Method for detecting the content of pro-resolving factors (SPMs) in microalgae oil

Abstract

The application provides a method for detecting the content of pro-resolution factors (SPMs) in microalgae oil, comprising: adding an isotopic internal standard to a prepared microalgae oil sample to obtain an internal standard sample solution; adding an alkaline reagent to the internal standard sample solution to perform a saponification hydrolysis reaction, and obtaining an alkaline saponification hydrolysis solution; adding an acidic reagent to the alkaline saponification hydrolysis solution to perform acidification treatment, adjusting the alkaline saponification hydrolysis solution to be acidic, and obtaining an acid hydrolysis solution; sequentially performing purification, concentration and redissolution on the acid hydrolysis solution to obtain a to-be-detected solution; performing UPLC-MS / MS detection on the to-be-detected solution to obtain a first response value of a target component and a second response value of the isotopic internal standard; and calculating the content of the target component in the microalgae oil sample according to the first response value, the second response value and an internal standard method standard curve. In this way, the accurate content of SPMs can be obtained, and the detection accuracy is improved.

Description

Technical Field

[0001] This application belongs to the field of analytical chemistry detection, specifically relating to a method for detecting the content of pro-inflammatory mitigating factors (SPMs) in microalgae oil. Background Technology

[0002] Specialized pro-resolving mediators (SPMs) are a class of highly bioactive lipid mediators produced by the metabolism of polyunsaturated fatty acids (such as EPA, DHA, AA, etc.). They include Resolvins (Resolution-phase interaction products), HEPEs (Hydroxyeicosapentaenoic Acids), and HETEs (Hydroxyeicosatetraenoic Acids), which play a key role in tissue repair and homeostasis restoration during the resolution phase of inflammation in the body.

[0003] Microalgal oil (especially *Micrococcus pseudocarpa* oil) is an important natural source of SPMs and their precursors. However, the accurate quantification of SPMs in microalgal oil currently faces significant technical barriers, mainly in the following aspects: (1) The microalgal oil matrix is ​​extremely complex and easily produces a strong "ion inhibition effect" (matrix effect) in LC-MS analysis, resulting in severe attenuation of the target analyte signal and making measurement difficult; (2) SPMs in microalgal oil usually exist at microgram / gram (μg / g) or even trace levels, with extremely low abundance of target analytes and highly similar structures, making it impossible to distinguish them on chromatography using conventional physicochemical analysis methods; (3) SPMs and their precursors in algal oil mostly exist in ester-bound form, and conventional extraction cannot completely release them, resulting in the measurement results not truly reflecting the total potential of SPMs in the product. Summary of the Invention

[0004] This application provides a method for detecting the content of pro-inflammatory mitigating factors (SPMs) in microalgae oil, with the aim of accurately detecting the SPMs content in microalgae oil.

[0005] Firstly, this application provides a method for detecting the content of pro-inflammatory mitigating factors (SPMs) in microalgae oil, including:

[0006] An isotopic internal standard was added to the prepared microalgae oil sample to obtain an internal standard sample solution.

[0007] An alkaline reagent is added to the internal standard sample solution to carry out a saponification and hydrolysis reaction, thereby preparing an alkaline saponification and hydrolysis solution.

[0008] An acidic reagent is added to the alkaline saponification hydrolysis solution to perform acidification treatment, thereby adjusting the alkaline saponification hydrolysis solution to acidity and preparing an acidified hydrolysis solution.

[0009] The acidified hydrolysate solution was purified, concentrated, and redissolved sequentially to prepare the test solution;

[0010] The test solution was analyzed by UPLC-MS / MS to obtain the first response value of the target component and the second response value of the isotope internal standard, respectively.

[0011] The content of the target component in the microalgae oil sample is calculated based on the first response value, the second response value, and the internal standard curve.

[0012] In conjunction with the first aspect, in one possible embodiment, the step of adding an alkaline reagent to the internal standard sample solution to perform a saponification and hydrolysis reaction to prepare an alkaline saponification and hydrolysis solution includes: adding ≥2 mL of an alkaline reagent with a concentration of ≥0.5 mol / L to the internal standard sample solution to obtain a first solution to be treated; the mass of the microalgae oil sample in the internal standard sample solution is ≥0.05 g, and the isotope internal standard is ≥100 μL with a concentration of ≥1 mg / L; subjecting the solution to be treated to a sealed vortex to obtain a second solution to be treated; and placing the second solution to be treated in a water bath at 75-95°C and shaking at 100-300 rpm for 20-60 minutes to perform saponification and hydrolysis to obtain the alkaline saponification and hydrolysis solution.

[0013] In conjunction with the first aspect, in one possible embodiment, the step of sequentially purifying, concentrating, and resolving the acidified hydrolysate to prepare the test solution includes: sequentially adding ≥5 ml of saturated saline solution and ≥5 ml of n-hexane to the acidified hydrolysate, and sequentially performing sealed vortexing, standing for layering, and removing the upper layer solution to obtain a purified solution; in the acidified hydrolysate, the acidic reagent is ≥0.2 mL of formic acid; transferring the purified solution to a nitrogen blowpipe to dry it, obtaining a concentrate; adding ≥1 ml of alcohol solution to the concentrate, performing sealed vortexing and resolving, to obtain the test solution.

[0014] In conjunction with the first aspect, in one possible embodiment, the step of performing UPLC-MS / MS detection on the test solution to obtain the first response value of the target component and the second response value of the isotope internal standard includes: determining the chromatographic and mass spectrometric conditions; injecting the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometry system, performing gradient separation of the target component under the chromatographic conditions to obtain multiple components; the chromatographic conditions are: using a C18 column, column temperature 30-40℃, mobile phase A is water, mobile phase B is acetonitrile, using a preset gradient elution program, and flow rate 0.375 mL / min-0.425 mL / min; sequentially inputting the multiple components into the mass spectrometry system, obtaining multiple first response values ​​of the multiple components and multiple second response values ​​of the corresponding multiple isotope internal standards under the mass spectrometric conditions; the mass spectrometric conditions include electrospray ionization source, negative ion mode, and multiple reaction monitoring mode scanning.

