A method for separating astaxanthin geometric isomers and their corresponding optical isomers

Abstract

This invention discloses a method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers, relating to the field of analytical technology. This invention can effectively separate four geometric isomers: all-trans astaxanthin, 9-cis astaxanthin, and 13-cis / 15-cis astaxanthin. Furthermore, it achieves the effective separation and detection of three optical isomers of all-trans astaxanthin, four optical isomers of 9-cis astaxanthin, and eight optical isomers of 13-cis / 15-cis astaxanthin. Therefore, it enables the effective separation and analysis of the optical isomers corresponding to multiple geometric isomers. This method has high specificity, good separation degree, and high accuracy in analyzing each geometric and optical isomer, which is of great significance for conducting research on the precise correlation between astaxanthin bioactivity and its isomers.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and more specifically, to a method or analytical method for separating or analyzing astaxanthin geometric isomers and their corresponding optical isomers. Background Technology

[0002] Astaxanthin is a precious natural antioxidant pigment belonging to the carotenoid family. Initially used to provide vibrant colors for farmed salmon and crustaceans, its various biological functions have been discovered, and it is now widely used globally in dietary supplements, fortified foods and beverages, and even pharmaceuticals. The carbon skeleton of astaxanthin consists of a central polyolefin chain and two β-ionone rings on either side, each aromatic ring containing one hydroxyl group and one ketone group. The C-3 and C-3' ends of the β-ionone rings are chiral centers. Based on the conformation of the terminal chiral carbons, astaxanthin has three optical isomers: levorotatory (3S, 3'S), dextrorotatory (3R, 3'R), and meso (3R, 3'S). Furthermore, astaxanthin contains 11 conjugated double bonds in its structure. Depending on the spatial arrangement of the double-bond linkages, astaxanthin also exists in various geometric isomers, mainly including all-trans astaxanthin, 9-cis-astaxanthin, 13-cis-astaxanthin, and 15-cis-astaxanthin. Studies have found that astaxanthin from different sources, such as microorganisms, marine organisms, and chemical synthesis, has different isomer compositions, and the biological functions of different isomers vary significantly.

[0003] Due to the widespread existence of astaxanthin isomers, analytical techniques are particularly important. Ultraviolet spectrophotometry, an early detection method, can only detect total astaxanthin content in simple media, not the content of individual isomers. Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy can provide structural characteristics of isomers, but are not suitable for analyzing complex matrices. Liquid chromatography (LC) is increasingly used for astaxanthin content and isomer analysis due to its high efficiency and sensitivity. However, existing LC methods can only analyze geometric or optical isomers in a matrix individually, and cannot achieve precise analysis of the optical isomers corresponding to a single geometric isomer, thus limiting in-depth research on the precise correlation between astaxanthin's biological function and chemical structure.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method or analysis method for separating or analyzing astaxanthin geometric isomers and their corresponding optical isomers, thereby simultaneously separating and analyzing multiple astaxanthin geometric isomers and their corresponding optical isomers.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a method or analytical method for separating or analyzing astaxanthin geometric isomers and their corresponding optical isomers, comprising separation or analysis according to any one of the following i-iv methods:

[0008] i: A portion of the sample to be tested is loaded onto a first-dimensional chromatographic column for one-dimensional chromatographic separation. The first-dimensional chromatographic column is a reversed-phase column. The mobile phase A of the first-dimensional chromatographic column is an aqueous solution of phosphoric acid, and the mobile phase B is a mixed solution of methyl tert-butyl ether, acetonitrile, and methanol, with isocratic elution. Using a center-cutting method, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are stored in a quantitative loop or loaded onto a trapping column at the first time. Through the flow path of the switching valve, all the components stored in the quantitative loop are sent to the second-dimensional chromatographic column for two-dimensional chromatographic separation, or the elution trapping column is used, and the elution product of the elution trapping column is switched to the second-dimensional chromatographic column for separation. The second-dimensional chromatographic column is a cellulose-type chiral chromatographic column. The preparation of the mobile phase for the second-dimensional chromatography is: mobile phase A is methanol, mobile phase B is acetonitrile, with gradient elution.

[0009] After separation, another portion of the sample is loaded onto the first-dimensional chromatographic column. Using a center-cutting method, at the second time point, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are stored in a quantitative loop or loaded onto a trapping column. Through the flow path of the switching valve, all the components stored in the quantitative loop are sent to the second-dimensional chromatographic column, or the elution trapping column is used. The elution products from the elution trapping column are then switched to the second-dimensional chromatographic column for separation.

