CN117491549B - Qualitative and quantitative method for phosphatidylcholine isomer - Google Patents
Qualitative and quantitative method for phosphatidylcholine isomer Download PDFInfo
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- 150000008105 phosphatidylcholines Chemical class 0.000 title claims abstract description 33
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 20
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 10
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- 238000001514 detection method Methods 0.000 claims description 30
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 28
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 7
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- QXDDDGCNWRUEFM-UMKMFDOBSA-N 1-octadecanoyl-2-[(5Z,8Z,11Z)-eicosatrienoyl]-sn-glycero-3-phosphocholine Chemical class CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/CCCCCCCC QXDDDGCNWRUEFM-UMKMFDOBSA-N 0.000 description 8
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- KLJBLQFSKZACGJ-MMKOEKFTSA-N 1,2-di-[(6Z)-octadecenoyl]-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCC\C=C/CCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCC\C=C/CCCCCCCCCCC KLJBLQFSKZACGJ-MMKOEKFTSA-N 0.000 description 4
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- VGSUMLIRUGDCTF-NSMRCOLJSA-N 1-octadecanoyl-2-[(8Z,11Z,14Z)-eicosatrienoyl]-sn-glycero-3-phosphocholine Chemical group CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCC\C=C/C\C=C/C\C=C/CCCCC VGSUMLIRUGDCTF-NSMRCOLJSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
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Abstract
The invention relates to the technical field of recognition of phosphatidylcholine isomers, in particular to a qualitative and quantitative method for phosphatidylcholine isomers. The invention provides a method for simultaneously carrying out structure identification and quantification on PCs based on negative mode MRM mass spectrometry. The method initiates a structure-driven carbon-carbon double bond position isomerism identification strategy, and comprehensive PCs qualitative and quantitative analysis is carried out through RPLC negative mode MRM. The method has the advantages of high sensitivity, good specificity and the like, and has higher practicability and reliability. In addition, the method is simple and quick, does not depend on high-resolution mass spectrum, can identify the phosphatidylcholine isomer under the condition of no standard substance, and can simultaneously perform quantitative analysis with high sensitivity and high selectivity.
Description
Technical Field
The invention relates to the technical field of recognition of phosphatidylcholine isomers, in particular to a qualitative and quantitative method for phosphatidylcholine isomers.
Background
Phosphatidylcholine (PCs) is the most abundant phospholipid in mammalian cells, an essential component of biological membranes, involved in many important biological processes including vesicle transport, cell signaling, energy metabolism and protein function regulation. The PCs family exhibits a great diversity in molecular structure, particularly in the positional isomers arising from the difference in the position of the carbon-carbon double bond (C ═ C) in its acyl chain. The positional isomerism of these C ═ C bonds has been shown to affect membrane fluidity, cell signaling, enzyme regulation, lipid metabolism, and various physiological and pathological processes. Therefore, it is important to identify the exact structure of the PCs double bond position and to quantify it accurately.
Over the last decade, efforts have been made to achieve comprehensive identification of PCs structures and to establish several methods. For example: gas phase ion activation/dissociation process. The gas-phase ion activation/dissociation method comprises ozone-induced dissociation, ultraviolet photolysis, electron collision excitation of organic ions, metastable atom activation dissociation, free radical directional dissociation and the like. These methods allow the identification of PCs isomers by generating MS2 fragments that are related to the double bond position, by means of filtering and matching algorithms.