[0015] In conjunction with the first aspect, in one possible embodiment, calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve includes: determining the final volume of the test solution, the solution dilution factor, and the first mass of the microalgae oil sample; calculating the first ratio of the corresponding isotopic internal standard to the concentration of the internal standard in the internal standard curve; determining the mass concentration of the target component from the internal standard curve; and determining the content of the target component based on the final volume, the solution dilution factor, the first mass, the first ratio, and the mass concentration.

[0016] In conjunction with the first aspect, in one possible embodiment, before calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve, the method further includes: preparing a standard solution of the target component, adding ethanol solution to the standard solution to make up to a first preset volume and mixing well to prepare a mixed solution of the target component; preparing a control group solution of the target component, adding ethanol solution to the control group solution to make up to a second preset volume and mixing well to prepare a mixed solution of the control group; preparing an internal standard solution of the isotope internal standard, adding ethanol solution to the internal standard solution to make up to a second preset volume and mixing well to prepare a mixed solution of the control group; and preparing an internal standard solution of the isotope internal standard, adding ethanol solution to the internal standard solution to make up to a second preset volume. The third preset volume is mixed thoroughly to prepare an isotope internal standard mixed solution; the target component mixed solution of the fourth preset volume and multiple control group mixed solutions of the fifth preset volume are respectively placed into corresponding volumetric flasks, and the internal standard solution of the fifth preset volume is added to each volumetric flask. Then, the solution in each volumetric flask is brought to a sixth preset volume with methanol solution to obtain multiple standard working solutions; the multiple standard working solutions are analyzed by UPLC-MS / MS, and the internal standard method standard curve is plotted with the mass concentration of the standard working solution as the abscissa and the ratio of the measured value of the corresponding compound to the response value of the internal standard compound as the ordinate.

[0017] In conjunction with the first aspect, in one possible embodiment, the gradient elution procedure includes: 0-1 min, the volume percentage of the mobile phase B is 40%; 1-3 min, the mobile phase B increases from 40% to 65%; 7-8 min, the mobile phase B increases from 65% to 90%.

[0018] In conjunction with the first aspect, in one possible embodiment, the specifications of the C18 chromatographic column are as follows: The particle size is 5μm.

[0019] In conjunction with the first aspect, in one possible embodiment, the microalgae oil includes *Micrococcus pseudocarpa* oil, which is derived from the Microecology and Nutrition Research Institute of Shenzhen Baoshijian Biotechnology Co., Ltd.

[0020] In conjunction with the first aspect, in one possible embodiment, the target component includes 12-hydroxyeicosaporaenoic acid, 15-hydroxyeicosaporaenoic acid, 18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosaporaenoic acid, 5-hydroxyeicosaporaenoic acid, 8-hydroxy-5Z,9E,11Z,14Z,17Z-eicosapene, 6,8,11,14,17-eicosaporaenoic acid, 5-hydroperoxy-, [S-(E,Z,Z,Z,Z)]-, (5Z,8Z,10E,12S,14Z,17Z)-12-hydroperoxy-5,8,10,14,17-eicosapentaenoic acid, (5Z,8Z,11Z,13E,15S,17Z)-15-hydroperoxy-5,8,11,13,17-eicosapentaenoic acid, (5Z,7E,11Z,14Z,17Z)-9-hydroxy-5,7,11,14,17-eicosapentaenoic acid, (5Z,8Z,12E,14Z,17Z)-11-hydroxy-5,8,12,14,17-eicosapentaenoic acid, 20-hydroxyeicosano-5Z ,8Z,11Z,14Z,17Z-pentaenoic acid, 5-hydroxy-6E,8Z,11Z,14Z eicosatetraenoic acid, (5E,9Z,11Z,14Z)-8-hydroxy-5,9,11,14-eicosatetraenoic acid, (5Z,8Z,12E,14Z)-11-hydroxy-5,8,12,14-eicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, (RS)-15-hydroxyeicosatetraenoic acid, 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosatetraenoic acid, 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosatetraenoic acid.

[0021] Compared with existing technologies, this invention exhibits groundbreaking performance indicators:

[0022] 1) Under standard sample weight, the limits of detection (LOD) of various SPMs are as low as 0.002-0.010 μg / g, and the limits of quantitation (LOQ) are uniformly set at 0.2 μg / g, which perfectly meets the monitoring needs of trace bioactive lipids in microalgae oil;

[0023] 2) By introducing an isotope internal standard and combining it with a dedicated liquid-liquid extraction system, the spiked recoveries of various target compounds remained stable between 80.2% and 109.9% (RSD 0.7%-5.8%) in spiked experiments at multiple different concentration levels, completely solving the ion inhibition effect caused by the oil matrix;

[0024] 3) The method exhibits excellent linearity within the range of 8–1200 μg / L (correlation coefficient R² ≥ 0.9987). The repeatability coefficient of variation (RSD) is 1.3%–5.8%, and the intermediate precision RSD across personnel / time is 2.9%–5.9%, demonstrating high reliability as a standard method for routine testing.

[0025] 4) The method was tested under extreme pressure. When there were slight fluctuations in hydrolysis time (2535 min), mobile phase flow rate (0.375-0.425 mL / min), and column temperature (30℃~40℃), the RSD of the results was all <10% (between 0.2% and 5.5%), which showed strong anti-interference ability and great value for industry promotion. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a flowchart of a method for detecting the content of pro-inflammatory mitigating factors (SPMs) in microalgae oil provided in the embodiments of this application. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0029] The terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, systems, products, or apparatuses.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] The following is a brief introduction to the relevant terminology used in this application.

[0032] UPLC-MS / MS: Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry; it is an analytical technique that combines ultra-high performance liquid chromatography with tandem mass spectrometry to achieve efficient separation, accurate qualitative and trace quantitative detection of target analytes in complex samples.

[0033] At present, the precise quantification of SPMs in microalgae oil faces great technical barriers, mainly in the following aspects: (1) The microalgae oil matrix is ​​extremely complex and is prone to strong "ion inhibition effect" (matrix effect) in LC-MS analysis, which leads to severe attenuation of the target signal and makes measurement difficult; (2) SPMs in microalgae oil usually exist at microgram / gram (μg / g) or even trace levels. The abundance of the target is extremely low and the structure is highly similar. Conventional physicochemical analysis methods cannot distinguish them on chromatography; (3) SPMs and their precursors in algae oil mostly exist in ester-bound state. Conventional extraction cannot completely release them, resulting in the measurement results not being able to truly reflect the total potential of SPMs in the product.

