Application of spiramycin in analysis of multiple bile acid isomers and analysis method of spiramycin

Abstract

The invention relates to the technical field of analysis and testing, in particular to application of spiramycin in analysis of multiple bile acid isomers and an analysis method of the spiramycin. According to the method, several bile acid isomers and spiramycin are prepared into a mixed solution in a certain proportion, gas-phase compound ions of bile acid molecules to be analyzed and spiramycin are prepared and generated by using an ionization device, and the ion mobility spectrometry of the gas-phase compound ions is measured by using an ion mobility spectrometry analysis device; the compound ions containing different bile acid isomers have different collision cross sections, so that the types of the isomers of the bile acid in the sample can be obtained according to the measurement result of ion migration, and the relative contents of the isomers in the mixture sample can be obtained according to the ion migration spectrum signal intensity of various bile acid isomer compounds; the analysis method provided by the invention is simple, accurate in analysis result and high in analysis speed, and can be applied to clinical medicine, life science, pharmacy, food and environmental science.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and testing, and in particular to an application of spiramycin in analyzing a variety of bile acid isomers and an analysis method thereof. Background Art

[0002] Bile acid is an important biological molecule that performs important biological functions in animals, especially digestion. Studies have shown that there are many types of bile acids in the body, such as isomers, and the content of each isomer is different. Changes in the content or type of bile acids in the body may not only cause certain diseases, but some diseases may also cause changes in the content or type of bile acids. Due to the important biological functions of bile acids, some bile acids have also become drugs for treating certain diseases, such as bear bile powder and pig bile powder, which are common drugs. Therefore, accurate analysis of the types, chemical structures and contents of bile acids is of great significance to life sciences, pharmaceuticals and other aspects.

[0003] Molecular isomers are molecules that have the same chemical formula but different chemical structures, such as Figure 1 The isomers of bile acid given in (a, b, c) all have the chemical formula C 28 H 43 NO6, and Figure 1 (d, e) Their chemical formula is C 26 H 45 NO6S, but the chemical structures of different isomers are significantly different. Since the chemical properties, physical properties and biological functions of molecules are closely related to the structure of molecules, it is also very important to understand the molecular structure of a molecule. In the past 100 years, molecular structural analysis has always been one of the goals of scientists, and various scientific methods for analyzing molecular structures have been developed, such as spectroscopy, mass spectrometry, and nuclear magnetic resonance. The structural analysis of molecular isomers is one of the difficulties in molecular structure analysis. This is because the molecules of isomers have exactly the same molecular mass, so they cannot be directly analyzed by mass spectrometry. Some isomers have very small structural differences, such as chiral isomers, and general spectroscopy and nuclear magnetic resonance cannot distinguish them. Due to the widespread existence of different isomer molecules in the fields of life sciences, pharmaceuticals, and chemicals, the accurate, rapid, and highly sensitive identification of the molecular structures of different isomers has always been one of the directions of interest in the field of analytical testing. Summary of the invention

[0004] The purpose of the present invention is to provide an application of spiramycin in analyzing multiple bile acid isomers and an analysis method thereof in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide a method for analyzing multiple bile acid isomers, comprising the steps of:

[0007] S1, mixing the sample to be tested with the spiramycin solution, wherein the bile acid in the sample to be tested is non-covalently bound to the spiramycin to prepare a mixed solution;

[0008] S2, treating the mixed solution with an ionization device to generate gas phase complex ions;

[0009] S3. Analyze the ion mobility spectrum of the gas phase complex ions using an ion mobility spectrometer to obtain ion mobility spectra or ion collision cross sections corresponding to different isomers of bile acid, and distinguish the isomers of bile acid.

[0010] Preferably, in step S1, the solvent of the spiramycin solution includes: methanol and formic acid water.

[0011] Preferably, in step S1, the bile acids in the sample to be tested include: Glycochenodeoxycholic acid, Glycoursodeoxycholic acid, Glycohyodeoxycholic acid, Taurochenodeoxycholicacid, and Tauroursodeoxycholic acid.

