Method for selectively enriching unsaturated fatty acid and positioning C / C bond of unsaturated fatty acid

Abstract

The invention relates to an unsaturated fatty acid selective enrichment and C = C bond positioning method, and belongs to the technical field of bioanalytical chemistry and lipidomics. The preparation method comprises the following steps: firstly preparing a silica gel material with a boric acid functional group, then converting a C = C bond in unsaturated fatty acid molecules into a vicinal diol structure through a cis-dihydroxylation reaction, and then realizing efficient selective enrichment of the derivatized unsaturated fatty acid by utilizing specific interaction between boric acid and vicinal diol, so as to obtain the derivative unsaturated fatty acid. The enriched sample is subjected to high-resolution mass spectrometry and further fragmentation to generate a secondary characteristic diagnosis ion pair, so that accurate positioning of C = C bonds in unsaturated fatty acid molecules is realized. The method has the advantages of simplicity and convenience in operation, high selectivity, high enrichment efficiency, wide application range and the like, can effectively solve the problems of difficulty in distinguishing isomeride and matrix interference in the traditional technology, and provides a new technology for fine structure analysis and lipid metabolism research of unsaturated fatty acid in a complex biological sample.

Description

Technical Field

[0001] The present invention belongs to the technical fields of bioanalytical chemistry and lipidomics, and particularly relates to a method for selectively enriching unsaturated fatty acids through chemical derivatization and solid-phase extraction techniques and determining the position of C=C bonds 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 signal transduction, inflammation regulation, energy metabolism, and disease occurrence. In recent years, with the in-depth study of lipidomics, more and more researchers have begun to focus on the influence of the double bond position and geometric configuration in unsaturated fatty acids on their biological functions. However, the existing analytical techniques have two major limitations:

[0003] Difficulty in distinguishing structural isomers: When traditional mass spectrometry techniques are used for qualitative analysis of unsaturated fatty acids, they often rely on retention time and accurate mass matching. 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 generate characteristic fragments with sufficient resolution to distinguish double bond positions, making it difficult to accurately identify isomers with different C=C bond positions.

[0004] Sample matrix interference and low abundance problem: The concentration of unsaturated fatty acids in biological samples is usually low, and at the same time, there are a large number of high-background interferents such as saturated fatty acids, phospholipids, and glycerides, resulting in serious impacts on the signal-to-noise ratio and sensitivity of traditional analytical methods and unable to ensure high selectivity and high recovery.

[0005] To address the above problems, developing a method that is efficient, highly selective, and capable of precisely determining 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 object of the present invention is to solve the limitations of the prior art in the analysis of unsaturated fatty acids, and to provide a method capable of simultaneously achieving selective enrichment of unsaturated fatty acids and C=C bond localization. First, a boric acid-functionalized material is prepared; secondly, the C=C bond of the unsaturated fatty acid is converted into a vicinal diol structure through cis-dihydroxylation reaction; then, through the covalent binding of boric acid-vicinal diol, the derivatized unsaturated fatty acids are selectively enriched from complex samples; finally, the precise localization of the double bond sites is achieved by using the characteristic diagnostic ion pairs generated by high-resolution mass spectrometry. Through this series of steps, the selective enrichment and fine structure identification of unsaturated fatty acids are realized. By optimizing the proportion of derivatization reagents and enrichment conditions, this method can be adapted to unsaturated fatty acids with different chain lengths (C14-C24), degrees of unsaturation (1-6 double bonds), and cis / trans configurations. By converting the C=C bond of unsaturated fatty acids into an identifiable chemical structure, enriching with specific materials, and combining high-resolution mass spectrometry technology, precise double bond localization is achieved.

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

[0008] Step 1: Preparation of boric acid-functionalized material: React a boric acid-functionalized reagent with an amino-functionalized material in an organic solvent at room temperature to obtain a boric acid-functionalized material SG-B; the boric acid-functionalized reagent is a compound capable of reacting with an amino material to introduce a boric acid group, and the amino-functionalized material is a silica gel material with amino functional groups on its surface;

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

[0010] .

