CN114689756B - Analysis method of triglyceride positional isomer - Google Patents

Analysis method of triglyceride positional isomer Download PDF

Info

Publication number
CN114689756B
CN114689756B CN202210352796.1A CN202210352796A CN114689756B CN 114689756 B CN114689756 B CN 114689756B CN 202210352796 A CN202210352796 A CN 202210352796A CN 114689756 B CN114689756 B CN 114689756B
Authority
CN
China
Prior art keywords
acid
triglyceride
triglycerides
abundance
palmitic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210352796.1A
Other languages
Chinese (zh)
Other versions
CN114689756A (en
Inventor
韦伟
张星河
金俊
金青哲
王兴国
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN202210352796.1A priority Critical patent/CN114689756B/en
Publication of CN114689756A publication Critical patent/CN114689756A/en
Application granted granted Critical
Publication of CN114689756B publication Critical patent/CN114689756B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • G01N30/02—Column chromatography
    • 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
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Abstract

The invention provides an analysis method of triglyceride positional isomer, belonging to the field of lipid analysis, and the method adopts a supercritical fluid chromatography-mass spectrometry combined method to carry out qualitative analysis on the triglyceride positional isomer; and quantitatively analyzing the triglyceride positional isomer by adopting an abundance method, which specifically comprises the following steps: obtaining the abundance of fragment ions by secondary mass spectrometry, and analyzing the relative content of triglyceride position isomers by the abundance; the triglycerides include ABC-type triglycerides and/or AAB-type triglycerides. The method can achieve the aim of accurately and quickly analyzing the content of the positional isomer of the triglyceride.

