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CN-121994974-A - Method for detecting content of pro-inflammatory fading factor (SPMs) in microalgae oil

CN121994974ACN 121994974 ACN121994974 ACN 121994974ACN-121994974-A

Abstract

The application provides a method for detecting the content of pro-inflammatory fading factors (SPMs) in microalgae oil, which comprises the steps of adding an isotope internal standard substance into a prepared microalgae oil sample to obtain an internal standard substance solution, adding an alkaline reagent into the internal standard substance solution, performing saponification hydrolysis reaction to obtain an alkaline saponification hydrolysis solution, adding an acidic reagent into the alkaline saponification hydrolysis solution, performing acidification treatment, regulating the alkaline saponification hydrolysis solution to be acidic to obtain an acidified hydrolysis solution, sequentially purifying, concentrating and redissolving the acidified hydrolysis solution to obtain a solution to be detected, performing UPLC-MS/MS detection on the solution to be detected to obtain a first response value of a target component and a second response value of the isotope internal standard substance, 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 curve. Therefore, the SPMs accurate content in the sample can be obtained, and the detection accuracy is improved.

Inventors

  • ZHANG SILU
  • PANG YUCHEN
  • Dai Yangyan
  • MA SHIJING
  • XIAO GUOXUN

Assignees

  • 深圳保时健生物工程有限公司

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. A method for detecting the content of pro-inflammatory resolution factor (SPMs) in microalgae oil, comprising the steps of: adding an isotope internal standard substance into the prepared microalgae oil sample to obtain an internal standard sample solution; Adding an alkaline reagent into the internal standard sample solution, and performing saponification hydrolysis reaction to prepare an alkaline saponification hydrolysis solution; adding an acidic reagent into the alkaline saponification hydrolysis solution, performing acidification treatment, and regulating the alkaline saponification hydrolysis solution to be acidic to prepare an acidified hydrolysis solution; Purifying, concentrating and redissolving the acidified hydrolysis solution in sequence to prepare a solution to be measured; performing UPLC-MS/MS detection on the solution to be detected to respectively obtain a first response value of a target component and a second response value of the isotope 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 curve.
  2. 2. The method according to claim 1, wherein the adding an alkaline reagent to the internal standard sample solution to perform saponification hydrolysis reaction, to prepare an alkaline saponification hydrolysis solution, comprises: Adding an alkaline reagent which is more than or equal to 2mL and more than or equal to 0.5mol/L into the internal standard sample solution to obtain a first solution to be treated, wherein the mass of the microalgae oil sample in the internal standard sample solution is more than or equal to 0.05g, and the isotope internal standard substance is more than or equal to 100 mu L and the concentration is more than or equal to 1mg/L; Sealing vortex is carried out on the solution to be treated to obtain a second solution to be treated; and placing the second solution to be treated in a water bath with the temperature of 75-95 ℃ and shaking at 100-300rpm to carry out saponification hydrolysis for 20-60 minutes, so as to obtain the alkaline saponification hydrolysis solution.
  3. 3. The method according to claim 2, wherein the sequentially purifying, concentrating and redissolving the acidified hydrolyzed solution to prepare a solution to be tested comprises: Sequentially adding saturated saline water and normal hexane which are more than or equal to 5mL into the acidified hydrolysis solution, sequentially carrying out sealing vortex, standing and layering, and removing an upper layer solution to obtain a purified solution, wherein in the acidified hydrolysis solution, the acidic reagent is formic acid which is more than or equal to 0.2 mL; Transferring the purified solution to a nitrogen blowing pipe for blow drying to obtain a concentrate; and adding more than or equal to 1ml of alcohol solution into the concentrate to carry out sealing vortex and redissolution to obtain the solution to be tested.
