A method for selective enrichment of unsaturated fatty acids and positioning of their C=C bonds

Abstract

The present invention relates to a method for selective enrichment of unsaturated fatty acids and positioning of their C=C bonds, belonging to the technical fields of bioanalytical chemistry and lipidomics. The present invention first prepares a silica gel material with a boronic acid functional group, then converts the C=C bond in the unsaturated fatty acid molecule into a vicinal diol structure through a cis-dihydroxylation reaction, and then utilizes the specific interaction between boric acid and the vicinal diol to achieve efficient and selective enrichment of the derivatized unsaturated fatty acids. The enriched sample is analyzed by high-resolution mass spectrometry and further fragmented to produce secondary characteristic diagnostic ion pairs, thereby achieving precise positioning of the C=C bond in the unsaturated fatty acid molecule. The present invention has the advantages of simple operation, strong selectivity, high enrichment efficiency and a wide range of applications. It can effectively solve the problems of traditional technologies in terms of difficulty in distinguishing isomers and matrix interference, and provides a new technology for the fine structure analysis of unsaturated fatty acids and lipid metabolism research in complex biological samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalytical chemistry and lipidomics, and specifically relates to a method for selectively enriching unsaturated fatty acids through chemical derivatization and solid-phase extraction technology and locating the C=C bond positions thereof using high-resolution mass spectrometry. Background Art

[0002] Unsaturated fatty acids, as a class of lipid molecules with important biological functions, play a key role in processes such as cell signaling, inflammation regulation, energy metabolism, and disease development. In recent years, with the deepening of lipidomics research, more and more researchers have begun to pay attention to the influence of double bond position and geometric configuration in unsaturated fatty acids on their biological functions. However, existing analytical techniques have two major limitations:

[0003] Difficulty in distinguishing structural isomers: Traditional mass spectrometry technology often relies on retention time and accurate mass matching for qualitative analysis of unsaturated fatty acids. However, since isomers have the same molecular weight, their primary mass spectra are difficult to distinguish. At the same time, conventional tandem mass spectrometry experiments are also difficult to produce characteristic fragments that are sufficient to distinguish the double bond position, making it difficult to accurately identify isomers at different C=C bond positions.

[0004] Sample matrix interference and low abundance issues: The concentration of unsaturated fatty acids in biological samples is usually low, and they are accompanied by a large number of high background interferents such as saturated fatty acids, phospholipids and glycerides. As a result, traditional analytical methods are seriously affected in terms of signal-to-noise ratio and sensitivity, and cannot guarantee high selectivity and high recovery rate.

[0005] To address the above problems, developing a method that is efficient, selective, and able to accurately locate C=C bonds can not only improve the accuracy of unsaturated fatty acid structure analysis, but also provide new technical means for lipidomics research, which has important scientific significance and application prospects. Summary of the Invention

[0006] The present invention aims to address the limitations of existing technologies in unsaturated fatty acid analysis by providing a method that simultaneously achieves selective enrichment of unsaturated fatty acids and localization of C=C bonds. First, a boronic acid-functionalized material is prepared. Second, the C=C bonds of unsaturated fatty acids are converted to vicinal diol structures through a cis-dihydroxylation reaction. Then, the derivatized unsaturated fatty acids are selectively enriched from complex samples through covalent binding of the boronic acid and vicinal diol. Finally, the double bond sites are precisely located using characteristic diagnostic ion pairs generated by high-resolution mass spectrometry. This series of steps enables the selective enrichment and fine structural characterization of unsaturated fatty acids. By optimizing the derivatization reagent ratio and enrichment conditions, this method is adaptable to unsaturated fatty acids of varying chain lengths (C14-C24), degrees of unsaturation (1-6 double bonds), and cis / trans configurations. By converting the C=C bonds of unsaturated fatty acids into recognizable chemical structures and enriching them using specific materials, precise double bond localization is achieved in conjunction with high-resolution mass spectrometry.