[0010] After separation, a portion of the sample to be tested is loaded onto the first-dimensional chromatographic column. Using a center-cutting method, at the third time point, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are stored in a quantitative loop or loaded onto a trapping column. Through the flow path of the switching valve, all the components stored in the quantitative loop are sent to the second-dimensional chromatographic column, or the elution trapping column. The elution products from the elution trapping column are switched to the second-dimensional chromatographic column for separation.

[0011] The first time, the second time, and the third time are selected from any one of 2.36-2.60 min, 3.55-3.85 min, and 4.10-4.50 min, and the first time, the second time, and the third time are not the same;

[0012] ii: Load the sample to be tested onto the first-dimensional chromatographic column, and use the center cutting method to store the components at 2.36-2.60 min, 3.55-3.85 min and 4.10-4.50 min respectively in the quantitative loop. Then, through the flow path of the switching valve, send each component stored in the quantitative loop into the second-dimensional chromatographic column for separation.

[0013] iii: Load the sample to be tested onto the first-dimensional chromatographic column, and collect the components from any one or two time periods using the center-cut method. Store the components stored in the quantitative loops separately, and then send the components stored in the quantitative loops into the second-dimensional chromatographic column for separation. The time periods are selected from: 2.36-2.60 min, 3.55-3.85 min, and 4.10-4.50 min.

[0014] iv: Load a portion of the sample to be tested onto the first-dimensional chromatographic column for one-dimensional chromatographic separation; using a center-cutting method, store the astaxanthin geometric isomers separated by the first-dimensional chromatographic column in a quantitative loop or load them onto a trapping column at any of the following time periods; through the flow path of the switching valve, send all the components stored in the quantitative loop into the second-dimensional chromatographic column for two-dimensional chromatographic separation, or, elute the trapping column, and switch the elution product of the elution trapping column to the second-dimensional chromatographic column for separation;

[0015] The time periods are selected from: 2.36-2.60 min, 3.55-3.85 min and 4.10-4.50 min.

[0016] The present invention has the following beneficial effects:

[0017] To address the technological gap in existing methods that cannot achieve precise analysis of optical isomers corresponding to a single geometric isomer, this invention provides a method for simultaneously analyzing astaxanthin geometric isomers and their corresponding optical isomers. It can effectively separate four geometric isomers: all-trans astaxanthin, 9-cis astaxanthin, and 13-cis / 15-cis astaxanthin. Furthermore, it enables the effective separation and detection of three optical isomers of all-trans astaxanthin, four optical isomers of 9-cis astaxanthin, and eight optical isomers of 13-cis / 15-cis astaxanthin. Therefore, it can achieve effective separation and analysis of optical isomers corresponding to multiple geometric isomers. This method is highly specific, has good separation accuracy, and provides high precision in the analysis of each geometric and optical isomer, making it significant for conducting research on the precise correlation between astaxanthin bioactivity and its isomers.

[0018] The method provided by this invention can be widely used in the separation and analysis of single geometric isomers and their corresponding optical isomers in the fields of nutritional supplements, fortified foods, beverages, and pharmaceuticals. It can also be applied to the qualitative analysis of single geometric isomers and their corresponding optical isomers. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A chromatogram of a one-dimensional geometric isomer;

[0021] Figure 2 The chromatograms of one-dimensional chromatography for cutting all-trans astaxanthin and the corresponding optical isomers in two-dimensional chromatography are shown.

[0022] Figure 3 The chromatograms of 9-cis-astaxanthin cut by one-dimensional chromatography and the corresponding optical isomers by two-dimensional chromatography are shown.

[0023] Figure 4 The chromatograms of 13-cis / 15-cis astaxanthin were cut into one-dimensional chromatograms and the corresponding optical isomers in two-dimensional chromatograms, with the center as the cut point.

[0024] Figure 5 The structural formulas are those of all-trans astaxanthin (a), 9-cis astaxanthin (b), 13-cis astaxanthin (c) and 15-cis astaxanthin (d);

[0025] Figure 6 The images show the results of one-dimensional chromatography qualitative analysis of the geometric isomers of all-trans astaxanthin standards (A: one-dimensional chromatographic result of all-trans astaxanthin standard; B: one-dimensional chromatographic result of all-trans astaxanthin standard after isomerization and mass spectrometry scan of 3 chromatographic peaks; C: spectral scan of 3 chromatographic peaks).

