CN113092643B - Method for detecting lipid oxygenation oxidation product - Google Patents

Abstract

The invention provides a method for detecting lipid oxygenation oxidation products, which comprises the following steps: preparing an oxidized sample of a lipid standard to be detected; the method comprises the steps of carrying out non-targeted detection on an oxidized oil sample by using a liquid chromatography tandem high-resolution mass spectrum to obtain compound information in the sample; the possible oxidation products are presumed according to lipid oxidation mechanism, and the mass-to-charge ratios of the excimer ions and characteristic fragment ions of the oxidation products are calculated according to the addition ions and fragmentation rules of the oxidation products in a mass spectrum; searching, checking and determining corresponding substances in the non-targeting data, thereby determining lipid oxygenation oxidation products and the content thereof in the oxidized oil standard sample. Aiming at lipid standard oxidation research, the invention develops a method for identifying lipid oxidation products through a mass spectrum according to lipid oxidation mechanism and the fragmentation rule of oxidation products in the mass spectrum and in combination with non-targeted detection of high-resolution mass spectrum, and can realize comprehensive detection of a plurality of low-abundance lipid oxygenation oxidation products in oxidized lipid standard samples.

Description

Method for detecting lipid oxygenation oxidation product

Technical Field

The invention relates to the field of detection of lipid oxygenation oxidation products, in particular to a method for detecting low-content lipid oxygenation oxidation intermediates by combining high-resolution mass spectrum.

Background

The existing lipid oxidation research mostly adopts ferric thiocyanate method, total carbonyl compound assay method, iodometry method, thiobarbituric acid method and other methods to detect lipid oxidation progress, but the monitoring methods can only evaluate the general condition of lipid oxidation, and are difficult to be used for more accurate oxidation research, such as research on lipid oxidation mechanisms of different structures and different types. Lipid standard oxidation is a commonly used lipid oxidation mechanism research mode in recent years, and is mainly researched to monitor the change of volatile products downstream of lipid oxidation by using a gas chromatography-mass spectrometry combined technology, but less research is conducted on lipid oxidation intermediates directly participating in lipid oxidation chain reaction.

Because of the wide variety of lipid oxidation intermediates, it is difficult to fully monitor lipid oxidation products in a common targeted monitoring mode, and thus few studies are conducted on lipid oxidation intermediates. The data volume of non-targeted detection is huge, 5000-6000 compounds with secondary mass spectrograms can be obtained for each detection by a Q exact HF-X mass spectrometer, the compounds are explained and identified, the existing method generally adopts spectrum library matching, however, the spectrum library of lipid oxygenation oxidation products is almost not available, so that the non-targeted detection is still rarely applied to the detection of lipid oxygenation oxidation products nowadays. In recent years, the advent of high resolution mass spectrometry has enabled the comprehensive detection of lipid oxidation intermediates. Non-targeted detection of high-resolution mass spectrometry can be used to detect lipid oxidation products comprehensively. However, lipid oxidation intermediates were lower in content, fewer in purity, and there was no rich library of lipid oxidation intermediates, therefore, it was difficult to annotate non-targeted data for lipid oxidation. Therefore, to take advantage of the non-targeted detection of high resolution mass spectra, it is necessary to develop a method for interpreting mass spectral data and identifying lipid oxidation products.

Disclosure of Invention

In order to solve the problem that the monitoring mode in the prior art can only evaluate the general condition of lipid oxidation, is difficult to be used for more accurate researches on lipid oxidation mechanisms of different structures and different types, for example, the comprehensive detection of lipid oxygenation oxidation products is realized.

The invention provides a method for detecting lipid oxygenation oxidation products, which comprises the following specific steps:

s1, preparing an internal standard sample: preparing a mixed internal standard working solution with the final concentration of 100 mug/mL;

s2, preparing a lipid oxidation sample: taking a lipid standard substance oxidation sample to be detected, adding methanol and the mixed internal standard working solution according to the proportion of 9:1 (v/v), re-dissolving to prepare a 0.5mg/mL sample, centrifuging for 10min at the temperature of 4-10 ℃ and the rotating speed of 8000-12000 r, and taking the supernatant to obtain the lipid oxidation sample;

s3, sample measurement: performing high-resolution mass spectrum non-targeted detection on the lipid oxidation sample prepared in the step S2 to obtain mass spectrograms and chromatograms of various compounds in the sample;

s4, supposing existing lipid oxygenation oxidation products, calculating the relative molecular mass of corresponding compounds, the excimer ion mass-to-charge ratio of each lipid oxygenation oxidation product and the characteristic fragment ion mass-to-charge ratio;

s5, searching the non-targeted detection data for a corresponding compound according to the calculated quasimolecule ion mass-charge ratio of the lipid oxidation product, and checking whether the lipid oxidation product contains the mass-charge ratio of the characteristic fragment ion or not so as to identify the lipid oxidation product.