Since these methods require more complicated equipment, the reliability thereof is lowered; and such methods require complicated gas phase reactions, resulting in poor sensitivity and specificity. In addition, none of the above strategies achieves accurate quantification.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for identifying and quantifying phosphatidylcholine isomers, which has excellent sensitivity and specificity and can achieve accurate quantification.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a qualitative and quantitative method for phosphatidylcholine isomers, which comprises the following steps: mixing an object to be detected containing phosphatidylcholine with an internal standard, and extracting to obtain an object to be detected;
performing reverse phase liquid chromatography tandem mass spectrometry detection on the liquid to be detected to obtain retention time of each unknown isomer, and determining fatty acid chains of each unknown isomer according to MRM precursor ions; during which any peaks associated with isotope distribution are effectively eliminated by the RT shift filter;
obtaining phosphatidylcholine isomers containing the fatty acid chains in a database, and predicting the oil-water partition coefficient LogP of each phosphatidylcholine isomer ow ；
Retention time of the same group of phosphatidylcholine isomers and of the respective unknown isomers and oil-water partition coefficient LogP ow All are ordered according to the same arrangement sequence to match each unknown isomer, so that the structure of each unknown isomer is obtained;
obtaining the mass of each unknown isomer according to the mass of the internal standard, the peak area of the internal standard and the peak area of each unknown isomer;
the conditions for the reverse phase liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution-methanol-acetonitrile system containing ammonium formate and formic acid; the mobile phase B is a methanol solution-isopropanol system containing ammonium formate and formic acid; the flow rate of the mobile phase system is 0.4mL/min; the elution mode is gradient elution;
the gradient elution procedure was:
0.0 to 0.5min: the volume percentage of the mobile phase B is 10%;
0.5 to 7.0min: the volume percentage of the mobile phase B is increased from 10% to 30% at a constant speed; 7.0 to 47.0min: the volume percentage of the mobile phase B is increased from 30% to 70% at a constant speed; 47.0-47.1 min: the volume percentage of the mobile phase B is reduced from 70% to 10% at a constant speed; 47.1 to 50.0min: the volume percentage of the mobile phase B is 10%;
the conditions for mass spectrometry detection include: an electrospray ion source; MRM mode negative ion mode scanning.
Preferably, in the mobile phase A, the concentration of ammonium formate in the aqueous solution containing ammonium formate and formic acid is 5mM; the volume concentration of formic acid was 0.05%.
Preferably, the mobile phase A contains an aqueous solution of ammonium formate and formic acid, and the volume ratio of methanol to acetonitrile is 1:1:1.
Preferably, in the mobile phase B, the concentration of ammonium formate in the methanol containing ammonium formate and formic acid is 5mM; the volume concentration of formic acid was 0.05%.
Preferably, the mobile phase B contains a methanol solution of ammonium formate and formic acid and isopropanol in a volume ratio of 1:1.
Preferably, the chromatographic column for the reversed phase liquid chromatography detection is a Waters ACQUITY UPLC BEH C column; the column temperature was 40 ℃.
Preferably, the gas curtain gas detected by the mass spectrum is nitrogen, and the temperature of the ion source is 500 ℃; the spray voltage was-4500V.
Preferably, the internal standard has a structure represented by formula I:
formula I.
Preferably, the extracted reagent is methyl tertiary butyl ether.
The invention provides a qualitative and quantitative method for phosphatidylcholine isomers, which comprises the following steps: mixing an object to be detected containing phosphatidylcholine with an internal standard, and extracting to obtain an object to be detected; performing reverse phase liquid chromatography tandem mass spectrometry detection on the liquid to be detected to obtain retention time of each unknown isomer, and determining fatty acid chains of each unknown isomer according to MRM precursor ions; during which any peaks associated with isotope distribution are effectively eliminated by the RT shift filter; obtaining phosphatidylcholine isomers containing the fatty acid chains in a database, and predicting the oil-water partition coefficient LogP of each phosphatidylcholine isomer ow The method comprises the steps of carrying out a first treatment on the surface of the Retention time of the same group of phosphatidylcholine isomers and of the respective unknown isomers and oil-water partition coefficient LogP ow All are ordered according to the same arrangement sequence to match each unknown isomer, so that the structure of each unknown isomer is obtained;obtaining the mass of each unknown isomer according to the mass of the internal standard, the peak area of the internal standard and the peak area of each unknown isomer; the conditions for the reverse phase liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution-methanol-acetonitrile system containing ammonium formate and formic acid; the mobile phase B is a methanol solution-isopropanol system containing ammonium formate and formic acid; the flow rate of the mobile phase system is 0.4mL/min; the elution mode is gradient elution; the gradient elution procedure was: 0.0 to 0.5min: the volume percentage of the mobile phase B is 10%;0.5 to 7.0min: the volume percentage of the mobile phase B is increased from 10% to 30% at a constant speed; 7.0 to 47.0min: the volume percentage of the mobile phase B is increased from 30% to 70% at a constant speed; 47.0-47.1 min: the volume percentage of the mobile phase B is reduced from 70% to 10% at a constant speed; 47.1 to 50.0min: the volume percentage of the mobile phase B is 10%; the conditions for mass spectrometry detection include: an electrospray ion source; MRM mode negative ion mode scanning. Compared with the prior art, the invention provides a method for simultaneously carrying out structural identification and quantification on PCs based on negative mode MRM mass spectrometry. The method initiates a structure-driven carbon-carbon double bond position isomerism identification strategy, and comprehensive PCs qualitative and quantitative analysis is carried out through RPLC negative mode MRM. The method has the advantages of high sensitivity, good specificity and the like, and has higher practicability and reliability. In addition, the method is simple and quick, does not depend on high-resolution mass spectrum, can identify single double bond and multiple double bond PCs isomers with positions under the condition of no standard substance, and can simultaneously perform quantitative analysis with high sensitivity and high selectivity.