Description

Analysis method of triglyceride positional isomer
Technical Field
The invention relates to the field of lipid analysis, in particular to an analysis method of triglyceride position isomers, which is particularly suitable for the qualitative and quantitative analysis of the triglyceride position isomers such as natural lipids, biological samples, complex samples, structural lipids and the like.
Background
The structure of triglycerides varies greatly among different species of biological origin in nature. In vegetable oils, saturated fatty acids are generally located at the sn-1/3 position and unsaturated fatty acids are located at the sn-2 position; in milk fat, especially human milk fat, saturated fatty acids are mainly located at the sn-2 position; in addition, there are also differences in the positional structures of Fatty Acids (FA) on milk fat triglycerides of different species.
The positional distribution of fatty acid types on triglycerides has a significant impact on the digestion and absorption of fat by infants and young children: the USU (U refers to unsaturated fatty acid, and S refers to saturated fatty acid) type triglyceride accounts for a relatively high proportion (about 48%), and studies show that sn-USU has the effects of promoting the absorption of energy and mineral substances of infants and reducing the hardness of excrement. Wherein the highest content of fatty acids at the sn-2 position is palmitic acid (P, C16: 0) accounting for more than 56% of all fatty acids at the sn-2 position, and the triglyceride having unsaturated fatty acids connected at the sn-1/3 position and palmitic acid connected at the sn-2 position is called 1, 3-di-unsaturated fatty acid-2-palmitic acid triglyceride (UPU). The sn-UPU has the highest content (about 32 percent) in the breast milk fat, and has special physiological action in the absorption and metabolism process of infants due to the unique molecular structure, so the development of research on the content distribution and physiological function of the breast milk UPU has important significance.
Although people know the benefit of UPU type triglyceride on the health of infants, functional structure fat 1, 3-dioleate 2-palmitic acid triglyceride (OPO) is more and more added into infant formula products at home and abroad, as a method for accurately quantifying triglyceride position isomer does not exist at present, the OPO content is only determined by a liquid chromatography combined with an evaporation light detector (HPLC-ELSD) method, and the index of milk powder is evaluated by combining a Gas Chromatography (GC) method for determining the content of sn-2 position palmitic acid. As a result, accurate quantification cannot be achieved due to the presence of OOP, which is a positional isomer of OPO, and other triglycerides such as OPP, which are each composed of palmitic acid bonded to the sn-2 position. The method currently applied to the separation and identification of positional isomers in human milk fat is silver ion chromatography (Ag) + HPLC) or off-line two-dimensional chromatography, but the method is only applied to the related research of two sets of positional isomers of OPO and OOP and OPP and POP, has the defects of long time consumption, poor reproducibility and the like, is not suitable for the analysis of a large number of samples, and is not widely applied at present.
In the related technology, a liquid chromatography-mass spectrometry combined detection method is adopted, and the triglyceride positional isomer can be analyzed by establishing a linear relation between the content of the triglyceride positional isomer and a secondary mass spectrum, so that the content of the triglyceride positional isomer in the vegetable oil can be analyzed simply.
Therefore, it is necessary to further study the analysis method of triglyceride, especially positional isomer of UPU type triglyceride.
Disclosure of Invention
Aiming at the problems and the defects in the prior art, the invention provides an analysis method of triglyceride position isomers, which uses a supercritical fluid chromatography-mass spectrometry combined method to carry out qualitative analysis on the triglyceride position isomers; quantitative analysis is carried out on the triglyceride positional isomer by adopting an abundance method, and the aim of accurately and quickly analyzing the content of the triglyceride positional isomer can be achieved.
The method is realized by the following technical scheme:
the invention provides an analysis method of triglyceride positional isomers, which adopts a supercritical fluid chromatography-mass spectrometry combined method to carry out qualitative analysis on the triglyceride positional isomers;
quantitative analysis is carried out on the triglyceride positional isomer by adopting an abundance method, which comprises the following steps:
obtaining the abundance of fragment ions by secondary mass spectrometry, and analyzing the relative content of triglyceride position isomers by the abundance;
the triglycerides include ABC-type triglycerides and/or AAB-type triglycerides.
Optionally, the supercritical fluid chromatography conditions are:
performing supercritical fluid chromatography; 2-ethyl pyrimidine or C18 is adopted as a chromatographic column of a filler; eluent A is supercritical CO 2 The eluent B is an ethanol/acetonitrile solution with the volume percentage of 50 percent to 50 percent; the compensation solvent is ammonium acetate/methanol solution, the concentration is 5mm/L-20mm/L, and the flow rate is 0.2mL/min; the gradient elution program is 0min, the proportion of B phase is 0.2%, B is 0.3% at 1min, B is 0.6% at 18min, B is 2% at 18.1min and is kept at 21min, B is 7% at 25min, B is 0.2% at 27min and is kept at 30min; the column temperature is 50 ℃; back pressure 2500psi; the sample injection volume is 1 mu L; the flow rate is 1mL/min; the sample was dissolved in n-hexane at a concentration of 1mg/mL.
Optionally, the mass spectrometry conditions are:
