Large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination

A liquid chromatography and profile analysis technology, applied in the field of large-scale profile analysis of plasma sphingolipids, can solve problems such as complex methods, achieve the effects of simple and rapid operation, low solvent toxicity, and improved detection sensitivity

Active Publication Date: 2014-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the diversity in structure and abundance of each subclass of sphingolipids, the introduction of multiple platforms

Method used

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  • Large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination
  • Large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination
  • Large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination

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Experimental program
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Embodiment 1

[0022] Thaw normal human plasma stored at -80°C in an ice bath, pipette 40 μL quantitatively, add 30 μL internal standard mixed solution, and vortex to mix. Internal standard solutions include Ceramide(d18:1 / 17:0) 0.42nmol / mL, SM(d18:1 / 12:0) 1.67nmol / mL, Sphingosine(d17:1) 0.17nmol / mL, Lactosylceramide(d18:1 / 12:0) 0.17nmol / mL, Glucosylceramide (d18:1 / 12:0) 0.03nmol / mL, Dihydroceramide (d18:0 / 12:0) 0.03nmol / mL. Add 225 μL of methanol and 75 μL of 1M sodium methoxide-methanol solution and vortex for 30 s. Add 1mL MTBE, vortex 30s. Shake on a shaker at 37°C for 2h. After taking it out and cooling it, add 25 μL of 20% (v / v) acetic acid-water solution and 250 μL of ultrapure water. Vortex for 30s. Centrifuge at 10°C at 12,000 rpm for 10 min, remove 800 μL of the supernatant quantitatively, and freeze-dry. With complex solution methanol / isopropanol / water=65:30:5 (MeOH / IPA / H 2 O=65:30:5) reconstituted to 50 μL, vortexed for 1 min, and used for LC-MS / MS analysis, the detection ...

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Abstract

The invention discloses a large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination, which is characterized in that sphingolipid in blood plasma is extracted by double-phase extraction with methyl tertiary butyl ether/methanol/water (MTBE/MeOH/H2O) combined with mild basic hydrolysis. Then rapid semi-quantitative analysis is realized within 15 min by combination of ultra-high performance liquid chromatography-electro-spray ionization-mass spectrometry. In the invention, the MTBE/MeOH/H2O extraction system is simple, rapid, and easy to operate, has less protein interference in the extract, and has good repeatability; interference of high-abundance phospholipid and glyceride in the lipid group is eliminated by hydrolysis; ion inhibition is reduced; therefore, the purpose of detecting low-abundance sphingolipid with high sensitivity is achieved. With the high resolution separation capability of subsequent ultra-high performance liquid chromatography (UHPLC) and the specificity of MRM, effective distinguishing of isomers is realized, and the introduction of complex isotope correction during quantification is avoided, which makes the method more rapid and accurate.

Description

technical field [0001] The invention relates to the fields of analytical chemistry and medicine, and is a large-scale plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry. Background technique [0002] Sphingolipids (SLs) are a class of lipid molecules with 1,3-hydroxy-2-aminoalkanes / alkenes as the common core. As the 2-amino group is connected to various fatty acids with different chain lengths and saturations through amide bonds, and the 1-hydroxy group is connected to various polar groups (such as phosphorylcholine, various simple and complex sugar groups, etc.), The molecular structure of sphingolipids is complex and diverse, and the chemical properties are different. At the same time, the abundance of each subclass of sphingolipids also varies greatly. Although the sphingolipid group is a small part of the whole lipid group, its function in living organisms cannot be underestimated. Sphingolipids are an essential component of th...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 许国旺李佳赵欣捷路鑫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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