Drawings
FIG. 1 is a liquid chromatogram of example 1 PC (18:1/18:1) for 4 isomer standards;
FIG. 2 is the LogP of example 1 PC (18:1/18:1) 4 isomer standards ow And linear correlation between RTs;
FIG. 3 is a liquid chromatogram of the PC (16:0/16:1) 2 isoforms of rat lung tissue of example 2;
FIG. 4 is a liquid chromatogram of 3 isomers of PC (18:0/20:3) from rat lung tissue of example 2;
FIG. 5 is a predicted LogP of 3 isoforms of PC (18:0/20:3) rat lung tissue of example 2 ow And linear correlation between RTs;
FIG. 6 is a heat map of PCs isoforms in lung tissue of CWP rats and control rats;
FIG. 7 is a volcanic plot of differential PCs isoforms in lung tissue of CWP rats and control rats.
Detailed Description
The invention provides a qualitative and quantitative method for phosphatidylcholine isomers, which is characterized by comprising the following steps of:
mixing an object to be detected containing phosphatidylcholine with an internal standard, and extracting to obtain an object to be detected;
performing reverse phase liquid chromatography tandem mass spectrometry detection on the liquid to be detected to obtain retention time of each unknown isomer, and determining fatty acid chains of each unknown isomer according to MRM precursor ions; during which any peaks associated with isotope distribution are effectively eliminated by the RT shift filter;
obtaining phosphatidylcholine isomers containing the fatty acid chains in a database, and predicting the oil-water partition coefficient LogP of each phosphatidylcholine isomer ow ；
Retention time of the same group of phosphatidylcholine isomers and of the respective unknown isomers and oil-water partition coefficient LogP ow All are ordered according to the same arrangement sequence to match each unknown isomer, so that the structure of each unknown isomer is obtained;
obtaining the mass of each unknown isomer according to the mass of the internal standard, the peak area of the internal standard and the peak area of each unknown isomer;
the conditions for the reverse phase liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution-methanol-acetonitrile system containing ammonium formate and formic acid; the mobile phase B is a methanol solution-isopropanol system containing ammonium formate and formic acid; the flow rate of the mobile phase system is 0.4mL/min; the elution mode is gradient elution;
the gradient elution procedure was:
0.0 to 0.5min: the volume percentage of the mobile phase B is 10%;
0.5 to 7.0min: the volume percentage of the mobile phase B is increased from 10% to 30% at a constant speed; 7.0 to 47.0min: the volume percentage of the mobile phase B is increased from 30% to 70% at a constant speed; 47.0-47.1 min: the volume percentage of the mobile phase B is reduced from 70% to 10% at a constant speed; 47.1 to 50.0min: the volume percentage of the mobile phase B is 10%;
the conditions for mass spectrometry detection include: an electrospray ion source; MRM mode negative ion mode scanning.
The invention mixes the object to be measured containing phosphatidylcholine with the internal standard substance to obtain the liquid to be measured.
In the present invention, the internal standard has a structure represented by formula I:
formula I.
In the present invention, the specimen containing phosphatidylcholine is preferably plasma or lung tissue.
In the present invention, the reagent for extraction is preferably methyl tertiary butyl ether.
