Quantification of ceramide levels in mammalian cells by high performance liquid chromatography coupled to tandem mass spectrometry with multiple-reaction-monitoring mode (HPLC-MS/MS-MRM)

Ceramides play an important role in a variety of cellular functions including cell differentiation and apoptosis, responses to DNA damage and stress, and transcriptional events. Detection of ceramides in mammalian cells is required for many biological studies. Here, we report a validated method using LC-MS/MS-MRM on an Agilent 6410 triple quadrupole mass spectrometer to simultaneously quantify six ceramides extracted from mammalian cell lysates following a 5 min HPLC gradient. This method demonstrated outstanding sensitivity, accuracy, reproducibility, and speed of analysis. © 2011 The Royal Society of Chemistry.