A reagent and method for the separation analysis of the position isomerism of c=c double bonds based on crown ethers

Abstract

The present application relates to the field of analytical test technology, and particularly provides a reagent and method for separating and analyzing C=C double bond position isomerism based on crown ether, which comprises: separating and analyzing 2 / 3-butenoic acid (BA), 2 / 3 / 4-pentenoic acid (PA) and 2 / 3 / 5-hexenoic acid (HA) isomers respectively, mixing the above unsaturated isomers with crown ethers including 18 crown 6 (18C6), 15 crown 5 (15C5), 12 crown 4 (12C4) and divalent metal ions to form a mixed solution, and then separating and analyzing the mixed solution by means of ion mobility mass spectrometry. The ion mobility mass spectrometry experiment shows that the molecules can form ternary complexes of crown ether-isomer-metal ion through non-covalent interaction, and the C=C position isomerism can be simultaneously separated through the formed complexes. The analytical reagent and method provided by the present application are simple, accurate and fast in analysis speed, and do not need chemical derivatization, chromatographic separation and other previous steps, and the method can be used for separating different C=C double bond position isomers.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and specifically to a reagent and method for the separation and analysis of C=C double bond positional isomerism based on crown ethers. Background Technology

[0002] Fatty acids are essential for the normal function of all organisms and have a significant impact on the properties and biological functions of biophysical membranes. Most fatty acids are unsaturated fatty acids, containing one or more carbon-carbon double bonds (C=C) in their structure. Importantly, the distribution of C=C isomers and geometric isomers greatly influences their biochemical properties. Butenoic acid (BA), pentenoic acid (PA), and hexenoic acid (HA) are all short-chain unsaturated fatty acids and can serve as important precursors for pharmaceutical applications. However, these compounds all have different C=C position isomers, and alterations in the distribution of C=C isomers are potential biomarkers for disease diagnosis. For example, 4-PA, a pentenoic acid with a double bond at position 4, may react with skin proteins but hardly reacts under physiological conditions; while 2-HA, a hexenoic acid with a double bond at position 2, was identified in screening as a fragrance material with no potential risk to the aquatic environment, but a risk level assessment for the aquatic environment is required. Therefore, it is urgent to distinguish the C=C isomers of these unsaturated fatty acids in order to elucidate their different biological functions, but this remains a technical challenge due to their similar molecular structures.

[0003] Mass spectrometry (MS) is a widely used analytical tool, offering advantages such as speed, accuracy, and sub-microgram requirements. Currently, the main methods for fatty acid detection are based on mass spectrometry, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), and thin-layer chromatography-MS (TLC-MS). However, traditional HPLC-MS or GC-MS methods are insufficient for structural resolution of the determined C=C position and are labor-intensive and time-consuming. In this context, methods for determining the C=C position in fatty acids, such as tandem mass spectrometry (MS / MS), have been reported. MS / MS is the most widely used method for distinguishing C=C positions in recent years, capable of characterizing detailed structural parts and identifying lipid isomers. However, the C=C bond breaking energy is high, rendering low-energy collision-induced dissociation (CID) ineffective in elucidating the C=C bond position. Therefore, ozone-induced dissociation (OzID), ultraviolet light dissociation (UVPD), and electron collision excitation of organic ions have been developed to distinguish C=C isomerism. In addition, other chemical derivative methods to reduce bond-breaking energies were investigated, such as ozone decomposition, methylthiolation, the Pasterno-Büchi reaction, and plasma-induced epoxidation. For example, specific fragment ions were identified using a Pasterno-Büchi chemical reaction combined with tandem mass spectrometry (MS / MS) to obtain information on the C=C isomer conformations of fatty acid isomers. However, while the strategy of combining chemical reactions with MS / MS is promising, it is also challenging, and chemical derivation is a mandatory step that often requires large sample volumes and can be complex and ambiguous. In such cases, other simple and convenient methods are worth exploring.

