Method for simultaneously identifying double-bond position, double-bond cis-trans configuration and fat acyl chain sn-position of double bond of unsaturated lipid

Abstract

The invention discloses a method for simultaneously identifying unsaturated lipid double-bond position isomerization, double-bond cis-trans-configuration and sn-position of a fatty acyl chain where double bonds are located, carbon-carbon double bonds are derived and converted into a product of an aza-ternary ring structure, and double-bond cis-trans isomers and sn-position isomers can be distinguished through liquid chromatography separation. The double-bond position in the double-bond position isomer can be judged through a tandem mass spectrometry method; the related aziridine acylation reaction is simple in reaction condition, fast in reaction and high in yield, and the problem of simultaneous identification of double bond position isomerization, double bond cis-trans isomerization and sn-position isomerization of a fatty acyl chain where double bonds are located of unsaturated lipid can be solved.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond and the sn-position of a fatty acyl chain where the double bond is located of an unsaturated lipid. Background Art

[0002] Lipids are one of the important components of organisms. There are a large number of isomers in organisms. The diversity of lipid structures gives them a variety of important biological and physical functions. Abnormal metabolism of lipid compounds is closely related to the occurrence and development of many major diseases. Therefore, the development of new methods for lipid isomer analysis has important biological significance and clinical value for clarifying its changes in the occurrence and development of diseases and the network mechanism of its metabolic regulation.

[0003] With the development of modern mass spectrometry technology, the structures of these lipid compounds can be systematically identified and analyzed. However, the current lipidomics analysis based on conventional mass spectrometry technology cannot resolve the double bond position and cis-trans configuration of unsaturated lipids, as well as the sn-position of the fatty acyl chain where the double bond is located. This is mainly because in the traditional lipidomics analysis workflow, conventional tandem mass spectrometry technology is used, which cannot break the target compound to produce characteristic ions related to the carbon-carbon double bond structure, including the sn-position.

[0004] In response to the above technical problems, various chemical derivatization strategies for carbon-carbon double bonds have been developed, including ozone decomposition, -Büchi reaction (PB) and epoxidation reaction form products containing oxygen heterocyclic structures, and reverse electron demand Diels-Alder reaction and aziridinization derivatization form products containing nitrogen heterocyclic structures. Mass spectrometry analysis of these derivatives can determine the double bond position of unsaturated lipids. However, there are still challenges in the multidimensional analysis of lipid isomerism. During the PB reaction, the cis-trans configuration of the double bond of unsaturated lipids will undergo mutual conversion. Combined with the chromatographic retention rules, only the double bond position and cis-trans configuration of unsaturated lipids can be analyzed simultaneously. The identification of the sn-position of the fatty acyl chain is very difficult, and even requires the use of special three-stage tandem mass spectrometry fragmentation analysis, including ozone-induced dissociation, ultraviolet light dissociation, and specific fragmentation induced by phosphoester metal ion complexes. However, there is currently no analytical strategy that can achieve multidimensional analysis of unsaturated lipids. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond and the sn-position of the fatty acyl chain where the double bond is located in an unsaturated lipid in view of the deficiencies in the prior art.

[0006] In one aspect of the present invention, the present invention provides a method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond, and the sn-position of the fatty acyl chain where the double bond is located of an unsaturated lipid. According to an embodiment of the present invention, the method comprises the following steps:

[0007] 1) dissolving a substrate containing an unsaturated double bond structure, a double bond cis-trans configuration and a sn-position structure, a reaction reagent containing a hydroxylamine structure and a catalyst in a solvent, and performing a mixing treatment to obtain a reaction solution to be detected;

[0008] 2) injecting the reaction solution to be detected into a mass spectrometer, and after chromatographic separation and ionization by an ion source, further undergoing mass spectrometry-induced collision dissociation and fragmentation to obtain characteristic fragment ions;

[0009] 3) Analyze and compare the product chromatographic behaviors of the reaction solution to be tested containing a double bond cis-trans isomer pair, and deduce the double bond cis-trans configuration of the unsaturated lipid;

[0010] 4) analyzing and comparing the product chromatographic behaviors of the reaction solution to be tested containing the sn-position isomer pair, and deducing the sn-position of the fatty acyl chain where the double bond is located;

[0011] 5) Analyze the characteristic fragment ions described in step 2) to reversely deduce the position of the unsaturated double bond. This method is simple to operate and can simultaneously identify the double bond position, double bond cis-trans configuration and sn-position of the fatty acyl chain where the double bond is located.

[0012] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the solvent is trifluoroethanol or hexafluoroisopropanol or a mixed solution thereof with other solvents.

