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- Title
Development and validation of a high-throughput LC-MS/MS assay for routine measurement of molecular ceramides.
- Authors
Kauhanen, Dimple; Sysi-Aho, Marko; Koistinen, Kaisa; Laaksonen, Reijo; Sinisalo, Juha; Ekroos, Kim
- 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 D-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 > 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. [Figure not available: see fulltext.]
- Subjects
INTERFERON gamma release tests; CORONARY arteries; HEART blood-vessels; CORONARY circulation; CERAMIDES
- Publication
Analytical & Bioanalytical Chemistry, 2016, Vol 408, Issue 13, p3475
- ISSN
1618-2642
- Publication type
Academic Journal
- DOI
10.1007/s00216-016-9425-z