[0011] Step 2: Cis-dihydroxylation reaction: Use a catalytic system composed of an osmium-based catalyst and an N-methylmorpholine-based oxidant to convert the C=C bond of the unsaturated fatty acid into a cis-vicinal diol structure to achieve complete derivatization of monounsaturated fatty acids; or selectively derivatize specific double bonds of polyunsaturated fatty acids by controlling the amount of oxidant used;

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

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

[0014] Step 3. Selective enrichment: Incubate the SG-B material prepared in Step 1 with the unsaturated fatty acids after cis-dihydroxylation reaction in Step 2 in a weakly alkaline solution, and use the covalent binding of boric acid and vicinal diols to enrich the target compounds, and then elute with a weakly acidic solution;

[0015] Preferably, the selective enrichment in Step 3 is carried out for adsorption and washing in weakly alkaline ethyl acetate (EA), and the elution is carried out with a weakly acidic methanol (FAM) solution, and the elution is repeated three times.

[0016] Furthermore, the methods described in Steps 1 to 3 are applicable to the selective enrichment of monounsaturated fatty acids and polyunsaturated fatty acids.

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

[0018] Furthermore, the high-resolution mass spectrometry analysis in Step 4 adopts the ESI- mode. First, scan the precursor ions of cis-diolated unsaturated fatty acids, and then generate secondary mass spectrometry ion fragments by fragmentation to find the diagnostic ion pairs and locate the C=C bond of unsaturated fatty acids.

[0019] The method described above 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] The present invention applies an affinity material with a boric acid group to the enrichment of unsaturated fatty acids, and utilizes the reversible binding property between the boronic acid affinity material and cis-dihydroxy fatty acids to achieve the efficient selective enrichment of unsaturated fatty acids. Compared with traditional separation methods, the boronic acid affinity material has higher selectivity and affinity, greatly improving the recovery efficiency of unsaturated fatty acids. Description of the Drawings

[0021] Figure 1 It is the high-performance liquid chromatography diagram for the detection of PBA-NHS in the supernatant before and after the reaction during the preparation process of the SG-B material in the embodiment of the present invention; Figure 2This is the liquid chromatography - mass spectrometry combined figure for the detection of 4 - carboxyphenylboronic acid in the supernatant after the hydrolysis of the modified groups on the SG - B material in the embodiments of the present invention; Figure 3 This is the infrared spectrum figure of PBA - NHS, SG - A material, SG - B material, and the material of SG - B combined with Diol - FA 18:1 in the embodiments of the present invention; Figure 4 This is the X - ray photoelectron spectroscopy figure of PBA - NHS, SG - A material, SG - B material, and the material of SG - B combined with Diol - FA 18:1 in the embodiments of the present invention; Figure 5 This is the mass spectrometry figure before and after the cis - dihydroxylation reaction of FA 18:1(9Z) in the embodiments of the present invention; Figure 6 This is the mass spectrometry figure after the cis - dihydroxylation reaction of FA 18:2(9Z,12Z) when the dosage of NMO catalyst is 0.3 eq., 0.5 eq., 0.7 eq. in the embodiments of the present invention; Figure 7 This is the mass spectrometry figure after the cis - dihydroxylation reaction of FA 22:6(4Z,7Z,10Z,13Z,16Z,19Z) when the NMO catalyst is 0.1eq., 0.5 eq., 1 eq. in the embodiments of the present invention; Figure 8 This is the recovery rate of the SG - B material for extracting the mixture of FA 19:0 and Diol - FA 18:1 in the embodiments of the present invention; Figure 9 This is the recovery rate of the SG - B material for extracting the eight - fatty - acid mixture in the embodiments of the present invention; Figure 10 This is the secondary mass spectrometry figure and the structural analysis figure after the cis - dihydroxylation of FA 18:1 in the embodiments of the present invention; Figure 11 This is the secondary mass spectrometry figure and the structural analysis figure after the cis - dihydroxylation of a single double bond of FA 18:2 in the embodiments of the present invention; Figure 12 This is the secondary mass spectrometry figure and the structural analysis figure after the cis - dihydroxylation of a single double bond of FA 22:6 in the embodiments of the present invention. Detailed implementation manners

[0022] To enable those skilled in the art to fully understand the present invention, the methods of the present invention are described in detail below in combination with embodiments, including the specific reaction conditions, operation methods, and detection and characterization processes of each step. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of the present invention.

