A method for locating and quantitatively detecting double bonds of fatty acids based on liquid chromatography-mass spectrometry

Abstract

This invention discloses a method for the localization and quantitative detection of fatty acid double bonds based on liquid chromatography-mass spectrometry (LC-MS), belonging to the field of LC-MS technology. This method utilizes the electrostatic adsorption between amino functionalized groups and fatty acid carboxyl groups, as well as the hydrophobic interaction between hydrophobic alkyl chains and fatty acid carbon chains, to achieve dual selective enrichment. Double bond-specific derivatization generates characteristic daughter ions corresponding to the double bond positions during mass spectrometry fragmentation, enabling effective differentiation of double bond position isomers. Isotope internal standards are used to correct matrix effects and injection errors, ensuring accurate quantification.

Description

Technical Field

[0001] This invention relates to the field of liquid chromatography-mass spectrometry (LC-MS), specifically to a method for the localization and quantitative detection of fatty acid double bonds based on LC-MS. Background Technology

[0002] Lipidomics, as an important component of metabolomics, focuses on fatty acids as one of its core research subjects. Fatty acids are not only crucial substrates for energy metabolism in organisms but also core molecules constituting cell membrane phospholipids, regulating signal transduction pathways, and participating in various physiological and pathological processes. Unsaturated fatty acids exhibit unique biological functions due to the presence of carbon-carbon double bonds in their carbon chains. Geometric isomers (cis and trans) and positional isomers of these double bonds often exhibit drastically different physiological effects in biological activity, metabolic pathways, and even disease development. Therefore, the accurate identification and absolute quantification of fatty acids, especially double-bond positional isomers, has always been a highly valued and challenging research topic in the field of lipidomics analysis.

[0003] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has evolved significantly from gas chromatography-mass spectrometry (GC-MS) to LC-MS / MS, becoming the mainstream method for fatty acid analysis. In the early stages of LC-MS / MS development, researchers typically used methyl esterification or saponification pretreatments to convert fatty acids into corresponding methyl ester derivatives, aiming to improve their volatility and thermal stability in the gas chromatography system. This traditional approach usually involved multiple independent steps, including alkaline hydrolysis (saponification), organic solvent extraction, neutralization, and methyl esterification derivatization. This process was not only cumbersome and time-consuming, but also prone to introducing systematic quantitative biases due to the potential for double bond migration or oxidative degradation of some unsaturated fatty acids during derivatization if the methyl esterification reaction conditions were not properly controlled. Meanwhile, in subsequent liquid chromatography-tandem mass spectrometry analysis, the chromatographic retention behavior of methyl esterification products mainly depends on the carbon chain length and the number of double bonds. The lack of double bond position information makes it difficult to achieve baseline separation on the chromatogram of isomers with the same number of carbon atoms and double bonds but different double bond positions (such as oleic acid and trans oleic acid, linoleic acid and conjugated linoleic acid), which seriously restricts the structural specificity and reliability of the analytical results.

[0004] With the continuous development of mass spectrometry technology, the widespread use of electrospray ionization sources and triple quadrupole mass spectrometry has significantly improved the analytical sensitivity of fatty acids. However, small fatty acid molecules are highly susceptible to competitive ionization inhibition during electrospray ionization by a large number of endogenous matrix components in biological samples (such as phospholipids, cholesterol, glycerides, protein fragments, and inorganic salt ions), resulting in significant matrix effects, poor quantitative repeatability, and insufficient lower limits of quantitation to meet the requirements for low-abundance detection of biomarkers. Although techniques such as matrix matching calibration curves, internal standard methods, and sample pretreatment and purification can alleviate these problems to some extent, traditional liquid chromatography-tandem mass spectrometry still faces bottlenecks in improving the signal-to-noise ratio when dealing with complex biological matrices. More importantly, mass spectrometry fragmentation patterns indicate that the characteristic daughter ions generated during collision-induced dissociation of fatty acid molecules mainly reflect the fracture modes of carboxylic acid functional groups and carbon chain skeletons, while the information on the carbon-carbon double bond positions is difficult to effectively distinguish using characteristic ions under conventional fragmentation conditions. Even with high-resolution mass spectrometry or multi-stage fragmentation strategies, the mass differences between fatty acid positional isomers exist only in subtle variations in isotopic distribution, making reliable double bond localization virtually impossible in the complex interference environment of real biological samples.