[0026] Figure 7 The graph shows the results of qualitative analysis of all-trans astaxanthin standards by one-dimensional chromatography and two-dimensional chromatography. Detailed Implementation

[0027] To make the objectives, 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] In a first aspect, the present invention provides a method or analytical method for separating or analyzing astaxanthin geometric isomers and their corresponding optical isomers, comprising separation or analysis according to any one of the following i-iv methods:

[0029] i: A portion of the sample to be tested is loaded onto a first-dimensional chromatographic column for one-dimensional chromatographic separation. The first-dimensional chromatographic column is a reversed-phase column. The mobile phase A of the first-dimensional chromatographic column is an aqueous solution of phosphoric acid, and the mobile phase B is a mixed solution of methyl tert-butyl ether, acetonitrile, and methanol, with isocratic elution. Using a center-cutting method, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are stored in a quantitative loop or loaded onto a trapping column at the first time. Through the flow path of the switching valve, all the components stored in the quantitative loop are sent to the second-dimensional chromatographic column for two-dimensional chromatographic separation, or the elution trapping column is used, and the elution product of the elution trapping column is switched to the second-dimensional chromatographic column for separation. The second-dimensional chromatographic column is a cellulose-type chiral chromatographic column. The preparation of the mobile phase for the second-dimensional chromatography is: mobile phase A is methanol, mobile phase B is acetonitrile, with gradient elution.

[0030] After separation, another portion of the sample is loaded onto the first-dimensional chromatographic column. Using a center-cutting method, at the second time point, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are stored in a quantitative loop or loaded onto a trapping column. Through the flow path of the switching valve, all the components stored in the quantitative loop are sent to the second-dimensional chromatographic column, or the elution trapping column is used. The elution products from the elution trapping column are then switched to the second-dimensional chromatographic column for separation.

[0031] After separation, a portion of the sample to be tested is loaded onto the first-dimensional chromatographic column. Using a center-cutting method, at the third time point, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are stored in a quantitative loop or loaded onto a trapping column. Through the flow path of the switching valve, all the components stored in the quantitative loop are sent to the second-dimensional chromatographic column, or the elution trapping column. The elution products from the elution trapping column are switched to the second-dimensional chromatographic column for separation.

[0032] The first time, the second time, and the third time are selected from any one of 2.36-2.60 min, 3.55-3.85 min, and 4.10-4.50 min, and the first time, the second time, and the third time are not the same;

[0033] ii: Load the sample to be tested onto the first-dimensional chromatographic column, and use the center cutting method to store the components at 2.36-2.60 min, 3.55-3.85 min and 4.10-4.50 min respectively in the quantitative loop. Then, through the flow path of the switching valve, send each component stored in the quantitative loop into the second-dimensional chromatographic column for separation.

[0034] iii: Load the sample to be tested onto the first-dimensional chromatographic column, and collect the components from any one or two time periods using the center-cut method. Store the components stored in the quantitative loops separately, and then send the components stored in the quantitative loops into the second-dimensional chromatographic column for separation. The time periods are selected from: 2.36-2.60 min, 3.55-3.85 min, and 4.10-4.50 min.

[0035] iv: Load a portion of the sample to be tested onto the first-dimensional chromatographic column for one-dimensional chromatographic separation; using a center-cutting method, store the astaxanthin geometric isomers separated by the first-dimensional chromatographic column in a quantitative loop or load them onto a trapping column at any of the following time periods; through the flow path of the switching valve, send all the components stored in the quantitative loop into the second-dimensional chromatographic column for two-dimensional chromatographic separation, or, elute the trapping column, and switch the elution product of the elution trapping column to the second-dimensional chromatographic column for separation;

[0036] The time periods are selected from: 2.36-2.60 min, 3.55-3.85 min and 4.10-4.50 min.

[0037] Methods i and ii can separate four geometric isomers of astaxanthin: all-trans-astaxanthin, 9-cis-astaxanthin, and 13-cis / 15-cis-astaxanthin. Methods iii and iv can separate a single astaxanthin geometric isomer or two astaxanthin geometric isomers. The chromatographic column and elution conditions used in method iii are the same as those in method ii. The chromatographic column and elution conditions used in method iv are the same as those in method i.

[0038] The method provided by this invention can separate and analyze a single geometric isomer of astaxanthin and its multiple optical isomers. It has the technical advantages of high specificity, good separation, and high accuracy in the analysis of each geometric isomer and optical isomer. It is of great significance for carrying out research on the precise correlation between the bioactivity of astaxanthin and its isomers.

[0039] During central cutting, the cutting time for all-trans astaxanthin is 2.36–2.60 min, for 9-cis astaxanthin it is 3.55–3.85 min, and for 13-cis / 15-cis astaxanthin it is 4.10–4.50 min. Therefore, valve switching technology can be used to transfer key components that could not be separated in one-dimensional chromatography online to a second-dimensional column for secondary separation. Central cutting allows for selective and intermittent transfer of target components, and the cutting method can be to cut one or several specific windows.

[0040] In this invention, 13-cis / 15-cis astaxanthin refers to a mixture of "13-cis-cis-astaxanthin and 15-cis-astaxanthin".

