CN112557520A - 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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Abstract
The invention provides a novel method for detecting isomers in a ticagrelor intermediate by adopting a high performance liquid separation mode, 1. a TGR-1-corresponding isomer detection method, which is characterized by comprising the following steps: after the isomer reference substance solution is subjected to pre-column derivatization, the isomer reference substance solution is analyzed and detected in a high performance liquid separation mode, and the method has the advantages of good stability, good reproducibility and simple operation, can accurately detect the isomer, and is favorable for quality control of the medicine.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and relates to a method for detecting TGR-1-enantiomer 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 synthetic route is as follows:
wherein TGR-1: 2- ((3aR,4S,6R,6aS) -6-amino-2, 2-dimethyltetrahydro-4H-cyclopenta [ d ] [1,3] dioxol-4-yl) oxy) ethyl-1-ol;
TGR-1-enantiomer: 2- ((3aR,4S, 6S, 6aR) -6-amino-2, 2-dimethyltetrahydro-4H-cyclopenta [ d ] [1,3] dioxol-4-yl) oxy) ethyl-1-ol.
TGR-1 and TGR-1-enantiomeric structural formulas are as follows:
the corresponding isomer of TGR-1 is a byproduct in the reaction, has low content, no ultraviolet absorption, similar structure with TGR-1, similar retention time and similar chromatographic behavior, is difficult to separate in a chromatographic column, and especially is difficult to detect by a conventional HPLC method (ultraviolet absorption), thereby causing great obstacle to actual detection. In order to effectively control the product quality, the impurity content of the product needs to be strictly controlled, and a set of analysis method suitable for detecting TGR-1-corresponding isomer in TGR-1 is developed. The method is rapid, simple and convenient, and has good stability and specificity; within a certain concentration range, the linear relation between the concentration and the peak area is good; the RSD of the recovery rate test is less than 10.0 percent; the lowest detectable amount (S/N ═ 3) was 0.003 μ g/ml (0.013%); when the temperature, the flow rate, the mobile phase proportion and different instruments or chromatographic columns of the chromatographic column are adopted, the detection results are consistent, and the durability of the method is good.
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: performing pre-column derivatization treatment on TGR-1 and TGR-1-corresponding isomers, and analyzing and detecting by adopting a high performance liquid separation mode, wherein the method specifically comprises the following steps:
1) derivatization treatment
And uniformly mixing the TGR-1 solution and the o-phthalaldehyde test solution according to a molar ratio of 1:1, and standing at room temperature for 5 minutes to obtain the TGR-1 derivative solution.
Taking TGR-1 corresponding isomer solution and o-phthalaldehyde test solution, mixing uniformly according to a molar ratio of 1:1, standing at room temperature for 5 minutes, and taking the mixture as TGR-1 corresponding isomer derivative solution.
The reaction mechanism is as follows: aldehyde compound (o-phthalaldehyde) containing carbonyl and primary amine compound (TGR-1) are subjected to 1:1 nucleophilic addition reaction, the nucleophilic reagent is an amine compound, nitrogen atoms with lone electron pairs in the structure of the amine compound attack carbon atoms with positive charges on carbonyl groups, the nucleophilic addition reaction is completed, intermediate alpha-hydroxylamine compound is formed, and then the Schiff base is formed through further dehydration.
TGR-1 reacts with o-phthalaldehyde according to the formula:
TGR-1 has no ultraviolet absorption, and can not be effectively detected by adopting high performance liquid separation; when TGR-1-corresponding isomer reacts with a derivatization reagent, Schiff base is generated, the ultraviolet absorption is strong, and the TGR-1-corresponding isomer and the TGR-1 are also corresponding isomers after derivatization, so that high performance liquid separation can be adopted for detection.