[0004] Ion mobility spectrometry (IMS) is a promising ion separation method that has emerged in recent years. The separation mechanism of IMS differs from that of chromatography. IMS is a gas-phase separation technique that identifies different ions based on differences in size, shape, and charge. Trapped ion mobility spectrometry (TIMS) is an IMS technique developed by Fernandez-Lima et al. in 2011. It uses a nitrogen flow to carry ions into the drift cell of the TIMS and separates them based on differences in size, charge, and shape. The combination of TIMS and MS (TIMS-MS) is potentially an exciting ion separation technique because the timescale (milliseconds) of TIMS separation and the fact that separation occurs in the gas phase make it ideal for coupling with MS, eliminating the need for chromatographic analysis and providing an additional separation dimension for molecular structure identification and characterization without affecting analytical throughput. The TIMS-MS method overcomes the limitation of single MS methods in separating ions with the same m / z ratio; it also enhances the sensitivity and m / z selectivity of TIMS analysis. Clearly, using mobility-based mass spectrometry (TIMS-MS) to analyze target enantiomers is a simple method that requires no prior separation or pretreatment of the target analyte. The chemical samples used are readily available, inexpensive, and non-toxic. This offers many advantages over currently used methods. However, directly separating unsaturated aliphatic isomers using TIMS-MS remains a very challenging area due to their highly similar chemical structures. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a reagent and method for the separation and analysis of C=C double bond positional isomerism based on crown ethers.

[0006] The technical solution to achieve the purpose of this invention is: a reagent for the separation and analysis of C=C double bond positional isomers based on crown ethers, wherein the reagent comprises C=C double bond isomers mixed with solvent, crown ethers and divalent metal ions.

[0007] The C=C double bond isomers include 2 / 3-butenoic acid (BA), 2 / 3 / 4-pentenoic acid (PA), and 2 / 3 / 5-hexenoic acid (HA) isomers.

[0008] The crown ethers include 18-crown 6 (18C6), 15-crown 5 (15C5), and 12-crown 4 (12C4).

[0009] The divalent metal ions include Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Sr2+, and Ba2+.

[0010] The preparation of the mixed solvent is not limited, and includes methanol, water, acetonitrile, etc.

[0011] The C=C double bond isomers are respectively mixed with three crown ethers and different divalent metal ions, and the molar ratio of the mixtures is arbitrary. As long as the solution includes C=C double bond isomers, crown ethers and divalent metal ions, a corresponding C=C isomer-crown ether-metal ion ternary complex can be formed.

[0012] A method for the separation and analysis of C=C double bond positional isomerism based on crown ethers, comprising the following steps:

[0013] S1. The C=C double bond isomers were mixed with three crown ethers and different divalent metal ions respectively to prepare corresponding C=C isomer-crown ether-metal ion mixed solutions.

[0014] S2 uses an ion source to generate ternary non-covalent complex ions of "isomer-crown ether-metal ion" from a mixture of C=C isomer-crown ether-metal ion, namely [18C6 / 15C5 / 12C4+2 / 3-BA+GH]+, [18C6 / 15C5 / 12C4+2 / 3 / 4-PA+GH]+, [18C6 / 15C5 / 12C4+2 / 3 / 5-HA+GH]+, (G is a divalent metal ion).

[0015] S3 uses ion mobility spectrometry to measure the ion mobility of the ternary non-covalent complex ions of the "isomer-crown ether-metal ion" structure. The structural information of the C=C position isomer molecule can be obtained from the measurement results of the ion mobility spectrum.

[0016] The ion source described in the above technical solution S2 is one of the following: electrospray ionization (ESI), laser-assisted desorption ionization (MALDI), desorption electrospray ionization (DESI), or other types of ion sources that can generate monovalent positive ions of "C=C isomer-crown ether-metal ion ternary complex".

[0017] The experimental device described in the above technical solution S3 is an ion mobility spectrometer or a combination of a composite experimental device including an ion mobility spectrometer, such as a mass spectrometer.