In the present invention, the obtaining of the liquid to be measured preferably includes the steps of:
mu.L of the test material was added to a 1.5 mL centrifuge tube at maximum speed vortex 10s, 3. Mu.L of internal standard (160. Mu.g/mL), 750. Mu.L of methyl tert-butyl ether, and at maximum speed vortex 10 s. Centrifuging at low temperature of 4deg.C for 15min at high speed, and collecting 700 μl of supernatant (sample divided into three layers, methyl tert-butyl ether phase (lipid phase), water phase, and solid residue from top to bottom) into new centrifuge tube. After the supernatant is dried by a nitrogen blower, 100 mu L of acetonitrile is added for re-dissolution, 10s vortex and 14000g (4 ℃) are centrifuged for 10min, and the obtained supernatant is the liquid to be detected.
After obtaining a liquid to be detected, the invention carries out reverse phase liquid chromatography tandem mass spectrometry detection on the liquid to be detected to obtain the retention time of each unknown isomer, and determines the fatty acid chain of each unknown isomer according to MRM precursor ions; during which any peaks associated with isotope distribution are effectively eliminated by the RT shift filter.
In the present invention, the conditions for the detection by reverse phase liquid chromatography include: the mobile phase system is a mobile phase A and a mobile phase B, the mobile phase A is an aqueous solution-methanol-acetonitrile system containing ammonium formate and formic acid, and the concentration of the ammonium formate in the aqueous solution containing the ammonium formate and the formic acid is preferably 5mM; the volume concentration of formic acid is preferably 0.05%;
the mobile phase B is a methanol solution-isopropanol system containing ammonium formate and formic acid; the concentration of ammonium formate in the methanol solution containing ammonium formate and formic acid is preferably 5mM; the volume concentration of formic acid is preferably 0.05%. The flow rate of the mobile phase system is 0.4mL/min; the elution mode is gradient elution;
the gradient elution is carried out by the following steps of;
0.0 to 0.5min: the volume percentage of the mobile phase B is 10%;
0.5 to 7.0min: the volume percentage of the mobile phase B is increased from 10% to 30% at a constant speed; 7.0 to 47.0min: the volume percentage of the mobile phase B is increased from 30% to 70% at a constant speed; 47.0-47.1 min: the volume percentage of the mobile phase B is reduced from 70% to 10% at a constant speed; 47.1 to 50.0min: the volume percentage of the mobile phase B is 10%.
The conditions for mass spectrometry detection include: an electrospray ion source; monitoring: MRM mode negative ion mode scanning; the gas 1 pressure is preferably 50 psi; the gas 2 pressure is preferably 50 psi; the gas curtain gas is preferably nitrogen, and the pressure of the gas curtain gas is preferably 30 psi; collision gas, medium; the ion source temperature is preferably 500 ℃; the spray voltage is preferably-4500V.
The fatty acid chain of each unknown isomer is determined, the phosphatidylcholine isomers containing the fatty acid chain are obtained in a database, and the oil-water distribution coefficient LogP of each phosphatidylcholine isomer is predicted ow ；
Retention time of the same group of phosphatidylcholine isomers and of the respective unknown isomers and oil-water partition coefficient LogP ow All ordered in the same order to match each unknown isomer, thereby obtaining the structure of each unknown isomer.
In the present invention, the oil-water fractionCoefficient of match LogP ow Preferably obtained by on-line software ALOGPS calculation.
After obtaining the structures of the unknown isomers, the invention obtains the mass of each unknown isomer according to the mass of the internal standard, the peak area of the internal standard and the peak area of each unknown isomer.
Specifically, according to formula II, the mass of each unknown isomer is calculated;
m=m 1 ×s/s 1 a formula II;
in the formula II: m is m 1 The mass of the internal standard; s is(s) 1 Peak area for internal standard;
s is the peak area of each isomer.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Conditions for reversed phase liquid chromatography tandem mass spectrometry detection:
the conditions for the reverse phase liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution-methanol-acetonitrile system containing ammonium formate and formic acid; the mobile phase B is a methanol solution-isopropanol system containing ammonium formate and formic acid; the flow rate of the mobile phase system is 0.4mL/min; the elution mode is gradient elution;
the gradient elution procedure was:
0.0 to 0.5min: the volume percentage of the mobile phase B is 10%;
0.5 to 7.0min: the volume percentage of the mobile phase B is increased from 10% to 30% at a constant speed; 7.0 to 47.0min: the volume percentage of the mobile phase B is increased from 30% to 70% at a constant speed; 47.0-47.1 min: the volume percentage of the mobile phase B is reduced from 70% to 10% at a constant speed; 47.1 to 50.0min: the volume percentage of the mobile phase B is 10%;
the conditions for mass spectrometry detection include: an electrospray ion source; monitoring: MRM mode negative ion mode scanning; the gas 1 pressure is preferably 50 psi; the gas 2 pressure is preferably 50 psi; the gas curtain gas is preferably nitrogen, and the pressure of the gas curtain gas is preferably 30 psi; collision gas, medium; the ion source temperature is preferably 500 ℃; the spray voltage is preferably-4500V.