Derivatizing reagent: 0.8mg/ml of o-phthalaldehyde test solution;
solvent: methanol;
TGR-1 solution: taking about 10mg of TGR-1, precisely weighing, placing in a 10ml measuring flask, adding a solvent for dissolving, diluting to scale, and shaking up to obtain TGR-1 solution;
TGR-1-enantiomer solution: precisely weighing about 10mg of TGR-1-enantiomer, placing in a 10ml measuring flask, adding a solvent for dissolving and diluting to scale, shaking up, precisely weighing 1ml, placing in a 100ml measuring flask, diluting to scale with the solvent, shaking up, precisely weighing 1ml, placing in a 10ml measuring flask, diluting to scale with the solvent, shaking up to obtain TGR-1-enantiomer solution;
TGR-1 derivatization solution: accurately measuring appropriate amounts of TGR-1 solution and o-phthalaldehyde test solution, mixing, standing at room temperature for 5min to obtain TGR-1 derivatization solution;
TGR-1-enantiomer derivatization solutions: accurately measuring appropriate amounts of TGR-1-enantiomer solution and o-phthalaldehyde test solution, mixing, standing at room temperature for 5min to obtain TGR-1-enantiomer derivatization solution;
blank solvent: precisely measuring a proper amount of the solvent and the phthalic dicarboxaldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to serve as a blank solvent;
2) chromatographic separation
A chromatographic column: chiral chromatographic column (filler is alpha 1-acid glycoprotein, 4.0mm x 100mm,5 μm);
mobile phase: water-acetonitrile (85:15) or water-acetonitrile (90:10) isocratic elution;
flow rate of mobile phase: 0.8-1.2 mL/min;
column temperature: 20-25 ℃;
detection wavelength: 220 nm;
detection time: 15 min;
3) analyzing and detecting: and (3) injecting 20 mu L of the TGR-1 derivatization solution, the TGR-1-corresponding isomer derivatization solution and the blank solvent in the step 1) into a liquid chromatograph to finish the detection of the TGR-1-corresponding isomer.
Further, we disclose the chromatographic column: chiral chromatography column, AGP 4.0mm x 100mm,5 μm.
Further, we disclose the mobile phase composition as: water-acetonitrile (85: 15).
Also, we disclose that the preferred column temperature is 25 ℃ and the flow rate of the mobile phase is 1.0 mL/min.
Finally, we further disclose the preparation of TGR-1 derivatized solution, TGR-1-enantiomer derivatized solution and blank solvent; TGR-1 derivatization solution: precisely measuring 0.2ml of TGR-1 solution and 0.4ml of o-phthalaldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain TGR-1 derivatization solution; TGR-1-enantiomer derivatization solutions: precisely measuring 0.2ml of TGR-1-corresponding isomer solution and 0.4ml of o-phthalaldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to obtain TGR-1-corresponding isomer derivatization solution; blank solvent: precisely measuring 0.2ml of solvent and 0.4ml of phthalic dicarboxaldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to serve as a blank solvent.
Advantageous effects
The invention effectively solves the problem of difficult detection of the TGR-1 isomer, 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 convenient and fast 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 good stability, reproducibility and accuracy, and is beneficial to the quality control of medicines.
Drawings
FIG. 1 shows the results of detection of a blank solvent in the examples;
FIG. 2 shows the results of measurement of TGR-1 derivatization solution in example;
FIG. 3 shows the results of measurement of TGR-1-enantiomer derivative solution in example
Detailed Description
In order to enable those skilled in the art to better understand the technical solution of the present invention, the following further discloses some non-limiting examples to further explain the present invention in detail.
The reagents used in the invention are all commercially available chromatographic pure-grade reagents.
