A simple and rapid method for extraction and measurement of circulating sphingolipids using LC-MS/MS: a targeted...

Sphingolipids are a class of lipids with high structural diversity and biological pleiotropy. Mounting evidence supports a role for sphingolipids in regulating pathophysiology of cardiometabolic diseases, and they have been proposed as potential cardiometabolic biomarkers. Current methods for quantifying sphingolipids require laborious pretreatment and relatively large sample volumes, and cover limited species, hindering their application in epidemiological studies. Herein, we applied a time-, labor-, and sample-saving protocol simply using methanol for plasma sphingolipid extraction. It was compared with classical liquid-liquid extraction methods and showed significant advantages in terms of simplicity, sphingolipid coverage, and sample volume. By coupling the protocol with liquid chromatography using a wide-span mobile phase polarity parameter and tandem mass spectrometry operated in dynamic multiple reaction monitoring mode, 37 sphingolipids from 8 classes (sphingoid base, sphingoid base phosphate, ceramide-1-phosphate, lactosylceramide, hexosylceramide, sphingomyelin, ceramide, and dihydroceramide) were quantified within 16 min, using only 10 microL of human plasma. The current method showed good performance in terms of linearity (R² > 0.99), intra- and interbatch accuracy (70-123%) and precision (RSD < 12%), matrix effect (91-121%), recovery (96-101%), analyte chemical stability (deviation < 19%), and carryover (< 16%). We successfully applied this method to quantify 33 detectable sphingolipids from 579 plasma samples of an epidemiological study within 10 days. The quantified sphingolipid concentrations were comparable with previous studies. Positive associations of ceramide C22:0/C24:0 and their precursors with homeostasis model assessment of insulin resistance suggested that the synthesis of the ceramides might be involved in insulin resistance. This novel method constitutes a simple and rapid approach to quantify circulating sphingolipids for epidemiological studies using targeted lipidomic analysis, which will help elucidate the sphingolipid-regulated pathways underlying cardiometabolic diseases.