CN107764890B

CN107764890B - Method for distinguishing and detecting ezetimibe enantiomers

Info

Publication number: CN107764890B
Application number: CN201710959742.0A
Authority: CN
Inventors: 左敏娟; 陈晓蕾; 何俏军; 吴洪海; 王鹭; 康玉; 曾苏
Original assignee: Hangzhou Leader Medical Science And Technology Co Ltd; Zhejiang University ZJU
Current assignee: Hangzhou Leader Medical Science And Technology Co Ltd; Zhejiang University ZJU
Priority date: 2017-10-16
Filing date: 2017-10-16
Publication date: 2019-12-17
Anticipated expiration: 2037-10-16
Also published as: CN107764890A

Abstract

the invention discloses a method for distinguishing and detecting ezetimibe enantiomers, which adopts beta-cyclodextrin and Cu ions as ligands to form complexes with different stable forms with ezetimibe (S, R, S), (R, S, R), and utilizes mass spectrometry to distinguish and distinguish the ezetimibe enantiomers. The method greatly shortens the detection time of the ezetimibe enantiomers (S, R, S) and (R, S, R), shortens the analysis time of the enantiomers (S, R, S) and (R, S, R) to 2-3min, simplifies the operation process and has good distinguishing repeatability; the amount of samples needed for distinguishing is very small, only about 200 mu l is needed, and the cost is very low. The invention provides a better method for distinguishing the enantiomers of the drug, and simultaneously provides a new idea for analyzing the multi-chiral-center enantiomer with higher distinguishing difficulty.

Description

Method for distinguishing and detecting ezetimibe enantiomers

Technical Field

The invention relates to the technical field of drug analysis, in particular to a method for distinguishing and detecting ezetimibe enantiomers (S, R, S) and (R, S, R).

background

Ezetimibe is cholesterol inhibiting absorbent and suitable for treating primary high cholesterolAlcoholism, homozygous familial hypercholesterolemia (HoFH) and homozygous sitosterolemia (or phytosterolemia) having the molecular formula C₂₄H₂₁F₂NO₃The chemical name is 1- (4-fluorophenyl) -3(R) - [3- (4-fluorophenyl) -3(S) -hydroxypropyl

A group ] -4(S) - (4-hydroxyphenyl) -2-azetidine (azetidine) one of the formula:

The molecule contains 3 chiral centers, a plurality of optical isomers exist, and for the optical isomer impurities of ezetimibe, quality control needs to be carried out in the process of drug synthesis. The distinction of optical isomers containing chiral carbon atoms is always a difficult point of quality control in the synthesis and preparation processes of chiral drugs, and the realization of the distinction of ezetimibe and the optical isomers thereof has practical significance in the synthesis of ezetimibe drugs and the quality control in the preparation processes. For molecules with multiple chiral centers, the difficulty in distinguishing diastereoisomers is low, and the difficulty in distinguishing enantiomers is high, so that the method has important significance in the research on the method for distinguishing the enantiomers of the drugs.

At present, the commonly used methods for distinguishing the enantiomers of ezetimibe are traditional chromatography methods, such as LC, HPLC, CE, CEC, SFC and the like, but the methods all need long distinguishing time and large sample amount, and bring inconvenience to practical operation. Mass spectrometry has the characteristics of rapidness, accuracy and sensitivity, and has been greatly developed since 1977 for the first time for chiral isomer differentiation, and the main methods for chiral isomer differentiation comprise mass spectrometry and tandem mass spectrometry. The former includes a host-guest method, an ion-molecule reaction method and a diastereomer dissociation method. At present, the distinction of ezetimibe enantiomers by mass spectrometry is not reported in the literature.

disclosure of Invention

The invention aims to provide a method for distinguishing and detecting ezetimibe enantiomers (S, R, S) and (R, S, R) so as to overcome the defects of the prior art.

The invention adopts the following technical scheme:

Beta-cyclodextrin and Cu ions are used as ligands for forming complexes with ezetimibe (S, R, S) and (R, S, R) in different stable forms, and the complexes are identified and distinguished by utilizing a mass spectrometry method.

Further, the final concentration of the beta-cyclodextrin is 20-25 mu g/ml; during preparation, a small amount of DMSO is added until the beta-cyclodextrin is completely dissolved, and then the mixture is diluted by methanol for later use.

Further, Cu ions are divalent, and the final concentration is 12-12.5 mu g/ml; distilled water is used as solvent in preparation.

Furthermore, the sample injection method of the mass spectrum is direct sample injection by a needle pump, so that the complex is prevented from being influenced by the external environment before collision dissociation is carried out.