[0041] When adjusting the isocratic elution conditions for one-dimensional chromatography or the elution conditions for two-dimensional chromatography, the time required to cut each geometric isomer at the center needs to be adjusted accordingly.

[0042] Compared to the quantitative loop method used in Method ii and Method i, Method i utilizes a trapping column to collect all components from a specific time period of the one-dimensional chromatographic column, without being limited by the volume of the quantitative loop. Furthermore, the trapping column method is less expensive than the quantitative loop method. Components from the first-dimensional chromatographic column at a specific time period are temporarily trapped on the trapping column, and the target components bound to the trapping column are eluted using the same flow ratio as in the second-dimensional chromatography, then directly introduced into the second-dimensional chromatographic column for chromatographic separation.

[0043] The reason for choosing a reversed-phase chromatography column for the trapping column is that astaxanthin is a highly lipid-soluble compound. The reversed-phase trapping column can effectively adsorb the astaxanthin eluted by one-dimensional chromatography, while the initial mobile phase of two-dimensional chromatography can completely elute the astaxanthin from the trapping column. This effectively connects the one-dimensional and two-dimensional chromatography elution programs, forming an overall matching.

[0044] In a preferred embodiment of the present invention, the flow rate of the one-dimensional chromatography is 0.5-1.0 mL / min, and the column temperature is 38-42℃. For example, the flow rate of the one-dimensional chromatography is 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL / min, and the column temperature is 38, 39, 40, 41, or 42℃. Those skilled in the art can adaptively adjust the flow rate according to the specific manufacturer and model of the column, and are not limited to the point values ​​exemplified above.

[0045] In a preferred embodiment of the present invention, the mobile phase A of the one-dimensional chromatography is an aqueous solution of phosphoric acid with a volume concentration of 0.05-0.2% (phosphoric acid:water = V:V), and the volume ratio of the mixture of methyl tert-butyl ether, acetonitrile and methanol in the mobile phase B is (18-22):(18-22):(56-64).

[0046] The mobile phase A of one-dimensional chromatography is, for example, an aqueous solution of phosphoric acid with a volume concentration of 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.18%, or 0.2%.

[0047] In mobile phase B, the volume ratio of the mixture of methyl tert-butyl ether, acetonitrile and methanol is 18:18:64, 18:20:62, 18:22:60, 20:20:60, 20:22:58 or 22:22:56.

[0048] In a preferred embodiment of the present invention, the isocratic elution conditions for one-dimensional chromatography are 0-35 min, the volume of mobile phase A is maintained at 25%, and the volume of mobile phase B is maintained at 75%.

[0049] In a preferred embodiment of the present invention, the flow rate of the two-dimensional chromatography is 0.5-1.0 mL / min, and the column temperature is 38-42°C. For example, the flow rate of the two-dimensional chromatography is 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL / min, and the column temperature is 38, 39, 40, 41, or 42°C. Those skilled in the art can adaptively adjust the flow rate according to the specific manufacturer and model of the column, and are not limited to the point values ​​exemplified above.

[0050] In a preferred embodiment of the present invention, the mobile phase A of the two-dimensional chromatography is 100% methanol and the mobile phase B is 100% acetonitrile.

[0051] In a preferred embodiment of the present invention, the two-dimensional chromatographic gradient elution conditions are as follows: for 0-5 min, the volume of mobile phase A is maintained at 100% and the volume of mobile phase B is maintained at 0%; for 5.1-30 min, the volume of mobile phase A decreases linearly to 60% and the volume of mobile phase B increases linearly to 40%; for 30.1-35 min, the volume of mobile phase A is maintained at 100% and the volume of mobile phase B is maintained at 0%.

[0052] In a preferred embodiment of the present invention, the trapping column is a reversed-phase chromatographic column, and the mobile phase of the trapping column is the same as that of the two-dimensional chromatographic column; keeping the mobile phases the same avoids unnecessary analytical errors in the chromatographic results due to differences in the mobile phases.

[0053] In a preferred embodiment of the present invention, the column temperature of the trapping column is 38-42 °C. For example, the column temperature of the trapping column is 38, 39, 40, 41 or 42 °C.

[0054] In a preferred embodiment of the present invention, the first-dimensional chromatographic column is a Poroshell 120 HPH-C18 (3.0×100 mm, 2.7 μm), the flow rate is 0.8 mL / min, and the column temperature is 40 °C.

[0055] The second-dimensional chromatographic column was a ValueLab LC Chiral RP-CD (4.6×250 mm, 5 μm), with a flow rate of 0.8 mL / min and a column temperature of 40 ℃.