[0007] To achieve the above object, the present invention specifically includes the following steps:

[0008] Step 1: Prepare a boronic acid functionalized material: react a boronic acid functionalized reagent with an amino functionalized material in an organic solvent at room temperature to prepare a boronic acid functionalized material SG-B; the boronic acid functionalized reagent is a compound that can react with the amino material to introduce boronic acid groups, and the amino functionalized material is a silica gel material containing amino functional groups on its surface;

[0009] Preferably, (4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)boronic acid (PBA-NHS) is reacted with amino silica gel (SG-A) in acetonitrile at room temperature to prepare SG-B material for subsequent enrichment. The reaction formula is:

[0010] .

[0011] Step 2, cis-dihydroxylation reaction: Using a catalytic system consisting of an osmium-based catalyst and an N-methylmorpholine-based oxidant, the C=C bond of the unsaturated fatty acid is converted into a cis-vicinal diol structure, achieving complete derivatization of the monounsaturated fatty acid; or selective derivatization of specific double bonds of polyunsaturated fatty acids by adjusting the amount of oxidant used;

[0012] Furthermore, the cis-dihydroxylation reaction in step 2 is carried out in a mixed solvent of tert-butanol, water and acetone.

[0013] Preferably, a catalytic system of potassium osmate dihydrate and N-methylmorpholine-N-oxide (NMO) is used to convert the C=C bond of the unsaturated fatty acid into a vicinal diol structure (Diol structure) to achieve complete derivatization of the monounsaturated fatty acid, or to derivatize a single double bond of the polyunsaturated fatty acid by controlling the amount of the oxidant NMO.

[0014] Step 3: Selective enrichment: The SG-B material prepared in step 1 is incubated with the unsaturated fatty acid after cis-dihydroxylation reaction in step 2 in a weak alkaline solution, and the target compound is enriched by covalent binding of boric acid and vicinal diol, and then eluted with a weak acidic solution;

[0015] Preferably, the selective enrichment in step 3 is performed by adsorption and washing in weakly alkaline ethyl acetate (EA), and elution is performed using weakly acidic methanol (FAM) solution, and the elution is repeated three times.

[0016] Furthermore, the method described in steps 1 to 3 is applicable to the selective enrichment of monounsaturated fatty acids and polyunsaturated fatty acids.

[0017] Step 4: High-resolution mass spectrometry analysis: The unsaturated fatty acids eluted in step 3 are analyzed using an ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometer. The fragmentation generates secondary mass spectrometry characteristic diagnostic ion pairs to accurately locate the position of the C=C bond of the unsaturated fatty acids.

[0018] Furthermore, the high-resolution mass spectrometry analysis described in step 4 adopts ESI-mode, first scanning the cis-diolated unsaturated fatty acid parent ion, and then fragmenting it to produce secondary mass spectrometry ion fragments, find the diagnostic ion pair, and locate the C=C bond of the unsaturated fatty acid.

[0019] The method can also be applied to lipidomics research, biological sample analysis, phospholipid detection and clinical sample analysis to analyze the structural isomers of unsaturated fatty acids and their biological functions.

[0020] This invention applies an affinity material with a boronic acid group to the enrichment of unsaturated fatty acids. By leveraging the reversible binding between the boron affinity material and cis-dihydroxy fatty acids, it achieves efficient and selective enrichment of unsaturated fatty acids. Compared with traditional separation methods, the boron affinity material exhibits higher selectivity and affinity, significantly improving the recovery efficiency of unsaturated fatty acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC chromatograms showing the detection of PBA-NHS in the supernatant before and after the reaction during the preparation of the SG-B material in an embodiment of the present invention;

[0022] Figure 2This is a liquid chromatography-mass spectrometry image of the detection of 4-carboxyphenylboronic acid in the supernatant after the modified groups on the SG-B material in the embodiment of the present invention are hydrolyzed;

