A mass spectrometry analysis method for compounds containing carbon-carbon double bonds

Abstract

The invention discloses a mass spectrometry analysis method for compounds containing carbon-carbon double bonds, comprising the following steps: a) subjecting light and heavy isotope labeling reagents to labeling reactions with compounds containing carbon-carbon double bonds in target analytes; b) subjecting the products after the labeling reactions to post-treatment and then subjecting them to mass spectrometry analysis; wherein the light isotope labeling reagent is [d0]-bis(pyridine)iodine tetrafluoroborate, and the heavy isotope labeling reagent is [d 10 ]-Bis(pyridine)iodine tetrafluoroborate. Experiments have shown that the present invention can not only significantly improve the mass spectrometry recognition of compounds containing carbon-carbon double bonds, but also has universality and a wide range of applications. It can be used for direct mass spectrometry analysis, liquid chromatography-mass spectrometry analysis, and can be used for quantitative analysis of compounds containing carbon-carbon double bonds in complex systems. It has important value and practicality for realizing rapid, efficient and accurate mass spectrometry analysis of compounds containing carbon-carbon double bonds in complex samples.

Description

Technical Field

[0001] The invention relates to a mass spectrometry analysis method for compounds containing carbon-carbon double bonds, belonging to the technical field of mass spectrometry analysis. Background Art

[0002] Biomarkers are objectively detected and evaluated, and can be used as indicators of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions. They have a wide range of uses, not only in exploring the pathogenesis at the molecular level, but also in accurately and sensitively evaluating early and low-level damage. They can provide a basis for early warning, prognosis and efficacy analysis, and accurate staging and typing of diseases, and can provide auxiliary diagnosis for clinicians to a large extent. Therefore, it is particularly important to be able to quickly and accurately perform qualitative and quantitative analysis on various biomarkers.

[0003] In addition, free unsaturated fatty acids are a very important class of energy metabolites and information molecules in organisms. They are not only a component of an important class of lipids, the main energy storage material, but also a signal molecule that regulates cell metabolism. In past studies, this type of compound has been shown to have a direct or indirect relationship with a variety of diseases, among which the difference in the content of free unsaturated fatty acids is more significant in cancer tissues and adjacent tissues. Therefore, studying a method that can quickly and accurately qualitatively and quantitatively analyze free unsaturated fatty acids in biological samples will play a very important role in studying physiological changes, disease diagnosis, and prognosis monitoring. However, modern detection and analysis methods have shown that the content of free fatty acids in the body only accounts for 5% of the total fatty acid content, and the content of free unsaturated fatty acids is relatively lower. At the same time, in the process of analysis and determination, in order to prevent the hydrolysis of lipids in the body to generate free unsaturated fatty acids, there are high requirements for tissue pretreatment and detection.

[0004] At present, the most commonly used method in clinical practice that can accurately detect free unsaturated fatty acids is the enzymatic method, but this enzymatic method requires the preparation of specific markers and antibodies for each component, which has the disadvantages of being costly, time-consuming and labor-intensive. In addition, the commonly used method in the laboratory is to convert free unsaturated fatty acids into fatty acid methyl esters and then perform GC-MS detection, but due to the low reaction efficiency of the methylation reagent and the harsh reaction conditions, this method is not ideal.

[0005] Therefore, developing a mass spectrometry analysis method for compounds containing carbon-carbon double bonds, especially a mass spectrometry analysis method that can achieve rapid, efficient and accurate analysis, will be of great help in studying the biological significance of free unsaturated fatty acids, and can provide a convenient way for high-throughput research on small molecule compounds containing carbon-carbon double bonds and mass spectrometry analysis of small molecule compounds containing carbon-carbon double bonds in complex matrix samples. Summary of the invention

[0006] In view of the above problems and needs in the prior art, an object of the present invention is to provide a mass spectrometry analysis method for compounds containing carbon-carbon double bonds that can be achieved quickly, efficiently and accurately.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0008] A mass spectrometry analysis method for compounds containing carbon-carbon double bonds comprises the following steps:

[0009] a) allowing light and heavy isotope labeling reagents to undergo labeling reactions with compounds containing carbon-carbon double bonds in target analytes;

[0010] b) performing post-treatment on the product after the labeling reaction and then performing mass spectrometry analysis;

[0011] Among them: the light isotope labeling reagent is [d0]-bis(pyridine) tetrafluoroborate iodine, and the heavy isotope labeling reagent is [d 10 ]-bis(pyridine)iodine tetrafluoroborate, the [d0]-bis(pyridine)iodine tetrafluoroborate and [d 10 The chemical structural formulas of ]-bis(pyridine)iodine tetrafluoroborate are as follows:

[0012]

[0013] In a preferred embodiment, the compound containing a carbon-carbon double bond is any one or more of a fatty acid containing a carbon-carbon double bond, a fatty alcohol containing a carbon-carbon double bond, a fatty acid ester containing a carbon-carbon double bond, or a terpene compound containing a carbon-carbon double bond.