[0056] In a preferred embodiment of the present invention, the trapping column is a Poroshell 120 EC C18 (4.6 × 5 mm, 4 μm), and the column temperature is 40 °C.

[0057] In a preferred embodiment of the present invention, during center cutting, the cutting time for all-trans astaxanthin is 2.36-2.60 min, the cutting time for 9-cis astaxanthin is 3.55-3.85 min, and the cutting time for 13-cis / 15-cis astaxanthin is 4.10-4.50 min; detection is performed using an ultraviolet detector with a detection wavelength of 474-476 nm.

[0058] In a preferred embodiment of the present invention, the sample to be tested is prepared by dissolving the astaxanthin sample in dichloromethane or dichloromethane-acetonitrile in a volume ratio of 3:2. The concentration of astaxanthin in the sample to be tested is 0.1-1 mg / mL, and the injection volume is 0.5-1.0 μL.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] Example 1

[0061] This embodiment provides a method or analytical method for separating or analyzing astaxanthin geometric isomers and their corresponding optical isomers, which includes the following steps:

[0062] (1) Preparation of test solution (i.e., the sample to be tested): Dissolve the astaxanthin sample to be tested in dichloromethane as solvent, put it into a brown sample vial to obtain the test solution, the concentration of the test solution is 0.1 mg / mL;

[0063] (2) Preparation of mobile phase for one-dimensional chromatography: Mobile phase A is a 0.1% volume concentration of phosphoric acid aqueous solution (phosphoric acid:water = V:V), and mobile phase B is a mixed solution of methyl tert-butyl ether, acetonitrile and methanol with a volume ratio of 20:20:60;

[0064] (3) Preparation of mobile phase for two-dimensional chromatography: Mobile phase A is 100% methanol, mobile phase B is 100% acetonitrile, gradient elution; the gradient elution conditions for two-dimensional chromatography are as follows: for 0-5 min, the volume of mobile phase A is maintained at 100%, and the volume of mobile phase B is maintained at 0%; for 5.1-30 min, the volume of mobile phase A decreases linearly to 60%, and the volume of mobile phase B increases linearly to 40%; for 30.1-35 min, the volume of mobile phase A is maintained at 100%, and the volume of mobile phase B is maintained at 0%.

[0065] The two-dimensional chromatographic column was a cellulose-type chiral column, specifically a ValueLab LC Chiral RP-CD (4.6 × 250 mm, 5 μm), with a flow rate of 0.8 mL / min and a column temperature of 40 ℃.

[0066] (4) The trapping column is a reversed-phase chromatographic column, specifically a Poroshell 120 EC C18 (4.6 × 5 mm, 4 μm), and the column temperature is 40℃;

[0067] (5) Sample loading, one-dimensional chromatography and trapping column for fraction collection: 0.5 μL of the test solution was loaded onto the first-dimensional chromatographic column described above for one-dimensional chromatography. The one-dimensional chromatographic column was a Poroshell 120 HPH-C18 (3.0×100 mm, 2.7 μm), the flow rate was 0.8 mL / min, and the column temperature was 40 ℃; isocratic elution was performed; the isocratic elution conditions for one-dimensional chromatography were 0-35 min, the volume of mobile phase A was maintained at 25%, and the volume of mobile phase B was maintained at 75%;

[0068] The all-trans astaxanthin fraction was extracted by central cutting within 2.36–2.60 min and loaded into the trapping column described above. Elution was performed using the two-dimensional chromatographic mobile phase and elution conditions described above.

[0069] The elution products from the elution trap column are then switched to a second-dimensional chromatographic column for separation.

[0070] (6) Perform two-dimensional chromatography:

[0071] The two-dimensional chromatographic gradient elution conditions were as follows: for 0-5 min, the volume of mobile phase A was maintained at 100%, and the volume of mobile phase B was maintained at 0%; for 5.1-30 min, the volume of mobile phase A decreased linearly to 60%, and the volume of mobile phase B increased linearly to 40%; for 30.1-35 min, the volume of mobile phase A was maintained at 100%, and the volume of mobile phase B was maintained at 0%.

[0072] The elution product from step (5) was loaded onto a two-dimensional chromatogram for separation, and the chromatogram was recorded. The geometric isomers of astaxanthin and their corresponding optical isomers were identified by the retention time comparison method. The detection wavelength of the ultraviolet detector was 475 nm. The optical isomers of all-trans astaxanthin include levorotatory (3S, 3'S) astaxanthin, dextrorotatory (3R, 3'R) astaxanthin, and meso-(3S, 3'R) astaxanthin.

[0073] (7) After the separation is completed, take 0.5 μL of the test solution and load it onto the first-dimensional chromatographic column for one-dimensional chromatography. The type of the one-dimensional chromatographic column and its elution conditions are the same as before.