The preparation method of the mixed internal standard working solution in the step S1 comprises the following steps: triacylglycerols (TG; 17:0/17:0/17:0) are selected; phosphatidylcholine (PC; 17:0/17:0); phosphatidylethanolamine (PE; 17:0/17:0) was used as an internal standard to prepare a mixed internal standard working solution with a final concentration of 100. Mu.g/mL, respectively.

The high-resolution mass spectrum non-targeted detection method in the step S3 comprises the following steps: determined by ultra-high performance liquid chromatography and mixed quadrupole-orbital ion trap mass spectrometry using a ACQUITY UPLC BEH C (2.1 x100mm 1.7 μm) column.

The conditions for the non-targeted detection of the high-resolution mass spectrum in the step S3 are as follows:

sample injection amount: 1 μl; acquisition mode: full ms+prm;

mobile phase: mobile phase a: acetonitrile/water (60:40, v/v) 0.1% ammonium formate;

mobile phase B: isopropanol/acetonitrile (90:10, v/v) 0.1% ammonium formate;

elution flow rate: 0.6mL/min;

elution gradient: 0-2min,15-30% B and 2-2.5min,30-48% B;2.5-11min,48-82% B;

11-11.5min，82-99％B；11.5-12min，99％B；12-12.1min，99-15％B；12.1-15

min，15％B；

mass spectrometry employs electrospray ion sources (ESI); detecting a positive ion mode and a negative ion mode; the acquisition range is as follows: 120-1200m/z; in the positive mode: sheath air flow is 60%, auxiliary air flow is 25%, and purge air flow is 2%;

the spraying voltage is 3.6kV, the capillary temperature is 380 ℃, and the temperature of the auxiliary gas heater is 370 ℃; the negative mode is similar to the positive mode and the spray voltage is 3.0kV.

Wherein, the method for identifying the lipid oxygenation oxidation product according to the characteristic fragment ions in the step S5 is as follows: compounds showing a secondary mass spectrum were checked in MSDIAL; from these compounds, the corresponding species are found from the deduced mass-to-charge ratios of the oxidation product excimer ions, which species are led to the msfield to look at the mass spectrum, checking if the characteristic fragment ions are present, which species present characteristic fragment ions are determined to be the correct oxygenated oxidation product.

Wherein, in the step S4, the calculation formula of the excimer ion mass-to-charge ratio of the lipid oxygenation oxidation product is as follows:

m _{excimer ions} ＝m _{Oxidation products} +m _{Adducts (+H, +Na, +NH4, -H, +CI, +HCOO)} 。

Wherein, in step S4, the characteristic fragment ion mass-to-charge ratio calculation formula is as follows:

negative ion mode m _{Characteristic ions} ＝m _{Oxidized fatty acid chains} -m _{Hydrogen atomic mass}

Positive ion mode m _{Characteristic ions} ＝m _{Oxidation products} -m _{Oxidized fatty acid chains} +m _{Adduct ions (+H, +Na, +NH4)} 。

The invention has the beneficial effects that:

1. the invention uses high-resolution mass spectrum to detect lipid oxidation products in a non-targeted way, can detect the lipid oxidation products more comprehensively, and ensures that the oxidation mechanism obtained by experiments is more accurate. In the unique mass spectrometry signal acquisition mode of Full ms+dd-MS, the quadrupole mass filter transmits only defined ions in the inclusion list to the intermediate C-trap, preparing for sample injection to more efficient high energy collision dissociation (HCD), and then performing high concentration analysis resolution in an Orbitrap analyzer. The C trap trapping part of the Q exact HF-X mass spectrometer replaces the final quadrupole rod part of the triple quadrupole rod instrument, and the transmission of ion products in the whole mass range can be completed. The Q exact HF-X mass spectrometer can provide accurate mass-to-charge ratio scanning, and is accurate to five bits after decimal point. The high accuracy of high resolution mass spectrometry will greatly improve the accuracy of mass spectrometry matching.

2. The invention summarizes the existing lipid oxidation mechanism and the fragmentation mechanism of lipid oxidation products in mass spectrum, and applies the lipid oxidation mechanism and the fragmentation mechanism to the interpretation of non-targeted data, so that the problem of lacking an oxidation product spectrum library is solved to a certain extent, the lipid oxidation products are well identified, and the advantage of non-targeted detection is exerted. Compared with the existing targeting research, the non-targeting method is quicker, a large amount of substances can be identified at one time to obtain a large amount of compound information, meanwhile, the oxidation products detected by the non-targeting detection are more comprehensive, and the non-targeting detection is more quicker, so that the method is very suitable for detecting a large amount of lipid oxidation products, therefore, the method can be used for explaining the non-targeting data, and the non-targeting detection can be applied to the field of lipid oxidation.