[0014] In a preferred embodiment, in step a), dichloromethane is used as the reaction solvent for the labeling reaction.

[0015] In a preferred embodiment, in step a), the labeling reaction is carried out on a shaker at room temperature for 1 to 3 hours.

[0016] In a preferred embodiment, in step a), the labeling reaction is quenched with an aqueous sodium thiosulfate solution.

[0017] In a preferred embodiment, in step b), the product after the labeling reaction is first dried with nitrogen, then redissolved with a methanol solution containing 0.1 wt % formic acid, and then subjected to mass spectrometry analysis.

[0018] In a preferred embodiment, the mass spectrometry analysis is LC-MS / MS tandem mass spectrometry analysis.

[0019] In a further preferred embodiment, the spectra of the LC-MS / MS tandem mass spectrometry analysis are collected in positive ion mode.

[0020] In one implementation, the lightly and heavily labeled target analytes are fragmented in a collision cell at a certain collision energy, and the MRM mode is used to select m / z 206 and m / z 211 as quantitative ions for MRM quantitative analysis.

[0021] In a preferred embodiment, the collision energy is 10 eV to 25 eV.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are:

[0023] Experimental results show that the present invention creatively selects [d0]-bis(pyridine) tetrafluoroborate iodine as a light isotope labeling reagent and [d 10 ]-bis(pyridine)iodine tetrafluoroborate is a heavy isotope labeling reagent used to specifically label the carbon-carbon double bond of the analyte, which not only reduces the complexity of mass spectrometry analysis of compounds containing carbon-carbon double bonds to a certain extent, but also significantly improves the mass spectrometry recognition of the analyte, and the isotope labeling reagent has good substrate compatibility and functional group tolerance, and can be labeled with a variety of substrates such as alcohols, carboxylic acids, esters containing carbon-carbon double bonds, and has good labeling effects, strong universality, and a wide range of applications;

[0024] Furthermore, the labeling reaction conditions are simple and mild, and can be performed at room temperature without the need for anhydrous and oxygen-free operation, and can be widely used in various occasions;

[0025] In addition, the method of the present invention can be applied to both direct mass spectrometry analysis and liquid chromatography combined with mass spectrometry analysis, which can effectively detect more low-abundance analytes, greatly improving the selectivity and practicality of the analysis;

[0026] In particular, by using the method of the present invention, the linear correlation coefficient R2 of the quantitative standard curve can be greater than 0.999, the linear range spans two orders of magnitude, and different samples can be analyzed. The intercept b is more than one order of magnitude smaller than the slope k, indicating that the quantitative method is less affected by factors such as the matrix, and can be used for the quantitative analysis of compounds containing carbon-carbon double bonds in complex systems.

[0027] Therefore, compared with the prior art, the present invention not only produces significant progress, but also produces unexpected technical effects, and has important value and practicality for achieving rapid, efficient and accurate mass spectrometry analysis of compounds containing carbon-carbon double bonds in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The mass spectrum obtained by analyzing palmitoleic acid using the mass spectrometry method described in Example 1 of the present invention;

[0029] Figure 2 The mass spectrum obtained by analyzing cis-10-heptadecenoic acid using the mass spectrometry analysis method described in Example 1 of the present invention;

[0030] Figure 3 The mass spectrum obtained by analyzing vaccenic acid using the mass spectrometry method described in Example 1 of the present invention;

[0031] Figure 4 is a mass spectrum obtained by analyzing oleic acid using the mass spectrometry analysis method described in Example 1 of the present invention;

[0032] Figure 5 The mass spectrum obtained by analyzing linoleic acid using the mass spectrometry method described in Example 1 of the present invention;