[0074] The 9-cis-astaxanthin fraction was extracted by center cutting within 3.55-3.85 min and loaded into the above-mentioned trapping column. Elution was performed using the above-mentioned two-dimensional chromatographic mobile phase and elution conditions.

[0075] The elution products from the elution trap column are switched to a second-dimensional chromatographic column for separation. The optical isomers of 9-cis-astaxanthin include 9-Z-OPT-1, 9-Z-OPT-2, 9-Z-OPT-3, and 9-Z-OPT-4.

[0076] (8) After the above separation is completed, take 0.5 μL of the test solution and load it onto the first-dimensional chromatographic column for one-dimensional chromatography. The type of the one-dimensional chromatographic column and its elution conditions are the same as before.

[0077] The 13-cis / 15-cis astaxanthin fraction was extracted by center cutting within 4.10-4.50 min and loaded into the above-mentioned trapping column. Elution was performed using the above-mentioned two-dimensional chromatographic mobile phase and elution conditions.

[0078] The elution products from the elution trap column are then switched to a second-dimensional chromatographic column for separation.

[0079] The optical isomers of 13-cis / 15-cis astaxanthin include 13 / 15-Z-OPT-1, 13 / 15-Z-OPT-2, 13 / 15-Z-OPT-3, 13 / 15-Z-OPT-4, 13 / 15-Z-OPT-5, 13 / 15-Z-OPT-6, 13 / 15-Z-OPT-7, and 13 / 15-Z-OPT-8.

[0080] The structural formulas of the geometric isomers of astaxanthin above are referenced. Figure 5 As shown.

[0081] Experimental Example 1

[0082] This experimental example uses one-dimensional chromatography to qualitatively identify the geometric isomers of astaxanthin, which facilitates the subsequent determination of the geometric isomers of astaxanthin samples.

[0083] First, the all-trans astaxanthin standard was loaded into the one-dimensional chromatographic column of Example 1, and isocratic elution was performed according to step (5) of the example to obtain the one-dimensional chromatogram of the standard. Figure 6 As shown in Figure A.

[0084] Then, the all-trans astaxanthin was isomerized (the isomerization method was as follows: 2 ml of a 0.1 mg / mL all-trans astaxanthin standard solution dissolved in acetone was taken into a 20 mL stoppered test tube, 3 mL of dichloromethane was added, mixed well, 0.5 mL of a 0.01 g / mL iodine-dichloromethane solution was added, vortexed thoroughly, sealed and placed under natural light for 15 min, then 8 mL of 0.1 mol / L sodium thiosulfate solution was added, and the mixture was shaken thoroughly to remove excess iodine. After standing and separating the phases, 3 mL of the lower phase was taken, dried under nitrogen, and then dissolved in 1 mL of dichloromethane). The isomerized sample was loaded into a one-dimensional chromatographic column of Example 1 and eluted isocratically according to step (5) of the Example. The chromatogram was referenced. Figure 6 As shown in Figure B, chromatographic peaks 1, 2, and 3 were obtained. Mass spectrometry scans of peaks 1, 2, and 3 in the one-dimensional chromatography were performed. The mass-to-charge ratio (M / Z) of the molecular ions in the three peak components was found to be 597.5. This preliminarily determined that components 2 and 3 are all-trans astaxanthin isomers (see Figure B). Figure 6 (Middle B)

[0085] Again, regarding Figure 6 In the one-dimensional chromatography of B, chromatographic peaks 1, 2, and 3 were spectrally scanned (see...). Figure 6 The maximum absorption wavelength in ultraviolet light was obtained from the C in the chromatogram. According to Yuan[1], when all-trans astaxanthin isomerizes to cis astaxanthin isomer, the maximum absorption wavelength in ultraviolet light will undergo a "blue shift". Chromatographic peak 1 is all-trans astaxanthin, and its maximum absorption wavelength in ultraviolet light is 480 nm. The maximum absorption wavelength in ultraviolet light of chromatographic peak 2 is 472 nm. Compared with all-trans astaxanthin, it underwent a "blue shift" of 8 nm and was identified as 9-cis astaxanthin isomer, which is consistent with Yuan's experimental results. Chromatographic peak 3 has two maximum absorption peaks at 374 and 470 nm and was identified as 13-cis astaxanthin isomer, which is consistent with Zhao[2]'s experimental results.

[0086] Furthermore, since the two-dimensional chromatogram of chromatographic peak 3 shows 8 peaks, which is inconsistent with the theoretical requirement that 13-cis-astaxanthin has only 4 isomers, it should be an addition of two geometric isomers of astaxanthin. Therefore, chromatographic peak 3 is determined to be a mixture of 13-cis-astaxanthin and 15-cis-astaxanthin.

