title: Development and validation of a high-throughput LC-MS/MS assay for routine measurement of molecular ceramides.
abstract: Monitoring the levels of the ceramides (Cer) d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1 and ratios thereof in human plasma empowers the prediction of fatal outcome of coronary artery disease (CAD). We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for clinical-scaled measurement of the four distinct ceramides. Rapid plasma precipitation was accomplished in 96-well format. Excellent extraction recoveries in the range of 98-109% were achieved for each ceramide. Addition of corresponding D7-labeled ceramide standards facilitated precise quantification of each plasma ceramide species utilizing a novel short 5-min LC-MS/MS method. Neither matrix interference nor carryover was observed. Robust intra- and inter-assay accuracy and precision <15% at five different concentrations were obtained. Linear calibration lines with regressions, R(2) > 0.99, were achieved for all analytes. Short-term bench top, long-term plasma, and extract stability demonstrated that the distinct ceramides were stable in the conditions evaluated. The validity of the methodology was demonstrated by determining the precise ceramide concentrations in a small CAD case-control study. Thus, our LC-MS/MS methodology features simple sample preparation and short analysis time for accurate quantification of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1, designed for routine analysis.
authors.affiliation: Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland.
authors.forename: Dimple
authors.initials: D
authors.lastname: Kauhanen
authors.affiliation: Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland.
authors.forename: Marko
authors.initials: M
authors.lastname: Sysi-Aho
authors.affiliation: Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland.
authors.forename: Kaisa M
authors.initials: KM
authors.lastname: Koistinen
authors.affiliation: Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland.
authors.forename: Reijo
authors.initials: R
authors.lastname: Laaksonen
authors.affiliation: Heart and Lung Center, Helsinki University Hospital, 00029 HUS, Helsinki, Finland.
authors.forename: Juha
authors.initials: J
authors.lastname: Sinisalo
authors.affiliation: Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland. kim.ekroos@zora.fi.
authors.forename: Kim
authors.initials: K
authors.lastname: Ekroos
mesh_terms: D002138:Calibration; D002518:Ceramides / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002518:Ceramides
keywords: Cardiovascular; Ceramides; Chromatography; Clinical; High throughput; Mass spectrometry