NP/HILIC-ELSD Separation of Phospholipid Classes and Application to Preliminary Analysis of Plasma Low Density Lipoproteins

Abstract

A new method for the analysis of phospholipids (Lysophosphatidylethanolamine, Phosphatidylethanolamine, Phosphatidylcholine, Sphingomyelin, and Lysophosphatidylcholine) by NP/HILIC-ELSD is described using a silica column. Addition of formic acid and triethylamine to a mobile phase based on acetonitrile/methanol/water delivered isocratically promoted a good and rapid separation of 5 phospholipid classes (15 min run). The use of an evaporative light scattering detector allowed the direct analysis of a mixture of phospholipids without complex sample pretreatment. Both power and linear regression showed satisfactory concordance with the relationship between response and concentration. The LOD and LOQ obtained were below 10 ng and 30 ng of injected mass for all cases, respectively. The method was validated with a standard mixture of phospholipids. This method has been applied to primarily characterize the phospholipid fraction of both native and oxidized low density lipoproteins.

Keywords:

• ELSD
• full factorial design
• low density lipoproteins (LDL)
• HILIC
• lysophosphatidylcholine
• normal phase
• phospholipid classes

Acknowledgments

The authors wish to thank Espectrociencia Ltda. for providing the Young-Lin LC – system and the LT-ELSD SEDEX 85 Evaporative Light Scattering Detector.

Notes

^a PtdEtn: Phosphatidylethanolamine.

*Statistically significant when p-value < 0.05. R ² = 97.2952.

^a n = 6/level 2.

^b n = 6.

*n = 10.

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