  4. 4. The method of claim 2, wherein said performing a UPLC-MS/MS test on said test solution to obtain a first response value for a target component and a second response value for said isotopic internal standard, respectively, comprises: Determining chromatographic conditions and mass spectrometry conditions; Injecting the liquid to be detected into an ultra-high performance liquid chromatography-tandem mass spectrometry system, and completing gradient separation of the target component under the chromatographic condition to obtain a plurality of components, wherein the chromatographic condition is that a C18 chromatographic column is adopted, the column temperature is 30-40 ℃, a mobile phase A is water, a mobile phase B is acetonitrile, and a preset gradient elution program is adopted, so that the flow rate is 0.375-0.425 mL/min; And sequentially inputting the components into a mass spectrum system, and obtaining a plurality of first response values of the components and a plurality of second response values of corresponding isotope internal standards under the mass spectrum condition, wherein the mass spectrum condition comprises electrospray ionization source, negative ion mode and multi-reaction monitoring mode scanning.
  5. 5. The method of claim 1, wherein 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 curve comprises: Determining the constant volume of the solution to be measured, the dilution factor of the solution and the first mass of the microalgae oil sample; Calculating a first ratio of the corresponding isotope 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 according to the constant volume, the dilution multiple of the solution, the first mass, the first ratio and the mass concentration.
  6. 6. The method of claim 4, wherein prior to calculating the content of the target component in the microalgae oil sample based on the first response value, the second response value and an internal standard curve, the method further comprises: preparing a standard solution of the target component, adding an ethanol solution into the standard solution to a first preset volume, and uniformly mixing to obtain a target component mixed solution; preparing a control group solution of the target component, adding an ethanol solution into the control group solution to a second preset volume, and uniformly mixing to prepare a control group mixed solution; Preparing an internal standard solution of the isotope internal standard substance, adding an ethanol solution into the internal standard solution to a third preset volume, and uniformly mixing to prepare an isotope internal standard substance mixed solution; Respectively filling the target component mixed solution with fourth preset capacity and the control group mixed solution with a plurality of fifth preset capacities into corresponding volumetric flasks, adding the internal standard solution with the fifth preset capacity into each volumetric flask, and fixing the volume of the solution in each volumetric flask to the sixth preset capacity through the methanol solution to obtain a plurality of standard working solutions; And respectively carrying out UPLC-MS/MS analysis on the plurality of standard working solutions, and drawing the standard curve of the internal standard method by taking the mass concentration of the standard working solution as an abscissa and the ratio of the measured value of the corresponding compound to the response value of the internal standard compound as an ordinate.
  7. 7. The method of claim 4, wherein the gradient elution procedure comprises: 0-1min, wherein the volume percentage of the mobile phase B is 40%; 1-3min, the mobile phase B rises from 40% to 65%; the mobile phase B increased from 65% to 90% for 7-8 min.
  8. 8. The method of claim 4, wherein the C18 column is of a format of The particle size was 5. Mu.m.
  9. 9. The method of any one of claims 1-8, wherein the microalgae oil comprises a pseudo-microalgae oil.
  10. 10. The method of any one of claims 1-8, wherein the target component comprises 12-hydroxyeicosapentaenoic acid, 15-hydroxyeicosapentaenoic acid, 18-hydroxy-5Z, 8Z,11Z,14Z, 16E-eicosapentaenoic acid, 5-hydroxyeicosapentaenoic acid, 8-hydroxy-5Z, 9E,11Z,14Z, 17Z-eicosapentaenoic acid, 6,8,11,14,17-eicosapentaenoic acid, 5-hydroperoxy- [ S- (E, Z, Z, Z, Z) ] -, (5Z, 8Z,10E, 12Z, 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-hydroxy-5Z, 8Z,11Z,14Z, 17Z-pentaenoic acid, 5-hydroxy-6E, 8Z,11Z,14Z eicosatetraenoic acid, (5E, 9Z, 11Z) -8-hydroxy-5,9,11,14-eicosatetraenoic acid, (5Z, 8Z) -11-hydroxy-5,8,12,14-eicosatetraenoic acid, 12-hydroxy-eicosatetraenoic acid, (5Z, 8Z, 12Z) -11-hydroxy-5,8,12,14,17-eicosapentaenoic acid, 20-hydroxy-eicosatetraenoic acid, 15, 178Z, 14Z, 17Z-pentaenoic acid, 5Z,8Z, 14Z, 178Z-eicosatetraenoic acid, 5-hydroxy-6Z, 178Z, 16Z, R-tetracosanoic acid.