[0012] Preferably, in step S2, the ionization device comprises: an electrospray ionization source, a laser-assisted desorption ionization source, and a desorption electrospray ionization source.

[0013] Preferably, in step S2, the gas phase complex ions include: positively charged ions of bile acid molecules and spiramycin molecules, and negatively charged ions of bile acid molecules and spiramycin molecules.

[0014] More preferably, in step S2, the gas phase complex ions include: [TCDCA / TUDCA+SPM+H] + 、[GUDCA / GCDCA / GHDCA+SPM+H] + 、[2GUDCA / GCDCA / GHDCA+SPM+H] + 、[2GCDCA+SPM+H] + 、[2GUDCA+SPM+H] + 、[2GHDCA+SPM+H] + 、[TCDCA / TUDCA+SPM-H] - 、[GUDCA / GCDCA / GHDCA+SPM-H] -、[2GUDCA / GCDCA / GHDCA+SPM-H] - .

[0015] More preferably, in step S2, the gas phase complex ions further include metal ions; the metal ions include: Li + 、Na + , K + , Rb + , Ca 2+ 、Ni 2+ , Sr 2+ , Cu 2+ .

[0016] Preferably, the steps further include:

[0017] S4. After drawing the standard curve, read the content of different bile acids in the sample to be tested by comparing with the standard curve.

[0018] More preferably, the step of drawing the standard curve includes: preparing a standard solution containing various bile acid concentrations and a certain concentration of spiramycin, measuring the ion mobility spectrum peak intensity of the bile acid complex under different bile acid sample concentrations, and obtaining the relationship between the bile acid concentration of each isomer and the corresponding complex ion mobility spectrum signal intensity, thereby obtaining a standard curve for quantitative analysis.

[0019] The second aspect of the present invention is to provide the use of spiramycin in analyzing multiple bile acid isomers.

[0020] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0021] The invention first prepares several bile acid isomers and spiramycin into a mixed solution of a certain proportion, then uses an ionization device to prepare and generate gas phase complex ions of bile acid molecules to be analyzed and spiramycin, and then uses an ion mobility spectrometry analysis device to measure the ion mobility spectra of these gas phase complex ions; since the complex ions containing different bile acid isomers have different collision cross sections, the types of bile acid isomers contained in the sample can be obtained according to the measurement results of ion mobility, and the relative contents of various bile acid isomer complexes in the mixture sample can also be obtained according to the ion mobility spectrometry signal intensity; compared with the existing bile acid isomer analysis method, the analysis method provided by the invention is simple, the analysis result is accurate, the analysis speed is fast, and it can be applied in clinical medicine, life science, pharmacy, food and environmental science. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The chemical structural formulas of various bile acid molecules and the molecular structure diagram of spiramycin; (a)

[0023] Glycochenodeoxycholic acid(GCDCA); (b)Glycoursodeoxycholic acid

[0024] (GUDCA); (c) Glycohyodeoxycholic acid (GHDCA); (d)

[0025] Taurochenodeoxycholic acid(TCDCA); (e)Tauroursodeoxycholic acid

[0026] (TUDCA); (f) Spiramycin (SPM);

[0027] Figure 2 The mass spectrometry analysis diagrams of mixed solutions of several bile acid molecules and the ion mobility spectrometry analysis results of the corresponding complex ions; (a) Mass spectrometry analysis results of mixed solutions of bile acid isomers, SPM and NaCl; (b) Mass spectrometry analysis results of mixed solutions of bile acid isomers and SPM; (c) Ion mobility spectrometry experimental results of complex ions of different bile acid isomers and SPM; (d) Ion mobility spectrometry experimental results of complex ions of different bile acid isomers and SPM; (e) Ion mobility spectrometry experimental results of complex ions of different bile acid isomers and SPM;