[0023] Figure 3 IR spectra of PBA-NHS, SG-A material, SG-B material, and the material combining SG-B and Diol-FA 18:1 in the embodiments of the present invention;

[0024] Figure 4 X-ray photoelectron spectra of PBA-NHS, SG-A material, SG-B material, and the material combining SG-B and Diol-FA 18:1 in the embodiments of the present invention;

[0025] Figure 5 The mass spectra of FA 18:1(9Z) before and after the cis-dihydroxylation reaction in the examples of the present invention are shown;

[0026] Figure 6 The mass spectra of FA 18:2 (9Z, 12Z) after cis-dihydroxylation reaction when the NMO catalyst dosage is 0.3 eq., 0.5 eq., and 0.7 eq. in the embodiment of the present invention are shown;

[0027] Figure 7 The mass spectra of FA 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) after cis-dihydroxylation reaction with 0.1 eq., 0.5 eq., and 1 eq. of NMO catalyst in the examples of the present invention are shown;

[0028] Figure 8 is the recovery rate of the FA 19:0 and Diol-FA 18:1 mixture extracted by the SG-B material in the embodiment of the present invention;

[0029] Figure 9 is the recovery rate of the eight fatty acid mixtures extracted by SG-B material in the embodiment of the present invention;

[0030] Figure 10 The MS and structural analysis diagram of FA 18:1 after cis-dihydroxylation in the embodiment of the present invention;

[0031] Figure 11 The MS and structural analysis diagram of FA 18:2 after cis-dihydroxylation of a single double bond in the embodiment of the present invention are shown;

[0032] Figure 12 This is the secondary mass spectrum and structural analysis diagram of FA 22:6 after single double bond cis-dihydroxylation in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to fully understand the present invention, the method of the present invention is described in detail below in conjunction with embodiment, including the specific reaction conditions, operating method and detection characterization process of each step. Obviously, the described embodiment is only an embodiment of a part of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise should fall within the scope of protection of the present invention.

[0034] A method for selectively enriching unsaturated fatty acids and locating their C=C bonds comprises the following steps:

[0035] Step 1: Prepare SG-B material

[0036] During the preparation of SG-B, SG-A (20 mg, 5 μm particle size, 100 Å pore size) was first rinsed twice with acetonitrile to remove surface impurities. Next, SG-A was suspended in a solution containing PBA-NHS (5 mg dissolved in 1 mL of acetonitrile). In this example, a 1:4 mass ratio of PBA-NHS to SG-A was used (a 1:3 to 1:5 ratio is recommended for practical applications). The reaction was allowed to proceed overnight (10-14 h) at room temperature using a tumble mixer to ensure sufficient grafting of PBA-NHS onto the SG-A surface. After completion of the reaction, the supernatant was discarded by centrifugation (8000 rpm, 5 min), and the material was washed twice with acetonitrile to remove unreacted PBA-NHS. The resulting SG-B material was stored in acetonitrile at 4°C to maintain its activity.

[0037] The reaction formula is:

[0038] .

[0039] The following experiments were performed to verify the SG-B material structure:

[0040] The raw material PBA-NHS used in the preparation of SG-B material in this embodiment has ultraviolet absorption. Figure 1 Figure a is the HPLC chromatogram of the supernatant before the reaction of preparing SG-B material, and figure b is the HPLC chromatogram of the supernatant after the reaction of preparing SG-B material. It can be seen that the peak area of ​​PBA-NHS is significantly reduced, indicating that the PBA-NHS substrate is consumed in large quantities.

[0041] Figure 2In the SG-B material, strong alkaline dissociation cleaves the amide bond. Following acidification, the hydrolysis product, 4-carboxyphenylboronic acid, is released. Its molecular formula is C7H7BO4 and its molecular weight is 165.94. The peak eluting around 11 minutes on the chromatogram corresponds to the UV absorption of 4-carboxyphenylboronic acid. In the negative ion mode (ESI-), its quasi-molecular ion peak [MH]⁻ is detected, indirectly indicating that PBA-NHS is grafted onto the SG-A surface. Figure 1 and Figure 2 The results preliminarily proved the successful preparation of SG-B material.

