title: A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS.
abstract: 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.
authors.affiliation: Genzyme Corporation, a Sanofi Company, One Mountain Road, Framingham, MA, USA.
authors.forename: Wei-Lien
authors.initials: WL
authors.lastname: Chuang
authors.affiliation: Genzyme Corporation, a Sanofi Company, One Mountain Road, Framingham, MA, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Pacheco
authors.affiliation: Genzyme Corporation, a Sanofi Company, One Mountain Road, Framingham, MA, USA. Kate.Zhang@genzyme.com.
authors.forename: Kate
authors.initials: K
authors.lastname: Zhang
mesh_terms: D053000:Analytic Sample Preparation Methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D059788:Dried Blood Spot Testing / Q000379:methods*; D005963:Glucosylceramides / Q000097:blood* / Q000302:isolation & purification; D006028:Glycosphingolipids / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005963:Glucosylceramides; D006028:Glycosphingolipids; C088880:ceramide trihexoside
keywords: Dried blood spot; Liquid chromatography; Mass spectrometry; Monophasic lipid extraction; Quantitation; Sphingolipids