title: Fractionation of complex lipid mixtures by hydroxyapatite chromatography for lipidomic purposes.
abstract: The comprehensive analysis of natural lipid mixtures is often challenging due to the simultaneous occurrence of functionally and structurally heterogeneous compounds. Modern analytical approaches in system-wide lipidomics essentially rely on mass spectrometry (MS). The overabundant amount of triacylglycerols (TAGs) in most samples hinders the direct detection of phospholipids (PLs) or other low-abundance lipids; therefore, a fractionation step is most often required to reduce sample complexity prior to MS analysis. In this work, we explore the use of hydroxyapatite (HAP) as a chromatographic stationary phase (gel HAP) or chemo-affinity sorbent material (ceramic HAP) to selectively enrich PLs and polar lipids from chicken egg yolk and buttermilk. Due to the affinity of phosphate-containing compounds for HAP, both in-column and in-batch HAP-based chromatography were effective to deplete TAGs before releasing PLs with an eluting ternary system containing sodium phosphate as the displacing agent. Buttermilk gangliosides and PLs co-eluted, indicating that HAP also exhibits a high affinity for sialylated glycolipids and that polar lipids are retained through a combined mechanism of chemo-affinity and hydrogen bonding. To the best of our knowledge, this is the first report in which HAP is proposed as an alternative stationary phase for separating both the PLs and sialylated glycolipids from TAGs in complex lipidomes. The HAP-based chromatography has potential to be improved for the separation of the polar lipid classes.
authors.affiliation: Dipartimento di Agraria, Università di Napoli "Federico II", Parco Gussone, 80055 Portici, NA, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Pinto
authors.affiliation: Istituto di Scienze dell'Alimentazione (ISA) - CNR, Via Roma 52, 83100 Avellino, Italy.
authors.forename: Simonetta
authors.initials: S
authors.lastname: Caira
authors.affiliation: Istituto di Scienze dell'Alimentazione (ISA) - CNR, Via Roma 52, 83100 Avellino, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Mamone
authors.affiliation: Dipartimento di Agraria, Università di Napoli "Federico II", Parco Gussone, 80055 Portici, NA, Italy.
authors.forename: Pasquale
authors.initials: P
authors.lastname: Ferranti
authors.affiliation: Istituto di Scienze dell'Alimentazione (ISA) - CNR, Via Roma 52, 83100 Avellino, Italy. Electronic address: addeo@unina.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Addeo
authors.affiliation: Istituto di Scienze dell'Alimentazione (ISA) - CNR, Via Roma 52, 83100 Avellino, Italy. Electronic address: picariello@isa.cnr.it.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Picariello
mesh_terms: D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D002645:Chickens; D002855:Chromatography, Thin Layer / Q000379:methods; D017886:Durapatite / Q000737:chemistry*; D004530:Egg Yolk / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D008055:Lipids; D017886:Durapatite
keywords: Buttermilk; Egg yolk; Gangliosides; Hydroxyapatite chromatography; Lipidomics; Phospholipids