[0018] The C=C double bond isomers described in the above technical solution S1 include 2 / 3-butenoic acid (BA), 2 / 3 / 4-pentenoic acid (PA), and 2 / 3 / 5-hexenoic acid (HA) isomers; the crown ethers described in S1 include 18-crown-6 (18C6), 15-crown-5 (15C5), and 12-crown-4 (12C4); the divalent metal ions include Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Sr2+, and Ba2+.

[0019] After adopting the above technical solution, the present invention has the following positive effects:

[0020] (1) This invention provides a new method for the separation and analysis of C=C double bond positional isomers based on crown ethers. It simply mixes the C=C double bond isomer sample with crown ether and divalent metal ions to form a mixed solution. Then, it uses an electrospray ionization source to generate a ternary non-covalent complex ion of "isomer-crown ether-metal ion". After that, it uses an experimental device containing ion mobility spectra to measure their ion mobility. The structural information of the C=C positional isomer molecule can be obtained based on the measurement results of the ion mobility spectra.

[0021] (2) The analytical reagent method provided by this invention is simple, accurate, and fast, with advantages such as no need for chemical derivatization or chromatographic separation. Furthermore, the three crown ethers and different divalent metal ions are widely applicable to the aforementioned C=C position isomers, and the method is rapid. This method can be used for the separation of isomers with different C=C double bond positions. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0023] Figure 1 Schematic diagrams of the structures of substances analyzed for C=C double bond isomers and three crown ethers;

[0024] Figure 2 Mass spectrometry of the mixture;

[0025] (a) BA, 18C6, 15C5, 12C4, Sr2+ and Ba2+;

[0026] (b) PA, 18C6, 15C5, 12C4, Sr2+ and Ba2+;

[0027] (c)HA, 18C6, 15C5, 12C4, Sr2+ and Ba2+;

[0028] Figure 3 For the mobility separation of isomer complexes;

[0029] (a) [BA+crown ether+Sr-H]+; from left to right, they are 3-BA and 2-BA;

[0030] (b) [BA + crown ether + Ba-H]+; from left to right, they are 3-BA and 2-BA;

[0031] (c)[PA + crown ether + Sr-H]+; from left to right, they are 4-PA, 3-PA, and 2-PA;

[0032] (d)[PA + crown ether + Ba-H]+; from left to right, they are 4-PA, 3-PA, and 2-PA;

[0033] (e)[HA + crown ether + Sr-H]+; from left to right, they are 5-HA, 3-HA, and 2-HA;

[0034] (f)[HA + crown ether + Ba-H]+; from left to right, they are 5-HA, 3-HA, and 2-HA;

[0035] Figure 4 The mobility separation degree of the three C=C double bond isomers. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0037] The unsaturated fatty acid carbon-carbon double bond (C=C) isomers used are 2 / 3-butenoic acid (BA), 2 / 3 / 4-pentenoic acid (PA), and 2 / 3 / 5-hexenoic acid (HA) isomers, with the following structural formulas: Figure 1 As shown.

[0038] The crown ethers used include 18-crown 6 (18C6), 15-crown 5 (15C5), and 12-crown 4 (12C4), with the following structural formulas: Figure 1 As shown.

[0039] First, the standard samples of the complex were prepared: 2 / 3-BA, 2 / 3 / 4-PA, and 2 / 3 / 5-HA were mixed with 18C6, 15C5, 12C4 and different divalent metal ions in a 1:1:1 ratio to form a concentration of 10. -5 mol L -1 The solution is a mixture of 50% methanol and 50% water.

[0040] Then, the mixed solution was analyzed using TIMS-TOF: the prepared solution was directly analyzed using TIMS-TOF, with the TIMS-TOF ion source voltage ranging from 2.5 to 4.5 kV and the injection flow rate from 0 to 5 μL / min. -1 The atomizer pressure is 0.3 bar; the dry gas concentration at 200°C is 3 L / min. -1 The test results were then analyzed.

