CN112098527B - Detection method of ticagrelor starting material - Google Patents
Detection method of ticagrelor starting material Download PDFInfo
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- CN112098527B CN112098527B CN201910526189.0A CN201910526189A CN112098527B CN 112098527 B CN112098527 B CN 112098527B CN 201910526189 A CN201910526189 A CN 201910526189A CN 112098527 B CN112098527 B CN 112098527B
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Abstract
The invention relates to the field of chemical substance analysis and detection, and particularly relates to a method for detecting a ticagrelor initial raw material. There is provided a method for detecting a chemical containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and/or an enantiomer thereof, comprising: (1) performing derivatization reaction on the chemical by using a nitro-substituted benzoyl chloride compound so as to obtain a derivative product; (2) and detecting the derivative product by using high performance liquid chromatography. The detection method provided by the invention is economic and reasonable, is simple and convenient to operate, and can accurately and sensitively detect the ticagrelor initial raw material and the impurity content thereof; the detection stability is good, and the repeatability is high.
Description
Technical Field
The invention relates to the field of chemical substance analysis and detection, and particularly relates to a method for detecting a ticagrelor initial raw material.
Background
Ticagrelor (Ticagrelor, trade name: brilnta), a selective Adenosine Diphosphate (ADP) receptor antagonist developed by AstraZeneca pharmaceutical limited (AstraZeneca AB), reversibly blocks ADP-mediated platelet activation and aggregation by activating the P2Y12 receptor.
Ticagrelor starting material 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid is an important starting material for the synthesis of ticagrelor, the enantiomeric impurity 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid typically being present in commercially available ticagrelor starting material products, the structural formulae of both being respectively aS follows:
the enantiomer impurity 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanol tartaric acid has simple molecular structure and small molecular weight, does not have chromogenic group on the molecule, does not absorb and/or has weak terminal absorption under ultraviolet illumination and does not respond under the common chromatographic condition, so the enantiomer impurity is difficult to directly detect by the common liquid chromatography. However, several other detection methods have different problems, for example, 1, the cost of the compound tested by a polarimeter is lower than that of the optical rotation, but the compound tested by the polarimeter is fast, but the compound tested by the polarimeter has poor accuracy, stability and reproducibility, is very easy to be influenced by external factors to cause larger deviation, and particularly, a racemate or a stereochiral mixture cannot be tested to obtain an accurate and reliable result, so that the content of an enantiomer in a starting material cannot be effectively controlled; 2. through X-ray determination, the method has high requirements on the purity and the crystal form of the compound, otherwise, the compound cannot be tested, and the test method is complicated, high in cost and not suitable for batch detection or detection at any time; 3. the nuclear magnetic resonance measurement needs expensive deuteration reagent to dissolve the compound, has certain requirements on the purity of the compound, and cannot obtain accurate analysis results on a spatial stereoisomeric mixture; 4. CD spectroscopy is not suitable for testing the optical purity of a spatial stereoisomer.
Chinese patent application No. 201711423224.3 discloses a method for liquid chromatography analysis of ticagrelor starting material, but it does not contain tartaric acid, the derivative FDAA is expensive, and it does not involve determination of enantiomeric impurity content, so it is not practical.
Therefore, the method has obvious defects in the aspect of detecting enantiomer impurity 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanol tartaric acid. The method is economic and reasonable, and can accurately and sensitively detect the content of the ticagrelor initial raw material and the impurity thereof.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide an economical and reasonable method capable of accurately and sensitively detecting the ticagrelor starting material and the impurity content thereof. The quality monitoring system can monitor the product quality of the ticagrelor raw material more comprehensively, so that the quality of the ticagrelor raw material medicine is improved, and the medication safety of a patient is further ensured.
According to the invention, the derivative agent is used for carrying out derivatization reaction on the ticagrelor initial raw material, so that a chromogenic group is formed in the obtained ticagrelor initial raw material derivative, and the derivative is absorbed by illumination under ultraviolet illumination, so that the detection can be carried out by using the high performance liquid chromatography, the product quality and quality of the ticagrelor initial raw material can be rapidly, simply and accurately determined, meanwhile, the condition of isomer impurities in the ticagrelor initial raw material can be determined, and guidance is provided for clinical medication and detection.
