Methods of separating lipids

Patent metadata

Field	Value
Publication number	US-9568454-B2
Application number	US-201414250622-A
Country	US
Kind code	B2
Filing date	Apr 11, 2014
Priority date	Apr 12, 2013
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

Abstract

The invention provides novel, simple, reliable and effective methods for separating various lipids from biological samples. In certain embodiments, the invention provides HILIC-based UHPLC separation methods. In one embodiment, the methods of the invention offer an efficient and rapid separation of ether glycophospholipids (e.g., plasmalogens) from their diacyl or monoacyl counterparts within the same lipid class. In certain instances, the invention relates to the use of a Waters ACQUITY UPLC® system.

First claim

We claim:

1. A method of separating lipids of different classes from a biological sample, said method comprising steps of
i) preparing the biological sample;
ii) loading said biological sample onto an ultrahigh performance liquid chromatography (UHPLC) system, wherein said UHPLC system comprises a hydrophilic interaction chromatography (HILIC) column;
iii) eluting said UHPLC column with an elution solvent, wherein the elution solvent is obtained by mixing a mobile phase A and a mobile phase B in situ, and wherein a volume ratio of the mobile phase B to the mobile phase A is at a gradient that increases during the elution step; and
iv) detecting lipids of different classes by using a mass spectrometer.

2. The method of claim 1 , wherein the mobile phase A and the mobile phase B, each independently, comprise acetonitrile and water.

3. The method of claim 2 , wherein said mobile phase A comprises 95:5 by volume of acetonitrile: water, and said mobile phase B comprises 50:50 by volume of acetonitrile: water.

4. The method of claim 1 , wherein said gradient is from 0 to 20% (v/v) and is completed within about 10 minutes.

5. The method of claim 1 , wherein a pH value of said elution solvent is higher than 7.

6. The method of claim 5 , wherein the pH value of said elution solvent is about 8.

7. The method of claim 1 , wherein said elution solvent comprises ammonium acetate.

8. The method of claim 1 , wherein said mass spectrometer is selected from the group consisting of LC-MS/MS, MALDI-MS, tandem quadrupole mass spectrometer, and ESI-MS, or a combination thereof.

9. The method of claim 1 , wherein said biological sample is selected from the group consisting of blood, plasma, urine, body tissue, and a lipid extract from cells or tissues, wherein said cells or tissues are from animals, bacteria, plants, or fungi.

10. The method of claim 1 , wherein said HILIC column is normal-phase.

11. The method of claim 10 , wherein said HILIC column is a 1.7 μm, 2.1 ×100 mm ethylene bridged hybrid HILIC column.

12. The method of claim 10 , wherein said HILIC column comprises a solid phase selected from the group consisting of silica, silica silanol, silica diol, amino, amide, anionic, cationic and zwitterionic materials, or a combination thereof.

13. A method of separating ether glycophospholipids from a biological sample, said method comprising steps of
i) preparing the biological sample;
ii) loading said biological sample onto an ultrahigh performance liquid chromatography (UPLC) system, wherein said UHPLC system comprises a hydrophilic interaction chromatography (HILIC) column;
iii) eluting said UHPLC system with an elution solvent, wherein the elution solvent is obtained by mixing a mobile phase A and a mobile phase B in situ, and wherein a volume ratio of the mobile phase B to the mobile phase A is at a gradient that increases during the elution step; and
iv) detecting the ether glycophospholipids by using a mass spectrometer.

14. The method of claim 13 , wherein said ether glycophospholipids comprise plasmalogens.

Assignees

• Waters Technologies Corp

Inventors

• Wong Stephen
• Ritchie Mark

Classifications

• G01N2030/8813

  biological materials · CPC title

• G01N30/34

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title

• H01J49/00

  Particle spectrometers or separator tubes · CPC title

• G01N2030/027

  Liquid chromatography · CPC title

• G01N30/36

  in high pressure liquid systems · CPC title