[0023] A method for selective enrichment of unsaturated fatty acids and determination of the position of their C=C bonds, specifically comprising the following steps:

[0024] Step 1: Preparation of SG-B material

[0025] In the preparation process of the SG-B material, first, SG-A (20 mg, particle size 5 μm, pore size 100 Å) is rinsed twice with acetonitrile to remove surface impurities. Then, SG-A is suspended in a solution containing PBA-NHS (5 mg, dissolved in 1 mL of acetonitrile). In this example, the SG-B material is prepared with PBA-NHS and SG-A at a mass ratio of 1:4 (the recommended ratio range in actual application is 1:3 to 1:5). The reaction is carried out overnight (10 - 14 h) at room temperature using a shaker to ensure that PBA-NHS is fully grafted onto the surface of SG-A. After the reaction is completed, the supernatant is discarded by centrifugation (8000 rpm, 5 min), and the material is washed twice with acetonitrile to remove the unreacted PBA-NHS. The finally prepared SG-B material is stored in acetonitrile at 4°C to maintain its activity.

[0026] The reaction formula is:

[0027] .

[0028] The following experiments are carried out to verify the structure of the SG-B material:

[0029] In this example, the raw material PBA-NHS used for preparing the SG-B material has ultraviolet absorption, Figure 1 where a is the high-performance liquid chromatography (HPLC) chromatogram of the supernatant before the reaction for preparing the SG-B material, and b is the HPLC chromatogram of the supernatant after the reaction for preparing the SG-B material. It can be seen that the peak area of PBA-NHS significantly decreases, indicating that a large amount of the PBA-NHS substrate is consumed.

[0030] Figure 2 In, the SG-B material is dissociated by a strong base to break the amide bond, and then acidified to release the hydrolysis product 4-carboxyphenylboronic acid, whose molecular formula is C7H7BO4 and molecular weight is 165.94. The peak at about 11 min on the chromatogram corresponds to the ultraviolet absorption of 4-carboxyphenylboronic acid. In the negative ion mode of mass spectrometry (ESI-), its quasi-molecular ion peak [M-H]⁻ is detected, indirectly indicating that PBA-NHS is grafted onto the surface of SG-A. Figure 1 and Figure 2 The results of preliminarily prove the successful preparation of the SG-B material.

[0031] Using a wavelength range of 400 - 4000 cm -1The infrared light was used to scan and analyze PBA-NHS, SG-A material, SG-B material, and the material formed by the combination of SG-B and Diol-FA 18:1.

[0032] As Figure 3 shown in a of -1 , in the infrared spectrum of PBA-NHS, the stretching vibration of B-O-H is at 3412.9 cm -1 , and the stretching vibration peak of B-O is at 1336.4 cm

[0033] As Figure 3 shown in b of -1 , in the infrared spectrum of SG-A material, the asymmetric stretching vibration of Si-O-Si corresponds to the peak at 1108.4 cm -1 , the symmetric stretching vibration corresponds to the peak at 811.9 cm -1 , the stretching vibration peak of N-H of the amino group corresponds to the peak at 3434.6 cm -1 , the stretching vibration peak of alkane C-H is at 2958.8 cm -1 , and the out-of-plane bending vibration peak of N-H is at 1627.6 cm

[0034] As Figure 3 shown in c of -1 , in the infrared spectrum of SG-B material, the peaks at 1496.5 cm -1 and 1543.8 cm -1 are attributed to the stretching vibration of aromatic ring skeleton C=C, and the peak at 1651.7 cm -1 is attributed to the stretching vibration of amide C=O, and the peak at 3422.1 cm -1 is attributed to the stretching vibration of B-O-H. These peaks confirm the successful preparation of SG-B;

[0035] As Figure 3 shown in d of -1 , in the infrared spectrum of the material formed by the combination of SG-B and Diol-FA 18:1, the characteristic peak of B-O-H at 3422.1 cm -1 completely disappears, which is attributed to the combination of boric acid group with the cis-diol structure of Diol-FA 18:1, resulting in the consumption of B-O-H group.

[0036] The functional group information revealed by Fourier transform infrared spectroscopy analysis is highly consistent with the synthetic route designed in this experiment, strongly proving the successful preparation of SG-B material. However, the SG-B material shows a strong absorption peak of asymmetric stretching vibration of Si-O-Si at 1108.4 cm -1 , and the characteristic peak of adjacent B-O bond may be masked due to the too high signal intensity. Therefore, XPS was used to further analyze the elemental chemical state on the surface of SG-B material to deeply study its surface chemical composition.