[0042] The wavelength range used is 400-4000 cm -1 PBA-NHS, SG-A material, SG-B material and the material in which SG-B is combined with Diol-FA 18:1 were scanned and analyzed using infrared light.

[0043] like Figure 3 As shown in a, in the infrared spectrum of PBA-NHS, 3412.9 cm -1 The stretching vibration of BO-H is at 1336.4 cm -1 The stretching vibration peak of BO is at

[0044] like Figure 3 As shown in b, in the infrared spectrum of SG-A material, at 1108.4 cm -1 、811.9 cm -1 The peaks correspond to the asymmetric stretching vibration and symmetric stretching vibration of Si-O-Si, 3434.6 cm -1 The NH stretching vibration peak corresponding to the amino group is 2958.8 cm -1 The stretching vibration peak of alkane CH is 1627.6 cm -1 The out-of-plane bending vibration peak of NH is at

[0045] like Figure 3 As shown in Figure c, in the infrared spectrum of SG-B material, at 1496.5 cm -1 and 1543.8 cm -1 The peak at 1651.7 cm is attributed to the stretching vibration of the aromatic ring skeleton C=C. -1 Assigned to the stretching vibration of amide C=O, 3422.1 cm -1 Assigned to the stretching vibration of BO-H, these peaks confirmed the successful preparation of SG-B;

[0046] like Figure 3 As shown in Figure d, in the infrared spectrum of the SG-B and Diol-FA 18:1 combination material, 3422.1 cm -1The BO-H characteristic peak at 18:1 completely disappeared, which was attributed to the combination of the boronic acid group with the cis-diol structure of Diol-FA 18:1, resulting in the consumption of the BO-H group.

[0047] The functional group information revealed by Fourier transform infrared spectroscopy is highly consistent with the synthetic route designed in this experiment, which strongly proves the successful preparation of SG-B material. -1 The strong Si-O-Si asymmetric stretching vibration absorption peak at the SG-B material is likely masked by the high signal intensity, which may have masked the adjacent BO bond characteristic peak. Therefore, XPS was used to further analyze the elemental chemical state of the SG-B material surface to further study its surface chemical composition.

[0048] like Figure 4 As shown in the figure, although element B is almost undetectable in the full XPS spectra of the SG-B material and its combination with Diol-FA 18:1, its presence is still observed in the pie chart content analysis, with contents of 2.23% and 1.47%, respectively. This phenomenon is attributed to the relatively low sensitivity of XPS technology for light elements (such as B, atomic number 5).

[0049] In summary, the results of HPLC, LC-MS, IR and X-ray photoelectron spectroscopy are consistent, which fully proves the successful preparation of SG-B material.

[0050] Step 2: Cis-dihydroxylation of unsaturated fatty acids

[0051] In the cis-dihydroxylation reaction of monounsaturated fatty acids, FA 18:1(9Z) was used as the substrate in this example. The reaction system contained NMO, potassium osmate dihydrate, water, and acetone. After stirring at room temperature for 5-10 minutes, the product was purified and recovered through the following post-treatment steps: 400 μL of EA was added and vortexed to mix. The mixture was then centrifuged at 12,000 rpm for 3 minutes. The supernatant was removed and the solvent was removed using a vacuum centrifugal concentrator. An additional 400 μL of EA was then added, and the vortexing and centrifugation steps were repeated. The organic layer was collected and transferred to a 1.5 mL EP tube. The solvent was again concentrated to remove the solvent, and the sample was dissolved in methanol (MeOH) for subsequent analysis.

