5 μm.
Still further, we disclose the mobile phase composition: water-acetonitrile (85:15).
At the same time we also disclose that the preferred column temperature is 25℃and the flow rate of the mobile phase is 1.0mL/min.
Finally, we further disclose the preparation of TGR-1 derivatization solutions, TGR-1-enantiomer derivatization solutions, and blank solvents; TGR-1 derivatization solution: precisely measuring 0.2ml of TGR-1 solution, 0.4ml of phthalic aldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain TGR-1 derivatization solution; TGR-1-enantiomer derivatization solution: precisely measuring 0.2ml of TGR-1-enantiomer solution, 0.4ml of phthalic aldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain TGR-1-enantiomer derivatization solution; blank solvent: precisely measuring 0.2ml of solvent, 0.4ml of phthalic aldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain a blank solvent.
Advantageous effects
The invention effectively solves the difficult problem of detecting the isomer of TGR-1, and the detection result shows that the TGR-1 and the TGR-1 isomer are completely separated, and the detection limit is improved; the invention utilizes a convenient and quick high-performance liquid chromatography, adopts a high-performance liquid separation mode to analyze and detect after pre-column derivatization treatment, effectively detects TGR-1-corresponding isomer, has very good stability, reproducibility and accuracy, and is beneficial to the quality control of medicines.
Drawings
FIG. 1 shows the results of detection of a white solvent in an example;
FIG. 2 shows the results of detection of TGR-1 derived solutions in examples;
FIG. 3 shows the results of detection of TGR-1-enantiomer derivatization solutions in examples.
Detailed Description
In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting examples are further disclosed below to further describe the present invention in detail.
The reagents used in the present invention are all commercially available chromatographic grade reagents.
Example 1:
chromatographic conditions:
instrument: island 20a, agilent 1260 detection wavelength: 220nm;
chromatographic column: chiral chromatographic column (filler is alpha 1-acid glycoprotein, 4.0mm x 100mm,5 μm);
mobile phase: isocratic elution with water-acetonitrile (85:15) or water-acetonitrile (90:10);
flow rate of mobile phase: 0.8-1.2 mL/min;
column temperature: 20-25 ℃;
detection wavelength: 220nm;
detection time: 15min;
assay:
derivatizing reagent: 0.8mg/ml of phthalic dicarboxaldehyde test solution;
solvent: methanol;
TGR-1 solution: taking about 10mg of TGR-1, precisely weighing, placing into a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain TGR-1 solution;
TGR-1-enantiomer solution: taking about 10mg of TGR-1-corresponding isomer, precisely weighing, placing into a 10ml measuring flask, adding a solvent for dissolution and dilution to a scale, shaking up, precisely weighing 1ml, placing into a 100ml measuring flask, diluting to the scale with the solvent, shaking up, precisely weighing 1ml, placing into a 10ml measuring flask, diluting to the scale with the solvent, shaking up, and taking as TGR-1-corresponding isomer solution;
TGR-1 derivatization solution: precisely measuring a TGR-1 solution and a phthalic aldehyde test solution (1:1 molar ratio), uniformly mixing, and standing at room temperature for 5 minutes to obtain a TGR-1 derivatization solution;
TGR-1-enantiomer derivatization solution: precisely measuring TGR-1-enantiomer solution and phthalic aldehyde test solution (1:1 molar ratio), uniformly mixing, and standing at room temperature for 5 minutes to obtain TGR-1-enantiomer derivatization solution;
blank solvent: precisely measuring a solvent and a phthalic aldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain a blank solvent;
precisely measuring 20 μl of each of the TGR-1 derivatization solution and the TGR-1-enantiomer derivatization solution, respectively injecting into a liquid chromatograph, recording the chromatograms, and calculating according to an external standard method by peak area, wherein the TGR-1-enantiomer is less than 0.10%.
From FIG. 2, it is clear that there is a large absorption peak around 6min after derivatization of TGR-1. The isomer corresponding to TGR-1 shown in figure 3 is not absorbed in about 6min after being derivatized, and small shoulder peak appears in about 3min, so that complete separation of TGR-1 and the corresponding isomer thereof can be realized after the derivatization, and the difficult problem of difficult measurement in the prior art is solved.
Claims (2)
- A method for detecting tgr-1-enantiomer, characterized by: after the isomer reference substance is derived before the solution column, the analysis and detection are carried out by adopting a high-efficiency liquid phase separation mode, and the specific steps are as follows:(1) Derivatization treatmentUniformly mixing a TGR-1 solution and a phthalic aldehyde test solution according to a molar ratio of 1:1, and standing at room temperature for 5 minutes to obtain a TGR-1 derivative solution; uniformly mixing TGR-1 corresponding isomer solution and phthalic aldehyde test solution according to a molar ratio of 1:1, and standing at room temperature for 5 minutes to obtain TGR-1 corresponding isomer derivatization solution; blank solvent: precisely measuring a solvent, mixing 0.4ml of phthalic aldehyde solution, and standing at room temperature for 5 minutes to serve as a blank solvent;(2) Chromatographic conditions:chromatographic column: chiral chromatographic column with filler of alpha 1-acid glycoprotein, 4.0mm x 100mm,5 μm; mobile phase: isocratic elution with water-acetonitrile volume ratio 85:15 or water-acetonitrile volume ratio 90:10;flow rate of mobile phase: 0.8-1.2 mL/min;column temperature: 20-25 ℃;detection wavelength: 220nm;detection time: 15min;(3) And (3) analysis and detection:taking 20 mu L of the TGR-1 derivatization solution, the TGR-1-enantiomer derivatization solution and the blank solvent in the step (1) and injecting into a liquid chromatograph to finish the detection of the isomer in the starting material;wherein TGR-1:2- [ [ (3 ar,4s,6r,6 as) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopenta-1, 3-dioxan-4-yl ] oxy ] -ethanol;TGR-1-enantiomer: 2- [ [ (3 as,4r,6s,6 ar) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopenta-1, 3-dioxan-4-yl ] oxy ] -ethanol;TGR-1 and TGR-1-enantiomer have the structural formula:
- 2. the TGR-1-enantiomer detection method according to claim 1, wherein: the column temperature was 25℃and the flow rate of the mobile phase was 1.0mL/min.
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