Drawings

FIG. 1 is a secondary mass spectrum of PE (18:1/18:1) identified in example 1;

FIG. 2 is a secondary mass spectrum of PE (18:1/18:1) + (Ke) identified in example 1;

FIG. 3 is a secondary mass spectrum of PE (18:1/18:1) + (OH or Ep) identified in example 1;

FIG. 4 is a secondary mass spectrum of PE (18:1/18:1) + (2 OH) identified in example 1;

FIG. 5 is a secondary mass spectrum of PE (18:1/18:1) + (OOH) identified in example 1;

FIG. 6 is a secondary mass spectrum of PC (18:1/18:1) identified in example 1;

FIG. 7 is a secondary mass spectrum of PC (18:1/18:1) + (Ke) identified in example 1;

FIG. 8 is a secondary mass spectrum of PC (18:1/18:1) + (OH or Ep) identified in example 1;

FIG. 9 is a secondary mass spectrum of PC (18:1/18:1) + (3 Ke) identified in example 1;

FIG. 10 is a secondary mass spectrum of PC (18:1/18:1) + (3 Ke) identified in example 1;

FIG. 11 is a secondary mass spectrum of PC (18:1/18:1) + (Ke+OH orKe+Ep) identified in example 1;

FIG. 12 is a secondary mass spectrum of PC (18:1/18:1) + (OOH) identified in example 1;

FIG. 13 is a secondary mass spectrum of PC (18:1/18:1) + (2 OH) identified in example 1;

FIG. 14 is a secondary mass spectrum of PC (18:1/18:1) + (Ke+OOH) identified in example 1;

FIG. 15 is a secondary mass spectrum of PC (18:1/18:1) + (OOH+OH or OOH+Ep) identified in example 1;

FIG. 16 is a secondary mass spectrum of TG (18:1/18:1/18:1) identified in example 1;

FIG. 17 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2 Ke) identified in example 1;

FIG. 18 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (Ke) identified in example 1;

FIG. 19 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OOH) identified in example 1;

FIG. 20 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OH orEp) identified in example 1;

FIG. 21 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OH orEp) identified in example 1;

FIG. 22 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2H+OH or 2H+Ep) identified in example 1;

FIG. 23 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2H+OH or 2H+Ep) identified in example 1;

FIG. 24 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (Ke+OOH) identified in example 1;

FIG. 25 is a secondary mass spectrum of TG (18:1/18:1/18:0) + (Ke+OH orKe+Ep) identified in example 1;

FIG. 26 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2 OH or 2 Ep) identified in example 1;

FIG. 27 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OOH) identified in example 1;

FIG. 28 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2Ke+OH or 2Ke+Ep) identified in example 1;

FIG. 29 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (3 OH or 3 Ep) identified in example 1;

FIG. 30 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (Ke+OOH+OH or Ke+OOH+Ep) identified in example 1;

FIG. 31 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OOH+2OH or OOH+2Ep) identified in example 1.

Detailed Description

Preparing an oxidized sample of a lipid standard to be detected; the method comprises the steps of carrying out non-targeted detection on an oxidized oil sample by using a liquid chromatography tandem high-resolution mass spectrum to obtain compound information in the sample; the possible oxidation products are presumed according to lipid oxidation mechanism, and the mass-to-charge ratios of the excimer ions and characteristic fragment ions of the oxidation products are calculated according to the addition ions and fragmentation rules of the oxidation products in a mass spectrum; searching, checking and determining corresponding substances in the non-targeting data, thereby determining lipid oxygenation oxidation products and the content thereof in the oxidized oil standard sample.

The method specifically comprises the following steps:

s1, preparing an internal standard sample: the internal standard is prepared into mixed internal standard working solution with the final concentration of 100 mug/mL;

s2, preparing a lipid oxidation sample: taking an oxidized sample of a lipid standard substance to be detected (the oxidation method is determined according to a specific experiment), adding methanol and mixed internal standard working solution according to the proportion of 9:1 (v/v), and re-dissolving to prepare a sample of 0.5 mg/mL; centrifuging the sample at 10deg.C and 12000 rpm for 10min, collecting 200 μl of supernatant, placing into a liquid sample injection vial, and immediately placing into a liquid quality detection device;

s3, sample measurement: performing high-resolution mass spectrum non-targeted detection on the lipid oxidation sample prepared in the step S2 to obtain mass spectrograms and chromatograms of various compounds in the sample;

s4, according to structural information, mass spectrograms and chromatograms of lipid standard substances and lipid oxidation mechanisms, supposing possible lipid oxygenation oxidation products, calculating relative molecular masses of corresponding compounds by using corresponding formulas, and according to the form of addition ions and fragmentation mechanisms of the lipid oxygenation oxidation products in mass spectrum, calculating the quasimolecule ion mass-to-charge ratio and characteristic fragment ion mass-to-charge ratio of each lipid oxygenation oxidation product by using corresponding formulas;

s5, searching corresponding compounds in non-targeted detection data by using MSDIAL software according to the calculated quasimolecule ion mass-charge ratio of the lipid oxygenation oxidation product, opening a mass spectrum of the compounds by using MSFINDER software, and checking whether the mass-charge ratio of the characteristic fragment ions is contained or not, thereby identifying the lipid oxygenation oxidation product.