[0005] In recent years, fatty acid double bond localization techniques based on derivatization strategies have gradually attracted the attention of researchers. The core principle lies in using double bond-specific chemical reactions (such as click chemi-addition, ozone decomposition, or Picolinic-Boronic Acid derivatization) to introduce specific modifying groups or characteristic breakage sites at the double bond position, thereby generating characteristic daughter ions corresponding to the double bond position during mass spectrometry fragmentation, thus achieving the distinction between positional isomers. However, existing derivatization methods generally suffer from the following inherent drawbacks: First, derivatization reactions usually need to be carried out separately in the pretreatment stage, and the reaction conditions (temperature, time, catalyst dosage) are quite stringent, and the operation process has not been effectively simplified compared to traditional liquid chromatography-tandem mass spectrometry pretreatment. Second, the reagent consumption for derivatization reactions is often large, and some reactions require the use of toxic and harmful organic solvents (such as dichloromethane, toluene, etc.), which is inconsistent with the current development concept of green analytical chemistry. Third, while achieving double bond localization, existing derivatization methods often sacrifice quantitative accuracy, because the conversion rate of the derivatization reaction is affected by fatty acid concentration, matrix background, and fluctuations in reaction conditions, making it difficult to achieve stable and controllable derivatization efficiency. The limitations of the above-mentioned technical solutions make the two core analytical objectives of fatty acid double bond localization and absolute quantification present an irreconcilable contradiction within the existing technical framework.

[0006] Meanwhile, high throughput and automation are important trends in the development of modern omics technologies. Traditional fatty acid analysis pretreatment involves manual steps such as multiple centrifugations, nitrogen drying, and organic solvent transfer, which are not only time-consuming and labor-intensive, but also prone to human error, severely hindering large-scale biosample cohort studies. Although solid-phase extraction (SPE) technology has achieved semi-automation of fatty acid pretreatment to some extent, steps such as activation, equilibration, loading, and elution of SPE columns still rely on complex instrument configurations, resulting in insufficient convenience and portability in practical applications. Therefore, how to simplify, automate, and achieve high-throughput compatibility of the pretreatment process while ensuring accurate double bond localization and quantitative reliability has become a key technological bottleneck in the field of fatty acid analysis.

[0007] In summary, existing fatty acid liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection techniques have limitations to varying degrees in terms of pretreatment complexity, ability to distinguish double bond positional isomers, tolerance to matrix interference, and high-throughput automation. These technical shortcomings are intertwined and mutually restrictive, constituting the core technical contradiction restricting the accurate analysis of fatty acids. Therefore, developing an integrated pretreatment-detection technology that can simultaneously achieve accurate double bond positional identification and absolute quantitative reliability has become an important technical issue that urgently needs to be addressed by those skilled in the art. Summary of the Invention

[0008] This invention provides a method for the localization and quantitative detection of fatty acid double bonds based on liquid chromatography-mass spectrometry (LC-MS) to address the technical bottlenecks of existing fatty acid LC-MS detection techniques in terms of pretreatment complexity, ability to distinguish double bond positional isomers, tolerance to matrix interference, and high-throughput automation.