[0033] Figure 6 is a mass spectrum obtained by analyzing α-linolenic acid using the mass spectrometry analysis method described in Example 1 of the present invention;

[0034] Figure 7 This is a mass spectrum obtained by analyzing arachidonic acid using the mass spectrometry analysis method described in Example 1 of the present invention;

[0035] Figure 8 The mass spectrum obtained by analyzing erucic acid using the mass spectrometry method described in Example 1 of the present invention;

[0036] Fig. 9 The mass spectrum obtained by analyzing nervonic acid using the mass spectrometry analysis method described in Example 1 of the present invention;

[0037] Fig.10 is a mass spectrum obtained by analyzing oleyl alcohol using the mass spectrometry analysis method described in Example 1 of the present invention;

[0038] Fig.11 The mass spectrum obtained by analyzing methyl erucate using the mass spectrometry method described in Example 1 of the present invention;

[0039] Fig.12 is a tandem mass spectrum of the oleic acid light labeled product obtained in Example 2 of the present invention;

[0040] Fig.13 is a tandem mass spectrum of the oleic acid heavy labeled product obtained in Example 2 of the present invention;

[0041] Fig.14 is a tandem mass spectrum of the light labeled product of linoleic acid obtained in Example 2 of the present invention;

[0042] Fig.15is a tandem mass spectrum of the linoleic acid heavy-mass labeled product obtained in Example 2 of the present invention;

[0043] Fig.16 is a tandem mass spectrum of the oleyl alcohol light labeled product obtained in Example 2 of the present invention;

[0044] Fig.17 is a tandem mass spectrum of the oleyl alcohol heavy labeled product obtained in Example 2 of the present invention;

[0045] Fig.18 is a tandem mass spectrum of the light labeled product of methyl erucate obtained in Example 2 of the present invention;

[0046] Fig.19 It is a tandem mass spectrum of the heavy-mass labeled product of methyl erucate obtained in Example 2 of the present invention. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described in detail and completely below in conjunction with the accompanying drawings and embodiments.

[0048] [d0]- or [d 10 ]-Bis(pyridine)iodine tetrafluoroborate (abbreviated as [d0]- / [d 10 ]-IPy2BF4) is prepared as follows:

[0049]

[0050] Dilute an aqueous solution of tetrafluoroboric acid (48 wt%, 1.90 mL, 14.5 mmol) with 15.0 mL of deionized water, and add silver carbonate (2.00 g, 7.25 mmol) in portions to the solution at room temperature while stirring;

[0051] After the addition of silver carbonate is complete, stir the mixture at room temperature until the solid in the solution is completely dissolved;

[0052] After the solution is clarified, silica gel (20-200 mesh, 3.00 g) is added to the reaction system, and then the reaction is continued to be vigorously stirred at room temperature. After the reaction is completed, the solvent in the reaction system is removed by rotary evaporation at 55° C. and 30 mmHg, and the silica gel is completely in a flowing powder state to obtain silica gel-supported silver tetrafluoroborate;

[0053] Subsequently, the obtained solid was dispersed in 80 mL of dichloromethane, and pyridine (2.25 mL, 28.0 mmol) and iodine (3.55 g, 14.0 mmol) were added in sequence at room temperature with vigorous stirring. After reacting at room temperature for 2 hours, the mixture was directly filtered, and the filter residue was washed twice with dichloromethane (30 mL×2). The organic phases were combined and the solvent was removed by rotary evaporation at 35° C. and 100 mmHg to obtain a red solid. The solid was recrystallized from dichloromethane and ether at room temperature (DCM / Et2O=1 / 2, 90 mL) to obtain a pale solid: [d0]-bis(pyridine)iodine tetrafluoroborate (2.71 g, 52%);

[0054] Deuterated pyridine (2.24 mL, 28.0 mmol) was used to replace the pyridine in the above step to synthesize [d 10 ]-Bis(pyridine)iodine tetrafluoroborate (2.79 g, 52%).