adopting a high-resolution mass spectrometer with an ionization source of ESI, wherein the ionization source is in a positive ion state, the ion source temperature is 100 ℃, the desolvation temperature is 400 ℃, the collision gas is argon, and the flow rate is 50L/h; desolvation gas is nitrogen, and the flow rate is 700L/h; real-time correction with leucine enkephalin; the mass spectrometry conditions comprise MS E DDA and SURVEY modes.
Optionally, the MS E The mode conditions are as follows: the taper hole voltage is 45V, the low energy collision energy is 6eV, and the high energy collision energy isThe amount is set to be 25V, the molecular weight scanning range is 200-1500m/z, and the data presentation format is Profile data;
the DDA mode conditions are as follows: cone voltage 45V, collision energy: 25eV, scan time interval 0.1s, molecular weight scan range: 200-1200m/z, and the data presentation format is Centroid data;
the SURVEY mode condition: cone voltage 45V, collision energy: 25eV, the scanning time of the primary mass spectrum is 1s, the scanning time interval is 0.02s, the component number of the secondary mass spectrum is 4, the scanning time is 0.2s, and the scanning time interval is 0.02s; molecular weight scan range: 100-1200m/z, and the data presentation format is Centroid data.
Alternatively, for ABC-type triglycerides, the relative contents of the triglyceride positional isomers sn-B-A-C, sn-A-B-C, sn-A-C-B are analyzed using equation (1):
Figure BDA0003575190350000031
wherein ase:Sub>A, B and C are the relative contents of sn-B-A-C, sn-A-B-C and sn-A-C-B respectively, and ase:Sub>A + B + C =1; y is A 、Y B 、Y C Are respectively fragment ions [ B-C] + 、[A-C] + 、[A-B] + The abundance of (A) is a percentage of the sum of the abundances of the three, Y A 、Y B 、Y C Can be obtained by secondary mass spectrometry; assuming that the relative abundance of fragment ions after neutral loss of sn-1 fatty acid and sn-3 fatty acid in triglyceride is 1, the relative abundance of fragment ions after neutral loss of sn-2 fatty acid is x i ，x i Obtained by secondary mass spectrometry.
Alternatively, for AAB-type triglycerides, the relative content of the triglyceride positional isomers sn-A-A-B, sn-A-B-A is analyzed using equation (2):
Figure BDA0003575190350000032
whereinbase:Sub>A and B are the relative contents of sn-A-A-B and sn-A-B-A respectively, andbase:Sub>A + B =1; z A 、Z B Are respectively fragment ions [ A-B] + 、[A-A] + The abundance of (a) is a percentage of the sum of the abundances of (b), Z A 、Z B Can be obtained by secondary mass spectrometry; assuming that the relative abundance of fragment ions after neutral loss of sn-1 fatty acid and sn-3 fatty acid in triglyceride is 1, the relative abundance of fragment ions after neutral loss of sn-2 fatty acid is x i ，x i Obtained by secondary mass spectrometry.
Optionally, the ABC-type triglycerides and AAB-type triglycerides are derived from vegetable oils, animal oils, milk fats, microbial oils, processed synthetic lipids, foods or pharmaceuticals containing lipids.
Alternatively, the ABC type triglycerides include, but are not limited to: 1-oleic acid-2-palmitic acid-3-lauric acid, 1-palmitic acid-2-oleic acid-3-lauric acid, 1-palmitic acid-2-lauric acid-3-oleic acid, 1-oleic acid-2-palmitic acid-3-stearic acid, 1-palmitic acid-2-oleic acid-3-stearic acid, 1-palmitic acid-2-stearic acid-3-oleic acid, 1-oleic acid-2-palmitic acid-3-linoleic acid, 1-palmitic acid-2-oleic acid-3-linoleic acid, 1-palmitic acid-2-linoleic acid-3-oleic acid.
Alternatively, the AAB-type triglycerides include, but are not limited to: 1, 2-dioleic-3-palmitic acid, 1, 3-dioleic-2-palmitic acid, 1, 2-dipalmitic-3-oleic acid, 1, 3-dipalmitic-2-oleic acid, 1, 2-dilinoleic-3-palmitic acid, 1, 3-dilinoleic-2-palmitic acid.
The invention has the following beneficial effects:
1. by using the supercritical fluid chromatography, triglyceride compounds can be well separated, the influence of interference factors such as co-effluent, isomer and the like on the analysis of the positional isomer is reduced, and the method has the advantages of quick analysis, good sample reproducibility and stability, and environmental protection.
2. By using the secondary mass spectrum in the mass spectrum, the molecular structure information of the triglyceride compound can be obtained, and the content of the triglyceride isomer can be accurately calculated by utilizing the relationship existing between the abundance of fragment ions in the secondary mass spectrum.
3. The ABC triglyceride and the AAB triglyceride are respectively calculated by a group of general equations to obtain the position isomer content, so that a large amount of standard test sample preparation and complex calculation processes required in the standard calibration curve manufacturing engineering can be avoided.
4. Generally, the qualitative and quantitative method for triglyceride isomers provided by the invention has the advantages of simple pretreatment operation, rapid analysis, higher qualitative and quantitative accuracy, stable and reliable result, less sample consumption, capability of batch processing of samples and capability of well solving the defect of poor sample repeatability in the prior art; the lipid compound which can be analyzed by the method provided by the invention is suitable for rapid analysis of various biological samples such as milk fat, animal fat, vegetable fat and the like
Of course, it is not necessary for any method of the invention to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1.MS E And the results of secondary mass spectrometry of O-P-O standard and O-P-O in breast milk in DDA and SURVEY modes ((A) - (C) are O-P-O standards and (D) - (F) are breast milk).
FIG. 2 is a total ion flow diagram of ABC type triglyceride standard (A) O-P-La, O-P-S, O-P-L and AAB type triglyceride standard (B) P-O-P, O-P-O and L-P-L.
FIG. 3 shows sn-O-P-O as an example, where (A) is the peak of the excimer ion [ M + NH ] at different cone voltages 4 ] + Abundance, (B) is the excimer peak [ M + NH ] under different collision energies 4 ] + (C) is fragment ion [ M + H-FA ] under different collision energies] + The abundance of (a).