[0034] To address the problem of accurately quantifying trace target compounds (i.e., SPMs) in microalgal oil, this application provides a method for detecting the content of pro-inflammatory reflux factors (SPMs) in microalgal oil. This method can be applied to scenarios involving the detection of SPM content in microalgal oil. The method involves adding an isotopic internal standard to a prepared microalgal oil sample to obtain an internal standard sample solution. An alkaline reagent is then added to the internal standard sample solution to perform a saponification hydrolysis reaction, resulting in an alkaline saponification hydrolysis solution. An acidic reagent is then added to the alkaline saponification hydrolysis solution to acidify it, resulting in an acidified hydrolysis solution. The acidified hydrolysis solution is then purified, concentrated, and reconstituted sequentially to obtain the test solution. The test solution is then analyzed by UPLC-MS / MS to obtain the first response value of the target component and the second response value of the isotopic internal standard. Based on the first response value, the second response value, and the internal standard curve, the content of the target component in the microalgal oil sample is calculated. This allows for the accurate determination of SPMs content in microalgae oil samples, improving detection accuracy. This method is applicable to various scenarios, including but not limited to those mentioned above.

[0035] The specific methods will be described in detail below.

[0036] Please see Figure 1 This application also provides a method for detecting the content of pro-inflammatory mitigating factors (SPMs) in microalgae oil, comprising:

[0037] Step S101: Add an isotopic internal standard to the prepared microalgae oil sample to obtain an internal standard sample solution.

[0038] In its specific implementation, the purpose of this application is to detect trace target substances in microalgae oil. Therefore, in this embodiment, a relatively small amount of microalgae oil sample is used as the detection limit to verify the lower limit of the target components that can be identified using the method of this application. Optionally, the mass of the microalgae oil sample is ≥0.05g, the isotope internal standard is ≥100μL with a concentration of ≥1mg / L, and the acidic reagent is ≥0.2mL of formic acid.

[0039] Optionally, the detection method of this application can detect 18 target components, including 12-hydroxyeicosaporaenoic acid (12-HEPE), 15-hydroxyeicosaporaenoic acid (15-HEPE), 18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosaporaenoic acid (18-HEPE), 5-hydroxyeicosaporaenoic acid (5-HEPE), 8-hydroxy-5Z,9E,11Z,14Z,17Z-eicosaporaenoic acid (8-HEPE), 6,8,11,14,17-eicosaporaenoic acid, 5-hydroperoxy-, [S-(E,Z,Z,Z,Z)]-(5-HpEPE), (5Z,8Z,10E,12S,14Z,17Z)-12-hydroperoxy-5,8,10,14,17-eicosapentaenoic acid (12-HpEPE), (5Z,8Z,11Z,13E,15S,17Z)-15-hydroperoxy-5,8,11,13,17-eicosapentaenoic acid (15-HpEPE), (5Z,7E,11Z,14Z,17Z)-9-hydroxy-5,7,11,14,17-eicosapentaenoic acid (9-HEPE), (5Z,8Z,12E,14Z,17Z)-11-hydroxy-5,8,12,14,17-eicosapentaenoic acid (11-HEPE). 20-Hydroxyeicosatetraenoic acid (20-HEPE), 5-Hydroxy-6E,8Z,11Z,14Z eicosatetraenoic acid (5-HETE), (5E,9Z,11Z,14Z)-8-Hydroxy-5,9,11,14-eicosatetraenoic acid (8-HETE), (5Z,8Z,12E,14Z)-11-Hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE), 12-Hydroxyeicosatetraenoic acid (12-HETE), (RS)-15-Hydroxyeicosatetraenoic acid (15-HETE), 5S,18R-Dihydroxy-6E,8Z,11Z,14Z,16E-eicosatetraenoic acid (Resolvin) E2 (RvE2), 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosalienoic acid (Resolvin E4, RvE4).

[0040] For example, a predetermined weight (accurate to 0.001 g) of microalgae oil is accurately weighed into an empty container to obtain a microalgae oil sample; then, according to the weight of the microalgae oil, an isotope internal standard of appropriate concentration and volume is prepared, and the isotope internal standard is added to the microalgae oil sample to obtain an internal standard sample solution.

[0041] Optionally, the microalgae oil includes *Micrococcus pseudocarpa* oil; the *Micrococcus pseudocarpa* oil is sourced from the Microecology and Nutrition Research Institute of Shenzhen Baoshijian Bioengineering Co., Ltd.

[0042] Since the isotopic internal standard and the target components (i.e., SPMs) have almost identical physicochemical properties, their chromatographic behavior, mass spectrometry response, and pretreatment recovery are also almost identical. During various subsequent treatments, the target components will be lost accordingly. However, due to the consistency between the isotopic internal standard and the target components, the loss ratio of the isotopic internal standard and the target components is the same. By tracking the various treatments of microalgae oil with the isotopic internal standard throughout the process, the matrix effect, pretreatment loss, and quantitative errors caused by instrument fluctuations can be effectively eliminated, thereby improving the accuracy and precision of the detection method.

[0043] Step S102: Add an alkaline reagent to the internal standard sample solution to carry out a saponification and hydrolysis reaction, and prepare an alkaline saponification and hydrolysis solution.

[0044] In this embodiment, the alkaline reagent can be a potassium hydroxide ethanol solution containing 0.1% antioxidant, which can be 2,6-di-tert-butyl-p-cresol (BHT), butylated hydroxyanisole (BHA), or 2,6-di-tert-butylhydroquinone (TBHQ).

[0045] In one possible embodiment, the step of adding an alkaline reagent to the internal standard sample solution to carry out a saponification and hydrolysis reaction to prepare an alkaline saponification and hydrolysis solution includes: adding ≥2 mL and ≥1 mol / L alkaline reagent to the internal standard sample solution to obtain a first solution to be treated; subjecting the solution to be treated to a sealed vortex to obtain a second solution to be treated; and placing the second solution to be treated in a 75℃-95℃ water bath and shaking at 100-300 rpm for 30-60 minutes to carry out saponification and hydrolysis to obtain the alkaline saponification and hydrolysis solution.