[0014] According to an embodiment of the present invention, the substrate is a lipid compound.

[0015] According to an embodiment of the present invention, the lipid compound is unsaturated glycerophospholipid.

[0016] According to an embodiment of the present invention, the hydroxylamine-containing reaction reagent is O-(sulfonyl)hydroxylamine, hydroxylamine-O-sulfonic acid, O-(2,4-dinitrophenyl)hydroxylamine or their corresponding derivatives.

[0017] According to an embodiment of the present invention, the catalyst is a rhodium catalyst.

[0018] According to an embodiment of the present invention, the catalyst is Rh(esp)2 or other rhodium catalysts.

[0019] According to an embodiment of the present invention, the molar ratio of the hydroxylamine-containing reaction reagent to the substrate is (0.01-100):1.

[0020] According to an embodiment of the present invention, the content of the catalyst is 0.1 to 10 (molar ratio) %.

[0021] According to an embodiment of the present invention, the mixing process takes longer than 10 seconds.

[0022] According to an embodiment of the present invention, the analysis step in step 3) is to compare and analyze the chromatographic elution time of the double bond cis-trans isomers to the product based on the liquid chromatogram of the solution to be detected obtained in step 1), so as to determine the cis-trans configuration of the substrate containing the unsaturated double bond cis-trans isomer structure.

[0023] According to an embodiment of the present invention, the analysis step in step 4) is to compare and analyze the chromatographic elution time of the product of the sn-position isomer pair of the fatty acyl chain where the unsaturated lipid double bond is located based on the liquid chromatogram of the solution to be detected obtained in step 1), so as to determine the sn-position of the fatty acyl chain where the unsaturated lipid double bond is located.

[0024] According to an embodiment of the present invention, in step 5), the characteristic fragment ions are analyzed, including, according to the secondary mass spectrum in step 2), analyzing and obtaining the molecular formula of the theoretical secondary fragment ions, comparing it with the product molecular formula of the substrate containing an unsaturated double bond structure or the reaction solution to be detected obtained in step 1), and combining the unsaturation of the characteristic ions to reversely deduce the position of the double bond.

[0025] In another aspect of the present invention, the present invention also provides a method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond and the sn-position of the fatty acyl chain where the double bond is located of an unsaturated lipid. According to an embodiment of the present invention, the method comprises the following steps:

[0026] S1) dissolving a substrate containing an unsaturated double bond, a reaction reagent containing a hydroxylamine structure and a catalyst in trifluoroethanol or hexafluoroisopropanol or other mixed solvents, and stirring to obtain a solution to be detected;

[0027] S2) mixing the solution to be detected in step S1) to obtain a reaction product;

[0028] S3) injecting the reaction product obtained in step S2) into a mass spectrometer, and after chromatographic separation and ionization by an ion source, further mass spectrometry-induced collision dissociation and fragmentation to obtain characteristic fragment ions;

[0029] S4) analyzing and comparing the chromatographic behaviors of the products of the substrates containing the unsaturated double bond cis and trans structures described in step S2) to deduce the cis and trans configurations of the unsaturated lipid double bonds;

[0030] S5) analyzing and comparing the chromatographic behaviors of the products of the substrates containing the sn-1 and sn-2 isomeric structures of the fatty acyl chains where the unsaturated double bonds are located in step S2), and deducing the sn-position of the fatty acyl chains;

[0031] S6) analyzing the characteristic fragment ions described in step S3) to reversely deduce the position of the unsaturated double bond.

[0032] In one or more embodiments of the present invention, in step S1), the substrate containing unsaturated double bonds is a lipid compound containing unsaturated double bonds and cis-trans isomers and having a sn-stereoconfiguration. Specifically, the substrate containing unsaturated double bonds, cis-trans isomers and sn-stereoconfiguration is a lipid compound such as unsaturated glycerophospholipids.

[0033] In one or more embodiments of the present invention, in step S1), the hydroxylamine-containing reaction reagent is O-(sulfonyl)hydroxylamine, hydroxylamine-O-sulfonic acid, O-(2,4-dinitrophenyl)hydroxylamine or their derivatives.

[0034] In one or more embodiments of the present invention, in step S1), the catalyst is a rhodium catalyst, preferably, the catalyst is Rh(esp)2 or other rhodium catalysts.

[0035] In one or more embodiments of the present invention, in the step S1), 5. the solvent is trifluoroethanol or hexafluoroisopropanol or a mixture of trifluoroethanol or hexafluoroisopropanol and other solvents.