The preparation method of the mixed internal standard working solution in the step S1 comprises the following steps: triacylglycerols (TG; 17:0/17:0/17:0) are selected; phosphatidylcholine (PC; 17:0/17:0); phosphatidylethanolamine (PE; 17:0/17:0) was used as an internal standard to prepare a mixed internal standard working solution with a final concentration of 100. Mu.g/mL, respectively.

The measurement method in step S3 is as follows: the measurement was performed by ultra-high performance liquid chromatography and mixed quadrupole-orbital mass spectrometry using ACQUITY UPLC BEH C (2.1 x100mm 1.7 μm) columns.

The specific detection conditions in the step S3 are as follows:

sample injection amount: 1 μl; acquisition mode: full ms+prm;

mobile phase: mobile phase a: acetonitrile/water (60:40, v/v) 0.1% ammonium formate; mobile phase B: isopropanol/acetonitrile (90:10, v/v) 0.1% ammonium formate;

elution flow rate: 0.6mL/min;

elution gradient: 0-2min,15-30% B; and 2-2.5min,30-48% B;2.5-11min,48-82% B;

11-11.5min，82-99％B；11.5-12min，99％B；12-12.1min，99-15％B；12.1-15

min，15％B.

the lipid oxidation mechanism in step S4 is: with an allyl unit as a core, the oxygenation and oxidation mainly comprises four cases of adding-OOH, -OH, =O and generating an epoxy compound at the double bond position. In addition, lipid oxidation products retaining the original chain length like this also have both an addition reaction of allyl units and unchanged. Thus, there are 6 oxidation reactions that may occur at each allylic position. The following is a mass calculation formula for the oxygenated oxidation product from which the relative molecular mass of the oxidation product that may occur can be inferred. The relative molecular mass of the oxidation product can be used for the calculation of the excimer ion mass-to-charge ratio and the characteristic fragment ion mass-to-charge ratio of the lipid.

Possible oxidation product mass calculations are:

m _{oxidation products} ＝m _{Original lipid} +m _{Oxidizing the added groups} -m _{Substituted groups}

Note that: m represents the mass. Among the possible oxidation products, OOH means addition of a hydroperoxide group, OH means addition of a hydroxyl group, ke means addition of an aldehyde group, ep means addition of an epoxy group at the double bond site, 2H means addition of a double bond by hydrogen, these groups being of the masses 32.9976, 17.0027, 15.9949, 15.9949,2.0156, respectively, and the substituted groups being H, H,2H, none, therefore m _{Substituted groups} 1.0078,1.0078,2.0156,0,0 respectively.

The fragmentation mechanism of the oxidized products of the oxygenation in the mass spectrum in step S4 is: the glyceride ester bond position is broken, OH is added after the main body part is broken or one H is lost to form a double bond, and the other half of the broken glyceride ester bond is separated in the form of a fatty acid chain. The main body part is combined with H+, na+, NH4+ in positive ion mode, and the fatty acid chain part exists mainly in [ FA-H ] -form in negative ion mode. The following formula is established based on the form of the adduct ions and the fragmentation mechanism of the oxygenate products in the mass spectrum, by which the excimer ion mass and the characteristic fragment ion mass (i.e., the excimer ion mass-to-charge ratio and the characteristic fragment ion mass-to-charge ratio of each possible oxygenate product can be calculated, since the adduct ions are all singly charged, their relative molecular masses are their mass-to-charge ratios).

The lipid oxygenation oxidation product excimer ion mass (i.e., the excimer ion mass to charge ratio of the lipid oxygenation oxidation product) is calculated by the formula:

m _{excimer ions} ＝m _{Oxidation products} +m _{Adducts (+H, +Na, +NH4, -H, +CI, +HCOO)}

Note that: m is respectively corresponding to +H, +Na, +NH4, -H, +CI, +HCOO _{Adduct ions} 1.0078, 22.9898, 18.0344, -1.0078, 34.9688, 44.9976.