[0055] Example 1

[0056] A mass spectrometry analysis method for compounds containing carbon-carbon double bonds comprises the following steps:

[0057] 1) preparing a series of dichloromethane solutions of compounds containing carbon-carbon double bonds (in this embodiment, palmitoleic acid, cis-10-heptadecenic acid, vaccenic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, erucic acid, nervonic acid, oleyl alcohol, erucic acid methyl ester, but not limited to these compounds, and can be any one or more of fatty acids containing carbon-carbon double bonds, fatty alcohols containing carbon-carbon double bonds, fatty acid esters containing carbon-carbon double bonds, or terpenoid compounds containing carbon-carbon double bonds), and the concentration of each solution is 1 mg / mL;

[0058] 2) Take 10 μL of each of the above solutions into different derivatization bottles, blow dry with nitrogen gas and set aside;

[0059] 3) 100 μL of 500 μg / mL [d0]-IPy2BF4 and 500 μg / mL [d 10 ]-IPy2BF4 in a methylene chloride mixed solution with a mass ratio of 1:1, and then vortexed at room temperature for 1 minute, and then labeled on a shaker at room temperature for 2 hours;

[0060] 4) 100 μL of 10% sodium thiosulfate aqueous solution was added to the above-mentioned labeling reaction systems to quench the labeling reaction, and then the lower organic phase solution was collected, dried with nitrogen, and redissolved in 100 μL of methanol containing 0.1 wt% formic acid;

[0061] 5) Perform LC-MS / MS analysis, wherein the analysis conditions of LC-MS / MS are as follows:

[0062] Agilent UPLC InfinityLab Poroshell 120HILIC, 4.6×5 mm, 2.7 μm, USA;

[0063] Agilent Zorbax HPLC Plus Narrow Bore RR, 2.1×100mm, 3.5μm, USA;

[0064] Agilent high performance liquid chromatography (HPLC 1260 Series), mass spectrometers are Agilent 6460A and Agilent 6560A, spray pressure is 30psi, nebulizing gas temperature is 350°C, voltage is 3500V; mobile phase is: phase A is 0.1wt% formic acid aqueous solution, phase B is 0.1wt% formic acid acetonitrile solution; elution gradient is: 0-3min, phase B 90%; 3-10min, phase B 90%-60%; 10-17min, phase B 60%; 17-19min, phase B 60%-90%; flow rate is 0.3mL / min, injection volume is 3μL.

[0065] Figures 1 to 11 The mass spectra are obtained by mass spectrometry analysis of compounds containing carbon-carbon double bonds (palmitoleic acid, cis-10-heptadecenic acid, vaccenic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, erucic acid, nervonic acid, oleyl alcohol, methyl erucate) using the mass spectrometry analysis method described in this embodiment, and then light and heavy labeling is performed. Figures 1 to 11 As shown in the spectrum, it can be seen that the [d0]-bis(pyridine) tetrafluoroborate iodine described in the present invention is used as a light isotope labeling reagent and [d 10 ]-Bis(pyridine)iodine tetrafluoroborate is a heavy isotope labeling reagent, which can specifically label the carbon-carbon double bond of the analyte and greatly improve the mass spectrometry response of the target molecule. Since the molecular weight is increased by about 200Da after derivatization, the mass spectrometry signals of the derivatization reagent and the derivatization product can be clearly distinguished; and the molecular weight of the light and heavy derivatization products is exactly 5Da different, showing a group of signals with a mass-to-charge ratio difference of 5 in the mass spectrum, which is very easy to identify; in addition, the signal intensity of the light and heavy derivatization products of each compound is close to 1:1, indicating that the reactivity of the light and heavy derivatization reagents is basically the same and there is no interference between them; in particular, the derivatization reaction is compatible with a variety of groups including hydroxyl, ester, and carboxyl groups, showing good functional group tolerance, and can be applied to the mass spectrometry analysis of various compounds containing carbon-carbon double bonds, such as alcohols, carboxylic acids, esters, etc., with strong universality and a wide range of applications.

[0066] Example 2

[0067] 1) Prepare dichloromethane solutions of oleic acid, linoleic acid, oleyl alcohol, and methyl erucate respectively, with the concentration of each solution being 1 mg / mL;

[0068] 2) Take 10 μL of each of the above solutions into different derivatization bottles, and take 2 portions of the dichloromethane solution of each compound at the same time, blow dry with nitrogen gas and set aside;

[0069] 3) Add 100 μL of 1000 μg / mL [d0]-IPy2BF4 dichloromethane solution to one substrate of the same compound after nitrogen drying, and add 100 μL of 1000 μg / mL [d0]-IPy2BF4 dichloromethane solution to another substrate of the same compound. 10 ]-IPy2BF4 dichloromethane solution, and then vortexed at room temperature for 1 minute, and then the labeling reaction was carried out on a shaker at room temperature for 2 hours;