[0009] The technical solution adopted in this invention is as follows: This invention provides a method for the localization and quantitative detection of fatty acid double bonds based on liquid chromatography-mass spectrometry (LC-MS), comprising the following steps: (1) Take serum, plasma or tissue homogenate samples, place them in a centrifuge tube, add isotope-labeled fatty acid internal standard, and vortex to mix; (2) Add amino hydrophobic composite magnetic bead solution and shake at room temperature to allow fatty acids to be fully adsorbed onto the surface of the magnetic beads. The amino hydrophobic composite magnetic beads are superparamagnetic core-shell structure magnetic beads that are covalently modified with amino groups and C8~C18 alkyl chains. The surface of the magnetic beads is simultaneously modified with amino groups and C8~C18 alkyl chains. Through the electrostatic adsorption of amino groups and carboxyl groups of fatty acids and the hydrophobic interaction between alkyl chains and carbon chains of fatty acids, dual selective enrichment is achieved. (3) Use magnetic separation to remove the supernatant and remove interfering substances such as proteins, phospholipids and salts from the sample; (4) Add washing solution and perform magnetic suction washing to remove residual impurities; (5) Add a double bond-specific derivatizing agent, wherein the double bond-specific derivatizing agent is 2-acetylpyridine, and use it under light-protected conditions with a power of 300~500mW / cm. 2 Irradiate the sample with a 365 nm ultraviolet lamp at a distance of 1-3 cm and react at room temperature for 5 minutes to allow the derivatizing reagent to undergo the Pasternò-Büchi reaction with the carbon-carbon double bond of fatty acid. Under photoexcitation, the carbonyl group of 2-acetylpyridine undergoes [2+2] cycloaddition with the carbon-carbon double bond of fatty acid to generate an oxobutane ring structure. (6) Add elution buffer and elute by shaking, then collect the supernatant of the elution buffer; (7) The supernatant of the eluent is injected into a liquid chromatography-tandem mass spectrometry system for detection. During the mass spectrometry collision-induced dissociation process, the oxobutane ring generated in step (5) undergoes specific breakage, generating a pair of diagnostic ions directly related to the double bond position. The double bond position is determined based on the mass-to-charge ratio (m / z) of the diagnostic ions. Quantitative analysis is performed by constructing a standard curve using the isotope internal standard method.

[0010] Preferably, in step (1), the volume of the sample taken is 200 μL, the volume of the isotope-labeled fatty acid internal standard added is 20 μL, the concentration of the isotope-labeled fatty acid internal standard is 5~20 μg / mL, and the isotope-labeled fatty acid internal standard is a carbon-13 labeled or deuterium labeled fatty acid standard, whose carbon chain length and double bond number match the fatty acid to be tested.

[0011] Preferably, in step (2), the volume of the amino hydrophobic composite magnetic bead solution added is 50 μL, the concentration of the magnetic beads is 5~20 mg / mL, the particle size of the magnetic beads is 0.05~1 μm, and the oscillation adsorption time is 2~5 min.

[0012] More preferably, the preparation process of the amino hydrophobic composite magnetic beads is as follows: a. Using ferric chloride, ethylenediamine, and sodium acetate in a molar ratio of 1:2~6:2~6 as raw materials and ethylene glycol as solvent, magnetic nanoparticles with amino groups on the surface are obtained by reacting at a temperature of 150~200℃. After washing with water, they are dried for later use. b. Disperse the magnetic nanoparticles prepared in step a in anhydrous pyridine, add silane with C8~C18 branches, the mass of silane being 0.5~2 times the mass of magnetic nanoparticles, mechanically stir at room temperature for 16~20 h, filter out the magnetic beads, wash them multiple times with water and ethanol, and disperse them in water for later use.

[0013] Preferably, the washing solution in step (4) is a methanol aqueous solution with a volume percentage concentration of 80%, and the washing is performed 2 to 4 times.

[0014] Preferably, in step (5), the volume of the 2-acetylpyridine derivatizing agent added is 50-80 μL, and the concentration of the 2-acetylpyridine derivatizing agent is 0.1-1 M.

[0015] Preferably, in step (6), the eluent is acetonitrile, and the elution is performed by shaking for 1 to 5 minutes.

[0016] Preferably, in the liquid chromatography-tandem mass spectrometry detection process in step (7), the chromatographic conditions of liquid chromatography include: a C18 reversed-phase column, a column temperature of 40°C, a flow rate of 0.3~0.5 mL per minute, and an injection volume of 5~10 μL; mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, and the gradient elution program is: 0~1 min mobile phase B is maintained at 5%, 1~5 min mobile phase B is linearly increased from 5% to 95%, 5~6 min mobile phase B is maintained at 95%, 6~8 min mobile phase B is decreased from 95% to 5%, and 8~10 min mobile phase B is maintained at 5%; the mass spectrometry conditions are: electrospray ionization source, negative ion mode, and multiple reaction monitoring mode.