[0037] As Figure 4 shown, although the B element is hardly detectable in the full XPS spectra of the SG-B material and the material after its combination with Diol-FA 18:1, the presence of the B element can still be observed in the pie chart content analysis, with its contents being 2.23% and 1.47% respectively. This phenomenon is attributed to the relatively low detection sensitivity of the XPS technique for light elements (such as B, with an atomic number of 5).

[0038] In summary, the results of high performance liquid chromatography, liquid chromatography-mass spectrometry, infrared spectrogram, and X-ray photoelectron spectrogram are consistent, fully demonstrating the successful preparation of the SG-B material.

[0039] Step 2: Cis-dihydroxylation reaction of unsaturated fatty acids

[0040] In the cis-dihydroxylation reaction of monounsaturated fatty acids, in this example, FA 18:1(9Z) is used as the substrate, and the reaction system contains NMO, potassium osmate dihydrate, water, and acetone. After the reaction is stirred at room temperature for 5 - 10 min, the product is purified and recovered through the following post-treatment steps: First, 400 μL of EA is added and vortex-mixed, then centrifuged at 12000 rpm for 3 min, the supernatant is taken, and the solvent is removed using a vacuum centrifugal concentrator; then 400 μL of EA is added again, the vortex and centrifugation steps are repeated, the organic layer is collected and transferred to a 1.5 mL EP tube, the solvent is concentrated again to remove it, and finally the sample is dissolved in methanol (MeOH) for subsequent analysis.

[0041] The reaction formula is:

[0042] ;

[0043] The following experiments are carried out to verify the cis-dihydroxylation reaction of FA 18:1(9Z):

[0044] Figure 5 are the mass spectra before (a) and after (b) the cis-dihydroxylation reaction of FA 18:1(9Z). Under the ESI-ionization source, FA 18:1(9Z) (m / z 281.24606) has successfully generated the derivatized product Diol-FA 18:1 (m / z 315.25281) after the cis-dihydroxylation reaction, and the reaction yield is almost 100%.

[0045] 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) are used as substrates. The reaction system contains NMO, potassium osmate dihydrate, water and acetone. The cis-dihydroxylation of a single double bond of polyunsaturated fatty acids is achieved by controlling the amount of the oxidant NMO. The reaction is stirred at room temperature for 20 - 60 min, and the post-treatment steps are the same as those for FA 18:1(9Z).

[0046] The reaction formula is:

[0047] ;

[0048] ;

[0049] The following experiments are carried out to verify the cis-dihydroxylation reaction of FA 18:2(9Z,12Z):

[0050] Figure 6 are the mass spectrometry diagrams after the cis-dihydroxylation reaction of FA 18:2(9Z,12Z) when the dosage of NMO oxidant is 0.3 eq., 0.5 eq., 0.7 eq. Under the ESI-ionization source, when NMO is 0.5 eq., the yield of the cis-dihydroxylation product Mono-Diol-FA 18:2 (m / z 313.2) of a single double bond is the highest, and this optimized condition is selected for subsequent high-resolution mass spectrometry structure analysis.

[0051] The following experiments are carried out to verify the cis-dihydroxylation reaction of FA 22:6(4Z,7Z,10Z,13Z,16Z,19Z): Figure 7 are the mass spectrometry diagrams after the cis-dihydroxylation reaction of FA 22:6(4Z,7Z,10Z,13Z,16Z,19Z) when NMO oxidant is 0.1 eq., 0.5 eq., 1 eq. When NMO is 0.5 eq., the proportion of the cis-dihydroxylation product Mono-Diol-FA 22:6 (m / z 361.2) of a single double bond is the highest, and this optimized condition is selected for subsequent high-resolution mass spectrometry structure analysis.

[0052] Step 3. Selective enrichment

[0053] In this example, the following two schemes are taken as examples for illustration.

