title: 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.
abstract: Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5-4 orders of magnitude (r(2)>0.99), a limit of detection of 0.01-0.17pmol/mL, and a limit of quantitation of 0.02-0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6-95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease.
authors.affiliation: CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.forename: Chunxiu
authors.initials: C
authors.lastname: Hu
authors.forename: Xinjie
authors.initials: X
authors.lastname: Zhao
authors.forename: Weidong
authors.initials: W
authors.lastname: Dai
authors.forename: Shili
authors.initials: S
authors.lastname: Chen
authors.forename: Xin
authors.initials: X
authors.lastname: Lu
authors.forename: Guowang
authors.initials: G
authors.lastname: Xu
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000097:blood; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood; D015203:Reproducibility of Results; D013107:Sphingolipids / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D013107:Sphingolipids
keywords: APCI; Cer; ESI; HILIC; HexCer; LacCer; Lipidomics; Liquid chromatography–mass spectrometry; MRM; MTBE; Multiple reaction monitoring; NPLC; RPLC; SM; Serum; Sph; Sphingolipid profiling; Tert-butyl methyl ether; atmospheric-pressure chemical ionization; ceramide; dHCer; dihydroceramide; electrospray ionization; free sphingoid base; hexosylceramide; hydrophilic interaction liquid chromatography; lactosylceramide; multiple reaction monitoring; normal phase liquid chromatography; reversed phase liquid chromatography; sphingomyelin; tert-butyl methyl ether