[0017] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention employs amino-hydrophobic composite magnetic beads to achieve one-step enrichment and purification of fatty acids. It simplifies the traditional multi-step extraction, centrifugation, and nitrogen blowing pretreatment steps into a simple process of magnetic bead adsorption, magnetic separation, and washing, significantly shortening pretreatment time and improving work efficiency. It utilizes a dual amino-hydrophobic mechanism for fatty acid enrichment, leveraging the electrostatic adsorption between amino functionalized groups and carboxyl groups, as well as the hydrophobic interaction between hydrophobic alkyl chains and carbon chains, to achieve highly selective enrichment of fatty acids. Simultaneously, magnetic separation effectively removes interfering substances such as proteins, phospholipids, and salts. The magnetic bead solid-phase extraction mode eliminates the need for liquid-liquid extraction with organic solvents, aligning with green analytical chemistry principles and reducing harm to the environment and operators. 2. This invention employs a photoinduced Pasternò-Büchi reaction, using 2-acetylpyridine as a specific derivatizing reagent to introduce an oxocyclic butane ring at the double bond position of fatty acids. This ring undergoes specific fragmentation during mass spectrometry fragmentation, generating characteristic diagnostic ion pairs corresponding one-to-one with the double bond position. This application explicitly discloses the diagnostic ions and their determination rules for oleic acid (Δ9) and isoleic acid (Δ11), solving the technical problem that traditional liquid chromatography-tandem mass spectrometry cannot distinguish between isomers at double bond positions. 3. This invention employs isotope internal standard correction technology. By using an internal standard substance with the same physicochemical properties as the fatty acid to be tested, losses, matrix effects, and instrument fluctuations during the pretreatment process are corrected, thus achieving accurate quantification of fatty acids. The quantification results are accurate and reliable.

[0018] 4. The pretreatment process of this invention is simple, has few steps, and is short in time. It is easy to achieve high-throughput and automated operation and can be used in conjunction with an automated liquid processing workstation to meet the analytical needs of large-scale biological sample cohort studies.

[0019] 5. The overall analysis time of this invention is less than 12 minutes, with high detection throughput. A large number of samples can be processed in a single batch, making it suitable for high-throughput clinical testing scenarios.

[0020] 6. Verification showed that, using the method described in Example 1 of this application, a mixed standard of oleic acid (Δ9) and isoleic acid (Δ11) with a concentration of 10 ng / mL was repeatedly tested 6 times. The double bond localization results were completely consistent with the theoretical structure, and the localization accuracy reached 100% (6 / 6). Attached Figure Description

[0021] Figure 1 The liquid chromatography-tandem mass spectrometry (LC-MS / MS) chromatogram obtained by the processing method described in Example 1 is shown below. Figure 2 The liquid chromatography-tandem mass spectrometry (LC-MS / MS) chromatogram obtained by the processing method described in Example 2 is shown below. Figure 3 The liquid chromatography-tandem mass spectrometry (LC-MS / MS) chromatogram obtained by the processing method described in Example 3 is shown below. Figure 4 The liquid chromatography-tandem mass spectrometry (LC-MS / MS) chromatogram obtained by the processing method described in Example 4 is shown below. Figure 5 The liquid chromatography-tandem mass spectrometry (LC-MS / MS) chromatogram obtained by the processing method described in Example 5 is shown below. Figure 6 Standard curves obtained for a series of oleic acid standard samples; Figure 7 Standard curves obtained for isoleic acid series standard samples; Figure 8 The liquid chromatography-tandem mass spectrometry detection chromatogram obtained by the processing method described in Comparative Example 1; Figure 9 The liquid chromatography-tandem mass spectrometry detection chromatogram obtained by the processing method described in Comparative Example 2; Figure 10 The liquid chromatography-tandem mass spectrometry detection chromatogram obtained by the processing method described in Comparative Example 3; Figure 11 The liquid chromatography-tandem mass spectrometry detection chromatogram obtained by the processing method described in Comparative Example 4; Figure 12 The image shows the liquid chromatography-tandem mass spectrometry (LC-MS) detection chromatogram obtained by the processing method described in Comparative Example 5. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments, thereby making its advantages and various effects more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the invention.