FIG. 4 is a standard curve of the concentration and peak area of triglyceride standards O-P-La, O-P-P, O-P-S, O-P-O, O-P-L and L-P-L.
FIG. 5 shows secondary mass spectrase:Sub>A of sn-A-A-B/sn-A-B-A ((A) - (C) are sn-O-P-O: sn-O-O-P = 1: 0, 1: 1, 0: 1) and sn-B-A-C/sn-A-B-C/sn-A-C/A-C-A-C-B ((D) - (G) are sn-O-P-L: sn-P-O-L: sn-P-L = 1: 1, 1: 0, 0: 1: 0, 0: 1) in different ratios.
FIG. 6.Sn-A-A-B and sn-A-B-A triglyceride isomer mixture the sn-A-B-A content of the standard substance and [ R ] in the secondary mass spectrum A-A ] + /[R A-A ] + +[R A-B ] + A linear fit curve between (P-O-P, (A); O-P-O, (B); L-P-L, (C)).
FIG. 7 content of fatty acids at sn-2 position in sn-B-A-C, sn-A-B-C and sn-A-C-B standard mixtures and [ R ] in secondary mass spectrase:Sub>A sn-1FA-sn-3FA ] + /[R B-C ] + +[R A-C ] + +[R A-B ] + A linear fit curve between (O-P-La, (A); O-P-S, (B); O-P-L, (C)).
FIG. 8.13 different ratios of [ R ] of fragment ions in secondary mass spectrase:Sub>A of sn-B-A-C/sn-A-B-C/sn-A-C-B triglyceride positional isomer mixtures sn-1FA-sn-3FA ] + /[R B-C ] + +[R A-C ] + +[R A-B ] + (the scale is shown on the projection view, O-P-La, (A); O-P-S, (B); O-P-L, (C)).
FIG. 9 Total ion flow diagrams of breast milk (A), cow milk (B), goat milk (C), donkey milk (D), yak milk (E), lard (F), and fish oil (G) triglycerides.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The sources of reagents used in the examples of the present invention are commercially available, except where otherwise specified.
Materials and reagents used in the present invention:
and (3) standard substance: the 15 triglyceride standards are classified into two types, AAB type and ABC type:
AAB type triglyceride refers to three kinds of fatty acids linked at the sn-1, sn-2 and sn-3 positions of triglyceride, wherein two kinds of fatty acid molecules are the same, and the other kind of fatty acid molecules are different, and two kinds of position isomer forms of sn-A-A-B and sn-A-B-A are provided (sn-1 and sn-3 are not distinguished). Including but not limited to: 1, 2-dioleo-3-palmitic acid (sn-O-O-P), 1, 3-dioleo-2-palmitic acid (sn-O-P-O), 1, 2-dipalmitoic-3-oleic acid (sn-P-P-O), 1, 3-dipalmitoic-2-oleic acid (sn-P-O-P), 1, 2-dilinoleic-3-palmitic acid (sn-L-L-P), 1, 3-dilinoleic-2-palmitic acid (sn-L-P-L);
ABC type triglyceride, which means three fatty acids linked at the sn-1, sn-2 and sn-3 positions of triglyceride, is ase:Sub>A type of triglyceride of fatty acid molecules different from each other, and has three positional isomer forms of sn-B-A-C, sn-A-B-C and sn-A-C-B (no distinction between sn-1 and sn-3), including but not limited to: 1-oleic-2-palmitic-3-lauric acid (sn-O-P-La), 1-palmitic-2-oleic-3-lauric acid (sn-P-O-La), 1-palmitic-2-lauric-3-oleic acid (sn-P-La-O), 1-oleic-2-palmitic-3-stearic acid (sn-O-P-S), 1-palmitic-2-oleic-3-stearic acid (sn-P-O-S), 1-palmitic-2-stearic-3-oleic acid (sn-P-S-O), 1-oleic-2-palmitic-3-linoleic acid (sn-O-P-L), 1-palmitic-2-oleic-3-linoleic acid (sn-P-O-L), 1-palmitic-2-linoleic-3-oleic acid (sn-P-L-O).
The organic reagents are all in chromatographic grade: n-hexane, methanol, acetonitrile, ethanol, chloroform; the purity of the high-purity carbon dioxide is more than or equal to 99.999 percent.
The specific implementation mode of the invention uses supercritical fluid chromatographic analysis, and the chromatographic conditions are as follows: performing supercritical fluid chromatography; 2-ethyl pyrimidine or C18 is adopted as a chromatographic column of a filler; eluent A is supercritical CO 2 The eluent B is an ethanol/acetonitrile solution with the volume percentage of 50 percent to 50 percent; the compensation solvent is ammonium acetate/methanol solution, the concentration is 5mm/L-20mm/L, and the flow rate is 0.2mL/min; the gradient elution program is 0min, the proportion of B phase is 0.2%, B is 0.3% at 1min, B is 0.6% at 18min, B is 2% at 18.1min and is kept to 21min, B is 7% at 25min, B is 0.2% at 27min and is kept to 30min; the column temperature is 50 ℃; back pressure 2500psi; the sample injection volume is 1 mu L; the flow rate is 1mL/min; the sample was dissolved in n-hexane at a concentrationIs 1mg/mL.
The mass spectrometry conditions of the embodiment of the invention are as follows: adopt ionization source to be the high resolution mass spectrometer of ESI, positive ion state, ion source temperature 100 ℃, desolvation temperature 400 ℃, collision gas is argon gas, velocity of flow 50L/h, desolvation gas is nitrogen gas, the velocity of flow: 700L/h. With leucine Enkephalin (ESI) + 556.2771m/z in mode) for real-time correction.
MS E The mode conditions are as follows: the cone hole voltage is 45V, the low energy collision energy is set to be 6eV, the high energy collision energy is set to be 25V, the molecular weight scanning range is 200-1500m/z, and the data presentation format is Profile data.
The DDA mode conditions are: cone voltage 45V, collision energy: 25eV, scan time interval 0.1s, molecular weight scan range: 200-1200m/z, and the data presentation format is Centroid data.
SURVEY mode conditions: taper hole voltage is 45V, collision energy: 25eV, the scanning time of the primary mass spectrum is 1s, the scanning time interval is 0.02s, the fraction of the secondary mass spectrum components is 4, the scanning time is 0.2s, and the scanning time interval is 0.02s. Molecular weight scan range: 100-1200m/z, and the data presentation format is Centroid data.
The analytical method of triglyceride positional isomers of the specific embodiment of the invention adopts a supercritical fluid chromatography-mass spectrometry combined method to carry out qualitative analysis on the triglyceride positional isomers; quantitative analysis is carried out on the triglyceride positional isomer by adopting an abundance method, which comprises the following steps: obtaining the abundance of fragment ions by secondary mass spectrometry, and analyzing the relative content of triglyceride position isomers by the abundance; the triglycerides include ABC-type triglycerides and/or AAB-type triglycerides. The analysis method can achieve the aim of accurately and quickly analyzing the content of the triglyceride positional isomer.