[0052] The reaction formula is:

[0053] ;

[0054] The following experiment was performed to verify the cis-dihydroxylation reaction of FA 18:1(9Z):

[0055] Figure 5The mass spectra of FA 18:1(9Z) before (a) and after (b) the cis-dihydroxylation reaction. Under the ESI-ionization source, FA 18:1(9Z) (m / z 281.24606) successfully generated the derivatization product Diol-FA 18:1 (m / z 315.25281) after the cis-dihydroxylation reaction, and the reaction yield was almost 100%.

[0056] In the cis-dihydroxylation reaction of polyunsaturated fatty acids, in this example, FA 18:2 (9Z, 12Z) and FA 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) were used as substrates. The reaction system contained NMO, potassium osmate dihydrate, water, and acetone. The single double bond of the polyunsaturated fatty acid was cis-dihydroxylated by controlling the amount of the oxidant NMO. The reaction was stirred at room temperature for 20-60 minutes. The post-treatment steps were the same as those for FA 18:1 (9Z).

[0057] The reaction formula is:

[0058] ;

[0059] ;

[0060] The following experiment was performed to verify the cis-dihydroxylation reaction of FA 18:2(9Z,12Z):

[0061] Figure 6 The following are the mass spectra of the cis-dihydroxylation reaction of FA 18:2(9Z,12Z) with NMO oxidant at 0.3 eq., 0.5 eq., and 0.7 eq. Under the ESI-ionization source, when NMO was 0.5 eq., the yield of the cis-dihydroxylation product Mono-Diol-FA 18:2 (m / z 313.2) with a single double bond was the highest. This optimized condition was selected for subsequent high-resolution mass spectrometry structure elucidation.

[0062] The following experiment was performed to verify the cis-dihydroxylation reaction of FA 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z): Figure 7 The following are the mass spectra of FA 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) after the cis-dihydroxylation reaction with NMO oxidant at 0.1 eq., 0.5 eq., and 1 eq. When NMO was 0.5 eq., the cis-dihydroxylation product Mono-Diol-FA 22:6 (m / z 361.2) with a single double bond accounted for the highest proportion. This optimized condition was selected for subsequent high-resolution mass spectrometry structure analysis.

[0063] Step 3: Selective Enrichment

[0064] In this embodiment, the following two solutions are used as examples for description.

[0065] Scheme 1: Selective enrichment of a mixture of Diol-FA 18:1 and FA 19:0 (two fatty acids or derivatized fatty acids), as follows:

[0066] First, 5 mg of SG-B material was rinsed twice with EA to activate the surface. The SG-B was then mixed with 1 mL of EA (pH 9) containing 60 μg / mL of a mixture of Diol-FA 18:1 and FA 19:0 and incubated in an ultrasonic bath for 10 minutes. Subsequently, the sample was washed twice with the loading reagent, and the target compound was eluted with 1 mL of 40 mM FAM solution. This elution was repeated three times, with each elution lasting 5 minutes. Finally, the solution was centrifuged (12,000 rpm, 3 minutes), and the supernatant was removed, evaporated to dryness, and reconstituted in MeOH for mass spectrometry analysis.

[0067] like Figure 8 As shown in the figure, by calculating the recovery rates of the SG-B material for the two fatty acids, it was found that the recovery rate of the SG-B material for FA 19:0 was lower, at 9.62%; the recovery rate for Diol-FA 18:1 was significantly higher, reaching 83.45%.