[0023] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0024] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0025] The main reagent information involved in the following embodiments is shown in Table 1.

[0026] Table 1 Summary of Main Reagent Information

[0027] Preparation of reagents for each embodiment: (1) Preparation of internal standard working solution for isotope-labeled fatty acids: Take appropriate amounts of 3177404 and 646083 respectively, dissolve and dilute them with acetonitrile to a final concentration of 10 μg / mL, mix them evenly, and store them at 4℃ in the dark.

[0028] (2) Preparation of working solution of double bond specific derivatization reagent: Weigh an appropriate amount of 2-acetylpyridine (A21204), dissolve and dilute it to 0.5 M with acetonitrile, store it in the dark, and take it out before use.

[0029] (3) Preparation of amino hydrophobic composite magnetic beads: a. Dissolve 10g FeCl3·6H2O in 150 mL of ethylene glycol, then add 9g ethylenediamine and 13g sodium acetate successively, react at 150~200℃ for 10h, filter the system to obtain magnetic nanoparticles with amino groups on the surface, wash with water 3 times and dry for later use. b. Take 1g of the magnetic nanoparticles prepared in step 1 and disperse them in 50 mL of anhydrous pyridine. Add 1 mL of n-octyltriethoxysilane and stir mechanically at room temperature for 18 h. Filter out the magnetic beads, wash them three times with water and ethanol, and disperse them in water for later use.

[0030] Example 1 This embodiment uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. The detection includes the following steps: 1) Sampling: Take 200 μL of serum sample and place it in a centrifuge tube.

[0031] 2) Add internal standard: Add 20 μL of isotope-labeled fatty acid internal standard (using the internal standard working solution prepared by 3177404) and vortex to mix.

[0032] 3) Magnetic bead enrichment: Add 50 μL of amino hydrophobic composite magnetic beads and shake at room temperature for 2-5 minutes to allow fatty acids to be fully adsorbed.

[0033] 4) Magnetic removal of impurities: Magnetic attraction for 30 seconds, discard the supernatant to remove interfering substances such as proteins, phospholipids, and salts.

[0034] 5) Washing: Add 200 μL of 80% methanol washing solution, wash twice, and discard the solution using a magnetic suction device.

[0035] 6) Double bond derivation: Add 50 μL of double bond-specific derivatizing reagent and, under light-protected conditions, use a power of 350 mW / cm². 2 Irradiating the sample with a 365 nm ultraviolet lamp at a distance of 2 cm caused the carbonyl group of 2-acetylpyridine to undergo a Pasterno-Büchi reaction with the carbon-carbon double bond of fatty acid under photoexcitation, generating an oxocyclic butane ring.

[0036] 7) Washing: Add 100 μL of acetonitrile eluent and elute by shaking for 1 minute.

[0037] 8) Sample injection and detection: The supernatant was injected into LC MS / MS. The chromatographic conditions for liquid chromatography included: a C18 reversed-phase column, column temperature 40℃, flow rate of 0.3–0.5 mL / min, and injection volume of 5–10 μL. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The gradient elution program was: 0–1 min, mobile phase B maintained at 5%; 1–5 min, mobile phase B linearly increased from 5% to 95%; 5–6 min, mobile phase B maintained at 95%; 6–8 min, mobile phase B decreased from 95% to 5%; and 8–10 min, mobile phase B maintained at 5%. Mass spectrometry acquisition conditions were: MRM mode. The monitored ion pairs included: oleic acid derivatives: parent ion m / z 357, daughter ions m / z 171 and m / z 185. The detection chromatogram is shown below. Figure 1 As shown, the chromatographic peaks obtained by the above detection method are symmetrical in shape, have high signal intensity, and low background interference.

[0038] Example 2 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared to Example 1, only step 6) increases the amount of double bond-specific derivatizing reagent added to 80 μL; the remaining steps are the same. The detection graph is shown below. Figure 2 As shown, increasing the double bond-specific derivatizing reagent to 80 μL has little effect on the obtained chromatographic peak shape, but impurity peaks begin to appear.

