阅读说明：本技术 基于离子淌度质谱仪高效识别青霉胺手性对映体的方法 (Method for efficiently identifying penicillamine chiral enantiomer based on ion mobility mass spectrometer ) 是由 丁传凡 杨淑童 吴芳玲 王欢欢 刘伊伊 方向 于 2021-06-29 设计创作，主要内容包括：本发明属于分析测试技术领域,具体涉及一种基于离子淌度质谱仪高效识别青霉胺手性对映体的方法。本发明将D-Pen和L-Pen分别与β-CD、LiCl等混合制备成混合溶液,再使用电喷雾电离离子源产生通过非共价作用形成的“[D-Pen+β-CD+Li]～+和[L-Pen+β-CD+Li]～+”三元复合物气相离子。最后使用可以测量离子碰撞截面的离子迁移谱装置进行离子迁移率的分析检测。由于不同异构体对应的“青霉胺-环糊精-Li～+”三元复合物的离子迁移谱不同,因此可以获得到对应于不同对映体的离子迁移谱,根据离子迁移谱实验结果可实现简单、快速、灵敏地对两种对映体的分析识别。(The invention belongs to the technical field of analysis and test, and particularly relates to a method for efficiently identifying a penicillamine chiral enantiomer based on an ion mobility mass spectrometer. The invention mixes D-Pen and L-Pen with beta-CD, LiCl, etc. to prepare mixed solution, then uses the electric spray ionization ion source to generate [ D-Pen + beta-CD + Li formed by non-covalent action] + And [ L-Pen + beta-CD + Li] + "ternary complex gas phase ions. And finally, carrying out analysis detection on the ion mobility by using an ion mobility spectrometry device capable of measuring the ion collision cross section. "Penicilliamine-cyclodextrin-Li due to different isomer correspondences + The ion mobility spectrums of the ternary complex are different, so that the ion mobility spectrums corresponding to different enantiomers can be obtained, and the two enantiomers can be simply, quickly and sensitively analyzed and identified according to the experimental result of the ion mobility spectrums.)

1. A method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer comprises a penicillamine sample of D-enantiomer and a penicillamine sample of L-enantiomer, wherein the molecular formula of the penicillamine sample is C₅H₁₁NO₂S, characterized by comprising the following steps:

s1, adding a solvent into a penicillamine sample to be subjected to enantiomer analysis, beta-CD and a compound containing lithium ions to prepare a mixture of Pen + beta-CD + lithium ions;

s2, using the ion source to generate the monovalent positive ion of the Pen + beta-CD + lithium ion, namely [ D-Pen + beta-CD + Li]⁺And [ L-Pen + beta-CD + Li]⁺The mass-to-charge ratio is m/z 1290.43;

s3, useAn experimental device comprising an ion mobility spectrometry measures the ion mobility spectrometry of univalent positive ions of the Pen + beta-CD + lithium ions, and the obtained measurement result of the ion mobility spectrometry is based on the sequence of collision cross sections [ L-Pen + beta-CD + Li ]]⁺>[D-Pen+β-CD+Li]⁺The structural information of the chiral enantiomer of the penicillamine molecule can be obtained.

2. The method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer as claimed in claim 1, wherein: the compound containing lithium ions is lithium chloride.

3. The method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer as claimed in claim 1, wherein: the solvent is a mixed solution of water and methanol.

4. The method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer as claimed in claim 1, wherein: the content ratio of the penicillamine sample, the beta-CD and the lithium ion-containing compound is any value greater than 0.

5. The method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer as claimed in claim 1, wherein: the beta-CD is beta-cyclodextrin.

6. The method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer as claimed in claim 1, wherein: the ion source is an electrospray ionization ion source.

Technical Field

The invention relates to the technical field of analytical testing, in particular to a method for efficiently identifying a penicillamine chiral enantiomer based on an ion mobility mass spectrometer.

Background

Chirality is a ubiquitous natural phenomenon, and chiral compounds are present in many organisms, environments, and compound products. Different chiral molecules often perform different functions, for example, the chiral drugs ofloxacin and levofloxacin may cause different pharmaceutical activities, functions, adverse reactions, etc. The study of the selectivity and stability of enantiomers currently makes it very important to understand the effect of individual enantiomers on pharmacological and toxicological activity. Therefore, there is a great deal of interest in the fields of analytical chemistry and the pharmaceutical industry in controlling the purity of enantiomers.