[0036] In one or more embodiments of the present invention, in step S1), 6. in the solution to be detected, the molar ratio of the reaction reagent containing hydroxylamine to the substrate containing an unsaturated double bond and a cis-trans isomer structure and having a sn-stereoconfiguration is (0.01-100):1, and the content of the catalyst is 0.1-10 (molar ratio)%.

[0037] In one or more embodiments of the present invention, the step S2) is characterized in that the reaction time is >10s.

[0038] In one or more embodiments of the present invention, the analysis step in step S4) is to compare and analyze the product chromatographic elution time of the double bond cis-trans isomer pair according to the liquid chromatogram of the product obtained in step S2), so as to determine the cis-trans configuration of the substrate containing the unsaturated double bond cis-trans isomer structure.

[0039] In one or more embodiments of the present invention, the analysis step in step S5) is to compare and analyze the chromatographic elution time of the product of the sn-position isomer pair of the fatty acyl chain where the unsaturated lipid double bond is located based on the liquid chromatogram of the product obtained in step S2), so as to determine the sn-position of the substrate containing the sn-position isomer structure of the fatty acyl chain where the unsaturated lipid double bond is located.

[0040] In one or more embodiments of the present invention, in the step S6), the characteristic fragment ions are analyzed, including obtaining the theoretical molecular formula of the secondary fragment ions based on the secondary mass spectrum of the product obtained in step S2), comparing it with the molecular formula of the substrate containing an unsaturated double bond structure or the reaction product obtained in step S2), and combining the unsaturation of the characteristic ions to reversely deduce the position of the double bond.

[0041] According to the embodiments of the present invention, the present invention proposes for the first time to utilize aziridine reaction, by simply mixing three reagents, namely, a substrate containing an unsaturated double bond, a probe containing a hydroxylamine structure and a catalyst, to form a nitrogen-containing three-membered heterocyclic derivative product at the unsaturated lipid double bond; then the obtained reaction product is separated by liquid chromatography and ionized by a mass spectrometry ion source, and then subjected to secondary fragmentation (such as collision-induced dissociation, etc.) in the mass spectrometer to generate characteristic fragment ions for determining the position of the unsaturated double bond, thereby achieving effective identification of the position of the carbon-carbon double bond; at the same time, due to the change from the substrate carbon-carbon double bond structure to the derivative product aziridine structure, the polarity of the compound changes accordingly, and the sn-isomer containing the unsaturated double bond is ionized by The same retention time of the original chromatographic peak is transformed into baseline separation of the chromatographic peak of the derivative product. The coupling effect of the sn-position isomer derivative product on the chromatographic column is different. The sn-1 isomer elutes earlier and the sn-2 isomer elutes later, thereby achieving the separation and identification of the sn-isomer; accordingly, since the lipid compounds containing unsaturated double bonds and cis-trans isomer structures, the coupling action group of the cis-trans isomer pair with the chromatographic column is transformed from the main carboxyl or phosphate head group to the secondary amine group, the action mode is changed, and the retention time on the chromatographic column is reversed, from the original substrate cis structure eluting earlier than the trans structure, to the cis derivative product eluting later than the trans derivative product, and the separation degree becomes larger.

[0042] Compared with the prior art, the present invention has at least one of the following advantages and beneficial effects:

[0043] The aziridination reaction of the unsaturated double bond proposed in the present invention has simple reaction conditions, fast reaction and high yield. The reaction product is detected and analyzed by liquid-mass mass spectrometry, and the double bond position, cis-trans configuration and sn-position of the double bond can be determined simultaneously, which can realize the multi-dimensional analysis of unsaturated lipid compounds by liquid-mass mass technology for the first time. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0045] Figure 1 The chromatographic separation diagram of the double bond position isomer FA 18:1 (9Z / 11Z) and the double bond cis-trans isomer FA 18:1 (9Z / 9E) derivative products of the fatty acid compound in Example 1, the diagnostic ion structure diagram corresponding to the double bond position and the corresponding secondary tandem mass spectrum diagram;

[0046] Figure 2 The diagram is a schematic diagram of the fragmentation of the double bond position diagnostic ions of the derivatization products of the sn-isomer pair of glycerophospholipid compounds (PC 16:0 / 18:1(9Z) and PC 18:1(9Z) / 16:0) in Example 2, and the chromatographic separation diagrams and triple tandem mass spectra before and after derivatization;

[0047] Figure 3 Schematic diagram of the fragmentation of the double bond position diagnostic ions of the double bond cis-trans isomers of the glycerophospholipid compounds in Example 2 for the PC 18:1 / 18:1(9Z) and PC18:1 / 18:1(9E) derivative products, the chromatographic separation diagram before and after derivatization, and the triple tandem mass spectrometry diagram. DETAILED DESCRIPTION