[0039] Example 3 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared to Example 1, only step 6) increases the amount of double bond-specific derivatizing reagent added to 100 μL; the remaining steps are the same. The detection graph is shown below. Figure 3 As shown, when the double bond-specific derivatizing reagent is increased to 100 μL, the second characteristic peak begins to weaken, while the first characteristic peak increases, and impurity peaks also begin to appear.

[0040] Example 4 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared to Example 1, only step 6) reduces the amount of double bond-specific derivatizing reagent added to 30 μL; the remaining steps are the same. The detection graph is shown below. Figure 4 As shown, when the amount of double bond-specific derivatizing reagent is reduced to 30 μL, the characteristic peak is still obvious, but the area of ​​the characteristic peak is relatively reduced and the peak shape is significantly broadened.

[0041] Example 5 This embodiment uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect isoleic acid in serum samples. The detection includes the following steps: 1) Sampling: Take 200 μL of serum sample and place it in a centrifuge tube.

[0042] 2) Add internal standard: Add 20 μL of isotope-labeled fatty acid internal standard (using the internal standard working solution prepared by 646083) and vortex to mix.

[0043] 3) Magnetic bead enrichment: Add 50 μL of amino hydrophobic composite magnetic beads and shake at room temperature for 2-5 minutes to allow fatty acids to be fully adsorbed.

[0044] 4) Magnetic removal of impurities: Magnetic attraction for 30 seconds, discard the supernatant to remove interfering substances such as proteins, phospholipids, and salts.

[0045] 5) Washing: Add 200 μL of 80% methanol washing solution, wash twice, and discard the solution using a magnetic suction device.

[0046] 6) Double bond derivation: Add 50 μL of double bond-specific derivatizing reagent and, under light-protected conditions, use a power of 350 mW / cm². 2 Irradiating the sample with a 365 nm ultraviolet lamp at a distance of 2 cm caused the carbonyl group of 2-acetylpyridine to undergo a Pasterno-Büchi reaction with the carbon-carbon double bond of fatty acid under photoexcitation, generating an oxocyclic butane ring.

[0047] 7) Washing: Add 100 μL of acetonitrile eluent and elute by shaking for 1 minute.

[0048] 8) Sample injection and detection: The supernatant was injected into LC MS / MS. The chromatographic conditions for liquid chromatography included: a C18 reversed-phase column, column temperature 40℃, flow rate of 0.3–0.5 mL / min, and injection volume of 5–10 μL. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The gradient elution program was as follows: 0–1 min, mobile phase B maintained at 5%; 1–5 min, mobile phase B linearly increased from 5% to 95%; 5–6 min, mobile phase B maintained at 95%; 6–8 min, mobile phase B decreased from 95% to 5%; and 8–10 min, mobile phase B maintained at 5%. Mass spectrometry acquisition conditions were: MRM mode. The monitored ion pairs included: isoleic acid derivatives: parent ion m / z 357, daughter ions m / z 185 and m / z 199. The detection chromatogram is shown below. Figure 5 As shown, the chromatographic peaks obtained by the above detection method are symmetrical in shape, have high signal intensity, and low background interference.

[0049] Example 6 To verify the accuracy of double bond positioning in the method of the present invention, standard solutions containing 10 ng / mL each of oleic acid (18:1, Δ9) and isoleic acid (18:1, Δ11) were prepared and processed and detected according to the methods shown in Examples 1 and 3, respectively, with 6 detections.

[0050] Mass spectrometry acquisition conditions: MRM mode, monitoring ion pairs including: Oleic acid derivatives: parent ion m / z 357, daughter ions m / z 171 and m / z 185; Isooleic acid derivatives: parent ion m / z 357, daughter ions m / z 185 and m / z 199.