Over the past several decades, a number of techniques and methods have been explored to identify and characterize chiral compounds, including High Performance Liquid Chromatography (HPLC), capillary electrophoresis, and supercritical fluid chromatography. The main thing in these methods is the efficient separation of enantiomers by selecting an appropriate Chiral Selector (CS) whose difference in binding capacity can be made. In HPLC, Qin et al distinguish chiral tryptophan by β -cyclodextrin (β -CD) and acrylamide. Shedania et al used pure acetonitrile instead of methanol to separate 14 chiral sulfides using 12 chiral columns of cellulose. Although these methods are widely used, these methods still have the disadvantages of long time, large sample amount, complicated sample pretreatment, and the like.

The beta-CD molecule is cone-shaped, forms a cavity, and is a common chiral selector with 35 stereocenters (FIGS. 1B and C). The 2-and 3-position secondary hydroxyl groups are distributed at cyclodextrin holes, and the 6-position primary hydroxyl group is arranged outside the cyclodextrin molecules to form a structure that the inside of the hole is relatively hydrophobic and the periphery of the hole is relatively hydrophilic. Recent studies have also demonstrated good chiral selectivity of cyclodextrins, and efficient separation on IM-MS can be achieved using binary complexes of cyclodextrins with isomers, such as aminobiphenyl and aminobenzenesulfonic acid isomers. Unlike the conventional methods, the reaction and derivatization between the enantiomer and CS are not used, the binary compound is combined with the enantiomer through non-covalent interaction, and the steps for preparing the sample are greatly simplified.

In recent years, ion mobility mass spectrometry (IM-MS) has become of increasing interest due to its ability to be rapid and efficient in enantiomer recognition. When passing through the ion drift tube, gas phase ions continuously collide with buffer gas in an electric field, and due to the difference of the size, the shape and the charge value, the difference of collision cross sections is caused, so that effective separation is achieved. In 2006, Dwivedi et al used (S) -2-butanol as a chiral selector to separate drug, amino acid and carbohydrate enantiomers, thereby gradually expanding the work based on IM-MS for efficient chiral identification of enantiomers by binding various CS. Shortly after, j.diana Zhang et al used proton-bound diastereomeric dimers to rapidly distinguish amino acids based on IM-MS. Wil et al developed a rapid amino acid chiral analysis method based on IM-MS chiral derivatization with chloronaproxen.

Penicillamine (Pen) is an important chiral compound obtained from penicillin and used for treating Wilson's disease, cystinuria and rheumatoid arthritis, and the D-Pen and L-Pen enantiomer structures of the penicillamine are shown in figure 1A. Wherein D-Pen has specific pharmacological activity, and L-Pen has adverse effect. Wang et al developed a glassy carbon electrode using bovine serum albumin as CS to identify Pen enantiomers. However, analysis and identification of the penicillamine (Pen) enantiomer has not been performed using IM-MS.

Disclosure of Invention

Penicilliamine (Pen) has two enantiomers, and only D-Pen is found to be used for treating cystinuria and rheumatoid arthritis, while L-Pen has adverse effect. In view of the wide demand for analyzing and separating penicillamine and the shortcomings of the separation techniques and methods used at present, the invention aims to solve the problems of rapid and effective analysis, identification and separation of penicillamine and provide a method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer.

The technical scheme for realizing the purpose of the invention is as follows: a method for efficiently identifying the chiral enantiomer of penicillamine based on an ion mobility mass spectrometer comprises a penicillamine sample of D-enantiomer and a penicillamine sample of L-enantiomer, wherein the molecular formula of the penicillamine sample is C₅H₁₁NO₂S, comprising the following steps:

s1, adding a solvent into a penicillamine sample to be subjected to enantiomer analysis, beta-CD and a compound containing lithium ions to prepare a mixture of Pen + beta-CD + lithium ions;

s2, using the ion source to generate the monovalent positive ion of the Pen + beta-CD + lithium ion, namely [ D-Pen + beta-CD + Li]⁺And [ L-Pen + beta-CD + Li]⁺The mass-to-charge ratio is m/z 1290.43;

s3, measuring ion mobility spectrum of monovalent positive ions of the Pen + beta-CD + lithium ions by using an experimental device comprising ion mobility spectrum, obtaining the measurement result of the ion mobility spectrum, and determining the collision cross-section sequence according to the L-Pen + beta-CD + Li]⁺>[D-Pen+β-CD+Li]⁺The structural information of the chiral enantiomer of the penicillamine molecule can be obtained.

