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

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

Analytical and bioanalytical chemistry (2016-02-29)
Dimple Kauhanen, Marko Sysi-Aho, Kaisa M Koistinen, Reijo Laaksonen, Juha Sinisalo, Kim Ekroos
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.

MATERIALS
Product Number
Brand
Product Description

Avanti
C22 Ceramide (d18:1/22:0), Avanti Research - A Croda Brand 860501P, powder
Avanti
C24:1 Ceramide (d18:2(4E,8Z)/24:1(15Z)), Avanti Research - A Croda Brand 860691P, powder
Avanti
C24:1 Ceramide-d7 (d18:1-d7/24:1(15Z)), Avanti Research - A Croda Brand 860679P, powder
Avanti
C16 Ceramide-d7 (d18:1-d7/16:0), Avanti Research - A Croda Brand 860676P, powder
Avanti
C18 Ceramide-d7 (d18:1-d7/18:0), Avanti Research - A Croda Brand 860677P, powder
Avanti
C24 Ceramide-d7 (d18:1-d7/24:0), Avanti Research - A Croda Brand 860678P, powder
Avanti
C10 Ceramide (d18:1/10:0), Avanti Research - A Croda Brand 860510P, powder
Avanti
C12 Ceramide (d18:1/12:0), Avanti Research - A Croda Brand