Further, the flow rate of direct sample injection by the needle pump is 20 μ l/min, the collection time is 1min, the mass spectrum vacuum temperature is 250 ℃, and the charge-to-mass ratio range is set to be 300-.

Further, when a secondary mass spectrum experiment is carried out in the mass spectrum, a target parent ion [ Cu (Ezetimibe) ] is directly input₂(β-CD)]²⁺The charge-to-mass ratio (m/z 1007.8) is entered into the secondary mass spectrum and the collision energy is adjusted to 0 to find the parent ion.

Further, the secondary mass spectrum experiment was carried out with a collision energy of 14-16 and a collection time of 40 ms.

The invention has the beneficial effects that:

The invention provides a novel method for distinguishing and detecting ezetimibe enantiomers (S, R, S) and (R, S, R), which adopts beta-cyclodextrin and divalent Cu ions as ligands, distinguishes the ezetimibe and the enantiomers thereof by mass spectrometry according to the dissocial characteristic difference of diastereomers, and adopts a computer simulation mode to construct the optimal configuration of the complex system, thereby deeply understanding the distinguishing mechanism.

The method greatly shortens the detection time of the ezetimibe enantiomers (S, R, S) and (R, S, R), shortens the analysis time of the enantiomers (S, R, S) and (R, S, R) to 2-3min, simplifies the operation process and has good distinguishing repeatability; the amount of samples needed for distinguishing is very small, only about 200 mu l is needed, and the cost is very low. The invention provides a better method for distinguishing the enantiomers of the drug, and simultaneously provides a new idea for analyzing the multi-chiral-center enantiomer with higher distinguishing difficulty.

Drawings

FIG. 1 is a schematic diagram of the three-dimensional structure of SRS-ezetimibe + beta-cyclodextrin + Cu ion system (A is a side view and B is a top view);

FIG. 2 is a schematic diagram of the three-dimensional structure of the RSR-ezetimibe + beta-cyclodextrin + Cu ionic system (A is a side view and B is a top view);

FIG. 3 is a secondary mass spectrum of the SRS-ezetimibe + beta-cyclodextrin + Cu ion system;

FIG. 4 is a secondary mass spectrum of the RSR-ezetimibe + beta-cyclodextrin + Cu ion system.

Detailed Description

The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

example 1

A. sample preparation: accurately weighing 1mg of RSR-ezetimibe, 1mg of SRS-ezetimibe and 1mg of CuSO₄Solids and 10mg beta-cyclodextrin. Diluting RSR-ezetimibe and SRS-ezetimibe to 50 μ g/ml with methanol respectively; CuSO₄Diluting the solid with pure water to 1 mg/ml; beta-cyclodextrin was first dissolved in a small amount of DMSO until no solids were visible (DMSO could not exceed 200. mu.l), diluted with methanol to 1mg/ml and then diluted with methanol to 50. mu.g/ml. Mixing 200 μ l 50 μ g/ml RSR-ezetimibe and 200 μ l 50 μ g/ml beta-cyclodextrin, adding 5 μ l 1mg/ml CuSO₄Obtaining a sample 1 by using the aqueous solution; sample 2 was prepared from SRS-ezetimibe using the above protocol.

B. Mass spectrum differentiation: respectively injecting the 2 samples into a mass spectrum continuously at a speed of 20 mu l/min in an Agilient Q-ToF 6550 high-resolution mass spectrometer in a manner of directly injecting samples through a needle pump, wherein the collection time is 1min, the vacuum temperature of the mass spectrum is 250 ℃, and the charge-to-mass ratio range is set to be 300-2000-; setting collision energy on secondary mass spectrogram to be 0 to find parent ion [ Cu (E)zetimibe)₂(β-CD)]²⁺(m/z 1007.8), and then gradually increasing the collision energy until a mass spectrum of ion fragments with obvious differences is obtained, wherein the collision energy is 14-16, and the collection time is 40ms, as shown in fig. 3 and 4. FIG. 3 is the second-order mass spectrum of SRS-ezetimibe + beta-cyclodextrin + Cu ion system, the parent ion is [ Cu (Ezetimibe) ]₂(β-CD)]²⁺(m/z-1007.8) the fragment ions produced were [ Ezetimibe-H ] respectively₂O+H]⁺(m/z＝392.15)，[Ezetimibe+H]⁺(m/z＝410.15)，[Cu(β-CD)-H]⁺(m/z-1196.3) and [ Cu (Ezetimibe) (. beta. -CD) -H]⁺(m/z-1605.5). FIG. 4 is a second mass spectrum of the RSR-Ezetimibe + beta-cyclodextrin + Cu ionic system, producing the same ion fragments as the SRS-Ezetimibe + beta-cyclodextrin + Cu ionic system, but [ Ezetimibe-H ]₂O+H]⁺