(2) Detection of
PC standards PC (18:1 (6Z)/18:1 (6Z)), PC (18:1 (8Z)/18:1 (8Z)), PC (18:1 (9Z)/18:1 (9Z)) and PC (18:1 (11Z)/18:1 (11Z)) were analyzed for the position of carbon-carbon double bonds on the fatty acid chains. Wherein nZ represents that a carbon-carbon double bond is located at the n-th carbon atom and is a cis structure.
10. Mu. MPC (18:1 (6Z)/18:1 (6Z)), PC (18:1 (8Z)/18:1 (8Z)), PC (18:1 (9Z)/18:1 (9Z)) and PC (18:1 (11Z)/18:1 (11Z)) were dissolved in acetonitrile, respectively
Then mixing to obtain a mixed solution;
mu.L of the mixture was added to a 1.5 mL centrifuge tube with maximum speed vortex 10s, 3. Mu.L of internal standard of formula I (160. Mu.g/mL), 750. Mu.L of methyl tert-butyl ether, and maximum speed vortex 10 s. Centrifuging at low temperature of 4deg.C for 15min at high speed, and collecting 700 μl of supernatant to a new centrifuge tube. The supernatant was blow-dried with a nitrogen blower. Adding 100 mu L of acetonitrile to recover lipid, swirling 10s, centrifuging 14000g (4 ℃) for 10min, and obtaining supernatant which is the standard liquid to be detected.
The standard liquid to be detected is detected according to the condition of "(1) reverse phase liquid chromatography tandem mass spectrometry detection", the chromatogram is shown in figure 1, and the figure 1 shows that: PC (18:1 (6Z)/18:1 (6Z)), PC (18:1 (8Z)/18:1 (8Z)), PC (18:1 (9Z)/18:1 (9Z)) and PC (18:1 (11Z)/18:1 (11Z)) are well separated under the detection conditions of the present invention.
LogP of PC (18:1 (6Z)/18:1 (6Z)), PC (18:1 (8Z)/18:1 (8Z)), PC (18:1 (9Z)/18:1 (9Z)) and PC (18:1 (11Z)/18:1 (11Z)) using the neural network program ALOGPS ow Predictions were made and the LogP of PC (18:1 (6Z)/18:1 (6Z)), PC (18:1 (8Z)/18:1 (8Z)), PC (18:1 (9Z)/18:1 (9Z)) and PC (18:1 (11Z)/18:1 (11Z)) ow And the retention time ordering thereof, a linear relationship is established, as can be seen from fig. 2, as follows: lo (Lo)gP ow And the retention time thereof has a good linear correlation.
Example 2
The example demonstrates the reliability of the application of the method to analysis and identification of PC isomers in biological samples (plasma), including reproducibility of analysis peak area and retention time of biological samples, precision and accuracy of labeled biological sample analysis.
Sample: PC (18:1 (11Z)/18:1 (11Z)), PC (18:1 (8Z)/18:1 (8Z)) standard substances are added into human plasma, and standard curves and quality control samples are prepared. The standard curve concentration is: 50 ng/mL, 200ng/mL, 500 ng/mL, 2000 ng/mL, 5000 ng/mL; the quality control concentration is 400 ng/mL, 4000 ng/mL.