The analysis method of positional isomers of triglycerides according to the embodiments of the present invention can perform qualitative and quantitative analysis of ABC-type triglycerides and/or AAB-type triglycerides, and as some alternative embodiments, ABC-type triglycerides include, but are not limited to, 1-oleic acid-2-palmitic acid-3-lauric acid, 1-palmitic acid-2-oleic acid-3-lauric acid, 1-palmitic acid-2-lauric acid-3-oleic acid, 1-oleic acid-2-palmitic acid-3-stearic acid, 1-palmitic acid-2-oleic acid-3-stearic acid, 1-palmitic acid-2-palmitic acid-3-linoleic acid, 1-palmitic acid-2-oleic acid-3-linoleic acid, 1-palmitic acid-2-linoleic acid-3-oleic acid; AAB type triglycerides include, but are not limited to: 1, 2-dioleic-3-palmitic acid, 1, 3-dioleic-2-palmitic acid, 1, 2-dipalmitic-3-oleic acid, 1, 3-dipalmitic-2-oleic acid, 1, 2-dilinoleic-3-palmitic acid, 1, 3-dilinoleic-2-palmitic acid. In theory, the analysis method of positional triglyceride isomers according to the embodiment of the present invention can perform qualitative and quantitative analysis of any one positional triglyceride isomer.
The invention is further illustrated by the following examples.
Example 1: comparison of Mass Spectrometry modes
Taking O-P-O standard and breast milk sample as examples, MS is used respectively E DDA and SURVEY patterns were tested at a concentration of 5. Mu.g/mL for the standards.
The results were as follows:
the secondary mass spectrum results of the three mass spectrum modes are similar. However, the SURVEY mode is superior to the other two in analyzing complex samples because the SURVEY mode has a higher secondary mass spectral signal intensity than under the DDA mode, whereas the MS mode E The mode cannot be realized, and the information specificity of the fragment ions is derived from the same parent ion. The O-P-O standard substance is in MS E The secondary mass spectrum results of the DDA and SURVEY patterns are shown in FIGS. 1 (A) - (C), and the secondary mass spectrum results of the breast milk sample O-P-O are shown in FIGS. 1 (D) - (F).
Example 2: screening of Mass Spectrometry conditions in concentration Range and SURVEY modes
Determination of the cone voltage: triglyceride standard concentrations were set at 2, 5 and 8 μ g/mL, respectively, cone voltage was set at 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60V, and collision energy was set at 25eV.
Determination of collision energy: triglyceride standard concentrations were set at 2, 5 and 8 μ g/mL, respectively, cone voltage was set at 45V, and collision energy was set at 6, 15, 20, 22, 25, 28, 30 and 35eV.
Determination of the linear range of the standard concentration: triglyceride standard concentrations were set at 0.4, 0.8, 1,2, 4, 5, 6, 8, 10 and 12 μ g/mL, respectively, the cone voltage was set at 45V, and the collision energy was set at 25eV.
The results are as follows
The total ion flow diagram of ABC type triglyceride standard products O-P-La, O-P-S and O-P-L is shown in figure 2 (A), and the total ion flow diagram of AAB type triglyceride standard products P-O-P, O-P-O and L-P-L is shown in figure 2 (B). Taking O-P-O as an example, when the cone hole voltage is 45V, the excimer ion peak [ M + NH ] in the primary mass spectrum of the triglyceride 4 ] + The highest signal intensity (FIG. 3 (A)). When the collision energy is 25eV, the peak of excimer ion [ M + NH4 ]] + The maximum dissociation was obtained (FIG. 3 (B)), but the fragment ion [ M + H-FA ]] + Without further decomposition (fig. 3 (C)). When the concentration ranges from 0.8 to 8 mug/mL, R of the curve is linearly fitted 2 Greater than 0.99 (fig. 4).
Example 3: establishment of AAB type triglyceride standard
The sn-A-A-B and sn-A-B-A triglyceride isomers are mixed inbase:Sub>A mass ratio of 1: 0, 3: 1, 1: 3 and 0: 1, and the concentration of the mixed standard is 5. Mu.g/mL.
Under the conditions that the taper hole voltage is 45V and the collision energy is 25eV, the SURVEY mode is selected for detection. Taking O-P-O as an example, the results of the secondary mass spectra of the mixture of sn-O-P-O: sn-O-O-P in the ratio of 1: 0, 1: 1 and 0: 1 are shown in FIGS. 5 (A) - (C).
The results are as follows
With the ratio of sn-A-B-A in the mixture as the abscissase:Sub>A, [ R ] A-A ] + /[R A-A ] + +[R A-B ] + Linear fitting is carried out for the ordinate to obtain a fitting curve and R 2 . The linear curve is shown in figure 6.
Example 4: establishment of ABC type triglyceride standard
<xnotran> sn-B-A-C, sn-A-B-C sn-A-C-B 1 ∶ 0 ∶ 0,0 ∶ 1 ∶ 0,0 ∶ 0 ∶ 1, 8 ∶ 1 ∶ 1,1 ∶ 8 ∶ 1,1 ∶ 1 ∶ 8, 14 ∶ 3 ∶ 3, 3 ∶ 14 ∶ 3, 3 ∶ 3 ∶ 14, 2 ∶ 1 ∶ 1,1 ∶ 2 ∶ 1,1 ∶ 1 ∶ 2 1 ∶ 1 ∶ 1 , 5 μ g/mL. </xnotran>
Under the conditions that the taper hole voltage is 45V and the collision energy is 25eV, the SURVEY mode is selected for detection. In the case of O-P-L, the secondary mass spectra results for the mixture of sn-O-P-L: sn-P-O-L: sn-P-L-O in the ratios of 1: 1, 1: 0, 0: 1: 0 and 0: 1 are shown in FIGS. 5 (D) - (G).
The results are as follows
With the ratio of sn-2 fatty acids in the mixture as the abscissa, [ R ] sn-1 FA-sn-3FA ] + /[R B-c ] + +[R A-C ] + +[R A-B ] + Linear fitting is carried out for the ordinate to obtain a fitting curve and R 2 . The linear curve is shown in FIG. 7, and the three-dimensional scattergram is shown in FIG. 8.
Example 5: establishment of AAB type triglyceride equation
The conditions for testing the AAB type triglyceride sample were the same as in example 3.
For AAB triglycerides, the general equation is
Figure BDA0003575190350000091
