A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS

A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 μg/mL with bias <15 %. The intra-assay coefficient of variation is less than 10 % for concentrations from 1.2 to 9 μg/mL, and less than 25 % for concentrations below 1.2 μg/mL. For glucosylceramide and ceramide trihexoside the linear range is 0.05-3 μg/mL with biases <10 % and <20 %, respectively. The intra-assay coefficient of variation for these analytes is less than 10 % at concentrations from 0.4 to 3 μg/mL, and less than 25 % for concentrations below 0.4 μg/mL.