[0046] In the specific implementation, after obtaining the internal standard sample solution, a strong base ethanol mixture is added to the internal standard sample solution. After sealing and vortexing, the container is placed in a water bath and shaken to carry out the saponification and hydrolysis reaction. Optionally, the strong base ethanol mixture can be a potassium hydroxide ethanol mixture or other strong base ethanol mixtures, and is not limited to a single type. More than 90% of HEPEs, HETEs, and efflux compounds in microalgae oil are bound to triglycerides by ester bonds, with very few in free state, making them undetectable by direct detection. Therefore, in this embodiment, saponification and hydrolysis are used to break the ester bonds, completely releasing the target components from the lipid backbone of microalgae oil into a detectable free form. This allows the target components and isotopic internal standards to be processed simultaneously in subsequent steps. In addition, microalgae oil is a viscous, lipid-soluble matrix, resulting in low extraction efficiency and many impurities when extracted directly. Therefore, after saponification of microalgae oil, the lipids become water-soluble fatty acid salts, and the system changes from an oil phase to an aqueous phase, making subsequent acidification and layered extraction more efficient and increasing the recovery rate of the target analyte. Furthermore, hydrolysis removes most of the non-target matrix, such as neutral oils and triglycerides, reducing the matrix inhibition effect in subsequent UPLC-MS / MS and making the trace target signal more accurate and stronger.

[0047] Step S103: Add an acidic reagent to the alkaline saponification hydrolysis solution to perform acidification treatment, adjust the alkaline saponification hydrolysis solution to acidity, and prepare an acidified hydrolysis solution.

[0048] In practice, saponified SPMs become carboxylic acid metal salts, which are hydrophilic and insoluble in ethyl acetate or n-hexane, making direct extraction impossible. Therefore, in this embodiment, the saponified SPMs are acidified to convert them into free fatty acids, which are lipophilic and completely soluble in organic solvents, thus allowing for extraction. Without acidification, subsequent extraction steps are completely ineffective, and the target analyte remains undetectable in the aqueous phase.

[0049] In addition, acidification can terminate the saponification and hydrolysis reaction, neutralize the strong alkali (KOH / NaOH) in the solution, stop the hydrolysis reaction immediately, prevent the target substance from being further damaged and degraded under high temperature and strong alkali, and protect trace substances.

[0050] Meanwhile, acidification can also reduce emulsification and make the stratification clearer. Alkaline systems are prone to oil-water emulsification and unclear stratification; an acidic environment can destroy the emulsion layer, allowing the aqueous phase and organic phase to separate quickly and improving extraction efficiency.

[0051] Finally, acidification can also preliminarily purify the sample so that water-soluble alkaline impurities remain in the aqueous phase, allowing the target analyte to enter the organic phase, reducing matrix impurities and minimizing mass spectrometry interference.

[0052] Step S104: The acidified hydrolysate solution is purified, concentrated and reconstituted sequentially to prepare the test solution.

[0053] Specifically, after saponification, hydrolysis, and acidification, the solution contains numerous impurities, such as oils, pigments, proteins, salts, BHT antioxidants, and residual acid and alkali reagents. Therefore, the acidified hydrolyzed solution needs to be extracted to obtain a purer solution. The extraction process includes purification, concentration, and reconstitution.

[0054] In one possible embodiment, the step of purifying, concentrating, and resolving the acidified hydrolysate solution sequentially to prepare the test solution includes: sequentially adding ≥5 ml of saturated saline solution and ≥5 ml of n-hexane to the acidified hydrolysate solution, and sequentially performing sealed vortexing, standing for layering, and removing the upper layer solution to obtain a purified solution; transferring the purified solution to a nitrogen blowpipe to dry it, and obtaining a concentrate; adding ≥1 ml of alcohol solution to the concentrate, performing sealed vortexing and resolving, and obtaining the test solution.

[0055] In practice, the purification step uses n-hexane and saturated saline solution. Hexane, being extremely weakly polar, dissolves only non-target impurities in microalgae oil, such as neutral oils, triglycerides, fat-soluble pigments, and BHT antioxidants; it does not dissolve target components like HEPEs, HETEs, and degradants (these are slightly more polar hydroxy fatty acids that remain in the aqueous phase). Essentially, n-hexane "absorbs" the oily impurities from the sample. The saturated saline solution, through the salting-out effect, reduces the solubility of the organic phase in the aqueous phase by high-concentration salt ions, allowing for rapid phase separation. Simultaneously, it solves the problem of oil-water emulsification and unclear phase separation after acidification, allowing impurities to more thoroughly enter the n-hexane phase, resulting in a purer target analyte in the aqueous phase.

[0056] Then, a sealed vortex is used to allow the aqueous phase and n-hexane to come into full contact and mix, ensuring that all fat-soluble impurities are transferred to n-hexane for more thorough impurity removal; the sealing is to prevent the evaporation of organic solvents and loss of the target substance.

[0057] Finally, the thoroughly mixed solution was allowed to stand and separate into two phases. The density difference allowed the two phases to separate automatically. The upper layer was the n-hexane phase (yellow / turbid, containing oily impurities), and the lower layer was the aqueous phase (clear, containing the target hydroxy fatty acid / degradant). The purified solution was obtained by aspirating the upper layer after standing. It can be seen that directly discarding the impurity phase after separation removes a large amount of oily matrix interference, completing the preliminary degreasing and purification, reducing the risk of oil clogging the C18 column and contaminating the mass spectrometer. Moreover, the fewer impurities, the more accurate the MRM signal and the better the peak shape of the target analyte.

[0058] Furthermore, the purified solution is transferred to a nitrogen blowpipe to dry, which removes excess water and yields a concentrate, thus achieving enrichment and concentration of the target substance. Moreover, nitrogen gas is used to isolate the air, preventing the hydroxy fatty acids and dehydrogenase from being oxidized and destroyed. Gentle drying without heating or boiling results in almost no loss of trace substances.