whereinbase:Sub>A and B are the relative contents of sn-A-A-B and sn-A-B-A respectively, andbase:Sub>A + B =1; z A 、Z B Are respectively fragment ions [ A-B] + 、[A-A] + The abundance of (a) is a percentage of the sum of the abundances of (b), Z A 、Z B Can be obtained by secondary mass spectrometry; assuming that the relative abundance of fragment ions after neutral loss of sn-1 fatty acid and sn-3 fatty acid in triglyceride is 1, the relative abundance of fragment ions after neutral loss of sn-2 fatty acid is x i ，x i Can be obtained by secondary mass spectrometry.
The results are as follows
The simplified rear equation is
Figure BDA0003575190350000092
X of AAB type triglyceride i The values are shown in Table 1.
Table 1.X of different fatty acids bound at the sn-2 position of triglycerides of the AAB and ABC types i Value of
Figure BDA0003575190350000093
Figure BDA0003575190350000101
Fatty acid abbreviation: la: 12: 0; p: 16: 0; s: 18: 0; o: 18: 1; l: 18: 2. Each sample was run in triplicate with SD less than or equal to 0.05%.
Example 6: establishment of equation for type ABC triglycerides
The ABC type triglyceride samples were tested under the same conditions as in example 4.
For ABC type triglycerides, the general equation is
Figure BDA0003575190350000102
Wherein ase:Sub>A, B and C are the relative contents of sn-B-A-C, sn-A-B-C and sn-A-C-B respectively, and ase:Sub>A + B + C =1; y is A 、Y B 、Y C Are respectively fragment ions [ B-C] + 、[A-C] + 、[A-B] + The abundance of (A) is a percentage of the sum of the abundances of the three, Y A 、Y B 、Y C Can be obtained by secondary mass spectrometry; assuming that the relative abundance of fragment ions after neutral loss of sn-1 fatty acid and sn-3 fatty acid in triglyceride is 1, the relative abundance of fragment ions after neutral loss of sn-2 fatty acid is x i ，x i Can be obtained by secondary mass spectrometry.
The results are as follows
The simplified rear equation is
Figure BDA0003575190350000103
X of ABC type triglycerides i The values are shown in Table 1.
Example 7: 13 correction of C isotope peak effects
When present, is 13 C isotope peak influence. To proceed with 13 C isotope peak correction, equation
Z＝1-(I M-2 /I M )0.011 2 m(m-1)/2
≈1-0.65×10 -5 m 2 (I M-2 /I M )
Wherein Z is an influencing factor, m is the total carbon number, I M-2 And I M The abundance values of the ion peaks with mass-to-charge ratios of M-2 and M, respectively.
Example 8: analysis of triglyceride isomers in breast milk
The detection conditions of the breast milk fatty triglyceride positional isomer were the same as in examples 3-4, the equation for the content calculation was the same as in examples 5-6, 13 the correction of the C isotope peak effect was the same as in example 7.
The results are as follows
The total ion flow diagram of the breast milk fatty triglyceride is shown in fig. 9 (a), the content of the breast milk fatty triglyceride isomer measured by the standard curve method is shown in table 2, and the comparison of the results of the standard curve method and the equation method is shown in table 3.
Example 9: analysis of triglyceride isomers in mammalian milk
The detection conditions of the positional isomers of the fat triglycerides of cow milk, goat milk, donkey milk and yak milk are the same as those in examples 3-4, the equation method for content calculation is the same as that in examples 5-6, 13 the correction of the effect of the C isotope peak was the same as in example 7.
The results are as follows
The total ion flow diagram of the fat triglyceride of cow milk, goat milk, donkey milk and yak milk is shown in fig. 9 (B) - (E), the contents of the fat triglyceride isomers of cow milk, goat milk, donkey milk and yak milk measured by the standard curve method are shown in table 2, and the comparison of the results of the standard curve method and the equation method is shown in table 3.
Example 10: analysis of triglyceride isomers in fish oil
The detection conditions of the fish oil triglyceride positional isomer were the same as in examples 3-4, the equation method for the content calculation was the same as in examples 5-6, 13 the correction of the C isotope peak effect was the same as in example 7.
The results are as follows
The total ion flow graph of the fish oil triglyceride is shown in fig. 9 (F), the content of the fish oil triglyceride isomers measured by the standard curve method is shown in table 2, and the comparison of the results of the standard curve method and the equation method is shown in table 3.
Example 11: analysis of triglyceride isomers in lard
The detection conditions of positional isomers of lard triglycerides are the same as in examples 3-4, the equation method for content calculation is the same as in examples 5-6, 13 the correction of the C isotope peak effect was the same as in example 7.
The results are as follows
The total ion flow diagram of the lard triglyceride is shown in figure 9 (G), the content of the lard triglyceride isomer measured by the standard curve method is shown in table 2, and the comparison of the results of the standard curve method and the equation method is shown in table 3.
TABLE 2 positional isomer content of the triglycerides O-P-La, O-P-P, O-P-S, O-P-O, O-P-L and L-P-L of breast milk, cow milk, goat milk, donkey milk, yak milk, lard and fish oil
Figure BDA0003575190350000131
Fatty acid abbreviation: la: 12: 0; p: 16: 0; s: 18: 0; o: 18: 1; l: 18: 2.nd: it was not detected.
TABLE 3 comparison of the positional isomer content of the triglycerides O-P-La, O-P-P, O-P-S, O-P-O, O-P-L and L-P-L of breast milk, cow milk, goat milk, donkey milk, yak milk, lard and fish oil by the two methods (standard curve method and equation method)
Figure BDA0003575190350000141
Figure BDA0003575190350000151
Fatty acid abbreviations: la: 12: 0; p: 16: 0; s: 18: 0; o: 18: 1; l: 18: 2. Each sample was run in triplicate with SD < 2%. nd: it was not detected.
The above examples are further illustrative of the present invention, and one of ordinary skill in the art would be able to perform the methods described herein, in conjunction with actual sample conditioning, to achieve analysis of positional triglyceride isomers in different samples. Therefore, any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims (6)