[0087] 1] Yuan, JP&Chen, F. (1999). Isomerization of trans-astaxanthin inorganic solvents. Journal of Agricultural&Food Chemistry, 47(9), 3656-3660.

[0088] [2] Zhao LY, Chen F, Zhao GH*, Wang ZF, Liao XJ, Hu XS. Isomerization of trans-astaxanthin induced by copper (II) ion in ethanol. J. Agric. FoodChem., 2005, 53 (24): 9620-9623.

[0089] Experimental Example 2

[0090] This experimental example uses two-dimensional chromatography to identify the geometric isomers of all-trans astaxanthin standards, which facilitates the subsequent determination of the geometric isomers of astaxanthin samples.

[0091] The two-dimensional chromatographic column was a tris(3,5-dimethylphenylcarbamate) cellulose chiral column, the same type used in the national standard "Determination of Astaxanthin Optical Isomers by Liquid Chromatography" (GB / T 38478-2021), both being cellulose chiral columns. This standard clearly specifies the chromatographic peak order of the astaxanthin optical isomers: levorotatory (3S,3'S) astaxanthin, mesotropic (3S,3'R) astaxanthin, and dextrorotatory (3R,3'R) astaxanthin.

[0092] This experimental example follows the one-dimensional chromatography method of Example 1 to analyze the all-trans astaxanthin standard by one-dimensional chromatography. The chromatogram is shown in the figure below. Figure 7 The image above was then cut into a two-dimensional chromatogram. The two-dimensional chromatographic column used was a tris(3,5-dimethylphenylcarbamate) cellulose chiral column, and the remaining chromatographic conditions were the same as step (6) in Example 1. The two-dimensional chromatographic results are shown in the figure above. Figure 7 The figure below shows the two-dimensional chromatograms of the three optical isomers of the all-trans astaxanthin standard.

[0093] Based on this, the present invention uses this as a basis to qualitatively identify the peaks of all-trans astaxanthin cut by one-dimensional chromatography in two-dimensional chromatography, which are in turn levorotatory (3S, 3'S) astaxanthin, meso-(3S, 3'R) astaxanthin, and dextrorotatory (3R, 3'R) astaxanthin.

[0094] Experimental Example 1

[0095] Comparison of chromatograms of geometric isomers of astaxanthin with and without central cleavage.

[0096] Figure 1 The results showed that one-dimensional chromatography alone could not separate a single geometric isomer of astaxanthin without center cutting, nor could it separate the optical isomers corresponding to the geometric isomers of astaxanthin.

[0097] Figure 2 The image shows the chromatograms of one-dimensional chromatography cutting all-trans astaxanthin and the corresponding optical isomers in two-dimensional chromatography for Example 1. Figure 2 The elution order of the three substances in the two-dimensional chromatogram, from front to back, is astaxanthin in the levorotatory form (3S, 3'S), meso-(3S, 3'R), and dextrorotatory (3R, 3'R) form.

[0098] Figure 3 The images show the chromatograms of one-dimensional chromatographic cutting of 9-cis-astaxanthin and the corresponding optical isomers in two-dimensional chromatography for Example 1. The results show that the method provided by this invention can separate four optical isomers of 9-cis-astaxanthin, including 9-Z-OPT-1, 9-Z-OPT-2, 9-Z-OPT-3 and 9-Z-OPT-4.

[0099] Figure 4 The images show the one-dimensional chromatograms of 13-cis / 15-cis astaxanthin and the corresponding optical isomers in two-dimensional chromatograms for Example 1. The results show that the method provided by this invention can separate eight optical isomers of 13-cis / 15-cis astaxanthin, named 13 / 15-Z-OPT-1, 13 / 15-Z-OPT-2, 13 / 15-Z-OPT-3, 13 / 15-Z-OPT-4, 13 / 15-Z-OPT-5, 13 / 15-Z-OPT-6, 13 / 15-Z-OPT-7, and 13 / 15-Z-OPT-8, respectively. The structural formulas of all-trans astaxanthin (a), 9-cis astaxanthin (b), 13-cis astaxanthin (c), and 15-cis astaxanthin (d) are shown below. Figure 5 As shown.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers, characterized in that, This includes separation or analysis using any one of the following methods i-ii: i: A portion of the sample to be tested is loaded onto a first-dimensional chromatographic column for one-dimensional chromatographic separation. The first-dimensional chromatographic column is a Poroshell 120 HPH-C18. The mobile phase A of the first-dimensional chromatographic column is an aqueous solution of phosphoric acid, and the mobile phase B is a mixed solution of methyl tert-butyl ether, acetonitrile, and methanol, with isocratic elution. The mobile phase A of the one-dimensional chromatography is an aqueous solution of phosphoric acid with a volume concentration of 0.05-0.2%. In the mobile phase B, the volume ratio of the mixed solution of methyl tert-butyl ether, acetonitrile, and methanol is (18-22):(18-22):(56-64). Using a center-cutting method, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are loaded onto a trapping column at the first time. The elution trapping column is then used to separate the elution products. The second-dimensional chromatographic column is a ValueLab LC Chiral RP-CD. The preparation of the mobile phase for the second-dimensional chromatography is as follows: mobile phase A is 100% methanol, and mobile phase B is 100% methanol. Acetonitrile % gradient elution; the trapping column is a Poroshell 120 EC C18, and the mobile phase of the trapping column is the same as the mobile phase of the two-dimensional chromatography. After the separation is completed, another portion of the sample to be tested is loaded onto the first-dimensional chromatographic column. Using a center-cutting method, at the second time, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are loaded onto the trapping column; the elution product of the trapping column is switched to the second-dimensional chromatographic column for separation. After separation, a portion of the sample to be tested is loaded onto the first-dimensional chromatographic column. Using a center-cut method, at the third time point, the astaxanthin geometric isomers separated by the first-dimensional chromatographic column are loaded onto the trapping column. The elution product from the trapping column is then switched to the second-dimensional chromatographic column for further separation. The first, second, and third times are selected from any one of 2.36-2.60 min, 3.55-3.85 min, and 4.10-4.50 min, and the first, second, and third times are not the same. 2.36-2.60 min is the cut-off time for all-trans astaxanthin, 3.55-3.85 min is the cut-off time for 9-cis astaxanthin, and 4.10-4.50 min is the cut-off time for 13-cis / 15-cis astaxanthin. ii: Load a portion of the sample to be tested onto the first-dimensional chromatographic column for one-dimensional chromatographic separation; using a center-cutting method, load the astaxanthin geometric isomers separated by the first-dimensional chromatographic column onto the trapping column at any of the following time periods; elute the trapping column by switching the flow path of the valve, and switch the elution product of the trapping column to the second-dimensional chromatographic column for separation; The time periods were selected from: 2.36-2.60 min, 3.55-3.85 min, and 4.10-4.50 min, where 2.36-2.60 min was the cleavage time for all-trans astaxanthin, 3.55-3.85 min was the cleavage time for 9-cis astaxanthin, and 4.10-4.50 min was the cleavage time for 13-cis / 15-cis astaxanthin.

2. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 1, characterized in that, The flow rate of the one-dimensional chromatography is 0.5-1.0 mL / min, and the column temperature is 38-42℃.

3. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 2, characterized in that, The isocratic elution conditions for the one-dimensional chromatography are 0-35 min, with the volume of mobile phase A maintained at 25% and the volume of mobile phase B maintained at 75%.

4. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 1, characterized in that, The flow rate of the two-dimensional chromatography is 0.5-1.0 mL / min, and the column temperature is 38-42℃.

5. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 4, characterized in that, The two-dimensional chromatographic gradient elution conditions are as follows: for 0-5 min, the volume of mobile phase A is maintained at 100% and the volume of mobile phase B is maintained at 0%; for 5.1-30 min, the volume of mobile phase A decreases linearly to 60% and the volume of mobile phase B increases linearly to 40%; for 30.1-35 min, the volume of mobile phase A is maintained at 100% and the volume of mobile phase B is maintained at 0%.

6. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 1, characterized in that, The chromatographic column temperature of the trapping column is 38-42 ℃.

7. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 1, characterized in that, The first-dimensional chromatographic column has dimensions of 3.0 × 100 mm and 2.7 μm, a flow rate of 0.8 mL / min, and a column temperature of 40 ℃. The second-dimensional chromatographic column has dimensions of 4.6 × 250 mm, 5 μm, a flow rate of 0.8 mL / min, and a column temperature of 40℃. The trapping column has dimensions of 4.6 × 5 mm and a diameter of 4 μm, and the column temperature is 40 °C.

8. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 1, characterized in that, The detection was performed using an ultraviolet detector with a wavelength of 474-476 nm.

9. The method or analytical method for separating or analyzing the geometric isomers of astaxanthin and their corresponding optical isomers according to claim 1, characterized in that, The test sample was prepared by dissolving astaxanthin in dichloromethane or a dichloromethane-acetonitrile mixture with a volume ratio of 3:

2. The concentration of astaxanthin in the test sample was 0.1-1 mg / mL, and the injection volume was 0.5-1.0 μL.

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