Lipid oxygenation oxidation product characteristic fragment ion mass (i.e., characteristic fragment ion mass to charge ratio of lipid oxygenation oxidation product) calculation formula:

negative ion mode m _{Characteristic ions} ＝m _{Oxidized fatty acid chains} -m _{Hydrogen atomic mass}

Positive ion mode m _{Characteristic ions} ＝m _{Oxidation products} -m _{Oxidized fatty acid chains} +m _{Adduct ions (+H, +Na, +NH4)}

Note that: m is m _{Hydrogen atomic mass} Is 1.0078

The method for identifying the lipid oxygenation oxidation product according to the characteristic fragment ions in the step S5 is as follows: substances exhibiting a secondary mass spectrum were checked in MSDIAL. Based on the estimated mass-to-charge ratio of the oxidation product excimer ions, the corresponding species is found from it, and the species is exported to a MSFINDER for a mass spectrum, checking whether the main characteristic fragment ions are present, and the species with the characteristic fragment ions are determined to be the correct oxidation product.

Example 1: detecting lipid oxygenation oxidation products of a sample to be tested

Lipid oxidation sample preparation: phosphatidylcholine (PC; 18:1/18:1) was selected; phosphatidylethanolamine (PE; 18:1/18:1), triacylglycerols (TG; 18:1/18:1/18:1) were subjected to lipid oxidation experiments. Three (3) lipid standards (PC (18:1/18:1), PE (18:1/18:1), TG (18:1/18:1/18:1)) were taken, 50mg each, and dissolved in 25mL of chloroform to prepare 2mg/mL lipid standard solutions. 100 mu L of lipid standard solution of 2mg/mL is taken in a 20mL brown sample bottle, and after the solvent is blown dry by nitrogen, the solution is placed in an oil bath pot for heating and oxidized for 40min at 140 ℃. And immediately after the oxidation is finished, placing the mixture in an ice bath for 5min to obtain an oxidized sample. 360. Mu.L of methanol and 40. Mu.L of mixed internal standard working solution (TG (17:0/17:0/17:0), PC (17:0/17:0) and PE (17:0/17:0)) are added into an oxidized sample, the mixture is redissolved for 1min, 360. Mu.L of the mixture is taken out from a centrifuge tube of 1.5mL, the mixture is centrifuged for 10min at 10 ℃ and 12000 revolutions, and 200. Mu.L of supernatant is taken out from a liquid phase sample injection vial after balancing and centrifuging. Immediately placing the mixture into a liquid for detection.

The preparation method of the mixed internal standard working solution comprises the following steps: triacylglycerols (TG; 17:0/17:0/17:0) are selected; phosphatidylcholine (PC; 17:0/17:0); phosphatidylethanolamine (PE; 17:0/17:0) was used as an internal standard to prepare a mixed internal standard working solution with a final concentration of 100. Mu.g/mL, respectively.

The oxidation products of the standard and the lipid composition of the biological samples were analyzed by ultra high performance liquid chromatography tandem and mixed quadrupole-orbital ion trap mass spectrometry (UHPLC-Q-actual HF-X) using ACQUITYUPLC BEH C (2.1X100 mm 1.7 μm) columns. (1) mobile phase A: acetonitrile/water (60:40, v/v) 0.1% ammonium formate, mobile phase B: isopropanol/acetonitrile (90:10, v/v) 0.1% ammonium formate; column temperature: 65 ℃; flow rate: 0.6mL/min; sample injection amount: 1 μl; elution gradient: 0-2min,15-30% B; and 2-2.5min,30-48% B;2.5-11min,48-82% B;11-11.5min,82-99% B;11.5-12min,99% B;12-12.1min,99-15% B;12.1-15min,15% B; (2) mass spectrometry using electrospray ion source (ESI); detecting a positive ion mode and a negative ion mode; the acquisition range is as follows: 120-1200m/z; in the positive mode: sheath air flow was 60%, auxiliary air flow was 25%, and purge air flow was 2%. The spraying voltage is 3.6kV, the capillary temperature is 380 ℃, and the temperature of the auxiliary gas heater is 370 ℃; the negative mode is similar to the positive mode and the spray voltage is 3.0kV.

Phosphatidylcholine (PC; 18:1/18:1) was speculated on the basis of lipid oxidation mechanism; phosphatidylethanolamine (PE; 18:1/18:1), triacylglycerols (TG; 18:1/18:1/18:1), it being possible for each allyl unit to be charged with OOH, OH, ke, ep,2H or unchanged, for example (PC; 18:1/18:1), there being more than 6 oxidation events for each chain, and thus 36 possible oxidation products.

