Large-scaled human serum sphingolipid profiling by using reversed-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: method development and application in hepatocellular carcinoma.

摘要: Abstract Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components cellular membrane, but also play pivotal roles in signaling other events. It is desirable for the development sensitive, robust structural-specific analytical approaches enabling rapid determination many sphingolipid species possible. Herein we present an method large-scaled profiling sphigolipids human serum, which consisted improved extraction protocol using tert -butyl methyl ether combined mild alkaline hydrolysis, ultra performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, sphingomyelin), were separated quantified single run within 10 min. A broad linear range over 2.5–4 orders magnitude ( r 2  > 0.99), limit detection 0.01–0.17 pmol/mL, quantitation 0.02–0.42 pmol/mL obtained each subcategory. Average recovery subcategory was 85.6–95.6%. Median values coefficient variation (CV) all detected sphingolipids 3.9% 6.8% intraday interday precision, respectively. This exemplarily applied study regarding dysregulated homeostasis hepatocellular carcinoma. The establishment this provides useful tool serum-based throughput screening biomarkers mechanism investigation metabolic regulation disease.