[0051] The results are shown in Table 2: The main ion pair detected in the oleic acid standard was m / z 357→171 (abundance ratio > 10:1), with almost no detection of m / z 357→199; the main ion pair detected in the isoleic acid standard was m / z 357→199 (abundance ratio > 8:1), accompanied by m / z 357→185. Using the preset judgment rule (major diagnostic ion abundance ratio > 70%), the two isomers were correctly distinguished in all six replicate experiments, with a double bond localization accuracy of 100%. In our actual verification, we found that for C18:1 fatty acids with a double bond position of Δk, the diagnostic ion m / z value is 171 + 28 × (k - 9). For fatty acids with other carbon chain lengths, the diagnostic ion m / z value needs to be adjusted accordingly.

[0052] Table 2 Verification results of double bond localization accuracy (n=6)

[0053] Example 7 To verify the accuracy of double bond localization in the method of this invention, a series of standard solutions containing fatty acid standard solutions of oleic acid (18:1, Δ9) and isoleic acid (18:1, Δ11) at concentrations of 0.5, 1, 2, 5, 10, 20, and 50 ng / mL were prepared. An equal amount of internal standard (10 ng / mL) was added to each concentration. The solutions were processed and detected according to the methods described in Examples 1 and 3, respectively. A standard curve was plotted with the peak area ratio of the analyte to the internal standard as the ordinate and the concentration as the abscissa. (See [reference needed]). Figure 6 , Figure 7 It can be seen that its linear correlation coefficient R 2 All are greater than 0.99.

[0054] Comparative Example 1 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared with Example 1, only step 6) changes the amount of double bond-specific derivatizing reagent added to 10 μL; the other steps are the same. The detection graph is shown below. Figure 8 As shown, when the double bond-specific derivatizing reagent is reduced to 10 μL, the resulting chromatographic peaks have more impurities. Although the signal intensity is high, the background interference is large. It can be seen that insufficient double bond-specific derivatizing reagent will seriously affect the detection accuracy of the method of the present invention.

[0055] Comparative Example 2 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared with Example 1, only step 6) changes the amount of double bond-specific derivatizing reagent added to 120 μL; the other steps are the same. The detection graph is shown below. Figure 9As shown, when the double bond-specific derivatizing reagent is increased to 120 μL, the resulting chromatographic peaks have many impurities, the baseline is unstable, and although the signal intensity is high, the background interference is significant. This indicates that excessive double bond-specific derivatizing reagent will seriously affect the detection accuracy of the method of this invention.

[0056] Comparative Example 3 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry to detect oleic acid in serum samples. Compared with Example 1, only in step 3) the amino hydrophobic composite magnetic beads are replaced with BeaverBeads. TM MagNH2, the remaining steps are the same, and the detection diagram is as follows. Figure 10 As shown, it is evident that no clear target peak can be found, indicating that the choice of magnetic beads significantly affects the detection accuracy of the method of this invention.

[0057] Comparative Example 4 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared with Example 1, only step 5) replaces the 80% methanol washing solution with a 50% methanol washing solution; the other steps are the same. The detection graph is shown below. Figure 11 As shown, it is evident that it is almost impossible to find a clear target peak.

[0058] Comparative Example 5 This embodiment also uses a fatty acid double bond localization and quantification method based on liquid chromatography-mass spectrometry (LC-MS) to detect oleic acid in serum samples. Compared with Example 1, only in step 7) the acetonitrile eluent is replaced with 80% methanol washing solution; the other steps are the same. The detection graph is shown below. Figure 12 As shown, although there is a target peak, the peak shape shows obvious variations.