The preparation method comprises the following steps:
(1) the EP tube was filled with 225. Mu.L of methanol, 20. Mu.L of plasma samples were added and vortexed at maximum speed for 10s, 20. Mu.L of mix was aspirated from each sample as sample QC;
(2) adding an internal standard; 3. mu.L of internal standard of formula I (160. Mu.g/mL);
(3) 750 μl of methyl tert-butyl ether was added, vortexed at maximum speed for 10s, and left standing at room temperature for 30 min;
(4) 188 μl of mass spectrometry grade water was added and vortexed for 20s;
(5) standing at room temperature for 10min;
(6) at 4℃and high speed, the mixture was centrifuged at 15000 rpm for 15min, and 700. Mu.L of the supernatant (sample divided into three layers, lipid phase, aqueous phase and solid residue from top to bottom) was taken into a new EP tube.
(7) The supernatant was blow-dried with a nitrogen blower.
(8) Adding 100 mu L of compound solution which is isopropanol to recover lipid: acetonitrile: water (volume ratio 30:65:5).
(9) Vortex for 10s and centrifuge for 10min at 14000g (4 ℃).
And sucking the supernatant on a sample tube for machine detection.
Sample detection: and (3) detecting the liquid to be detected according to the condition of "(1) reverse-phase liquid chromatography tandem mass spectrometry detection", wherein the detection results are shown in tables 1-3.
Table 1 biological sample PC isomer analysis peak area reproducibility
Table 2 biological sample PC isomer analysis retention time reproducibility
TABLE 3 precision and accuracy of PC isomer analysis of labeled biological samples (PC (18:1/18:1) for example)
Example 3
PC isomer in lung tissue of a rat model with lung injury caused by coal dust particles is analyzed by the method.
12 male SD rats (180-200 g) were randomly divided into a control group and a coal dust exposure group, each group having 6 male SD rats. A CWP (coal dust lung) rat model was established by single tracheal instillation of saline (1 mL) followed by instillation of coal dust particles (50 mg/mL,1 mL). Rats were sacrificed at week 4 post exposure. Lung tissue was isolated from rats and frozen in liquid nitrogen for later use.
Sample preparation: the lung tissue sample 10 mg was mixed with 100 μl acetonitrile and the resulting solution extracted with 1mL methyl tert-butyl ether and vortexed vigorously for 10 minutes. The methyl tert-butyl ether phase was collected and dried with a vacuum concentrator. Prior to LC-MS analysis, the dried sample was reconstituted with 30. Mu.L acetonitrile containing an internal standard (200 ng/mL) to give the test solution.
Detecting the liquid to be detected according to the condition of "(1) reversed-phase liquid chromatography tandem mass spectrometry detection", and obtaining a chromatogram of each isomer;
FIG. 3 is a liquid chromatogram of the PC (16:0/16:1) 2 isoforms of rat lung tissue of example 2. FIG. 4 is a liquid chromatogram of 3 isomers of PC (18:0/20:3) of example 2 rat lung tissue of example 2, as can be seen from FIGS. 3-4: isomers of either monounsaturated PC (PC (16:0/16:1)) or polyunsaturated PC (PC (18:0/20:3)) were successfully separated.
LogP prediction of PC (18:0/20:3) 3 isomers using the neural network program ALOGGS ow LogP of 3 isomers of PC (18:0/20:3) ow And after the retention time ordering, a linear relationship is established, as can be seen from FIG. 5: PC (18:0/20:3) 3 isomers LogP ow And RTs, and it can be inferred that in FIG. 4 RT is PC (18:0/20:3 (8Z, 11Z, 14Z)) for 26.55min, RT is PC (18:0/20:3 (5Z, 8Z, 14Z)) for 27.27min, and RT is PC (18:0/20:3 (5Z, 8Z, 11Z)) for 27.95 min.
Figure 6 is a heat map of PCs isomers of CWP rats and control rats showing significant differences in PC isomer composition between the two groups. Further statistical analysis showed that PC with unsaturated fatty chains showed the most significant changes, as shown in fig. 7. This structure-driven strategy helps to efficiently, sensitively, accurately identify PC isomers and further facilitates further research into PC biological functions.
Example 4
5mg of lung tissue mill grind was added to a 1.5 mL centrifuge tube, 1mL of methyl tert-butyl ether was added, vortexed for 10min, centrifuged at 13300rpm at 4℃for 10min, 800. Mu.L of supernatant was taken, concentrated to dryness by vacuum centrifugation, and 30. Mu.L of acetonitrile: the solution containing the internal standard (200 ng/ml) of isopropanol (1:1) is redissolved to dry a sample, 10s vortex and 13300rpm (4 ℃) are centrifuged for 10min, and the obtained supernatant is the liquid to be detected.