[0054] Scheme 1. Selective enrichment of the mixture of Diol-FA 18:1 and FA 19:0 (two fatty acids or derivatized fatty acids), specifically as follows:

[0055] First, take 5 mg of SG-B material and rinse it twice with EA to activate the surface. Then, mix SG-B with 1 mL of pH = 9 EA solution containing a mixture of 60 μg / mL of Diol-FA 18:1 and FA 19:0, and incubate it in an ultrasonic bath for 10 min. Subsequently, wash it twice with the loading reagent. After that, elute the target compound with 1 mL of 40 mM FAM solution, repeat the elution three times, and incubate for 5 min each time. Finally, centrifuge the solution (12,000 rpm, 3 min), take the supernatant, evaporate to dryness, and redissolve it with MeOH to prepare for mass spectrometry analysis.

[0056] As Figure 8 shown, by calculating the recovery rates of the two fatty acids by the SG-B material, it is found that the recovery rate of the SG-B material for FA 19:0 is relatively low, at 9.62%; the recovery rate for Diol-FA 18:1 is significantly higher, reaching 83.45%.

[0057] 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), specifically as follows:

[0058] First, take 5 mg of SG-B material and rinse it twice with EA to activate the surface. Then, mix the SG-B material with 1 mL of pH = 9 EA solution containing a mixture of 60 μg / mL 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, and incubate it in an ultrasonic bath for 10 min. Subsequently, wash it twice with the loading reagent. After that, elute the target compound with 1 mL of 40 mM FAM solution, repeat the elution three times, and incubate for 5 min each time. Finally, centrifuge the solution (12,000 rpm, 3 min), take the supernatant, evaporate to dryness, and redissolve it with MeOH to prepare for mass spectrometry analysis.

[0059] As Figure 9As shown, by calculating the recovery rates of SG-B material for eight fatty acids, it is found that SG-B material has relatively high recovery rates for Diol-FA18:1, Diol-FA 22:1, Mono-Diol-FA 18:2, and Mono-Diol-FA 18:3, which are 69.73%, 71.46%, 66.36%, and 84.77% respectively, while the recovery rates for FA 11:0, FA 13:0, FA 15:0, and FA 17:0 are low, which are 5.88%, 4.44%, 3.43%, and 2.51% respectively.

[0060] The results of the selective enrichment experiment prove that for the derivatized monounsaturated fatty acids and polyunsaturated fatty acids, SG-B material shows significant adsorption effects, demonstrating good selectivity for the target compounds. Moreover, in a complex fatty acid sample system, SG-B material can also specifically recognize and enrich the derivatized unsaturated fatty acids while avoiding the competitive interference of saturated fatty acids.

[0061] Step Four: High-resolution mass spectrometry analysis

[0062] In this example, a UHPLC-LTQ Orbitrap Elite instrument is used for high-resolution mass spectrometry analysis, and the analysis conditions are as follows: the detection mode is ESI-; the CID energy is set to 35 eV; the resolution is set to 30000; the scanning range is m / z 85 - 380. The chromatographic conditions are: mobile phase A is water, mobile phase B is acetonitrile, and an isocratic elution mode is adopted. The proportion of acetonitrile remains at 50% within 0 - 1 min, and the flow rate is 0.5 mL / min.

[0063] As Figure 10 shown, two pairs of significant diagnostic ions are observed for FA 18:1(9Z) after cis-dihydroxylation in the high-resolution mass spectrum: one pair of diagnostic ions from the alkyl end, which are m / z 141.12781 and m / z 171.13818 respectively; the other pair of diagnostic ions from the carboxyl end, which are m / z 171.10181 and m / z 201.11211 respectively. Based on the exact masses and relative abundances of these ion pairs, the position of the C=C bond in this compound can be determined to be Δ9.

[0064] As Figure 11As shown, after cis-dihydroxylation of a single double bond, FA 18:2(9Z,12Z) shows two pairs of significant diagnostic ions in the high-resolution mass spectrum: one pair of diagnostic ions from the carboxyl terminus of Δ9, with m / z values of 171.10181 and m / z 201.11230 respectively; the other pair of diagnostic ions from the alkyl terminus of Δ12, with m / z values of 99.08125 and m / z 129.09152 respectively. Based on the exact masses and relative abundances of these ion pairs, the position of the C=C bond in the compound can be determined.

