Lipoxin A₄: problems with its determination using reversed phase chromatography-tandem mass spectrometry and confirmation with chiral chromatography

Abstract

Lipoxins belong to the family of so-called pro-resolving endogenous lipid mediators which are derived from arachidonic acid and play a key role in the counter-regulation of inflammation. Arachidonic acid is also precursor of multiple pro-inflammatory lipid mediators, such as prostaglandins and leukotrienes, which are simultaneously present in biological compartments. The close structural relationship between several of these lipid mediators and the absence of blank matrix samples enormously complicates the unequivocal identification of these compounds. The determination of lipoxin A4 has been accomplished by chromatographic separation using a C18 reversed phase column and tandem mass spectrometry detection. Samples were liquid-liquid extracted with ethyl acetate before injection. Identification of the analyte was done based on three criteria: retention time, ratio of the m/z transitions and MS/MS spectrum. To avoid false positive results due to endogenous interferences, the extracted samples were re-injected into a chiral Lux Amylose-2 chromatographic column. The authors recommend the use of chiral chromatography in the determination of pro-resolving lipid mediators, together with transition area ratio and fragmentation spectra to improve selectivity for identification and quantitation purposes.

Keywords: Chiral chromatography; Lipid mediators; Lipoxin; Selectivity.