The compound containing lithium ions in the technical scheme is lithium chloride.

The solvent is mixed liquid of water and methanol.

The content ratio of the penicillamine sample, the beta-CD and the lithium ion-containing compound in the technical scheme is any value larger than 0.

The beta-CD is beta-cyclodextrin.

The ion source in the technical scheme is an electrospray ionization ion source.

After the technical scheme is adopted, the invention has the following positive effects:

respectively mixing D-Pen and L-Pen with beta-CD, LiCl and the like to prepare a mixed solution, and then generating a 'D-Pen + beta-CD + Li' formed by non-covalent interaction by using an electrospray ionization ion source]⁺And [ L-Pen + beta-CD + Li]⁺"ternary complex gas phase ions. And finally, carrying out analysis detection on the ion mobility by using an ion mobility spectrometry device capable of measuring the ion collision cross section. "Penicilliamine-cyclodextrin-Li due to different isomer correspondences⁺The ion mobility spectrums of the ternary complex are different, so that the ion mobility spectrums corresponding to different enantiomers can be obtained, and the two enantiomers can be simply, quickly and sensitively analyzed and identified according to the experimental result of the ion mobility spectrums.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1(A) is a schematic diagram of the chemical structures of D-Pen and L-Pen;

FIG. 1(B) is a schematic diagram of the chemical structure of beta-cyclodextrin;

FIG. 1(C) is a schematic diagram showing the structure of a glucose unit in beta-cyclodextrin;

FIG. 2 is a mass spectrometric detection of mixed solutions of D-Pen and L-Pen, lithium chloride, beta-cyclodextrin;

FIG. 3 is the TIMS separation chart of the mixed solution of D-Pen and L-Pen, lithium chloride and beta-cyclodextrin (A) [ D-Pen + beta-CD + H ]]⁺；(B)[L-Pen+β-CD+H]⁺；(C)[D/L-Pen+β-CD+Li]⁺。

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

(example 1)

To verify the feasibility and the advancement of the method of the present invention, the inventors carried out a "penicillamine-cyclodextrin-Li solution" containing chiral penicillamine molecules in positive ion mode using a commercial ion mobility spectrometry-mass spectrometer (TIMS-TOF MS, manufactured by Bruke corporation)⁺"ion mobility of ternary complex ions and mass spectrometry experiments. The primary mass spectrum result is shown in FIG. 2, and a ternary complex [ Pen-beta-CD-Li ] formed by the two isomers, beta-CD and LiCl can be seen]⁺Separation was not obtained due to the same mass to charge ratio. After TIMS analysis, the ternary complexes of the two isomers were well separated due to their different ion mobilities, and the results are shown in FIG. 3。

(1) Preparation of sample solution

Firstly, weighing a certain amount of D-Pen and L-Pen standard substances, beta-cyclodextrin and LiCl solid on an analytical balance, dissolving with a proper solvent to prepare the mixture with the concentration of 10^-2M, mother liquor. Respectively taking 10uL of mother liquor, and diluting to 1mL by using methanol/water/formic acid (1: 1: 0.001, v/v/v) as a solvent to obtain 10^-4"D-Pen + beta-CD + Li" of M]⁺"and" [ L-Pen + beta-CD + Li]⁺"mix sample solution and shake well for use.

(2) Sample detection

The TIMS-TOF was first calibrated and the sample solution was detected by manual sample injection. The instrument parameters are as follows: an electrospray ion source; a positive ion mode; sample introduction flow rate: 3 mu L/min; electrospray voltage: 3600V; atomizing: 0.3 bar; flow rate of drying gas: 3.0L/min; temperature of the drying gas: 200 ℃; the ramp time is 500 ms. And analyzing the detection result.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.