[0068] Scheme 2: Selective enrichment of a mixture of FA 11:0, FA 13:0, FA 15:0, FA 17:0, Diol-FA 18:1, Diol-FA 22:1, Mono-Diol-FA 18:2, and Mono-Diol-FA 18:3 (eight fatty acids or derivatized fatty acids), as follows:

[0069] First, 5 mg of SG-B material was rinsed twice with EA to activate the surface. The SG-B material was then mixed with 1 mL of EA (pH 9) containing a 60 μg / mL mixture of FA 11:0, FA 13:0, FA 15:0, FA 17:0, Diol-FA 18:1, Diol-FA 22:1, Mono-Diol-FA 18:2, and Mono-Diol-FA 18:3. The mixture was incubated in an ultrasonic bath for 10 minutes. Subsequently, the sample was washed twice with loading reagent, and the target compound was eluted with 1 mL of 40 mM FAM solution three times, with each elution lasting 5 minutes. Finally, the solution was centrifuged (12,000 rpm, 3 minutes), and the supernatant was removed, evaporated to dryness, and reconstituted in MeOH for mass spectrometry analysis.

[0070] like Figure 9As shown in the figure, by calculating the recoveries of eight fatty acids of SG-B material, it was found that SG-B material had higher recoveries of Diol-FA18:1, Diol-FA 22:1, Mono-Diol-FA 18:2, and Mono-Diol-FA 18:3, which were 69.73%, 71.46%, 66.36%, and 84.77%, respectively, while the recoveries of FA 11:0, FA 13:0, FA 15:0, and FA 17:0 were low, which were 5.88%, 4.44%, 3.43%, and 2.51%, respectively.

[0071] Results from selective enrichment experiments demonstrate that the SG-B material exhibits significant adsorption for both derivatized monounsaturated and polyunsaturated fatty acids, demonstrating excellent selectivity for the target compounds. Furthermore, in complex fatty acid sample systems, the SG-B material is able to specifically identify and enrich derivatized unsaturated fatty acids while avoiding competitive interference from saturated fatty acids.

[0072] Step 4: High-resolution mass spectrometry analysis

[0073] In this example, high-resolution mass spectrometry analysis was performed using a UHPLC-LTQ Orbitrap Elite instrument under the following conditions: ESI- detection mode, CID energy set to 35 eV, resolution set to 30,000, and scan range m / z 85-380. Chromatographic conditions were: mobile phase A consisting of water and mobile phase B consisting of acetonitrile, in isocratic elution mode, with an acetonitrile ratio maintained at 50% between 0 and 1 minute, and a flow rate of 0.5 mL / min.

[0074] like Figure 10 As shown, two pairs of prominent diagnostic ions were observed in the high-resolution mass spectrum of cis-dihydroxylated FA 18:1(9Z): one pair originating from the alkyl terminus, at m / z 141.12781 and m / z 171.13818; the other pair originating from the carboxyl terminus, at m / z 171.10181 and m / z 201.11211. Based on the accurate masses and relative abundances of these ion pairs, the position of the C=C bond in this compound was determined to be Δ9.

[0075] like Figure 11As shown, two pairs of prominent diagnostic ions were observed in the high-resolution mass spectrum of FA 18:2 (9Z,12Z), a single double bond cis-dihydroxylated compound: one pair of diagnostic ions from the carboxyl terminus of Δ9, at m / z 171.10181 and m / z 201.11230, respectively; and another pair of diagnostic ions from the alkyl terminus of Δ12, at m / z 99.08125 and m / z 129.09152, respectively. The accurate masses and relative abundances of these ion pairs allow the location of the C=C bond in this compound to be determined.

[0076] like Figure 12 As shown in Figure 3, eight pairs of significant diagnostic ions were observed in the high-resolution mass spectrum of FA 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) after single double bond cis-dihydroxylation: a pair of alkyl terminal diagnostic ions from Δ4, m / z 257.18979 and m / z 287.19940, respectively; a pair of alkyl terminal diagnostic ions from Δ7, m / z 217.15871 and m / z 247.16875, respectively; a pair of carboxyl terminal diagnostic ions from Δ7, m / z 141.05507 and m / z 171.06519, respectively; a pair of alkyl terminal diagnostic ions from Δ10, m / z 177.12732 and m / z 207.13773, respectively; a pair of carboxyl terminal diagnostic ions from Δ10, m / z 181.08583 and m / z The following ions are from the alkyl group of Δ13: m / z 211.09633; another pair of diagnostic ions from the alkyl group of Δ13: m / z 137.09633 and m / z 167.10675; another pair of diagnostic ions from the carboxyl group of Δ16: m / z 261.14792 and m / z 291.16018; and finally, a pair of diagnostic ions from the carboxyl group of Δ19: m / z 301.17902 and m / z 331.19049. Based on the accurate masses and relative abundances of these ion pairs, the position of the C=C bond in this compound can be determined. Similarly, for other unsaturated fatty acids, the corresponding diagnostic ion pairs can also accurately locate the double bond position.