PC (18:1/18:1) + (OOH) means (PC; 18:1/18:1) that one strand is added to OOH. Taking oxidation product PC (18:1/18:1) + (OOH) as an example, the excimer ion mass-to-charge ratio and the characteristic fragment ion mass-to-charge ratio are calculated as follows:

the addition of OOH groups to PC (18:1/18:1) yields PC (18:1/18:1) + (OOH), PC (18:1/18:1) + (OOH) being one of the putative oxidation products according to the oxidation mechanism, the following is a procedure for identifying this substance:

and (3) calculating the mass of an oxygenation oxidation product:

the original lipid of PC (18:1/18:1) + (OOH) is PC (18:1/18:1), so m _{Original lipid} The mass of (2) is 785.5935, m _{Oxidizing the added groups} Is OOH, mass is 32.9976, m _{Is substituted withIs a group of (2)} H, mass 1.0078.

According to m _{Oxidation products} ＝m _{Original lipid} +m _{Oxidizing the added groups} -m _{Substituted groups} Can be given m _{Oxidation products} 817.5833.

The oxygen-added oxidation product excimer ion mass (i.e., the excimer ion mass to charge ratio of the lipid oxygen-added oxidation product) was calculated:

the adduct ions of PC (18:1/18:1) + (OOH) are +H, +Na, +NH4, -H, +CI, +HCOO, m _{Adduct ions} 1.0078, 22.9898, 18.0344, -1.0078, 34.9688, 44.9976, m _{Oxidation products} As is apparent from the calculation of the foregoing formula, 817.5833. Wherein +H, +Na, +NH4 is an adduct ion in positive ion mode, +CI, +HCOO is an adduct ion in negative ion mode.

According to m _{Excimer ions} ＝m _{Oxidation products} +m _{Adducts (+H, +Na, +NH4, -H, +CI, +HCOO)} It was found that PC (18:1/18:1) + (OOH) with 6 adduct ions added, respectively, had excimer ion mass to charge ratios of 818.5911, 840.5731, 835.6177, 816.5755, 852.5521, 862.5809, respectively.

The mass of characteristic ions of the oxidation products of oxygenation (i.e. the characteristic fragment ion mass to charge ratio of the lipid oxidation products of oxygenation) is calculated:

PC (18:1/18:1) + (OOH) has two fatty acid chains, the added group OOH being on one chain, so that the two fatty acid chains contained by PC (18:1/18:1) + (OOH) are FA 18:1 and FA 18:1+OOH, respectively, and thus m _{Oxidized fatty acid chains} 282.2589, 314.2487, respectively. m is m _{Hydrogen atomic mass} Is 1.0078.

According to the negative ion mode m _{Characteristic ions} ＝m _{Oxidized fatty acid chains} -m _{Hydrogen atomic mass} Two characteristic fragment ions can be derived, the mass to charge ratios being 281.2511, 313.2409 respectively.

In the positive ion mode, +H is more often the case. m is m _{Adduct ions (+H)} Is 1.0078.

According to positive ion mode m _{Characteristic ions} ＝m _{Oxidation products} -m _{Oxidized fatty acid chains} +m _{Adduct ions (+H)} Can obtain 2 kinds ofThe mass to charge ratios of the characteristic fragment ions were 536.3322, 504.3424, respectively.

From this, the oxidation product PC (18:1/18:1) of PC (18:1/18:1) + (OOH) has an excimer ion mass to charge ratio of 818.5911, 840.5731, 835.6177, characteristic ions of 536.3322, 504.3424 in the positive ion mode. The mass-to-charge ratio of the excimer ions in the negative ion mode is 816.5755, 852.5521, 862.5809, 281.2511, 313.2409 for the characteristic ions.

After mass spectra and chromatogram peak areas of internal standard, lipid and lipid oxidation products in a sample are obtained through ultra-high performance liquid phase serial mixed quadrupole-orbitrap mass spectrometry detection, corresponding substances are searched in MS-DIAL according to the predicted oxidation product excimer ion mass-charge ratio by utilizing MS-DIAL opening data, then the mass spectra of the substances are transferred into MSFINDER software, the mass spectra of the substances are checked, and the substances capable of finding characteristic fragment ions are determined to be correct lipid oxidation products. In order to further understand the content of the substance in the different samples, the integrated peak areas of the substance in the different samples were derived by MS-DIAL software. Lipid oxidation products were searched and identified separately, and three lipids matched to 4, 9, 15, respectively, oxygenation oxidation products.

Note that: OOH represents a hydroperoxide group, OH represents a hydroxyl group, ke represents a carbonyl group, ep represents an epoxy group, and 2H generally refers to double bond addition. Lipid oxidation product name writing: "lipid name before oxidation+group added by oxidation".

TABLE 1 identification of lipid oxygenation oxidation products

As shown in table 1, three standards identified 28 lipid oxidation products altogether, which were more comprehensive than the targeted study.

