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A High Coverage Lipidomics Analysis Method Based on Liquid Chromatography-Mass Spectrometry

A lipid group, high-coverage technology, applied in the fields of analytical chemistry, biochemistry and medicine, can solve problems such as poor linear response of mass spectrometry, influence of quantitative accuracy, and difficult data repetition, so as to simplify the data processing process and improve repeatability , the effect of accurate quantitative ability

Active Publication Date: 2021-08-10
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain limitations in non-targeted methods: for example, due to the simultaneous scanning of a wide range, the number of ions detected at the same time is too large, which makes the linear response of mass spectrometry poor, low-abundance lipids are difficult to detect, and the quantitative accuracy is also low. will be affected; in addition, subsequent data processing is cumbersome, and the peak matching process is easily affected by peak matching parameters, which will introduce errors. Difficult to repeat
However, targeted lipidomics analysis is aimed at a certain class of lipid metabolites with important biological functions or key lipid metabolites on a specific pathway. The number of lipids detected is limited, and it is difficult to meet the detection requirements in lipidomics possible multiple lipid metabolite requirements

Method used

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  • A High Coverage Lipidomics Analysis Method Based on Liquid Chromatography-Mass Spectrometry
  • A High Coverage Lipidomics Analysis Method Based on Liquid Chromatography-Mass Spectrometry
  • A High Coverage Lipidomics Analysis Method Based on Liquid Chromatography-Mass Spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 High coverage lipidomics analysis method based on UPLC / QQQ-MS-DMRM

[0025] The flowchart of the establishment of the high-coverage lipidomics analysis method based on UPLC / QQQ-MS-DMRM is as follows figure 1 Shown in A, the specific implementation steps are as follows:

[0026] 1. Preparation of different types of biological samples.

[0027] Eight exogenous metabolites were added to the extraction solutions of lipid metabolites in different types of samples as internal standards, internal standard mixture PC38:0, 6.7μg / ml; LPC19:0, 3.3μg / ml; TG45:0, 5.3 μg / ml; PE34:0 / PE30:0, 3.3μg / ml; FFA16:0-d3, 6.7μg / ml; FFA18:0-d3, 6.7μg / ml; Cer35:1, 1.7μg / ml; SM30: 1, 1.7 μg / ml.

[0028] 1) Tissue samples: operate on ice, weigh 10mg rat brain tissue into a 2ml EP tube, add grinding beads, add 30μl internal standard mixture, add 400μl methanol solution, grind for 25HZ*1min*2times, add 800μl chloroform , shake for 10 minutes, add 240 μl of ultra-pure water, shake for 5 ...

Embodiment 2

[0044] Example 2. Analysis of plasma high coverage lipidomics

[0045] The specific process of plasma high-coverage lipidomics analysis is as follows: figure 1 Shown in B, concrete implementation steps are as follows:

[0046] 1. Plasma sample pretreatment

[0047]First, mix the plasma samples to be tested in equal amounts, as the quality control sample QC, take 40 μl QC, add 300 μl methanol solution (containing 8 internal standards, PC38:0, 0.67 μg / ml; LPC19:0, 0.33 μg / ml; TG45: 0, 0.53μg / ml; PE34:0 / PE30:0, 0.33μg / ml; FFA16:0-d3, 0.67μg / ml; FFA18:0-d3, 0.67μg / ml; Cer35:1, 0.17μg / ml ;SM30:1, 0.17μg / ml), vortex for 10s, add 1ml MTBE, shake for 10min, add 300μl ultrapure water, vortex for 30s, stand at 4℃ for 10min, centrifuge at 10000rpm*4℃*10min, take the upper layer 400μl frozen Dry, redissolve (reconstituted solution: dichloromethane / methanol=2:1; diluent: acetonitrile / isopropanol / water=65:30:5; reconstituted solution / diluted=1:3, V / V) to 120μl, used for mass spectrometr...

Embodiment 3

[0050] Example 3. Methodological investigation of high-coverage lipidomics analysis based on UPLC / QQQ-MS-DMRM

[0051] The above-mentioned plasma samples to be tested were mixed in equal volumes to prepare a quality control sample (QC), which was pretreated according to the following different investigation objects.

[0052] 1. Repeated inspection

[0053] Pipette 40μl QC, add 300μl methanol solution (containing 8 internal standards, PC38:0, 0.67μg / ml; LPC19:0, 0.33μg / ml; TG45:0, 0.53μg / ml; PE34:0 / PE30:0 , 0.33μg / ml; FFA16:0-d3, 0.67μg / ml; FFA18:0-d3, 0.67μg / ml; Cer35:1, 0.17μg / ml; SM30:1, 0.17μg / ml), vortex 10s , add 1ml MTBE, shake for 10min, add 300μl ultrapure water, vortex for 30s, stand at 4℃ for 10min, centrifuge at 10000g*4℃*10min, take 400μl of the upper layer to freeze-dry. Redissolve (reconstituted solution: dichloromethane / methanol=2:1; diluent: acetonitrile / isopropanol / water=65:30:5; reconstituted solution / diluted=1:3, V / V) to 200 μl, For negative ion analysis,...

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Abstract

The invention discloses a high-coverage lipidomics analysis method based on liquid chromatography-mass spectrometry. Based on the extraction of various types of matrices to make separate or mixed samples, use the ultra-high performance liquid chromatography-high resolution mass spectrometry data-dependent acquisition mode to automatically collect and combine the secondary mass spectrometry of lipid metabolites in each sample, and use qualitative analysis software to extract lipid retention time, Precursor ion and its product ion information; screen the characteristic product ions corresponding to the parent ion according to the lipid mass spectrometry structure characteristics to obtain the characteristic ion pair; add the characteristic ion pair information obtained from different types of samples, and further base on the lipid database, lipid structure The characteristics and chromatographic retention rules were used to expand the lipid ion pairs to obtain the final lipid ion pair library. Lipid transitions were scanned using UPLC‑QQQ‑MS in dynamic multiple reaction monitoring mode. Compared with the traditional non-targeted lipidomics analysis method, the present invention has better repeatability and lipid coverage.

Description

technical field [0001] The invention relates to the fields of analytical chemistry, biochemistry and medicine, and is a method for high-coverage lipidomics analysis based on ultra-high performance liquid chromatography / triple quadrupole mass spectrometry using a dynamic multiple reaction monitoring mode. Background technique [0002] Lipid is an important part of cell membrane and plays an important role in cell signal transduction, material transportation, energy storage and so on. Abnormal lipid metabolism is closely related to the occurrence and development of many diseases such as obesity, hypertension, diabetes, cardiovascular disease, Parkinson's disease, and cancer. Lipids have various components and complex structures, and the difference between high and low lipid content in the human body is as high as 6 to 7 orders of magnitude. Therefore, establishing a reliable, stable, high-coverage, and highly sensitive lipid metabolite analysis method has positive guiding sig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88G01N30/86G01N30/06
Inventor 许国旺轩秋慧胡春秀路鑫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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