[0028] Figure 3 The analysis results of various bile acid isomers in pig bile powder and bear bile powder; (a) pig bile powder; (b) pig bile powder m / z 1292.99; (c) bear bile powder; (d) bear bile powder m / z 1343.78;

[0029] Figure 4 Schematic diagram of the quantitative analysis method and results of the contents of multiple bile acids in a mixture. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0033] Example

[0034] This embodiment provides a method for analyzing multiple bile acid isomers, the steps comprising:

[0035] S1. Mix the sample to be tested with the spiramycin solution and prepare it to a concentration of 10 -5 mol L -1 A mixed solution, wherein the solvent used is 50% methanol and 50% formic acid water, the formic acid water is an aqueous solution with a formic acid content of 0.1%, and in the mixed solution, bile acid and spiramycin are non-covalently bound;

[0036] S2. Perform TIMS-TOF experiment on the mixed solution to detect and analyze the mass spectra and ion mobility spectra of various gas phase ion complexes. The ion source voltage is 2.5-4.5 kV and the injection flow rate is 1-5 μL min -1 ; Atomizer pressure 0.3 bar; Dry gas 3 L min at 200 °C -1 , and analyze the test results.

[0037] S3. Analyze the ion mobility spectrometry experimental results of various ion complexes to obtain the molecular species of bile acid isomers in the analyzed samples.

[0038] S4. Prepare standard solutions containing various bile acid concentrations and a certain concentration of spiramycin, measure the ion mobility spectroscopic peak intensity of the bile acid complex under different bile acid sample concentrations, obtain the relationship between the bile acid concentration of each isomer and the corresponding complex ion mobility spectroscopic signal intensity, and obtain a standard curve for quantitative analysis. Compare with the standard curve, read the relative content of the bile acid isomer molecules in the sample to be tested.

[0039] The results are as follows Figure 2-4 As shown, the isomers of different bile acids are clearly distinguished.

[0040] In summary, the analysis method of the present invention overcomes the difficulty that mass spectrometry, spectrum and the like cannot analyze and identify bile acid isomers; compared with traditional structural analysis techniques and methods of bile acid isomers, the present method is simple, rapid and accurate, and can analyze each isomer and its content in a mixed sample; the present invention requires a small amount of sample and has high sensitivity.

[0041] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing multiple bile acid isomers, characterized in that the steps include: S1, mixing the sample to be tested with the spiramycin solution, wherein the bile acid in the sample to be tested is non-covalently bound to the spiramycin to prepare a mixed solution; S2, treating the mixed solution with an ionization device to generate gas phase complex ions; S3. Analyze the ion mobility spectrum of the gas phase complex ions using an ion mobility spectrometer to obtain ion mobility spectra or ion collision cross sections corresponding to different isomers of bile acid, and distinguish the isomers of bile acid.

2. The analysis method according to claim 1, characterized in that In step S1, the solvent of the spiramycin solution includes methanol and formic acid water.

3. The analysis method according to claim 1, characterized in that In step S2, the ionization device includes: an electrospray ionization source, a laser-assisted desorption ionization source, and a desorption electrospray ionization source.

4. The analysis method according to claim 1, characterized in that In step S2, the gas phase complex ions include: positively charged ions of bile acid molecules and spiramycin molecules, and negatively charged ions of bile acid molecules and spiramycin molecules.

5. The analysis method according to claim 1, characterized in that The steps also include: S4. After drawing the standard curve, read the content of different bile acids in the sample to be tested by comparing with the standard curve.

6. The analysis method according to claim 5, characterized in that The steps of drawing the standard curve include: preparing a standard solution containing various bile acid concentrations and a certain concentration of spiramycin, measuring the ion mobility spectrum peak intensity of the bile acid complex under different bile acid sample concentrations, obtaining the relationship between the bile acid concentration of each isomer and the corresponding complex ion mobility spectrum signal intensity, and obtaining a standard curve for quantitative analysis.

7. Application of spiramycin in the analysis of various bile acid isomers.

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