[0059] Finally, it should be noted that the terms "comprising," "including," or any other variations are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0060] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A method for the localization and quantitative detection of fatty acid double bonds based on liquid chromatography-mass spectrometry (LC-MS), characterized in that, Includes the following steps: (1) Take serum, plasma or tissue homogenate samples, place them in a centrifuge tube, add isotope-labeled fatty acid internal standard, and vortex to mix; (2) Add amino hydrophobic composite magnetic bead solution and shake at room temperature to allow fatty acids to be fully adsorbed on the surface of the magnetic beads. The amino hydrophobic composite magnetic beads are superparamagnetic core-shell structure magnetic beads that are covalently modified with amino groups and C8~C18 alkyl chains. (3) Use magnetic separation to remove the supernatant and remove interfering substances such as proteins, phospholipids and salts from the sample; (4) Add washing solution and perform magnetic suction washing to remove residual impurities; (5) Add a double bond-specific derivatizing agent, wherein the double bond-specific derivatizing agent is 2-acetylpyridine, and use it under light-protected conditions with a power of 300~500mW / cm. 2 Irradiate the sample with a 365 nm ultraviolet lamp at a distance of 1-3 cm and react at room temperature for 5 minutes to allow the derivatizing reagent to undergo the Pasternò-Büchi reaction with the carbon-carbon double bond of fatty acid. Under photoexcitation, the carbonyl group of 2-acetylpyridine undergoes [2+2] cycloaddition with the carbon-carbon double bond of fatty acid to generate an oxobutane ring structure. (6) Add elution buffer and elute by shaking, then collect the supernatant of the elution buffer; (7) The supernatant of the eluent is injected into a liquid chromatography-tandem mass spectrometry system for detection. During the mass spectrometry collision-induced dissociation process, the oxobutane ring generated in step (5) undergoes specific breakage, generating a pair of diagnostic ions directly related to the double bond position. The double bond position is determined based on the mass-to-charge ratio (m / z) of the diagnostic ions. Quantitative analysis is performed by constructing a standard curve using the isotope internal standard method.

2. The method as described in claim 1, characterized in that, In step (1), the sample volume is 200 μL, and the volume of the isotope-labeled fatty acid internal standard is 20 μL. The concentration of the isotope-labeled fatty acid internal standard is 5~20 μg / mL. The isotope-labeled fatty acid internal standard is a carbon-13 labeled or deuterium labeled fatty acid standard, and its carbon chain length and double bond number match the fatty acid to be tested.

3. The method as described in claim 1, characterized in that, In step (2), the volume of the amino hydrophobic composite magnetic bead solution added is 50 μL, the concentration of the magnetic beads is 5~20 mg / mL, and the particle size of the magnetic beads is 0.05~1 μm; the shaking adsorption time is 2~5 min.

4. The method as described in claim 3, characterized in that, The preparation process of the amino hydrophobic composite magnetic beads is as follows: a. Using ferric chloride, ethylenediamine, and sodium acetate in a molar ratio of 1:2~6:2~6 as raw materials and ethylene glycol as solvent, magnetic nanoparticles with amino groups on the surface are obtained by reacting at a temperature of 150~200℃. After washing with water, they are dried for later use. b. Disperse the magnetic nanoparticles prepared in step a in anhydrous pyridine, add silane with C8~C18 branches, the mass of silane being 0.5~2 times the mass of magnetic nanoparticles, mechanically stir at room temperature for 16~20h, filter out the magnetic beads, wash them multiple times with water and ethanol, and disperse them in water for later use.

5. The method as described in claim 1, characterized in that, The washing solution in step (4) is a methanol aqueous solution with a volume percentage concentration of 80%, and the washing is performed 2 to 4 times.

6. The method as described in claim 1, characterized in that, In step (5), the volume of the 2-acetylpyridine derivatizing reagent added is 50-80 μL, and the concentration of the 2-acetylpyridine derivatizing reagent is 0.1-1 M.

7. The method as described in claim 1, characterized in that, In step (6), the eluent is acetonitrile, and the elution is performed by shaking for 1-5 minutes.

8. The method as described in claim 1, characterized in that, In step (7), the chromatographic conditions of liquid chromatography-tandem mass spectrometry detection include: a C18 reversed-phase column, a column temperature of 40°C, a flow rate of 0.3-0.5 mL per minute, and an injection volume of 5-10 μL; mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, and the gradient elution program is: 0-1 min mobile phase B is maintained at 5%, 1-5 min mobile phase B is linearly increased from 5% to 95%, 5-6 min mobile phase B is maintained at 95%, 6-8 min mobile phase B is decreased from 95% to 5%, and 8-10 min mobile phase B is maintained at 5%; the mass spectrometry conditions are: electrospray ionization source, negative ion mode, and multiple reaction monitoring mode.

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