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.
Highlights
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
For the purpose of demonstrating the separation, all lipids, except the FFAs were analyzed in positive ion mode; it should be mentioned that multiple lipid classes [e.g., phosphatidylinositol (PI), phosphoethanolamine (PE), phosphatidic acid (PA), etc.] ionize much more efficiently in negative ion mode
We have coupled the chromatographic system to a traveling wave ion-mobility-enabled hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer
Summary
HPLC-grade methanol originated from Actu-All Chemicals BV (Oss, The Netherlands). HPLC-grade chloroform, ultra liquid chromatography (ULC)-MS-grade water, 2-propanol (IPA), acetonitrile (ACN), and 99% pure ULC-MS formic acid were purchased from Biosolve BV (Valkenswaard, The Netherlands). Ammonium formate, dichloromethane (DCM), leucine enkephalin (Leu-Enk), and poly-DL-alanine (product number P9003) were from Sigma Aldrich (St. Louis, MO). All lipid standards (as shown in supplementary Table I), and extracts of bovine heart, liver, and brain were purchased from Avanti Lipids (Alabaster, AL) and from Nu-Chek Prep (Elysian, MN). Human plasma with heparin as anticoagulant was obtained from healthy volunteers who had given their informed consent for use of their plasma for method development
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