[0065] As Figure 12 shown, after cis-dihydroxylation of a single double bond, FA 22:6(4Z,7Z,10Z,13Z,16Z,19Z) shows eight pairs of significant diagnostic ions in the high-resolution mass spectrum: one pair of diagnostic ions from the alkyl terminus of Δ4, with m / z values of 257.18979 and m / z 287.19940 respectively; one pair of diagnostic ions from the alkyl terminus of Δ7, with m / z values of 217.15871 and m / z 247.16875 respectively, one pair of diagnostic ions from the carboxyl terminus of Δ7, with m / z values of 141.05507 and m / z 171.06519 respectively; one pair of diagnostic ions from the alkyl terminus of Δ10, with m / z values of 177.12732 and m / z 207.13773 respectively, one pair of diagnostic ions from the carboxyl terminus of Δ10, with m / z values of 181.08583 and m / z 211.09633 respectively; another pair of diagnostic ions from the alkyl terminus of Δ13, with m / z values of 137.09633 and m / z 167.10675 respectively; and one pair of diagnostic ions from the carboxyl terminus of Δ16, with m / z values of 261.14792 and m / z 291.16018 respectively; the last pair of diagnostic ions from the carboxyl terminus of Δ19, with m / z values of 301.17902 and m / z 331.19049 respectively. Based on the exact masses and relative abundances of these ion pairs, the position of the C=C bond in the compound can be determined. Similarly, for other unsaturated fatty acids, the position of the double bond can also be accurately located through the corresponding diagnostic ion pairs.

[0066] Through chemical derivatization, boronic acid affinity enrichment, and high-resolution mass spectrometry analysis, the recovery rate of derivatized unsaturated fatty acids in complex biological samples is significantly higher than that of saturated fatty acids in the present invention, realizing the efficient and selective enrichment of unsaturated fatty acids and the accurate positioning of C=C bonds, and can be extended to phospholipid analysis and clinical sample detection.

Claims

1. A method for selective enrichment of unsaturated fatty acids and determination of the position of their C=C bonds, characterized in that: It includes the following steps: Step 1. Preparation of boric acid-functionalized material: React a boric acid-functionalizing reagent with an amino-functionalized material in an organic solvent at room temperature to obtain a boric acid-functionalized material SG-B; the boric acid-functionalizing reagent is a compound capable of reacting with an amino material to introduce a boric acid group, and the amino-functionalized material is a silica gel material with amino functional groups on its surface; Step 2. Cis-dihydroxylation reaction: Use a catalytic system composed of an osmium-based catalyst and an N-methylmorpholine-based oxidant to convert the C=C bond of unsaturated fatty acids into a cis-vicinal diol structure to achieve complete derivatization of monounsaturated fatty acids; or selectively derivatize specific double bonds of polyunsaturated fatty acids by regulating the amount of oxidant used; Step 3. Selective enrichment: Incubate the SG-B material prepared in Step 1 with the unsaturated fatty acids after the cis-dihydroxylation reaction in Step 2 in a weakly basic solution, and use the covalent binding of boric acid and vicinal diol to enrich the target compound, and then elute with a weakly acidic solution; Step 4. High-resolution mass spectrometry analysis: Analyze the unsaturated fatty acids eluted in Step 3 using an ultra-high performance liquid chromatography-linear ion trap orbitrap mass spectrometer, and then generate secondary mass spectrometry characteristic diagnostic ion pairs through fragmentation to accurately locate the position of the C=C bond of 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 selected to react with amino silica gel material in acetonitrile at room temperature to prepare the SG-B material, and the reaction formula is: 。 3. The method according to claim 1, characterized in that: 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, characterized in that: The selective enrichment in Step 3 is carried out for adsorption and washing in weakly basic ethyl acetate, and the elution is carried out using a weakly acidic methanol solution.

5. The method according to claim 1, characterized in that: The high-resolution mass spectrometry analysis in Step 4 adopts the ESI- mode. First, scan the parent ions of the cis-dihydroxylated unsaturated fatty acids, and then generate secondary mass spectrometry ion fragments through fragmentation to find the diagnostic ion pairs and locate the C=C bond of the unsaturated fatty acids.

6. The method according to claim 1 or 5, characterized in that: The fragmentation selected is collision-induced dissociation CID fragmentation.

7. The method according to claim 1 or 4, characterized in that: The methods described in Steps 1 to 3 are applicable to the selective enrichment of monounsaturated fatty acids and polyunsaturated fatty acids.

8. Application of the method according to 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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