[0070] 4) 100 μL of 10% sodium thiosulfate aqueous solution was added to the above-mentioned labeling reaction systems to quench the labeling reaction, and then the lower organic phase solution was collected, dried with nitrogen, and redissolved in 100 μL of methanol containing 0.1 wt% formic acid;

[0071] 5) performing LC-MS / MS analysis, wherein the analysis conditions of LC-MS / MS are the same as those described in Example 1;

[0072] Fig.12 is the tandem mass spectrum of the oleic acid light labeled product obtained in this example, Fig.13 is a tandem mass spectrum of the oleic acid heavy labeled product obtained in this example; combined with Fig.12 and Fig.13 As shown, the sequences are pyridine plus proton (m / z=80, 85), monopyridinium iodonium ion (m / z=206, 211) and a pair of characteristic cations of unsaturated fatty acid head group fragmentation (m / z=M-19, M-37, where M is the molecular weight of substrate unsaturated fatty acid) in descending order of mass-to-charge ratio. The signals in the tandem mass spectra of light and heavy derivatization products are strong, and the fragment ions have obvious mass labels, which are very easy to identify. Since oleic acid represents a monounsaturated fatty acid containing a carbon-carbon double bond, it is shown that the isotope labeling reagent of the present invention has a good labeling effect on monounsaturated fatty acids containing a carbon-carbon double bond and has good mass spectrometry recognition.

[0073] Fig.14 is the tandem mass spectrum of the linoleic acid light labeled product obtained in this example, Fig.15 is a tandem mass spectrum of the heavy labeled product of linoleic acid obtained in this example; combined with Fig.14 and Fig.15As shown, in the tandem mass spectrum of linoleic acid after derivatization with the isotope labeling reagent, in addition to retaining the fragment information of the mass label of the monounsaturated fatty acid derivatization product, there is also a strong cation signal of the polyunsaturated fatty acid substrate minus the hydrogen anion (m / z=M'-1, where M' is the molecular weight of the substrate polyunsaturated fatty acid); because linoleic acid represents a polyunsaturated fatty acid containing a carbon-carbon double bond, it is shown that the isotope labeling reagent described in the present invention also has a good labeling effect on polyunsaturated fatty acids containing a carbon-carbon double bond, and has good mass spectrometry recognition.

[0074] Fig.16 is the tandem mass spectrum of the light labeled product of oleyl alcohol obtained in this example, Fig.17 is the tandem mass spectrum of the heavy labeled product of oleyl alcohol obtained in this example; Fig.16 and Fig.17 It can be seen that the isotope labeling reagent of the present invention also has a good labeling effect on unsaturated fatty alcohols containing carbon-carbon double bonds and has good mass spectrometry recognition.

[0075] Fig.18 is the tandem mass spectrum of the light labeled product of methyl erucate obtained in this example, Fig.19 is the tandem mass spectrum of the heavy labeled product of methyl erucate obtained in this example, Fig.18 and Fig.19 It can be seen that the isotope labeling reagent described in the present invention also has a good labeling effect on unsaturated fatty acid esters containing carbon-carbon double bonds and has good mass spectrometry recognition.

[0076] And, by Fig.12 , 14 , 16, 18 and Fig.13 , 15 , 17, 19 also show that: the reporter ions used in the MRM analysis of the light isotope-labeled derivatization products described in the present invention are all fragment ions with a mass-to-charge ratio of 206, while the reporter ions used in the MRM analysis of the heavy isotope-labeled derivatization products described in the present invention are all fragment ions with a mass-to-charge ratio of 211.

[0077] Example 3

[0078] The operation of using the mass spectrometry method of the present invention to quantitatively analyze compounds containing carbon-carbon double bonds is as follows:

[0079] 1. Prepare a series of compounds containing carbon-carbon double bonds (in this embodiment, palmitoleic acid, cis-10-heptadecenic acid, vaccenic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, erucic acid, nervonic acid, oleyl alcohol, methyl erucate, but not limited to these compounds, and can be any one or more of fatty acids containing carbon-carbon double bonds, fatty alcohols containing carbon-carbon double bonds, fatty acid esters containing carbon-carbon double bonds, or terpenoid compounds containing carbon-carbon double bonds) in dichloromethane solutions, the concentration of each solution is 10 nmol / mL, take 500 μL of each solution in different derivatization bottles, blow dry with nitrogen for later use;

[0080] 2. Add a) 500 μL of 1000 nmol / mL [d0]-IPy2BF4 dichloromethane solution; b) 500 μL of 1000 nmol / mL [d 10 ]-IPy2BF4 dichloromethane solution; c) 500 μL of 1000 nmol / mL [d0]-IPy2BF4 and 1000 nmol / mL [d 10 ]-IPy2BF4 mixed solution of dichloromethane in a mass ratio of 1:1;

[0081] 3. After vortexing for 1 min at room temperature, labeling reaction was performed on a shaker at room temperature for 2 h;

[0082] 4. After the reaction is completed, 200 μL of 10% sodium thiosulfate aqueous solution is used to quench the labeling reaction, and the lower organic phase solution is collected, dried with nitrogen, and then redissolved in 500 μL of methanol containing 0.1 wt% formic acid;

[0083] 5. Accurately weigh about 0.1 mmol of standard samples of the above 11 compounds of different types into different derivatization bottles, and add 1.0 eq. 0.1 mmol / mL of [d 10 ]-bis(pyridine) tetrafluoroborate iodine dichloromethane solution, labeling reaction was carried out at room temperature for 2h. After the reaction was completed, 1mL of 10% sodium thiosulfate aqueous solution was added to quench the labeling reaction, the aqueous phase was extracted three times with 2mL of dichloromethane, and then the organic phases were combined, the organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to concentrate, and the derivatized product was purified by thin layer chromatography, and the purified product was dissolved in dichloromethane to prepare a 1mg / mL (based on the derivatized substrate, the same below) deuterated standard internal standard stock solution;

[0084] 6. The concentration of the standard samples of the above 11 compounds was gradually increased from 1 pg / mL, and a derivatization labeling reaction was performed. The MRM detection was performed by HPLC-ESI-TSQ-MS. The software MassHunter WorkstationAgtQual in the instrument can automatically calculate the signal-to-noise ratio (SNR). The concentration corresponding to SNR ≥ 3 was used as the concentration of the minimum detection limit (LOD) after the standard was derivatized; the concentration corresponding to SNR ≥ 10 was used as the concentration of the minimum quantitative detection limit (LOQ) after the standard was derivatized. The experiment was repeated three times for each concentration.

[0085] 7. The concentration of the standard samples of the above 11 compounds was gradually increased from 1 ng / mL, and SIM detection was performed using HPLC-ESI-TSQ-MS in negative ion mode. The software MassHunter Workstation AgtQual in the instrument can automatically calculate the signal-to-noise ratio (SNR). The concentration corresponding to SNR ≥ 3 was used as the minimum detection limit (LOD) concentration of the standard sample for direct detection. The experiment was repeated three times for each concentration.

[0086] 8. For the above different compounds, according to the difference in the minimum quantitative detection limit (LOQ) concentration after derivatization, starting from the sample concentration n of 5-10 times the LOQ, 500 μL of standard samples with sample concentrations of n, 5n, 10n, 20n, 40n, 60n, and 100n were prepared in sequence. After nitrogen drying, 500 μL of 1000nmol / mL [d0]-IPy2BF4 dichloromethane solution was added, and the labeling reaction was carried out at room temperature for 2h. After the reaction, 1mL of 10% sodium thiosulfate aqueous solution was used to quench the labeling reaction, and the lower organic phase solution was collected. 500 μL of a deuterated standard internal standard stock solution with a concentration of 10n was added, and the solution was dried by nitrogen and redissolved in 500 μL of methanol containing 0.1wt% formic acid, and MRM detection was performed by HPLC-ESI-TSQ-MS;

[0087] 9. Draw the y=ax+b standard curves respectively, where y is the ratio of the integrated peak areas of the light and heavy derivatization products in the MRM mode (d0 / d5), x represents the sample concentration, and the quantitative range given in Table 1 is the sample concentration range. Then, the data processing software (MassHunter Workstation AgtQual and OriginPro, Version 2021, OriginLabCorporation, Northampton, MA, USA.) can automatically generate the standard curve through linear regression without forcing the origin;