(m/z-392.15) the abundance of this fragment ion is different from it. From the results of the mass spectrum experiments in FIGS. 3 and 4, it can be seen that [ Ezetime-H ] is observed at the same collision energy₂O+H]⁺(m/z-392.15) this fragment ion is less abundant than ezetimibe (S, R, S), indicating that the former binds more stably, confirming the results of the theoretical calculations described below.

C. Constructing the optimal configuration of the complex by a computer simulation method: the calculation was performed by DFT (density functional method) in a computer simulation method, wherein the selected multiplicity is 2, the system charge number is 2, the Lanl2DZ pseudopotential group is used for Cu ions, and the 6-31G group is used for other atoms. The optimal configuration of the 2 complexes possibly possessed in the vacuum environment of the mass spectrum is calculated, and the mechanism of separating the ezetimibe enantiomers is intuitively shown, as shown in figures 1 and 2. FIG. 1 is a schematic diagram of a three-dimensional structure of an SRS-ezetimibe + beta-cyclodextrin + Cu ion system calculated by a computer simulation experiment, wherein 2 molecules of SRS-ezetimibe penetrate through a beta-cyclodextrin cavity to form Cu-O bond complexation with Cu ions; one is located at the periphery of the beta-cyclodextrin and forms hydrogen bonds with its hydroxyl groups. Fig. 2 is a schematic diagram of a three-dimensional structure of an RSR-ezetimibe + β -cyclodextrin + Cu ion system calculated by a computer simulation experiment, and 2 molecules of RSR-ezetimibe are all located at a β -cyclodextrin cavity and form hydrogen bond bonding with a hydroxyl group thereof. Comparing fig. 1 and 2, it can be seen that ezetimibe (S, R, S), (R, S, R) is not bound to the ligand in the same manner, but ezetimibe (S, R, S) forms a coordinate bond with Cu ions, which is stronger and more stable than that of ezetimibe (R, S, R) simply bound by a hydrogen bond.

In the embodiment, the mass spectrometry is adopted to distinguish the enantiomers of ezetimibe (S, R, S) and (R, S, R), the time for use is only 2-3min, the speed is very high, and in addition, the experimental method simulated by a computer is used for researching the mechanism of the enantiomers, so that a new thought is provided for the distinction of chiral isomers.

Claims

1. a method for distinguishing and detecting ezetimibe enantiomers is characterized in that beta-cyclodextrin and Cu ions are used as ligands for forming complexes with different stable forms with ezetimibe (S, R, S) and ezetimibe (R, S, R), and the complexes are distinguished and distinguished by mass spectrometry.

2. The method for the differential detection of the enantiomers of ezetimibe according to claim 1, wherein the final concentration of β -cyclodextrin is 20-25 μ g/ml; during preparation, a small amount of DMSO is added until the beta-cyclodextrin is completely dissolved, and then the mixture is diluted by methanol for later use.

3. The method for the differential detection of ezetimibe enantiomers according to claim 1, wherein the Cu ion is divalent and has a final concentration of 12-12.5 μ g/ml; distilled water is used as solvent in preparation.

4. The method for distinguishing and detecting the ezetimibe enantiomer according to claim 1, wherein the sample injection method of the mass spectrum is needle pump direct sample injection, so that the complex is prevented from being influenced by the external environment before collision dissociation.

5. The method for detecting the enantiomers of ezetimibe according to claim 1, wherein the flow rate of direct injection by a needle pump is 20 μ l/min, the collection time is 1min, the mass spectrum vacuum temperature is 250 ℃, and the charge-to-mass ratio range is set to be 300-2000.

6. The method for discriminating and detecting an ezetimibe enantiomer according to claim 1, wherein a target parent ion [ Cu (Ezetimibe) ] is directly inputted when performing a secondary mass spectrometry experiment in the mass spectrometry₂(β-CD)]²⁺The charge-to-mass ratio (m/z 1007.8) is entered into the secondary mass spectrum and the collision energy is adjusted to 0 to find the parent ion.

7. The method for the differential detection of the enantiomers of ezetimibe according to claim 1, wherein the secondary mass spectrometry is performed with a collision energy of 14-16 and a collection time of 40 ms.