Detecting the liquid to be detected according to the condition of "(1) reverse-phase liquid chromatography tandem mass spectrometry detection", obtaining the retention time of each unknown isomer, and determining the fatty acid chain of each unknown isomer according to the detection ion pair; during which any peaks associated with isotope distribution are effectively eliminated by the RT shift filter.
Obtaining phosphatidylcholine isomers containing the fatty acid chains from a database, and predicting the oil-water partition coefficient LogP of each phosphatidylcholine isomer ow The method comprises the steps of carrying out a first treatment on the surface of the Retention time of the same group of phosphatidylcholine isomers and of the respective unknown isomers and oil-water partition coefficient LogP ow Are all ordered according to the same orderObtaining LogP ow And RTs, aligning the observed isomer peaks with the respective structures to match each unknown isomer, thereby obtaining the structures of each unknown isomer. Table 4 shows the phosphatidylcholine isomers identified in example 4.
Table 4 Phosphatidylcholine isomers identified in tissues
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The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A qualitative and quantitative method for phosphatidylcholine isomers, comprising the steps of:
mixing an object to be detected containing phosphatidylcholine with an internal standard, and extracting to obtain an object to be detected;
performing reverse phase liquid chromatography tandem mass spectrometry detection on the liquid to be detected to obtain retention time of each unknown isomer, and determining fatty acid chains of each unknown isomer according to MRM precursor ions; during which any peaks associated with isotope distribution are effectively eliminated by the RT shift filter;
acquiring phosphatidylcholine isomers containing the fatty acid chains from a database, and predicting the oil-water partition coefficient LogP of each phosphatidylcholine isomer based on adopting a neural network program ALOGGS ow ；
Retention time of the same group of phosphatidylcholine isomers and of the respective unknown isomers and oil-water partition coefficient LogP ow All are ordered according to the same arrangement sequence to match all unknown isomers, so that the structure of each unknown isomer is obtained, and the position isomerism identification of the carbon-carbon double bond of the phosphatidylcholine isomer is realized;
obtaining the mass of each unknown isomer according to the mass of the internal standard, the peak area of the internal standard and the peak area of each unknown isomer;
the conditions for the reverse phase liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution-methanol-acetonitrile system containing ammonium formate and formic acid; the mobile phase B is a methanol solution-isopropanol system containing ammonium formate and formic acid; the flow rate of the mobile phase system is 0.4mL/min; the elution mode is gradient elution;
the gradient elution procedure was:
0.0 to 0.5min: the volume percentage of the mobile phase B is 10%;
0.5 to 7.0min: the volume percentage of the mobile phase B is increased from 10% to 30% at a constant speed;
7.0 to 47.0min: the volume percentage of the mobile phase B is increased from 30% to 70% at a constant speed;
47.0-47.1 min: the volume percentage of the mobile phase B is reduced from 70% to 10% at a constant speed;
47.1 to 50.0min: the volume percentage of the mobile phase B is 10%;
the conditions for mass spectrometry detection include: an electrospray ion source; MRM mode negative ion mode scanning; in the mobile phase A, the concentration of ammonium formate in the aqueous solution containing ammonium formate and formic acid is 5mM; the volume concentration of formic acid is 0.05%; the mobile phase A contains an aqueous solution of ammonium formate and formic acid, and the volume ratio of methanol to acetonitrile is 1:1:1; in the mobile phase B, the concentration of ammonium formate in the methanol solution containing ammonium formate and formic acid is 5mM; the volume concentration of formic acid is 0.05%; the mobile phase B contains methanol solution of ammonium formate and formic acid and isopropanol with the volume ratio of 1:1; the chromatographic column for the reversed phase liquid chromatography detection is Waters ACQUITY UPLC BEH C column; the column temperature was 40 ℃.
2. The qualitative and quantitative method according to claim 1, wherein the gas curtain gas detected by mass spectrometry is nitrogen, and the ion source temperature is 500 ℃; the spray voltage was-4500V.
3. The qualitative and quantitative method according to claim 1, wherein the internal standard has a structure according to formula I:
formula I.
4. The qualitative and quantitative method according to claim 1, wherein the extracted reagent is methyl tert-butyl ether.
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