Description

Method for detecting content of pro-inflammatory fading factor (SPMs) in microalgae oil Technical Field The application belongs to the field of analytical chemistry detection, and particularly relates to a method for detecting the content of pro-inflammatory fading factors (SPMs) in microalgae oil. Background Specific pro-inflammatory regressions (Specialized Pro-resolving Mediators, SPMs) are a class of highly bioactive lipid mediators produced by the metabolism of polyunsaturated fatty acids (e.g., EPA, DHA, AA, etc.), including Resolvins (Resolution-phase interaction products, resolvins), HEPEs (Hydroxyeicosapentaenoic Acids, hydroxyeicosapentaenoic acid), HETEs (Hydroxyeicosatetraenoic Acids, hydroxyeicosatetraenoic acid), etc., which exert critical tissue repair and steady state restoration effects during the regressive phase of body inflammation. Among these, microalgae oils (particularly pseudo-microalgae oils) are an important natural source of SPMs and its precursors. However, the accurate quantification of SPMs in microalgae oils is currently faced with a great technical barrier, and is mainly characterized in that (1) microalgae oils are extremely complex, a strong ion inhibition effect (matrix effect) is easy to generate in LC-MS analysis, so that a target signal is seriously attenuated, measurement is difficult, SPMs is usually present in the microalgae oils at microgram/gram (mug/g) or even trace level, the target abundance is extremely low and the structures are highly similar, a conventional physicochemical analysis method cannot distinguish the target from the microalgae oil on the chromatogram at all, SPMs and precursors thereof in the microalgae oil are usually present in an ester bonding state, and conventional extraction cannot completely free the target signal, so that the measurement result cannot truly reflect the total potential of a product SPMs. Disclosure of Invention The application provides a method for detecting the content of pro-inflammatory fading factors (SPMs) in microalgae oil, so as to accurately detect the SPMs content in the microalgae oil. In a first aspect, the application provides a method for detecting the content of pro-inflammatory resolution factor (SPMs) in microalgae oil, comprising: adding an isotope internal standard substance into the prepared microalgae oil sample to obtain an internal standard sample solution; Adding an alkaline reagent into the internal standard sample solution, and performing saponification hydrolysis reaction to prepare an alkaline saponification hydrolysis solution; adding an acidic reagent into the alkaline saponification hydrolysis solution, performing acidification treatment, and regulating the alkaline saponification hydrolysis solution to be acidic to prepare an acidified hydrolysis solution; Purifying, concentrating and redissolving the acidified hydrolysis solution in sequence to prepare a solution to be measured; performing UPLC-MS/MS detection on the solution to be detected to respectively obtain a first response value of a target component and a second response value of the isotope 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 curve. In combination with the first aspect, in one possible embodiment, the alkaline reagent is added into the internal standard sample solution to perform saponification hydrolysis reaction to prepare an alkaline saponification hydrolysis solution, wherein the alkaline hydrolysis solution is prepared by adding the alkaline reagent which is more than or equal to 2mL and more than or equal to 0.5mol/L into the internal standard sample solution to obtain a first to-be-treated solution, the mass of the microalgae oil sample in the internal standard sample solution is more than or equal to 0.05g, the isotopic internal standard is more than or equal to 100 mu L and the concentration is more than or equal to 1mg/L, the to-be-treated solution is subjected to sealing vortex to obtain a second to-be-treated solution, and the second to-be-treated solution is placed into a water bath at 75-95 ℃ to perform saponification hydrolysis for 20-60 minutes by shaking at 100-300rpm to obtain the alkaline saponification hydrolysis solution. In combination with the first aspect, in one possible embodiment, the steps of sequentially purifying, concentrating and redissolving the acidified hydrolysis solution to prepare a solution to be tested comprise sequentially adding more than or equal to 5mL of saturated saline and more than or equal to 5mL of normal hexane into the acidified hydrolysis solution, sequentially carrying out sealed vortex and standing delamination, removing an upper layer solution to obtain a purified solution, wherein the acidic reagent is more than or equal to 0.2mL of formic acid in the acidified hydrolysis solution, moving the purified solution to a nitrogen b