Format

Send to

Choose Destination
Anal Bioanal Chem. 2016 May;408(13):3475-83. doi: 10.1007/s00216-016-9425-z. Epub 2016 Feb 27.

Development and validation of a high-throughput LC-MS/MS assay for routine measurement of molecular ceramides.

Author information

1
Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland.
2
Heart and Lung Center, Helsinki University Hospital, 00029 HUS, Helsinki, Finland.
3
Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland. kim.ekroos@zora.fi.

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.

KEYWORDS:

Cardiovascular; Ceramides; Chromatography; Clinical; High throughput; Mass spectrometry

PMID:
26922344
DOI:
10.1007/s00216-016-9425-z
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center