[0059] Furthermore, after nitrogen blowing, the target analyte is a dry solid (i.e., a concentrate). Liquid chromatography / mass spectrometry (LC / MS) cannot directly detect solids; it must be reconstituted into a homogeneous liquid before injection analysis. Therefore, in this embodiment, a reconstitution solution is added to dissolve the concentrate, transforming it into a liquid phase. This allows for subsequent UPLC-MS / MS analysis. Optionally, the reconstitution solution can be a solvent with the same initial ratio as the mobile phase, such as an ethanol solution.

[0060] Step S105: Perform UPLC-MS / MS detection on the test solution to obtain the first response value of the target component and the second response value of the isotope internal standard.

[0061] In one possible embodiment, the step of performing UPLC-MS / MS detection on the test solution to obtain the first response value of the target component and the second response value of the isotope internal standard includes: determining the chromatographic and mass spectrometric conditions; injecting the test solution into an ultra-high performance liquid chromatography-tandem mass spectrometry system, performing gradient separation of the target component under the chromatographic conditions to obtain multiple components; the chromatographic conditions are: using a C18 column, column temperature 30-40℃, mobile phase A is water, mobile phase B is acetonitrile, using a preset gradient elution program, and flow rate 0.375 mL / min-0.425 mL / min; sequentially inputting the multiple components into the mass spectrometry system, obtaining multiple first response values ​​of the multiple components and multiple second response values ​​of the corresponding multiple isotope internal standards under the mass spectrometric conditions; the mass spectrometric conditions include electrospray ionization source, negative ion mode, and multiple reaction monitoring mode scanning.

[0062] For example, the gradient elution procedure includes: 0-1 min, the volume percentage of mobile phase B is 40%; 1-3 min, the volume percentage of mobile phase B increases from 40% to 65%; 7-8 min, the volume percentage of mobile phase B increases from 65% to 90%.

[0063] For example, the specifications of the C18 chromatographic column are as follows: The particle size is 5μm.

[0064] In practice, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) is used to detect the sample. Liquid chromatography is used to achieve efficient separation of the target compound, and tandem mass spectrometry is used to complete the accurate qualitative and quantitative analysis of trace target substances through multiple reaction monitoring mode, effectively eliminating matrix interference and ensuring the sensitivity, accuracy and repeatability of the detection results.

[0065] Specifically, UPLC uses a C18 column and gradient elution to sequentially separate the target components into peaks without interference, achieving baseline separation. The target analytes are present in extremely low concentrations in microalgal oil, undetectable by ordinary instruments. However, the electrospray ionization (ESI) and multiple reaction monitoring (MRM) modes of MS / MS offer sensitivities down to the nanogram or even picogram level, accurately capturing minute signals of the target analytes. Simultaneously, UPLC's primary mass spectrometry locks the mass of the target component's precursor ion, while secondary mass spectrometry screens for characteristic daughter ions. Through specific ion pairs of "precursor ion and daughter ion," the analyte is precisely identified as a target, not an impurity, resulting in reliable and accurate results. Furthermore, a single injection can simultaneously detect dozens of hydroxy fatty acids and desensitizing agents, and UPLC has a short run time (5-10 minutes), resulting in extremely high detection efficiency.

[0066] Step S106: Calculate the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve.

[0067] In practice, after detecting the first response value of the target component and the second response value of the isotope internal standard, the content of the target component in the microalgae oil sample can be directly calculated by combining the internal standard method standard curve.

[0068] In one possible embodiment, calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve includes: determining the fixed volume of the test solution, the solution dilution factor, and the first mass of the microalgae oil sample; calculating the first ratio of the concentration of the isotopic internal standard to the concentration of the internal standard in the internal standard curve; determining the mass concentration of the target component from the internal standard curve; and determining the content of the target component based on the fixed volume, the solution dilution factor, the first mass, the first ratio, and the mass concentration.

[0069] In practice, an internal standard curve is plotted based on the concentration of the standard series solutions and the response ratio, and the absolute content of each SPM in the sample is calculated.

[0070] For each SPM in the target component, the corresponding SPM content can be calculated using formula (1).

[0071] X=(C×10×V×DF) / (m×1000) Formula (1);

[0072] In the formula:

[0073] X is the content of a single SPM in the sample, in μg / g, and the result is rounded to one decimal place.

[0074] C is the mass concentration of the analyte in the sample solution obtained from the standard curve, expressed in micrograms per liter (μg / L).

[0075] V is the final volume of the sample solution, expressed in milliliters (mL);

[0076] DF is the dilution factor of the sample solution;

[0077] m is the mass of the sample, in grams (g);

[0078] 10 represents the ratio of the sample internal standard concentration to the curve internal standard concentration;

[0079] 1000 is the conversion factor from μg / kg to μg / g.

[0080] Once X, C, V, DF, and m are determined, the corresponding SPMs content can be calculated.

[0081] As can be seen, in this embodiment, by pre-treating the microalgae oil sample to obtain the test solution, the target components and isotope internal standard components in the test solution are detected by UPLC-MS / MS, and finally the accurate content of each SPM in the microalgae oil is calculated based on the detection results, thereby improving the detection accuracy.

[0082] In one possible embodiment, before calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve, the method further includes: preparing a standard solution of the target component, adding ethanol solution to the standard solution to make up to a first preset volume and mixing well to prepare a mixed solution of the target component; preparing a control solution of the target component, adding ethanol solution to the control solution to make up to a second preset volume and mixing well to prepare a mixed solution of the control solution; preparing an internal standard solution of the isotope internal standard, adding ethanol solution to the internal standard solution to make up to a third preset volume. The solutions were mixed thoroughly to prepare a mixed solution of isotope internal standards. The target component mixed solution of a fourth preset volume and multiple control group mixed solutions of a fifth preset volume were respectively placed into corresponding volumetric flasks. An internal standard solution of a fifth preset volume was added to each volumetric flask, and the solution in each volumetric flask was then brought to a sixth preset volume using methanol solution to obtain multiple standard working solutions. UPLC-MS / MS analysis was performed on the multiple standard working solutions. A standard curve for the internal standard method was plotted with the mass concentration of the standard working solution as the abscissa and the ratio of the measured value of the corresponding compound to the response value of the internal standard compound as the ordinate.

[0083] Before testing microalgae oil samples, it is necessary to first plot an internal standard curve, and then calculate the content of SPMs based on the internal standard curve.