[0041] To verify the feasibility and advancement of the method presented in this invention, the inventors used a commercially available ion mobility spectrometry-mass spectrometry (IMS-TOFMS) instrument manufactured by Bruke to analyze a total of eight C=C double bond positional isomers. The first-order mass spectra results are as follows: Figure 2 As shown, the C=C position isomers can form corresponding ternary complexes with 18C6, 15C5, 12C4, and divalent metal ions Sr2+ and Ba2+. TIMS analysis revealed that all eight C=C position isomers in the three groups were well separated. Figure 3 As shown in the figure. Meanwhile, complexes formed by other divalent metal ions also exhibited good separation effects on their C=C position isomers, as shown in the figure. Figure 4 As shown, the resolution can reach as high as 2.44. Here, the algorithm for calculating the resolution is as follows: Where A1 is the drift value of the preceding peak among two adjacent peaks, A2 is the mobility drift data of the following peak among two adjacent peaks, and W1 and W2 are the peak widths of the two adjacent peaks. The experimental results clearly show that BA, PA, and HA, which have C=C positional isomers, can be easily distinguished.

[0042] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reagent for the separation and analysis of C=C double bond positional isomerism based on crown ethers, characterized in that: The reagents include C=C double bond isomers mixed with solvents, crown ethers, and divalent metal ions; The divalent metal ion mentioned is Mg 2+ Ca 2+ Mn 2+ Fe 2+ Co 2+ Ni 2+ Zn 2+ 、Sr 2+ Or Ba 2+ ; The C=C double bond isomer is 2 / 3-butenoic acid (BA), and the crown ether is 18-crown-6 (18C6) or 15-crown-5 (15C5); or, The C=C double bond isomer is 2 / 3 / 4-pentenoic acid (PA) or 2 / 3 / 5-hexenoic acid (HA), and the crown ether is 18-crown-6 (18C6), 15-crown-5 (15C5), or 12-crown-4 (12C4).

2. The reagent for the separation and analysis of C=C double bond positional isomerism based on crown ethers according to claim 1, characterized in that: The solvents include methanol, water, or acetonitrile.

3. A method for the separation and analysis of C=C double bond positional isomerism based on crown ethers, characterized in that, It includes the following steps: S1. Mix the C=C double bond isomer with crown ether and divalent metal ion to prepare a corresponding C=C isomer-crown ether-metal ion mixed solution; The divalent metal ion mentioned is Mg 2+ Ca 2+ Mn 2+ Fe 2+ Co 2+ Ni 2+ Zn 2+ 、Sr 2+ Or Ba 2+ ; The C=C double bond isomer is 2 / 3-butenoic acid (BA), and the crown ether is 18-crown-6 (18C6) or 15-crown-5 (15C5); or, The C=C double bond isomer is 2 / 3 / 4-pentenoic acid (PA) or 2 / 3 / 5-hexenoic acid (HA), and the crown ether is 18-crown-6 (18C6), 15-crown-5 (15C5), or 12-crown-4 (12C4). S2 uses an ion source to generate a ternary non-covalent complex ion of "isomer-crown ether-metal ion", namely [18C6 / 15C5+2 / 3-BA+GH], from a mixture of C=C isomers, crown ethers, and metal ions. + , [18C6 / 15C5 / 12C4+2 / 3 / 4-PA+GH] + , [18C6 / 15C5 / 12C4+2 / 3 / 5-HA+GH] + G represents the aforementioned divalent metal ions; S3 uses an experimental setup to measure the ion mobility of the ternary non-covalent complex ion of "isomer-crown ether-metal ion". The structural information of the C=C position isomer molecule can be obtained from the measurement results of the ion mobility spectrum.

4. The method for separation and analysis of C=C double bond positional isomerism based on crown ethers according to claim 3, characterized in that: The ion source mentioned in S2 includes one of the following: electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), or desorption electrospray ionization (DESI).

5. The method for separation and analysis of C=C double bond positional isomerism based on crown ethers according to claim 3, characterized in that: The experimental apparatus described in S3 is an ion mobility spectrometer or a composite experimental apparatus that includes an ion mobility spectrometer.

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