FIG. 1 is a PE (18:1/18:1) secondary mass spectrum identified in example 1, characterized by fragment ion 281.2480, in which the corresponding fragment ion peak (mass deviation < 0.005) can be found, thereby identifying the compound; FIG. 2 is a secondary mass spectrum of PE (18:1/18:1) + (Ke) identified in example 1, which is characterized by fragment ions 281.2480, 295.2273, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 3 is a secondary mass spectrum of PE (18:1/18:1) + (OH or Ep) identified in example 1, characterized by fragment ions 281.2480, 297.2429, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 4 is a secondary mass spectrum of PE (18:1/18:1) + (2 OH) identified in example 1, characterized fragment ion of 297.2429, in which the corresponding fragment ion peak (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 5 is a secondary mass spectrum of PE (18:1/18:1) + (OOH) identified in example 1, characterized by fragment ions 281.2480, 313.2379, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 6 is a secondary mass spectrum of PC (18:1/18:1) identified in example 1, characterized by fragment ion 281.2480, in which the corresponding fragment ion peak (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 7 is a secondary mass spectrum of PC (18:1/18:1) + (Ke) identified in example 1, which is characterized by fragment ions 281.2480, 295.2273, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 8 is a secondary mass spectrum of PC (18:1/18:1) + (OH or Ep) identified in example 1, which is characterized by fragment ions 281.2480, 297.2429, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 9 is a secondary mass spectrum of PC (18:1/18:1) + (3 Ke) identified in example 1, which is characterized by fragment ions 295.2273, 309.2066, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 10 is a secondary mass spectrum of PC (18:1/18:1) + (3 Ke) identified in example 1, which is characterized by fragment ions 295.2273, 309.2066, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 11 is a secondary mass spectrum of PC (18:1/18:1) + (Ke+OH or Ke+Ep) identified in example 1, which is characterized by fragment ions 295.2273, 297.2429, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 12 is a secondary mass spectrum of PC (18:1/18:1) + (OOH) identified in example 1, which is characterized by fragment ions 281.2480, 313.2379, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 13 is a secondary mass spectrum of PC (18:1/18:1) + (2 OH) identified in example 1, which is characterized by fragment ion 297.2429, in which the corresponding fragment ion peak (mass deviation < 0.005) can be found, thereby identifying the compound; FIG. 14 is a secondary mass spectrum of PC (18:1/18:1) + (Ke+OOH) identified in example 1, which is characterized by fragment ions 295.2273, 313.2379, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 15 is a secondary mass spectrum of PC (18:1/18:1) + (OOH+OH or OOH+Ep) identified in example 1, which is characterized by fragment ions 297.2429, 313.2379, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 16 is a secondary mass spectrum of TG (18:1/18:1/18:1) identified in example 1, characterized by fragment ion 603.5354, in which the corresponding fragment ion peak (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 17 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2 Ke) identified in example 1, which is characterized by fragment ion peaks 617.5146, 631.4939, 617.5146, where the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 18 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (Ke) identified in example 1, which is characterized by fragment ions 603.5354, 617.5147, 617.5147, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 19 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OOH) identified in example 1, characterized by fragment ion peaks 603.5354, 635.5252, 635.5252, corresponding to fragment ion peaks (mass deviation < 0.005) found in the mass spectrum, whereby the compound can be identified; FIG. 20 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OH or Ep) identified in example 1, characterized by fragment ion peaks 603.5354, 619.5303, 619.5303, corresponding to fragment ion peaks (mass deviation < 0.005) found in the mass spectrum, whereby the compound can be identified; FIG. 21 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OH or Ep) identified in example 1, characterized by fragment ion peaks 603.5354, 619.5303, 619.5303, where the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 22 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2H+OH or 2H+Ep) identified in example 1, which is characterized by fragment ions 605.551, 621.546, 619.5303, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 23 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2H+OH or 2H+Ep) identified in example 1, which is characterized by fragment ions 605.551, 621.546, 619.5303, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 24 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (Ke+OOH) identified in example 1, characterized by fragment ions 635.5252, 617.5146, 649.5045 in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 25 is a secondary mass spectrum of TG (18:1/18:1/18:0) + (Ke+OH or Ke+Ep) identified in example 1, characterized by fragment ions 617.5147, 633.5096, 619.5303, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 26 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2 OH or 2 Ep) identified in example 1, characterized by fragment ions 619.5303, 635.5252, 619.5303, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 27 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OOH) identified in example 1, characterized by fragment ion peaks 603.5354, 635.5252, 635.5252, corresponding to fragment ion peaks (mass deviation < 0.005) found in the mass spectrum, whereby the compound can be identified; FIG. 28 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (2Ke+OH or 2Ke+Ep) identified in example 1, which is characterized by fragment ions 633.5096, 631.4939, 633.5096, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 29 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (3 OH or 3 Ep) identified in example 1, characterized by fragment ions 635.5252, 635.5252, 635.5252 in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified; FIG. 30 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (Ke+OOH+OH orKe+OOH+Ep) identified in example 1, characterized fragment ions of the material were 673.5021, 655.4915, 671.4864, and corresponding fragment ion peaks (mass deviation < 0.005) could be found in the mass spectrum, whereby the compound could be identified; FIG. 31 is a secondary mass spectrum of TG (18:1/18:1/18:1) + (OOH+2OH or OOH+2Ep) identified in example 1, which is characterized by fragment ions 657.5072, 673.5021, 673.5021, in which the corresponding fragment ion peaks (mass deviation < 0.005) can be found, whereby the compound can be identified.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.