Example 1:
chromatographic conditions are as follows:
the instrument comprises the following steps: shimadzu 20A, Agilent 1260 detection wavelength: 220 nm;
a chromatographic column: chiral chromatographic column (filler is alpha 1-acid glycoprotein, 4.0mm x 100mm,5 μm);
mobile phase: water-acetonitrile (85:15) or water-acetonitrile (90:10) isocratic elution;
flow rate of mobile phase: 0.8-1.2 mL/min;
column temperature: 20-25 ℃;
detection wavelength: 220 nm;
detection time: 15 min;
the determination method comprises the following steps:
derivatizing reagent: 0.8mg/ml of o-phthalaldehyde test solution;
solvent: methanol;
TGR-1 solution: taking about 10mg of TGR-1, precisely weighing, placing in a 10ml measuring flask, adding a solvent for dissolving, diluting to scale, and shaking up to obtain TGR-1 solution;
TGR-1-enantiomer solution: precisely weighing about 10mg of TGR-1-enantiomer, placing in a 10ml measuring flask, adding a solvent for dissolving and diluting to scale, shaking up, precisely weighing 1ml, placing in a 100ml measuring flask, diluting to scale with the solvent, shaking up, precisely weighing 1ml, placing in a 10ml measuring flask, diluting to scale with the solvent, shaking up to obtain TGR-1-enantiomer solution;
TGR-1 derivatization solution: accurately measuring TGR-1 solution and o-phthalaldehyde test solution (1:1 molar ratio), mixing, standing at room temperature for 5min to obtain TGR-1 derivatization solution;
TGR-1-enantiomer derivatization solutions: accurately measuring TGR-1-corresponding isomer solution and o-phthalaldehyde test solution (1:1 molar ratio), mixing, standing at room temperature for 5min, and making into TGR-1-corresponding isomer derivatization solution;
blank solvent: precisely measuring a solvent and an o-phthalaldehyde test solution, uniformly mixing, and standing at room temperature for 5 minutes to serve as a blank solvent;
accurately measuring 20 mul of TGR-1 derivatization solution and TGR-1-corresponding isomer derivatization solution respectively, respectively injecting the solutions into a liquid chromatograph, recording a chromatogram, and calculating according to an external standard method by peak area, wherein the TGR-1-corresponding isomer can not exceed 0.10%.
From FIG. 2, it is clear that TGR-1 has a large absorption peak around 6min after derivatization. However, as shown in FIG. 3, the isomer corresponding to TGR-1 is not absorbed in about 6min after derivatization, and a small shoulder appears in about 3min, it can be seen that complete separation of TGR-1 and its corresponding isomer can be realized after derivatization, and the problem of difficult measurement in the prior art is solved.
Claims (3)
- A TGR-1-enantiomer detection method, characterized by: after pre-column derivatization of an isomer reference substance solution, analyzing and detecting by adopting a high performance liquid separation mode, and specifically comprising the following steps:(1) derivatization treatmentTaking TGR-1 solution and o-phthalaldehyde test solution, mixing uniformly according to a molar ratio of 1:1, standing at room temperature for 5 minutes to obtain TGR-1 derivative solution;taking TGR-1 corresponding isomer solution and o-phthalaldehyde test solution, uniformly mixing according to a molar ratio of 1:1, and standing at room temperature for 5 minutes to obtain TGR-1 corresponding isomer derivative solution;blank solvent: precisely measuring a solvent and 0.4ml of an o-phthalaldehyde solution, uniformly mixing, and standing at room temperature for 5 minutes to serve as a blank solvent;(2) chromatographic conditions are as follows:a chromatographic column: a chiral chromatographic column, wherein the filler is alpha 1-acid glycoprotein, 4.0mm x 100mm and 5 mu m;mobile phase: isocratic elution with water-acetonitrile volume ratio of 85:15 or water-acetonitrile volume ratio of 90: 10;flow rate of mobile phase: 0.8-1.2 mL/min;column temperature: 20-25 ℃;detection wavelength: 220 nm;detection time: 15 min;(3) analyzing and detecting: and (2) injecting 20 mu L of the TGR-1 derivatization solution, the TGR-1-corresponding isomer derivatization solution and the blank solvent in the step (1) into a liquid chromatograph respectively to finish the detection of the isomers in the starting material.
- 2. The TGR-1-enantiomer detection method of claim 1, wherein: the mobile phase is as follows: the volume ratio of water to acetonitrile is 85: 15.
- 3. The TGR-1-enantiomer detection method of claim 1, wherein: the column temperature was 25 ℃ and the flow rate of the mobile phase was 1.0 mL/min.
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