[0077] Through chemical derivatization, boric acid affinity enrichment and high-resolution mass spectrometry analysis, the present invention achieves a significantly higher recovery rate of derivatized unsaturated fatty acids than saturated fatty acids in complex biological samples, achieving efficient selective enrichment of unsaturated fatty acids and precise positioning of C=C bonds, and can be expanded to phospholipid analysis and clinical sample detection.

Claims

1. A method for selectively enriching unsaturated fatty acids and locating their C=C bonds, characterized in that: The following steps are involved: Step 1: Preparation of a boronic acid functionalized material: (4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)boronic acid PBA-NHS is reacted with amino silica gel material SG-A in an organic solvent at room temperature to prepare a boronic acid functionalized material SG-B; Step 2, cis-dihydroxylation reaction: Using a catalytic system consisting of NMO and potassium osmate dihydrate, the C=C bond of the unsaturated fatty acid is converted into a cis-vicinal diol structure, achieving complete derivatization of the monounsaturated fatty acid; or selective derivatization of specific double bonds of polyunsaturated fatty acids by adjusting the amount of oxidant; Step 3: Selective enrichment: The SG-B material prepared in step 1 is incubated with the unsaturated fatty acid after cis-dihydroxylation reaction in step 2 in a weak alkaline solution, and the target compound is enriched by covalent binding of boric acid and vicinal diol, and then eluted with a weak acidic solution; Step 4. High-resolution mass spectrometry analysis: The unsaturated fatty acids eluted from step 3 were analyzed using a UHPLC-LTQ Orbitrap Elite instrument under the following analysis conditions: ESI- detection mode; chromatographic conditions: mobile phase A: water, mobile phase B: acetonitrile, isocratic elution mode; and fragmentation to generate secondary mass spectrometry characteristic diagnostic ion pairs to precisely locate the positions of the C=C bonds of the unsaturated fatty acids.

2. The method according to claim 1, wherein: In step 1, (4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)boric acid is reacted with amino silica gel material in acetonitrile at room temperature to prepare SG-B material. The reaction formula is: .

3. The method according to claim 1, wherein: The cis-dihydroxylation reaction in step 2 is carried out in a mixed solvent of tert-butanol, water and acetone.

4. The method according to claim 1, wherein: The selective enrichment in step 3 is adsorbed and washed in weakly alkaline ethyl acetate, and eluted with a weakly acidic methanol solution.

5. The method according to claim 1, wherein: The high-resolution mass spectrometry analysis described in step 4 adopts ESI-mode, first scanning the cis-dihydroxylated unsaturated fatty acid parent ion, then fragmenting to produce secondary mass spectrometry ion fragments, finding the diagnostic ion pair, and locating the C=C bond of the unsaturated fatty acid.

6. The method according to claim 1 or 5, wherein: The fragmentation is collision induced dissociation (CID) fragmentation.

7. The method according to claim 1 or 4, wherein: Steps 1 to 3 are suitable for the selective enrichment of monounsaturated fatty acids and polyunsaturated fatty acids.

8. Use of the method of claim 1 in lipidomics research, biological sample analysis, phospholipid detection, and clinical sample analysis for analyzing the structural isomers of unsaturated fatty acids and their biological functions.

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