[0084] The technical solutions proposed in this application will be described in more detail below with specific embodiments, but this application is not limited to these embodiments.

[0085] In its specific implementation, this embodiment includes the following steps:

[0086] 1. Prepare the following materials:

[0087] Water: Grade I water as specified in GB / T6682;

[0088] 2,6-Di-tert-butyl-4-methylphenol (C15H24O), analytical grade;

[0089] Sodium hydroxide (NaOH), analytical grade;

[0090] Sodium chloride (NaCl), analytical grade;

[0091] Methanol (CH3OH), chromatographic grade;

[0092] Anhydrous ethanol (CH3CH2OH), chromatographic grade;

[0093] n-Hexane (C6H14), chromatographic grade;

[0094] Formic acid (CH2O2), analytical grade;

[0095] 18 standards and isotope internal standards for pro-inflammatory and anti-inflammatory factors;

[0096] 2 mol / L sodium hydroxide ethanol (containing 0.1% BHT): Weigh 1.6 g of sodium hydroxide and 0.02 g of 2,6-di-tert-butyl-4-methylphenol and dissolve them in 20 mL of anhydrous ethanol. Prepare fresh and use immediately.

[0097] Ethanol (containing 0.1% BHT): Weigh 0.02 g of 2,6-di-tert-butyl-4-methylphenol and dissolve it in 20 mL of anhydrous ethanol;

[0098] 2. Prepare the following equipment:

[0099] Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) system with electrospray ionization (ESI) source;

[0100] Analytical balance: accurate to 0.01 mg and 0.1 mg;

[0101] Vortex mixer;

[0102] Thermostatic water bath shaker;

[0103] Automatic nitrogen blowing device;

[0104] Syringe filter, 0.22μm PTFE membrane.

[0105] 3. Preparation of standard solutions:

[0106] Preparation of a mixed stock solution (1 mg / L) of 18 pro-inflammatory remission factor reference standards: 100 μL of each pro-inflammatory remission factor standard solution (concentration 100 mg / L) was accurately transferred into the same 10 mL volumetric flask, dissolved in ethanol (containing 0.1% BHT) and diluted to the first preset volume, mixed well, and the target component mixed solution was prepared and stored at ≤18℃.

[0107] Preparation of a mixed stock solution of 18 pro-inflammatory remission factors (concentration of 0.1 mg / L): Accurately transfer 100 μL of the mixed stock solution of 18 pro-inflammatory remission factors (concentration of 1 mg / L) into a 10 mL volumetric flask, dissolve it with ethanol (containing 0.1% BHT) and dilute to the second preset volume, mix well to prepare the control group mixed solution, and store it at ≤18℃.

[0108] Preparation of internal standard solution (concentration of 1 mg / L): Accurately transfer 100 μL of HDHA-D5 internal standard solution (concentration of 100 mg / L) into a 10 mL volumetric flask, dissolve it with ethanol (containing 0.1% BHT) and dilute to the third preset volume, mix well to prepare the isotope internal standard mixed solution, and store it at ≤18℃.

[0109] Preparation of mixed working solutions of 18 pro-inflammatory remission factors: Accurately pipette 100 μL (i.e., the fourth preset volume) of mixed stock solution of reference standard (concentration of 0.1 mg / L) and multiple mixed stock solutions of reference standard (concentration of 1 mg / L) of the fifth preset volume (e.g., 100 μL, 200 μL, 500 μL and 1000 μL respectively) into five 1.0 mL volumetric flasks. Add 10 μL of internal standard solution (1 mg / L) to each flask, and dilute to the sixth preset volume with methanol. After mixing, prepare a series of standard working solutions with mass concentrations of 10 μg / L, 100 μg / L, 200 μg / L, 500 μg / L and 1000 μg / L (internal standard concentration of 10 μg / L). Store at 0℃-4℃.

[0110] 4. Preparation of the test solution:

[0111] Weigh 0.05 g of the sample (accurate to 0.001 g) and place it in a 30 mL glass headspace vial. Add 100 μL of internal standard (1 mg / L), 2 mL of 2 mol / L NaOH ethanol (containing 0.1% BHT), cap the vial, vortex to mix, place in a 90 °C water bath, shake at 150 rpm for 30 min, add 0.2 mL of formic acid, 5 mL of saturated saline solution, and 5 mL of n-hexane. Seal and vortex for 1 min. Allow to stand and separate into layers. Take the upper n-hexane layer into a 15 mL nitrogen blow-off tube, blow dry with nitrogen, add 1 mL of methanol, vortex for 1 min, and filter through a 0.22 μm PTFE membrane for instrument testing.

[0112] 5. Instrument conditions:

[0113] The reference conditions for ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) are shown in Table 1:

[0114] Table 1

[0115]

[0116] Column: Poroshell 120 EC-C;

[0117] Column temperature: 35 ℃;

[0118] Injection volume: 5 μL;

[0119] Mass spectrometry parameters: Electrospray ionization source (ESI), negative ion mode. Multiple reaction monitoring (MRM) mode was used for scanning.

[0120] Analysis was performed using UPLC-MS / MS. A standard curve was plotted with the mass concentration of the standard series solutions as the x-axis and the ratio of the measured value of the corresponding compound to the response value of the internal standard compound as the y-axis. Blank solutions and sample solutions were separately injected into the UPLC-MS / MS for measurement, and the concentrations of each SPM in the solution were obtained by calibration using the standard curve.

[0121] 6. Result Calculation:

[0122] The content of individual SPMs (μg / g) in the microalgae oil sample was calculated according to formula (1):

[0123] X=(C×10×V×DF) / (m×1000) Formula (1);

[0124] In the formula:

[0125] X is the content of a single SPM in the sample, in μg / g, and the result is rounded to one decimal place.

[0126] C is the mass concentration of the analyte in the sample solution obtained from the standard curve, expressed in micrograms per liter (μg / L).

[0127] V is the final volume of the sample solution, expressed in milliliters (mL);

[0128] DF is the dilution factor of the sample solution;

[0129] m is the mass of the sample, in grams (g);

[0130] 10 represents the ratio of the sample internal standard concentration to the curve internal standard concentration;

[0131] 1000 is the conversion factor from μg / kg to μg / g.

[0132] The experimental procedures and data for the above six experimental steps are described below.