1. A method for analyzing positional isomers of triglycerides, characterized in that,
performing qualitative analysis on the triglyceride positional isomer by using a supercritical fluid chromatography-mass spectrometry combined method;
and quantitatively analyzing the triglyceride positional isomer by adopting an abundance method, which specifically comprises the following steps:
obtaining the abundance of fragment ions by secondary mass spectrometry, and analyzing the relative content of triglyceride position isomers by the abundance;
the supercritical fluid chromatographic conditions are as follows:
performing supercritical fluid chromatography; 2-ethyl pyrimidine or C18 is adopted as a chromatographic column of a filler; eluent A is supercritical CO 2 The eluent B is 50 percent of ethanol/acetonitrile solution with the volume percentage of 50 percent; the compensation solvent is ammonium acetate/methanol solution, the concentration is 5mm/L-20mm/L, and the flow rate is 0.2mL/min; the gradient elution program is 0min, the proportion of B phase is 0.2%, B is 0.3% at 1min, B is 0.6% at 18min, B is 2% at 18.1min and is kept to 21min, B is 7% at 25min, B is 0.2% at 27min and is kept to 30min; the column temperature is 50 ℃; back pressure 2500psi; the sample injection volume is 1 mu L; the flow rate is 1mL/min; dissolving a sample in normal hexane, wherein the concentration is 1mg/mL;
the triglycerides comprise ABC type triglycerides and/or AAB type triglycerides;
the abundance method specifically comprises the following steps:
for ABC type triglycerides, positional isomers of triglycerides are analyzed using equation (1)
sn-B-A-C、sn-A-B-C、snRelative content of A-C-B:
Figure QLYQS_1
wherein a, b and c are respectivelysn-B-A-C、sn-A-B-C、sn-relative content of a-C-B, a + B + C =1; y is A 、Y B 、Y C Are respectively fragment ions [ B-C] + 、[A-C] + 、[A-B] + The abundance of (A) is a percentage of the sum of the abundances of the three, Y A 、Y B 、Y C Can be obtained by secondary mass spectrometry; assuming neutral loss of triglyceridessn-fatty acid at position 1 andsnthe relative abundance of fragment ions after fatty acid at position-3 is 1, and neutral losssnThe relative abundance of fragment ions after fatty acid-2 is x i ，x i Obtained by secondary mass spectrometry;
for AAB-type triglycerides, positional triglyceride isomers are analyzed using equation (2)sn-A-A-B、sn-relative content of ase:Sub>A-B-ase:Sub>A:
Figure QLYQS_2
wherein a and b are respectivelysn-A-A-B、sn-relative content of ase:Sub>A-B-ase:Sub>A, ase:Sub>A + B =1; z A 、Z B Are fragment ions [ A-B ] respectively] + 、[A-A] + The abundance of (a) is a percentage of the sum of the abundances of (b), Z A 、Z B Can be obtained by secondary mass spectrometry; hypothesis of neutral loss of triglyceridessn-fatty acid at position 1 andsnthe relative abundance of fragment ions after fatty acid at position-3 is 1, and neutral losssnThe relative abundance of fragment ions after fatty acid-2 is x i ，x i Obtained by secondary mass spectrometry.
2. The analytical method of claim 1, wherein the mass spectrometry conditions are:
a high-resolution mass spectrometer with an ionization source of ESI is adopted, the mass spectrometer is in a positive ion state, the temperature of an ion source is 100 ℃, the temperature of a desolvation is 400 ℃, the flow rate of a collision gas is 50L/h; desolventizing gas is nitrogen, and the flow rate is 700L/h; real-time correction with leucine enkephalin; the mass spectrometry conditions comprise MS E DDA and surveyy.
3. The analytical method of claim 2,
the MS E The mode conditions are as follows: the taper hole voltage is 45V, the low energy collision energy is set to be 6eV, the high energy collision energy is set to be 25V, the molecular weight scanning range is 200-1500m/z, and the data presentation format is Profile data;
the DDA mode conditions are as follows: cone voltage 45V, collision energy: 25eV, scan time interval 0.1s, molecular weight scan range: 200-1200m/z, and the data presentation format is Centroid data;
the SURVEY mode condition: cone voltage 45V, collision energy: 25eV, the scanning time of the primary mass spectrum is 1s, the scanning time interval is 0.02s, the number of the secondary mass spectrum components is 4, the scanning time is 0.2s, and the scanning time interval is 0.02s; molecular weight scan range: 100-1200m/z, and the data presentation format is Centroid data.
4. The assay of claim 1, wherein the ABC-type triglycerides and AAB-type triglycerides are derived from vegetable oils, animal oils, milk fats, microbial oils, processed synthetic lipids, foods or pharmaceuticals containing lipids.
5. The assay of any one of claims 1 to 4, wherein the ABC-type triglyceride comprises: 1-oleic acid-2-palmitic acid-3-lauric acid, 1-palmitic acid-2-oleic acid-3-lauric acid, 1-palmitic acid-2-lauric acid-3-oleic acid, 1-oleic acid-2-palmitic acid-3-stearic acid, 1-palmitic acid-2-oleic acid-3-stearic acid, 1-palmitic acid-2-stearic acid-3-oleic acid, 1-oleic acid-2-palmitic acid-3-linoleic acid, 1-palmitic acid-2-oleic acid-3-linoleic acid, 1-palmitic acid-2-linoleic acid-3-oleic acid.
6. The assay of any one of claims 1 to 4, wherein the AAB-type triglyceride comprises: 1, 2-dioleic-3-palmitic acid, 1, 3-dioleic-2-palmitic acid, 1, 2-dipalmitic-3-oleic acid, 1, 3-dipalmitic-2-oleic acid, 1, 2-dilinoleic-3-palmitic acid, 1, 3-dilinoleic-2-palmitic acid.
CN202210352796.1A 2022-03-31 2022-03-31 Analysis method of triglyceride positional isomer Active CN114689756B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210352796.1A CN114689756B (en) 2022-03-31 2022-03-31 Analysis method of triglyceride positional isomer