It should be noted that the detection method provided by the present invention is suitable for detecting chemicals containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanolic tartaric acid; or for the detection of chemicals containing 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid; it is also suitable for detecting chemicals containing the two substances. The 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanol tartaric acid is used aS an important starting material of ticagrelor, and the content of the starting material is monitored, so that guidance can be provided for obtaining ticagrelor in subsequent preparation.
Specifically, the invention provides the following technical scheme:
according to a first aspect of the present invention, there is provided a method for detecting a chemical containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and/or 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid, comprising: (1) performing derivatization reaction on the chemical by using a nitro-substituted benzoyl chloride compound so as to obtain a derivative product; (2) and detecting the derivative product by using high performance liquid chromatography. The inventor of the invention finds that a nitro-substituted benzoyl chloride compound can be subjected to a derivative reaction with a functional group on 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanolic tartaric acid or an enantiomer thereof, a generated derivative product contains a chromogenic group and can be absorbed under the irradiation of purple light, therefore, the derivative product can be detected by high performance liquid chromatography to reflect the condition of 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanol tartaric acid or enantiomer thereof in the chemical. For example, the amounts of the respective components in the chemical can be determined by subjecting a 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid standard or a 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid to the same derivatization reaction, plotting a standard curve, and comparing the results of high performance liquid chromatography measurement of each standard and chemical.
According to an embodiment of the present invention, the above-mentioned method for detecting a chemical may further include the following technical features:
in some embodiments of the invention, the chemical contains 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid, which is used aS a ticagrelor starting material.
In some embodiments of the invention, step (1) further comprises: (1-1) mixing the nitro-substituted benzoyl chloride compound, the chemical, dichloromethane and triethylamine, performing the derivatization reaction, and drying to obtain a dried product; (1-2) dissolving the dried product with a C1-C5 alcohol to obtain the derivative product. Therefore, redundant impurities are not introduced into the prepared derivative product, and the high performance liquid chromatography is convenient for gradient elution and determination.
In some embodiments of the invention, the molar ratio of the nitro-substituted benzoyl chloride compound to the chemical is 1-3: 1. thus, accurate measurement results can be obtained by high performance liquid chromatography.
In some embodiments of the invention, the nitro-substituted benzoyl chloride compound is 3, 5-dinitrobenzoyl chloride or p-nitrobenzoyl chloride. The nitro-substituted benzoyl chloride compounds are used as derivatization agents, so that the method is economic and reasonable, the derivatization method is simple, convenient and easy to operate, the stability of the derivatization agent solution is good, and an ideal derivatization effect can be obtained.
In some embodiments of the invention, the hplc detection conditions are: chromatographic column CHIRALPAK AD-H, the column temperature is 20-40 ℃. When the chromatographic column is used for detection, if the column temperature is too high, the filler loss can be caused to reduce the column efficiency, and the retention time can be shortened due to the increase of the column temperature, so that the separation degree between a main peak and a hetero peak is influenced; on the contrary, if the column temperature is too low compared with the room temperature, the temperature of the column oven is difficult to control accurately. Under the condition of 20-40 ℃, for example, 28-32 ℃, an ideal distinguishing result can be obtained.
In some embodiments of the present invention, the detection wavelength is 215-225nm, preferably 220 nm. The ticagrelor initial raw material derivative has the maximum absorption at the wavelength and has no interference of other substances, so that a spectrogram can be better distinguished and analyzed, and the aim of accurate detection is fulfilled.
In some embodiments of the invention, step (2) further comprises: and (3) carrying out gradient elution on the chemicals by using a mobile phase, wherein the mobile phase comprises an A phase and a B phase, the A phase comprises C1-C5 alcohol, n-hexane and an additive, the additive is selected from at least one of diethylamine, triethylamine, triethanolamine and ammonia water, and the B phase is C1-C5 alcohol. Thus, a good peak pattern and a high degree of separation can be obtained.
In some embodiments of the invention, the C1-C5 alcohol is selected from at least one of methanol, ethanol, propanol.
In some embodiments of the invention, the flow rate of the mobile phase is 0.7-1.2mL/min, with a flow rate of 1mL/min being preferred. Too high a flow rate may result in too high a column pressure, too low a flow rate may result in prolonged peak time to the detriment of rapid detection, and good separation may be achieved at flow rates of 0.7-1.2mL/min, e.g. at 1 mL/min.
In some embodiments of the invention, the C1-C5 alcohol, n-hexane, and additive are present in the a phase at a volume ratio of 20-40:60-80:0.02, preferably 30:70: 0.02. Thus, a peak type measurement result with good reproducibility and high resolution can be obtained.
In some embodiments of the invention, the gradient elution conditions are:
| time (min) | Flow rate (ml/min) | Mobile phase A phase (%) | Mobile phase B phase (%) |
| 0 | 1.0 | 100 | 0 |
| 40 | 1.0 | 100 | 0 |
| 41 | 0.8 | 0 | 100 |
| 71 | 0.8 | 0 | 100 |
| 75 | 1.0 | 100 | 0 |
| 95 | 1.0 | 100 | 0 |
In some embodiments of the invention, the method described above further comprises: a first standard which is a 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid standard, said first standard being assayed according to the detection method described in any one of the above embodiments by replacing the chemical with the first standard; determining the content of the 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid in the chemical based on the assay result of the first standard.
In some embodiments of the invention, the method described above further comprises: a second standard, which is a 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid standard, measured by replacing the chemical with the second standard according to the detection method described in any one of the above embodiments; determining the content of the 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid in the chemical based on the assay result of the second standard.
The invention has the beneficial effects that:
the invention uses nitro-substituted benzoyl chloride compound, especially 3, 5-dinitrobenzoyl chloride, as derivatization agent, which is economic and reasonable, the derivatization method is simple and easy to operate, and the stability of the derivative solution is good; the enantiomer impurity concentration and the peak area have a good linear relationship, so that the ticagrelor initial raw material impurity and the content thereof can be accurately and sensitively detected; the ticagrelor initial raw material and the impurity content thereof have good detection stability and high repeatability;
the method disclosed by the invention is scientific and reasonable in design and simple and convenient to operate, and can be used for accurately and effectively detecting the enantiomer through a high performance liquid chromatography detection mode, so that the problems of special property and sensitivity limitation in the prior art are well solved.
Drawings
FIG. 1 is a high performance liquid chromatogram of the derived solution of the test sample in the experimental group 1 of example 1 at an absorption value ranging from-2000-6000 mAU;
FIG. 2 is a high performance liquid chromatogram of the derived solution of the test sample in experimental group 1 of example 1 with an absorption value ranging from-20 mAU to 200 mAU;
FIG. 3 is a high performance liquid chromatogram of a solution derived from a starting material standard for ticagrelor in Experimental group 1 of example 1;
FIG. 4 is a high performance liquid chromatogram of a high concentration enantiomer derivative solution in experimental group 1 of example 1;
FIG. 5 is a high performance liquid chromatogram of a low concentration enantiomer derivative solution in experimental group 1 of example 1;
FIG. 6 is a high performance liquid chromatogram of the derived solution of the test sample in the experimental group 2 of example 1 when the absorption value range is-2000-5000 mAU;
FIG. 7 is a high performance liquid chromatogram of a sample-derived solution of Experimental group 2 of example 1, with an absorption value ranging from-20 to 200 mAU;
FIG. 8 is a high performance liquid chromatogram of the derived solution of the test sample in the experimental group 3 of example 1 at the absorption value range of-2000-5000 mAU;
FIG. 9 is a high performance liquid chromatogram of a sample-derived solution of Experimental group 3 of example 1, with absorption values ranging from-20 to 200 mAU;
FIG. 10 is a high performance liquid chromatogram of the sample derived solution in the control group 1 when the absorption value range is-2000-5000 mAU;
FIG. 11 is a high performance liquid chromatogram of the sample-derived solution in control 1 at an absorption value ranging from-20 to 200 mAU.
In fig. 1 to 11, the abscissa represents Time (Time) and the ordinate represents Absorbance (Absorbance).
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
The invention provides a method for detecting a chemical containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanolic tartaric acid and/or an enantiomer thereof, which comprises the following steps: (1) performing derivatization reaction on the chemical by using a nitro-substituted benzoyl chloride compound so as to obtain a derivative product; (2) and detecting the derivative product by using high performance liquid chromatography. Taking 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and 3, 5-dinitrobenzoyl chloride aS examples, 3, 5-dinitrobenzoyl chloride can react with the former to generate a chromophore, and can be detected by a high performance liquid chromatography method under ultraviolet light.
In particular, the method is suitable for the detection of chemicals containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid. The method is also suitable for detecting the chemicals containing 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanol tartaric acid. It is also suitable for the detection of chemicals containing both 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and its enantiomer 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid. Wherein "chemical" herein may refer to any substance containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and/or containing an enantiomer thereof.
Among them, a chemical containing 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid can be used aS a starting material for ticagrelor. By detecting the chemical, the amount of the ticagrelor starting material and the amount of the enantiomer thereof can be determined, so that the quality of the ticagrelor starting material product can be evaluated by the method.
In some embodiments of the invention, step (1) further comprises: (1-1) mixing the nitro-substituted benzoyl chloride compound, the chemical, dichloromethane and triethylamine, performing the derivatization reaction, and drying to obtain a dried product; (1-2) dissolving the dried product with a C1-C5 alcohol to obtain the derivative product.
Wherein the C1-C5 alcohol refers to alcohol with 1-5 carbon atoms, and the usable C1-C5 alcohol can be methanol, ethanol, propanol, isopropanol, etc., preferably ethanol.
In at least some embodiments of the present invention, the drying is blow drying with nitrogen in a water bath. Therefore, other oxidation reactions can not occur in the drying process, and the accuracy of subsequent high-performance liquid phase measurement is ensured.
In at least some embodiments of the invention, step (2) further comprises: performing gradient elution on the chemical by using a mobile phase, wherein the mobile phase comprises an A phase and a B phase, the A phase comprises ethanol, n-hexane and an additive, and the additive is selected from at least one of diethylamine, triethylamine, triethanolamine and ammonia water; the phase B is ethanol. This makes it possible to obtain a good peak pattern and a high degree of separation.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Example 1 provides identification of ticagrelor starting material and detection of enantiomeric impurities using high performance liquid chromatography. The method is specifically divided into the following experimental groups and control groups:
experimental group 1
Chromatographic conditions are as follows:
a chromatographic column:
AD-H(250mm×4.6mm，5μm)
a detector: ultraviolet detector
Detection wavelength: 220nm
Mobile phase A: ethanol-n-hexane-diethylamine (volume ratio 30:70: 0.02); mobile phase B: ethanol
Gradient elution was performed as in table 1 below:
TABLE 1 gradient elution conditions
Column temperature: 30 deg.C
Sample introduction amount: 10 μ l
Preparing a sample derivative solution:
preparing a sample derivative solution: taking 200mg of a test sample, accurately weighing, placing in a 10ml measuring flask, adding 200mg of 3, 5-dinitrobenzoyl chloride and 2ml of dichloromethane 5ml triethylamine, discharging gas, stirring for 1h at room temperature, drying by using nitrogen in a water bath at 40 ℃, adding ethanol for dissolving, diluting to scale, and shaking up.
Enantiomer control (standards) stock: taking 10mg of enantiomer reference substance, placing the enantiomer reference substance in a 10ml measuring flask, precisely weighing, adding ethanol for dissolving, diluting to scale, and shaking up.
Preparation of low-concentration enantiomer impurity reference substance derivative solution: precisely measuring 0.2ml of enantiomer reference substance stock solution, placing the enantiomer reference substance stock solution into a 10ml measuring flask, drying the enantiomer reference substance stock solution by using nitrogen in water bath at 40 ℃, adding 200mg of 3, 5-dinitrobenzoyl chloride and 2ml of dichloromethane 5ml triethylamine, discharging gas, stirring the mixture for 1h at room temperature, drying the mixture by using nitrogen in water bath at 40 ℃, adding ethanol to dissolve and dilute the mixture to scale, and shaking the mixture evenly.
Preparation of high-concentration enantiomer impurity reference substance derivative solution: 6ml of enantiomer reference substance stock solution is precisely measured, and the preparation method is as the preparation of low-concentration enantiomer impurity reference substance derivative solution.
Preparing a standard derivative solution of a ticagrelor starting material: taking 200mg of a ticagrelor initial raw material standard product, and preparing the standard product by a preparation method such as a sample derivative solution.
Blank solution: the operation is carried out according to the method without adding the substance to be detected to be used as a blank solution;
the measurement method is as follows:
and precisely measuring 10 mu l of each of the sample derivative solution, the enantiomer impurity reference substance derivative solution, the ticagrelor starting material standard solution and the blank solution by using a low-temperature sample injector (6 ℃), respectively injecting into a liquid chromatograph, and recording the chromatogram.
Wherein, the high performance liquid chromatogram of the derivative solution of the ticagrelor starting material standard is shown in fig. 3, and the result in fig. 3 shows that the retention time of the derivative is 5.847min, and the retention time of the ticagrelor starting material derivative is 9.213 min.
The high performance liquid chromatogram of the high concentration enantiomer control derived solution is shown in FIG. 4; the retention time of the derivatizing agent was 6.133min and the retention time of the enantiomeric impurity derivative was 30.420 min.
The high performance liquid chromatogram of the low concentration enantiomer control derived solution is shown in FIG. 5; the retention time of the enantiomeric impurity derivative was 30.403 min.
The HPLC chromatogram of the sample derivative solution at an absorption value range of-2000-6000 mAU is shown in FIG. 1, the retention time of the derivative is 6.007min, the retention time of the ticagrelor starting material 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolane-4-yl ] oxy ] ethanolic tartaric acid derivative is 9.423min, the enantiomeric impurity, a derivative of 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid, has a retention time of 30.073 min.
The high performance liquid chromatogram of the derived solution of the test sample with absorption value ranging from-20 mAU to 200mAU is shown in FIG. 2; from FIG. 2, it can be seen that the enantiomeric impurities of FIG. 2 have a better peak shape.
The optical configuration of the ticagrelor starting material is identified by the difference of the retention time t (min) of the main component of the ticagrelor starting material and the peak of the enantiomer impurity in the main component in an HPLC (high performance liquid chromatography) diagram. aS can be seen from fig. 1 and 2, most of the test samples were 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid, which is a main ingredient of the desired ticagrelor starting material.
When the chromatogram of the test solution is calculated by an external standard method, if an enantiomer peak exists, the content of the enantiomer peak cannot exceed 0.1 percent.
The enantiomeric content was calculated as follows:
enantiomeric content (wt%) (a-like × C-like)/(a-pair × C-like) × 100%
In the above formula:
sample A-peak area of enantiomer in the sample derived solution;
peak area of enantiomer in the derivative solution of the A-enantiomer control;
the concentration of enantiomer, μ g/ml, in the derived solution of C para-enantiomer control;
sample C-concentration of sample, μ g/ml.
A sample A: 6.392, respectively; a pair: 7.017
C, pair: 20 mu g/ml; and C, sample C: 20000 ug/ml
The content of the enantiomer in the test sample is calculated according to the formula: 0.091 wt%. The value is consistent with the theoretical value of the content of the enantiomer in the test sample, and the result shows that the Ticagrelor starting material can be accurately measured under the liquid chromatography condition. And the content of the enantiomer is below 0.1 percent, which indicates that the quality of the test sample meets the requirement.
Experimental group 2
Chromatographic conditions
A chromatographic column:
AD-H(250mm×4.6mm，5μm)
a detector: ultraviolet detector
Detection wavelength: 220nm
Mobile phase A: ethanol-n-hexane-triethylamine (volume ratio 35: 65: 0.02); mobile phase B: ethanol
Column temperature: 32 deg.C
Sample introduction amount: 10 μ l
Elution was performed according to the same gradient elution conditions as in table 1 of experimental group 1.
Solution preparation:
preparing a sample derivative solution: taking 200mg of the product, accurately weighing, placing in a 10ml measuring flask, adding 200mg of 3, 5-dinitrobenzoyl chloride and 2ml of dichloromethane 5ml triethylamine, discharging gas, stirring for 1h at room temperature, drying by using nitrogen in a water bath at 40 ℃, adding ethanol for dissolving, diluting to scale, and shaking up.
The determination method comprises the following steps: a low-temperature sample injector (6 ℃) is adopted, 10 mu l of each of the sample derivative solution, the low-concentration isomer reference derivative solution (preparation method such as experimental group 1) and the blank solution (preparation method such as experimental group 1) is precisely measured, injected into a liquid chromatograph, and the chromatogram is recorded.
The HPLC chromatogram of the sample derivative solution at an absorption value range of-2000-5000 mAU is shown in FIG. 6, wherein the retention time of the derivative is 6.613min, the retention time of the ticagrelor starting material derivative is 9.600min, and the retention time of the enantiomer impurity derivative is 31.470 min.
The high performance liquid chromatogram of the sample derived solution with absorption value ranging from-20 mAU to 200mAU is shown in FIG. 7.
As can be seen from the results given in experimental group 2, the volume ratio of 35: 65: elution with 0.02 ethanol-n-hexane-triethylamine as mobile phase a and ethanol as mobile phase B gave good peak results.
The content of the enantiomer in the test sample is calculated according to the formula: 0.088 wt%. The value is consistent with the theoretical value of the content of the enantiomer in the test sample, and the result shows that the Ticagrelor starting material can be accurately measured under the liquid chromatography condition. And the content of the enantiomer is below 0.1 percent, which indicates that the quality of the test sample meets the requirement.
Experimental group 3
Chromatographic conditions
A chromatographic column:
AD-H(250mm×4.6mm，5μm)
a detector: ultraviolet detector
Detection wavelength: 220nm
Mobile phase A: ethanol-n-hexane-ammonia (volume ratio 20: 80: 0.02); mobile phase B: ethanol
Column temperature: 28 deg.C
Sample introduction amount: 10 μ l
Gradient elution was performed under the same conditions as in table 1 in experimental group 1.
Solution preparation:
preparing a sample derivative solution: taking 200mg of the product, accurately weighing, placing in a 10ml measuring flask, adding 200mg of 3, 5-dinitrobenzoyl chloride and 2ml of dichloromethane 5ml triethylamine, discharging gas, stirring for 1h at room temperature, drying by using nitrogen in a water bath at 40 ℃, adding ethanol for dissolving, diluting to scale, and shaking up.
The determination method comprises the following steps: a low-temperature sample injector (6 ℃) is adopted, 10 mu l of each of the sample derivative solution, the low-concentration isomer reference derivative solution (preparation method such as experimental group 1) and the blank solution (preparation method such as experimental group 1) is precisely measured, injected into a liquid chromatograph, and the chromatogram is recorded.
The high performance liquid chromatogram of the derived solution of the test sample when the absorption value range is-2000-5000 mAU is shown in figure 8; the retention time of the derivatizing agent was 6.707min, the retention time of the ticagrelor starting material derivative was 10.897min, and the retention time of the enantiomeric impurity derivative was 36.413 min.
The high performance liquid chromatogram of the sample derived solution with absorption value ranging from-20 mAU to 200mAU is shown in FIG. 9.
As can be seen from the results given in experimental group 3, the volume ratio of 20: 80: elution with 0.02 ethanol-n-hexane-ammonia as mobile phase a and ethanol as mobile phase B gave good peak results.
The content of the enantiomer in the test sample is calculated according to the formula: 0.092 wt%. The value is consistent with the theoretical value of the content of the enantiomer in the test sample, and the result shows that the Ticagrelor starting material can be accurately measured under the liquid chromatography condition. And the content of the enantiomer is below 0.1 percent, which indicates that the quality of the test sample meets the requirement.
Control group 1
Chromatographic conditions
A chromatographic column:
AD-H(250mm×4.6mm，5μm)
a detector: ultraviolet detector
Detection wavelength: 220nm
Mobile phase A: ethanol-n-hexane-diethylamine (volume ratio 10: 90: 0.02); mobile phase B: ethanol
Column temperature: 30 deg.C
Sample introduction amount: 10 μ l
Gradient elution was performed under the same conditions as in Table 1 in Experimental group 1.
Preparing a test product derivative solution: taking 200mg of the product, accurately weighing, placing in a 10ml measuring flask, adding 200mg of 3, 5-dinitrobenzoyl chloride and 2ml of dichloromethane 5ml triethylamine, discharging gas, stirring for 1h at room temperature, drying by using nitrogen in a water bath at 40 ℃, adding ethanol for dissolving, diluting to scale, and shaking up.
The determination method comprises the following steps: a low-temperature sample injector (6 ℃) is adopted, 10 mu l of each of the sample derivative solution, the low-concentration isomer reference derivative solution (preparation method such as experimental group 1) and the blank solution (preparation method such as experimental group 1) is precisely measured, injected into a liquid chromatograph, and the chromatogram is recorded.
The HPLC chromatogram of the sample derivative solution at an absorption value range of-2000-5000 mAU is shown in FIG. 10, the retention time of the derivative is 5.840min, the retention time of the ticagrelor starting material derivative is 9.470min, the retention times of the enantiomer impurities and the side small peaks are together and cannot be separated, and the retention time is 29.850 min.
The high performance liquid chromatogram of the sample derived solution with absorption value ranging from-20 mAU to 200mAU is shown in FIG. 11.
As can be seen from the results given in comparative example 1, the volume ratio of 10: 90: 0.02 ethanol-n-hexane-diethylamine is used as a fluidity A and ethanol is used as a mobile phase B for elution, and the obtained peak pattern cannot well distinguish the enantiomer from a side small peak, so the effect is not ideal.
Example 2
To further validate the detection method provided by the present invention, the following tests were provided:
wherein the sample of run No. TG-1-20170801 had been subjected to HPLC analysis as described in Experimental group 1 of example 1, and contained no enantiomeric impurities.
1. Stability test of derivative solution
A sample of TG-1-20170801 lot was used as a test sample. Precisely weighing a proper amount of a test sample, adding a standard (20 mu g/ml of enantiomer impurity), preparing a test sample derivative solution according to the method in example 1, standing, precisely taking 10uL of each test sample derivative solution in different standing times, injecting the solution into a high performance liquid chromatograph, recording a chromatogram, and determining the peak area of the enantiomer impurity, wherein the result is shown in table 2:
table 2 results of solution stability test of derivatives
The results show that: the sample derivative solution was allowed to stand at 6 ℃ for 27 hours, and the peak area RSD% of the enantiomer was 3.69% at each time point. The test derivative solution was stable for 27 hours.
2. Linear relationship between enantiomeric impurity concentration and peak area
An appropriate amount of an enantiomer reference substance is precisely weighed to prepare enantiomer reference substance solutions with the following concentrations, a series of enantiomer reference substance derivative solutions with a series of concentrations are prepared according to the method of the experimental group 1 in the example 1, 10uL of the enantiomer reference substance derivative solutions with different concentrations are respectively and precisely taken and injected into a high performance liquid chromatograph, a chromatogram is recorded, and the peak area is measured, wherein the results are shown in a table 3:
table 3 isomer impurity linearity test results
The results show that: when the concentration of the enantiomer impurity is in the range of 2.0325-30.4877 mu g/ml, the enantiomer impurity and the peak area form a good linear relation.
3. Repeatability of
A sample of TG-1-20170801 lot was used as a test sample. Adding a standard (enantiomer impurity), preparing a standard sample derivative solution according to the method of the experimental group 1 in the example 1, precisely taking 10uL, injecting into a high performance liquid chromatograph, and continuously injecting for 6 times, wherein the verification result is shown in a table 4:
TABLE 4 results of the repeatability tests
The results show that: the enantiomeric content RSD% was 1.30%, indicating that the method was highly reproducible.
4. Accuracy of
A sample of TG-1-20170801 lot was used as a test sample. Adding a standard (enantiomer impurity), preparing a standard sample derivative solution with the concentration according to the method of the experimental group 1 in the example 1, precisely taking 10uL, injecting into a high performance liquid chromatograph, and verifying the results as shown in the following table: the recovery rate calculation formula is as follows: percent recovery ═ 100% (measured amount of isomer impurity/added amount) ×
Table 5 accuracy experimental results for isomer 1
The results show that: the recovery rate of the enantiomer impurities is 90-108%, the average recovery rate is 94.57%, and the RSD% is 6.21%.
5. Durability
And changing the proportion of the mobile phase A, the wavelength, the column temperature and the proportion of the mobile phase A, and determining the content of the enantiomer of the solution of the standard sample. The results are shown in Table 6:
table 6: durability test results
As can be seen from Table 6, the chromatographic conditions were slightly varied to give an enantiomeric content RSD% of 10% or less.
In summary, based on the detection method for the ticagrelor starting material provided by the application, an identification experiment and an enantiomer content detection experiment for the ticagrelor starting material are respectively performed, so that the method can be used for identifying chiral isomers and detecting enantiomer content of the ticagrelor starting material, and enantiomer impurities are strictly controlled.
Furthermore, an attempt to derive the substance of the present application, namely 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and/or an enantiomer thereof, using OPA (o-phthalaldehyde) was made, but the solution for its derivation was unstable and affected the detection. Similarly, when FDAA (N-alpha- (5-fluoro-2, 4-dinitrophenyl) -L-alaninamide) is used for derivative detection, the obtained derivative has a peak shape and a separation degree which do not meet the detection requirement.
The terms "first", "second" and "first" are used herein for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (11)
1. A method for detecting a chemical, which comprises 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid and/or 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid,
the method comprises the following steps:
(1) performing derivatization reaction on the chemical by using a nitro-substituted benzoyl chloride compound so as to obtain a derivative product;
(2) detecting the derivative product by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography are as follows:
the chromatographic column is CHIRALPAK AD-H; performing gradient elution on the chemical by using a mobile phase, wherein the mobile phase comprises an A phase and a B phase,
the phase A comprises C1-C5 alcohol, n-hexane and an additive, wherein the additive is at least one of diethylamine, triethylamine, triethanolamine and ammonia water, and the volume ratio of the C1-C5 alcohol, the n-hexane and the additive in the phase A is 20-40:60-80: 0.02;
the phase B is C1-C5 alcohol, and the C1-C5 alcohol is at least one selected from methanol, ethanol and propanol;
the flow rate of the mobile phase is 0.7-1.2 mL/min;
the gradient elution conditions were:
。
2. The detection method according to claim 1, wherein the chemical contains 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid, and the chemical is used aS a starting material for ticagrelor.
3. The detection method according to claim 1, wherein the step (1) further comprises:
(1-1) mixing the nitro-substituted benzoyl chloride compound, the chemical, dichloromethane and triethylamine, performing the derivatization reaction, and drying to obtain a dried product;
(1-2) dissolving the dried product with a C1-C5 alcohol to obtain the derivative product.
4. The detection method according to claim 3, wherein the molar ratio of the nitro-substituted benzoyl chloride compound to the chemical is 1 to 3: 1.
5. the detection method according to claim 1, wherein the nitro-substituted benzoyl chloride compound is 3, 5-dinitrobenzoyl chloride or p-nitrobenzoyl chloride.
6. The detection method according to claim 1, wherein the column temperature of the chromatographic column is 20-40 ℃; the detection wavelength is 215-225 nm.
7. The detection method according to claim 6, wherein the detection wavelength is 220 nm.
8. The detection method according to claim 1, wherein the flow rate of the mobile phase is 1 mL/min.
9. The detection method according to claim 1, wherein the volume ratio of the C1-C5 alcohol, n-hexane and additive in the A phase is 30:70: 0.02.
10. The detection method according to any one of claims 1 to 9, further comprising:
a first standard which is a 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid standard, said first standard being assayed in place of said chemical;
determining the content of the 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid in the chemical based on the assay result of the first standard.
11. The detection method according to any one of claims 1 to 9, further comprising:
a second standard, which is a 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid standard, which is assayed in place of the chemical;
determining the content of the 2- [ [ (3aS,4R,6S,6aR) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopentene-1, 3-dioxolan-4-yl ] oxy ] ethanolic tartaric acid in the chemical based on the assay result of the second standard.
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