title: Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection.
abstract: An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a flow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofluids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipids in biological samples of different matrices.
authors.affiliation: Netherlands Metabolomics Centre Leiden University, 2300 RA Leiden, The Netherlands Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands.
authors.forename: Carola W N
authors.initials: CW
authors.lastname: Damen
authors.affiliation: Waters Corporation, Milford, MA 01757.
authors.forename: Giorgis
authors.initials: G
authors.lastname: Isaac
authors.affiliation: Waters Corporation, Wilmslow, SK9 4AX, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Langridge
authors.affiliation: Netherlands Metabolomics Centre Leiden University, 2300 RA Leiden, The Netherlands Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hankemeier
authors.affiliation: Netherlands Metabolomics Centre Leiden University, 2300 RA Leiden, The Netherlands Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands.
authors.forename: Rob J
authors.initials: RJ
authors.lastname: Vreeken
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008055:Lipids / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D008055:Lipids
keywords: charge surface hybrid column; ion-mobility/high-resolution mass spectrometry; structural elucidation; ultraperformance liquid chromatography