[0133] 1. Durability:

[0134] The method should be robust to interference from variable experimental factors, and should be able to withstand minor changes in measurement conditions without affecting the results. This validation investigated the impact of varying pretreatment hydrolysis time, mobile phase flow rate, and column temperature on the detection results, providing a basis for the application of the established method in routine testing.

[0135] 1.1 The effect of pretreatment hydrolysis time on test results:

[0136] For the same batch of samples, the saponification and hydrolysis times can be selected as 25 min, 30 min, and 35 min, respectively. These three hydrolysis times are used as examples for illustration. The remaining steps are performed according to the test solution preparation process in step 4. The effect of sampling amount on the test results is investigated, and the results are shown in Table 2.

[0137] Table 2. Effect of hydrolysis time on test results

[0138]

[0139] The above results indicate that different hydrolysis times have little impact on the test results of the 18 SPMs, with RSDs of 0.2% to 4.2% < 10%, which meets the requirements of the General Rules for Validation of Chemical Analysis Methods in the National Food Safety Standard GB5009.295-2023.

[0140] 1.2 The impact of mobile phase flow rate on test results:

[0141] For the same sample, the mobile phase flow rate was set to 0.375 mL / min, 0.400 mL / min, and 0.425 mL / min during instrument analysis, with other conditions remaining the same as the instrument conditions in step 5. The effect of the mobile phase flow rate on the test results was investigated, and the results are shown in Table 3.

[0142] Table 3. Influence of mobile phase flow rate on test results

[0143]

[0144] The above results indicate that different mobile phase flow rates have little impact on the test results of the 18 SPMs, with RSDs of 0.4% to 4.4% < 10%, which meets the requirements of the General Rules for Validation of Chemical Analysis Methods in the National Food Safety Standard GB5009.295-2023.

[0145] 1.3 The effect of column temperature on test results during analysis:

[0146] For the same sample, the column temperature was set to 30℃, 35℃, and 40℃ during instrument analysis, with other conditions remaining the same as the instrument conditions in step 5. The effect of column temperature on the test results was investigated, and the results are shown in Table 4.

[0147] Table 4. Effect of column temperature on test results

[0148]

[0149] The above results indicate that different ultrasonic extraction times have little impact on the test results of the content of 18 SPMs, with RSDs of 0.5%~5.5% <10%, which meets the requirements of the General Rules for Validation of Chemical Analysis Methods in the National Food Safety Standard GB5009.295-2023.

[0150] 2. Linear:

[0151] Linearity refers to the degree to which the measured response value is proportional to the concentration of the analyte within the designed range.

[0152] In this validation, standard solutions with concentrations of 10 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L were prepared. A standard curve was plotted with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis. The experimental results showed that within the concentration range of 10-1000 μg / L, the response value of the target component had a good linear relationship with the concentration, which could meet the requirements of quantitative analysis.

[0153] 3. Limit of detection and limit of quantitation:

[0154] 3.1 Detection limit:

[0155] The lowest concentration or amount of an analyte required to reliably identify or distinguish its measurement signal from a specific matrix background using a particular method is called the method detection limit. GB / T27417-2017, the "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods" of the People's Republic of China, specifies that the detection limit is calculated using the following formula:

[0156] Detection limit = 3 × SD / b × x / 1000;

[0157] In the formula:

[0158] SD: Standard deviation of the response ratios of 18 SPMs analytes and internal standard;

[0159] b: The slope in the standard curve regression equation;

[0160] x: Conversion factor applied to the sample;

[0161] 1000: Conversion factor from mg / kg to μg / g;

[0162] In this verification, standard solutions with a concentration of 100 μg / L were prepared and the detection limit was tested 10 times. The results are shown in Table 5.

[0163] Table 5 shows that when the sample weight is 0.05 g, the final volume is 1.0 mL, and the injection volume is 3 μL, the method detection limit is 0.002 μg / g to 0.010 μg / g. Table 5 is as follows:

[0164] Table 5. Results of detection limit tests for 18 SPMs

[0165]

[0166] 4. Accuracy:

[0167] Accuracy refers to the degree to which the results measured by the established method are close to the true or reference values, and is generally expressed as recovery rate (%).

[0168] Right now ;

[0169] In the formula:

[0170] P—Recovery rate of the added standard substance;

[0171] C1—Test concentration of the spiked sample (μg / L);

[0172] C—theoretical spiking concentration (μg / L);

[0173] In this validation, the method blank was spiked at three concentration levels: low (100 μg / L), medium (200 μg / L), and high (500 μg / L). The spiked solutions were calculated based on the spiking amounts of 18 different SPMS. Each concentration level was performed in triplicate. Recovery rates were calculated based on theoretical spiking concentrations, and the data are shown in Table 6. The results show that the recoveries at the low, medium, and high concentration levels ranged from 80.2% to 109.9%, with RSDs ranging from 0.7% to 5.8%. The accuracy was good and met the requirements of GB5009.295-2023 National Food Safety Standard, General Rules for Validation of Chemical Analysis Methods. Table 6 is as follows:

[0174] Table 6. Accuracy experimental data of 18 SPMs

[0175]

[0176] Based on the comprehensive analysis of the above experimental results, it can be seen that the method of this application for determining the content of 18 SPMs in microalgae oil (derived from *Micrococcus pseudocarpa*) has demonstrated good accuracy, precision, linearity, and robustness through methodological validation. The established method is suitable for determining the content of 18 SPMs in microalgae oil (derived from *Micrococcus pseudocarpa*).

[0177] While this application discloses the above information, it is not limited thereto. Any person skilled in the art can easily conceive of variations or substitutions without departing from the spirit and scope of this application, and can make various alterations and modifications, including combinations of the different functions and implementation steps described above, as well as software and hardware implementation methods, all of which are within the protection scope of this application.

Claims

1. A method for detecting the content of pro-inflammatory mitigating factors (SPMs) in microalgae oil, characterized in that, include: An isotopic internal standard was added to the prepared microalgae oil sample to obtain an internal standard sample solution. An alkaline reagent is added to the internal standard sample solution to carry out a saponification and hydrolysis reaction, thereby preparing an alkaline saponification and hydrolysis solution. This includes: adding ≥2 mL of an alkaline reagent with a concentration of ≥0.5 mol / L to the internal standard sample solution to obtain a first solution to be treated; the mass of the microalgae oil sample in the internal standard sample solution is ≥0.05 g, and the isotopic internal standard is ≥100 μL with a concentration of ≥1 mg / L; the solution to be treated is then subjected to a sealed vortex to obtain a second solution to be treated; the second solution to be treated is then placed in a 75℃-95℃ water bath and shaken at 100-300 rpm for 20-60 minutes to undergo saponification and hydrolysis, thereby obtaining the alkaline saponification and hydrolysis solution. An acidic reagent is added to the alkaline saponification hydrolysis solution to perform acidification treatment, thereby adjusting the alkaline saponification hydrolysis solution to acidity and preparing an acidified hydrolysis solution. The acidified hydrolysis solution is purified, concentrated, and reconstituted sequentially to prepare the test solution, including: adding ≥5 ml of saturated saline and ≥5 ml of n-hexane sequentially to the acidified hydrolysis solution, and then sequentially performing sealed vortexing, standing for layering, and removing the upper layer solution to obtain a purified solution; the acidic reagent in the acidified hydrolysis solution is ≥0.2 mL of formic acid; the purified solution is transferred to a nitrogen blowpipe to dry, obtaining a concentrate; ≥1 ml of alcohol solution is added to the concentrate, and the solution is sealed vortexed and reconstituted to obtain the test solution; The test solution was analyzed by UPLC-MS / MS to obtain the first response value of the target component and the second response value of the isotope internal standard, respectively. The content of the target component in the microalgae oil sample is calculated based on the first response value, the second response value, and the internal standard curve.

2. The method according to claim 1, characterized in that, The step of performing UPLC-MS / MS detection on the test solution to obtain the first response value of the target component and the second response value of the isotope internal standard includes: Determine the chromatographic and mass spectrometric conditions; The test solution was injected into an ultra-high performance liquid chromatography-tandem mass spectrometry system, and the target components were separated by gradient under the chromatographic conditions to obtain multiple components. The chromatographic conditions were as follows: a C18 column was used, the column temperature was 30-40℃, mobile phase A was water, mobile phase B was acetonitrile, a preset gradient elution program was used, and the flow rate was 0.375 mL / min-0.425 mL / min. The multiple components are sequentially input into a mass spectrometry system, and multiple first response values ​​of the multiple components and multiple second response values ​​of the corresponding multiple isotope internal standards are obtained under the mass spectrometry conditions; the mass spectrometry conditions include electrospray ionization source, negative ion mode and multiple reaction monitoring mode scanning.

3. The method according to claim 1, characterized in that, The step of calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve includes: Determine the final volume of the test solution, the dilution factor of the solution, and the first mass of the microalgae oil sample; Calculate the first ratio of the concentration of the corresponding isotopic internal standard to the concentration of the internal standard in the internal standard method standard curve; The mass concentration of the target component is determined from the internal standard curve. The content of the target component is determined based on the fixed volume, solution dilution factor, first mass, first ratio, and mass concentration.

4. The method according to claim 3, characterized in that, Before calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value, and the internal standard curve, the method further includes: Prepare a standard solution of the target component, add ethanol solution to the standard solution and dilute to a first preset volume and mix well to obtain a mixed solution of the target component; Prepare a control group solution for the target component, add ethanol solution to the control group solution and make up to a second preset volume and mix well to prepare a control group mixed solution; Prepare an internal standard solution of the isotope internal standard, add ethanol solution to the internal standard solution and dilute to a third preset volume and mix well to prepare a mixed solution of isotope internal standard; The target component mixed solution of the fourth preset capacity and the control group mixed solutions of the fifth preset capacity are respectively put into the corresponding volumetric flasks, and the internal standard solution of the fifth preset capacity is added to each volumetric flask. Then, the solution in each volumetric flask is adjusted to the sixth preset capacity with methanol solution to obtain multiple standard working solutions. Multiple standard working solutions were analyzed by UPLC-MS / MS. The standard curve of the internal standard method was plotted with the mass concentration of the standard working solution as the x-axis and the ratio of the measured value of the corresponding compound to the response value of the internal standard compound as the y-axis.

5. The method according to claim 2, characterized in that, The gradient elution procedure includes: From 0 to 1 minute, the volume percentage of the mobile phase B is 40%. Within 1-3 minutes, the mobile phase B increased from 40% to 65%. Within 7-8 minutes, the mobile phase B increased from 65% to 90%.

6. The method according to claim 2, characterized in that, The specifications of the C18 chromatographic column are as follows: The particle size is 5μm.

7. The method according to any one of claims 1-6, characterized in that, The microalgae oil includes Micrococcus pseudochlorella oil.

8. The method according to any one of claims 1-6, characterized in that, The target components include 12-hydroxyeicosaporaenoic acid, 15-hydroxyeicosaporaenoic acid, 18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosaporaenoic acid, 5-hydroxyeicosaporaenoic acid, 8-hydroxy-5Z,9E,11Z,14Z,17Z-eicosapene, 6,8,11,14,17-eicosaporaenoic acid, and 5-hydroperoxy-. [S-(E,Z,Z,Z,Z)]-, (5Z,8Z,10E,12S,14Z,17Z)-12-hydroperoxy-5,8,10,14,17-eicosapentaenoic acid, (5Z,8Z,11Z,13E,15S,17Z)-15-hydroperoxy-5,8,11,13,17-eicosapentaenoic acid, (5Z,7E,11Z,14Z,17Z)-9-hydroxy-5,7,11,14,17-eicosapentaenoic acid, (5Z,8Z,12E,14Z,17Z)-11-hydroxy-5,8,12,14,17-eicosapentaenoic acid, 20-hydroxyeicosano-5Z ,8Z,11Z,14Z,17Z-pentaenoic acid, 5-hydroxy-6E,8Z,11Z,14Z eicosatetraenoic acid, (5E,9Z,11Z,14Z)-8-hydroxy-5,9,11,14-eicosatetraenoic acid, (5Z,8Z,12E,14Z)-11-hydroxy-5,8,12,14-eicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, (RS)-15-hydroxyeicosatetraenoic acid, 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosatetraenoic acid, 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosatetraenoic acid.

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