[0048] The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0049] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0050] Example 1

[0051] A method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond and the sn-position of the fatty acyl chain where the double bond is located of an unsaturated lipid comprises the following steps:

[0052] 1) Dissolving fatty acid compounds containing unsaturated double bonds, including double bond cis-trans isomers FA 18:1 (9Z / 9E) and double bond position isomers FA 18:1 (9Z / 11Z), probe N-benzyl-O-(2,4-dinitrophenyl)hydroxylamine, and catalyst Rh(esp)2 in trifluoroethanol solution respectively, mixing the three, and subjecting the obtained reaction product to liquid chromatography tandem mass spectrometry for detection and analysis;

[0053] 2) The product ion chromatogram m / z 388.3 was extracted in the positive ion mode. The chromatographic peaks of both the double bond cis-trans isomer pair and the positional isomers could achieve baseline separation, and the chromatographic peak elution time of the cis isomer derivative product was longer than that of the trans isomer derivative product. The chromatographic elution time of the isomer derivative product with the double bond position closer to the carboxyl end was longer than that of the isomer with the double bond farther from the carboxyl end ( Figure 1 );

[0054] 3) At the same time, the CID technology is used to fragment the quasi-molecular ion peak of the reaction product to obtain the corresponding characteristic fragment ions and the corresponding secondary mass spectrum;

[0055] 4) The secondary mass spectrum obtained in step 3) was compared with the theoretical secondary fragment ions, and the characteristic ion m / z 232 (C 16 H 26 N) and 262(C 16 H 24 NO2), corresponding to the FA 18:1 double bond position at position Δ9, and the characteristic ion m / z 204 (C 14 H 22 N) and 290(C 18 H 28 NO2), corresponding to the FA 18:1 double bond position at Δ11, indicating that both the double bond position isomer pair and the cis-trans isomer pair of the fatty acid reacted with N-benzyl-O-(2,4-dinitrophenyl)hydroxylamine to form a nitrogen-containing three-membered heterocyclic structure, and the reaction products can be separated by chromatographic baseline, which can be used to indicate the separation and identification of double bond cis-trans isomers, and the fragment ions of the products can be used to indicate the double bond position.

[0056] Example 2

[0057] A method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond and the sn-position of the fatty acyl chain where the double bond is located of an unsaturated lipid comprises the following steps:

[0058] 1) dissolving glycerol phospholipid compounds containing unsaturated double bonds, including sn-positional isomer pairs PC 16:0 / 18:1(9Z) and PC 18:1(9Z) / 16:0, and double bond cis-trans isomer pairs PC 18:1 / 18:1(9Z) and PC 18:1 / 18:1(9E), a probe N-benzyl-O-(2,4-dinitrophenyl)hydroxylamine, and a catalyst Rh(esp)2 in a trifluoroethanol solution, mixing the three, and subjecting the obtained reaction products to liquid chromatography tandem mass spectrometry for detection and analysis;

[0059] 2) In the positive ion mode, the product ion chromatogram m / z 865.6 was extracted. The elution time of the chromatographic peaks of the sn-isomers before derivatization was the same (32.59 and 32.54 min), and the chromatographic peaks of the products after derivatization could be baseline separated (22.10 and 22.49 min). The chromatographic elution time of the sn-1 isomer was shorter than that of the sn-2 isomer ( Figure 2 ).

[0060] 3) In the positive ion mode, the product ion chromatogram m / z 891.6 was extracted. The double bond cis-trans isomer pair had only one chromatographic peak before derivatization, and the chromatographic peaks of the cis-trans isomers just reached the baseline separation (32.51 and 33.32 min). After derivatization, there were two product chromatographic peaks, corresponding to the double bonds of the derivatized groups on different fatty acyl chains (i.e., sn-positional isomers), and the chromatographic elution time difference became larger (19.01 / 19.50 min for the trans isomer, 22.56 / 22.90 mi n corresponds to the cis isomer), the chromatographic peak elution time of the cis isomer derivative product is greater than that of the trans isomer (22.90 and 22.56 min corresponding to the cis isomer, 19.50 and 19.01 min corresponding to the trans isomer), and the chromatographic elution time of the sn-1 isomer is less than that of the sn-2 isomer (19.01 and 22.56 min corresponding to the sn-1 isomer, 19.50 and 22.90 min corresponding to the sn-2 isomer) ( Figure 3 ).

