An LC-MS/MS Multiplexed Method to Quantify 14 Sphingolipids in Human Cerebrospinal Fluid (CSF)

Sphingolipids are bioactive lipids comprised of a sphingoid base linked to various long-chain fatty acids and phosphocholine and, in some cases, may also contain one or more sugars. A few species of sphingolipids share similar structures and may even have identical chemical composition but differ in their biological function. For this reason, they can only be accurately quantified using liquid chromatography coupled with mass spectrometry (LC-MS). This technique can separate sphingolipids based on their hydrophobicity and quantify them based on their mass-to-charge ratio (m/z). This is especially important when measuring isomeric species that have identical m/z but differ in their retention time. Here, we describe the development of a new LC-MS method to measure 14 sphingolipid species, including 7 ceramides (C14, C16, C18, C20, C24, C18:1, C24:1), 4 glycosphingolipids (C16 Glucosyl(ß) Ceramide (d18:1/16:0, C16 Galactosyl(ß) Ceramide (d18:1/16:0), C16 Lac Cer, C18 Lac Cer) and 3 sphingosines (Sphingosine (Sph), Glucosyl(ß) Sphingosine (d18:1), Galactosyl(ß) Sphingosine (d18:1)) in human cerebrospinal fluid (CSF). Using this 7-min method, chromatographic resolution of isomeric species glucosylceramide (Glc Cer) and galactosylceramide (Gal Cer) was achieved. This resolution is important as these two sphingolipid classes have distinct biological functions with important implications in neurological diseases like multiple sclerosis.