[0088] 10. The analysis conditions of HPLC-ESI-TSQ-MS were as follows: Agilent UPLC InfinityLabPoroshell 120HILIC, 4.6×5mm, 2.7μm, USA; Agilent Zorbax HPLC Plus Narrow BoreRR, 2.1×100mm, 3.5μm, USA; Agilent high performance liquid chromatography (HPLC 1260 Series), mass spectrometers are Agilent6460A and Agilent6560A; spray pressure is 30psi, nebulizing gas temperature is 350°C, voltage is 3500V; mobile phase is: phase A is 0.1wt% formic acid aqueous solution, phase B is 0.1wt% formic acid acetonitrile solution; elution gradient is: 0-3min, phase B 90%; 3-10min, phase B 90%-60%; 10-17min, phase B 60%; 17-19min, phase B 60%-90%; flow rate is 0.3mL / min, injection volume is 3μL.

[0089] Table 1 shows the quantitative standard curves and detection limits obtained for the 11 standard compounds.

[0090] Table 1 Quantitative standard curves and detection limits of 11 standard compounds

[0091]

[0092]

[0093] Note: [a] The standard curve is the relationship between the standard sample concentration (x) and the ratio of the integrated peak area of ​​the light and heavy derivatized products in the MRM mode (y), where x is the sample concentration × 10 / (μg·mL -1 ); [b] the concentration of heavy internal standard was 0.005 μg / mL; [c] the concentration of heavy internal standard was 0.02 μg / mL.

[0094] From the results shown in Table 1, it can be seen that: by adopting the method of the present invention, the linear correlation coefficient R2 of the quantitative standard curve of the compound containing carbon-carbon double bond can be greater than 0.999, the linear range spans two orders of magnitude, and different samples can be analyzed, and the intercept b is more than one order of magnitude smaller than the slope k, indicating that the quantitative method of the present invention is less interfered by factors such as the matrix, and can be used for the quantitative analysis of compounds containing carbon-carbon double bonds in complex systems; in addition, by comparing the changes in the detection limit before and after derivatization, it can be seen that the method of the present invention can reduce the minimum detection limit of the compound containing carbon-carbon double bond by 2-3 orders of magnitude, greatly improving the detection efficiency and detection accuracy, and can meet the detection requirements in many different situations. It has important value and practicality for realizing fast, efficient and accurate mass spectrometry analysis of compounds containing carbon-carbon double bonds in complex samples.

[0095] Finally, it is necessary to point out here that the above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto, and any changes or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A mass spectrometry method for compounds containing carbon-carbon double bonds, comprising the following steps: a) allowing light and heavy isotope labeling reagents to undergo labeling reactions with compounds containing carbon-carbon double bonds in target analytes; b) performing post-treatment on the product after the labeling reaction and then performing mass spectrometry analysis; Features: The light isotope labeling reagent is [d0]-bis(pyridine)iodine tetrafluoroborate, and the heavy isotope labeling reagent is [d 10 ]-bis(pyridine)iodine tetrafluoroborate, the [d0]-bis(pyridine)iodine tetrafluoroborate and [d 10 The chemical structural formulas of ]-bis(pyridine)iodine tetrafluoroborate are as follows:

2. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: The compound containing a carbon-carbon double bond is any one or more of a fatty acid containing a carbon-carbon double bond, a fatty alcohol containing a carbon-carbon double bond, a fatty acid ester containing a carbon-carbon double bond, or a terpene compound containing a carbon-carbon double bond.

3. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: In step a), dichloromethane is used as the reaction solvent for the labeling reaction.

4. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: In step a), the labeling reaction is carried out on a shaker at room temperature for 1 to 3 hours.

5. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: In step a), the labeling reaction is quenched with an aqueous sodium thiosulfate solution.

6. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: In step b), the product after the labeling reaction is first dried with nitrogen, then redissolved with a methanol solution containing 0.1 wt % formic acid, and then subjected to mass spectrometry analysis.

7. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: The mass spectrometry analysis is LC-MS / MS tandem mass spectrometry analysis.

8. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 7, characterized in that: The spectra of the LC-MS / MS tandem mass spectrometry analysis were collected in positive ion mode.

9. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 1, characterized in that: The lightly and heavily labeled target analytes were fragmented in the collision cell at a certain collision energy, and the MRM mode was used to select m / z 206 and m / z 211 as quantitative ions for MRM quantitative analysis.

10. The mass spectrometry analysis method for compounds containing carbon-carbon double bonds according to claim 9, characterized in that: The collision energy is 10 eV to 25 eV.

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