[0061] 4) At the same time, the CID technology is used to perform tertiary fragmentation on the head group loss ion of the quasi-molecular ion peak of the reaction product to obtain the corresponding characteristic fragment ions and the corresponding tertiary mass spectrum;

[0062] 5) The secondary mass spectrum obtained in step 4) was compared with the theoretical tertiary fragment ions, and the characteristic ion m / z 556 (C 35 H 58 NO4) and 451(C 28 H 51 O4), the double bond position of the corresponding sn-positional isomer pair is at position Δ9, and the characteristic ion m / z 582 (C 33 H 52 NO4) and 477(C 26 H 44O4), the double bond position of the corresponding double bond cis-trans isomer pair is at Δ9, indicating that both the sn-isomer pair and the double bond cis-trans isomer pair of the glycerol phospholipid react with N-benzyl-O-(2,4-dinitrophenyl)hydroxylamine to form a nitrogen-containing three-membered heterocyclic structure, and the sn-position isomerization reaction product can achieve chromatographic baseline separation, and the chromatographic retention time difference of the double bond cis-trans isomerization derivative products is large, which is conducive to the qualitative analysis of the isomer pair, and the fragment ions of the product can be used to indicate the double bond position, that is, to achieve the simultaneous analysis of the sn-position, double bond cis-trans configuration and double bond position isomerism.

[0063] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0064] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for simultaneously identifying the double bond position, the cis-trans configuration of the double bond and the sn-position of the fatty acyl chain where the double bond is located of an unsaturated lipid, characterized in that: The steps include: 1) dissolving a substrate containing an unsaturated double bond structure, a double bond cis-trans configuration and a sn-position structure, a reaction reagent containing a hydroxylamine structure and a catalyst in a solvent, and performing a mixing treatment to obtain a reaction solution to be detected; 2) injecting the reaction solution to be detected into a mass spectrometer, and after chromatographic separation and ionization by an ion source, further undergoing mass spectrometry-induced collision dissociation and fragmentation to obtain characteristic fragment ions; 3) Analyze and compare the product chromatographic behaviors of the reaction solution to be tested containing a double bond cis-trans isomer pair, and deduce the double bond cis-trans configuration of the unsaturated lipid; 4) analyzing and comparing the product chromatographic behaviors of the reaction solution to be tested containing the sn-position isomer pair, and deducing the sn-position of the fatty acyl chain where the double bond is located; 5) Analyze the characteristic fragment ions described in step 2) and reversely deduce the position of the unsaturated double bond.

2. The method according to claim 1, characterized in that The solvent is trifluoroethanol or hexafluoroisopropanol or a mixed solution with other solvents.

3. The method according to claim 1, characterized in that The substrate is a lipid compound; Optionally, the lipid compound is an unsaturated glycerophospholipid.

4. The method according to claim 1, characterized in that: The hydroxylamine-containing reaction reagent is O-(sulfonyl)hydroxylamine, hydroxylamine-O-sulfonic acid, O-(2,4-dinitrophenyl)hydroxylamine or their corresponding derivatives.

5. The method according to claim 1, characterized in that The catalyst is a rhodium catalyst; Preferably, the catalyst is Rh(esp)2 or other rhodium catalysts.

6. The method according to claim 1, characterized in that The molar ratio of the hydroxylamine-containing reaction reagent to the substrate is (0.01-100):1; Optionally, the content of the catalyst is 0.1 to 10 (molar ratio) %.

7. The method according to claim 1, characterized in that The mixing time is greater than 10 s.

8. The method according to claim 1, characterized in that The analysis step in step 3) is to analyze the chromatographic elution time of the double bond cis-trans isomer products according to the liquid chromatogram of the solution to be detected obtained in step 1), so as to determine the cis-trans configuration of the substrate containing the unsaturated double bond cis-trans isomer structure.

9. The method according to claim 1, characterized in that: The analysis step in step 4) is to analyze the chromatographic elution time of the isomer product of the sn-position of the fatty acyl chain where the unsaturated lipid double bond is located based on the liquid chromatogram of the solution to be tested obtained in step 1), thereby determining the sn-position of the fatty acyl chain where the unsaturated lipid double bond is located.

10. The method according to claim 1, characterized in that In step 5), the characteristic fragment ions are analyzed, including, according to the secondary mass spectrum in step 2), analyzing the theoretical molecular formula of the secondary fragment ions, comparing it with the product molecular formula of the substrate containing an unsaturated double bond structure or the reaction solution to be detected obtained in step 1), and combining the unsaturation of the characteristic ions to reversely deduce the position of the double bond.

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