Claims (3)

1. A method for detecting lipid oxygenation oxidation products, comprising the steps of:

s1, preparing an internal standard sample: preparing a mixed internal standard working solution with the final concentration of 100 mug/mL; the preparation method of the mixed internal standard working solution comprises the following steps: triacylglycerols 17:0/17:0/17:0; phosphatidylcholine 17:0/17:0; phosphatidylethanolamine 17:0/17:0 is used as an internal standard to prepare mixed internal standard working solution with the final concentration of 100 mug/mL respectively;

s2, preparing a lipid oxidation sample: taking a lipid standard substance oxidation sample to be detected, adding methanol and the mixed internal standard working solution according to the proportion of 9:1v/v, re-dissolving to prepare a 0.5mg/mL sample, centrifuging for 10min at the temperature of 4-10 ℃ and the rotating speed of 8000-12000 r, and taking the supernatant to obtain the lipid oxidation sample;

s3, sample measurement: performing high-resolution mass spectrum non-targeted detection on the lipid oxidation sample prepared in the step S2 to obtain mass spectrograms and chromatograms of various compounds in the sample;

s4, supposing existing lipid oxygenation oxidation products, calculating the relative molecular mass of corresponding compounds, the excimer ion mass-to-charge ratio of each lipid oxygenation oxidation product and the characteristic fragment ion mass-to-charge ratio;

s5, searching corresponding compounds in the non-targeting detection data according to the calculated quasimolecule ion mass-charge ratio of the lipid oxygenation oxidation product, and checking whether the corresponding compounds contain the mass-charge ratio of characteristic fragment ions or not so as to identify the lipid oxygenation oxidation product;

the manner of identifying the lipid oxygenation oxidation product according to the characteristic fragment ion in the step S5 is as follows: compounds showing a secondary mass spectrum were checked in MSDIAL; finding corresponding substances from the compounds according to the estimated mass-to-charge ratio of the oxidation product excimer ions, leading the substances to a MSFINDER to check a mass spectrum, and checking whether characteristic fragment ions exist, wherein the substances with the characteristic fragment ions are determined to be the correct oxygenation oxidation products;

in the step S4, the calculation formula of the excimer ion mass-to-charge ratio of the lipid oxygenation oxidation product is as follows:

m _{excimer ions} ＝m _{Oxidation products} +m _{Adduct ions} (+H,+Na,+NH4,-H,+CI,+HCOO)；

The characteristic fragment ion mass-to-charge ratio calculation formula in step S4:

negative ion mode m _{Characteristic ions} ＝m _{Oxidized fatty acid chains} -m _{Hydrogen atomic mass}

Positive ion mode m _{Characteristic ions} ＝m _{Oxidation products} -m _{Oxidized fatty acid chains} +m _{Adduct ions} (+H,+Na,+NH4)。

2. The method of claim 1, wherein the high resolution mass spectrometry non-targeted detection method in step S3 is:

determined by ultra high performance liquid chromatography and mixed quadrupole-orbitrap mass spectrometry using a ACQUITY UPLC BEH C2.1x100mm 1.7 μm column.

3. The method of claim 1, wherein the conditions for the high resolution mass spectrometric non-targeted detection in step S3 are:

sample injection amount: 1 μl; acquisition mode: full ms+prm;

mobile phase: mobile phase a: acetonitrile/water 60:40, v/v 0.1% ammonium formate;

mobile phase B: isopropanol/acetonitrile 90:10, v/v 0.1% ammonium formate;

elution flow rate: 0.6mL/min;

elution gradient: 0-2min,15-30% B and 2-2.5min,30-48% B;2.5-11min,48-82% B;11-11.5min,82-99% B;11.5-12min,99% B;12-12.1min,99-15% B;12.1-15min,15% B;

the mass spectrum adopts an electrospray ion source ESI; detecting a positive ion mode and a negative ion mode; the acquisition range is as follows: 120-1200m/z; in the positive mode: sheath air flow is 60%, auxiliary air flow is 25%, and purge air flow is 2%;

the spraying voltage is 3.6kV, the capillary temperature is 380 ℃, and the temperature of the auxiliary gas heater is 370 ℃; the negative mode is similar to the positive mode and the spray voltage is 3.0kV.

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