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210352796.1A CN114689756B (en) 2022-03-31 2022-03-31 Analysis method of triglyceride positional isomer

Publications (2)

Publication Number Publication Date
CN114689756A CN114689756A (en) 2022-07-01
CN114689756B true CN114689756B (en) 2023-04-07

Family

ID=82143545

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210352796.1A Active CN114689756B (en) 2022-03-31 2022-03-31 Analysis method of triglyceride positional isomer

Country Status (1)

Country Link
CN (1) CN114689756B (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104698104B (en) * 2015-03-20 2016-05-25 中国农业科学院油料作物研究所 A kind of method of liquid chromatography-mass spectrometry long-chain polyunsaturated fatty acid triglycerides
JP6991021B2 (en) * 2017-09-08 2022-01-12 月島食品工業株式会社 Methods for Producing Positional and / or Enantiomers of Triacylglycerol
CN108181395B (en) * 2017-12-28 2021-01-08 广州白云山汉方现代药业有限公司 Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography

Also Published As

Publication number Publication date
CN114689756A (en) 2022-07-01

Similar Documents

Publication Publication Date Title
Villaverde et al. Trends in analysis of pesticide residues to fulfil the European Regulation (EC) No. 1107/2009
Hancock et al. Advances and unresolved challenges in the structural characterization of isomeric lipids
Triebl et al. Lipidomics by ultrahigh performance liquid chromatography-high resolution mass spectrometry and its application to complex biological samples
Zhao et al. Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry: the determination of choline containing compounds in foods
Nakazawa et al. Simultaneous determination of residual tetracyclines in foods by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry
Huang et al. Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography–tandem mass spectrometry
Guo et al. Direct and sensitive determination of glyphosate and aminomethylphosphonic acid in environmental water samples by high performance liquid chromatography coupled to electrospray tandem mass spectrometry
Kataoka et al. Determination of patulin in fruit juice and dried fruit samples by in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry
CN107037149B (en) Fipronil and its metabolite residue amount method for measuring in a kind of egg
Li et al. Development and validation of a semi-automated method for L-dopa and dopamine in rat plasma using electrospray LC/MS/MS
Guo et al. Paper spray ionization mass spectrometry for rapid quantification of illegal beverage dyes
Ai et al. Determination of diclazuril, toltrazuril and its two metabolites in poultry tissues and eggs by gel permeation chromatography–liquid chromatography–tandem mass spectrometry
Chou et al. Solid phase microextraction with liquid chromatography–electrospray ionization–tandem mass spectrometry for analysis of amphetamine and methamphetamine in serum
Bian et al. Progress in the pretreatment and analysis of N-nitrosamines: an update since 2010
CN109781883A (en) A method of based on QuEChERS- dispersive liquid-liquid microextraction trace detection 5 hydroxymethyl furfural
Meng et al. Direct analysis in real time coupled with quadrupole-Orbitrap high-resolution mass spectrometry for rapid analysis of pyrethroid preservatives in wooden food contact materials
CN113791146B (en) Method for detecting organic phosphate pollutants in food and sample treatment method thereof
CN112730710B (en) Detection method for rapid real-time quantification of target analytes in a sample by introducing a series of different isotopic labels
Franklin et al. Structural elucidation of triacylglycerol using online acetone Paternò–Büchi reaction coupled with reversed-phase liquid chromatography mass spectrometry
Piovesana et al. Investigation of free seleno-amino acids in extra-virgin olive oil by mixed mode solid phase extraction cleanup and enantioselective hydrophilic interaction liquid chromatography-tandem mass spectrometry
CN114689756B (en) Analysis method of triglyceride positional isomer
CN108535395A (en) A method of using 32 kinds of free fatties in UPLC-QTof Rapid Simultaneous Determination health liquors
Nadaoka et al. Quantitative determination of total L-carnitine in infant formula, follow-up formula, and raw materials by liquid chromatography with tandem mass spectrometry
Leveque et al. Determination of triacylglycerol regioisomers using electrospray ionization-quadrupole ion trap mass spectrometry with a kinetic method
Corbini et al. Polarographic determination of total pyrethroid residues in stored cereals

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant