title: Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography-tandem mass spectrometry.
abstract: D-erythro-sphingosine (Sph) and its phosphorylated product, d-erythro-sphingosine 1-phosphate (S1P) are sphingolipids mediating numerous cellular processes. Imbalance of Sph/S1P levels contributes to many diseases. Given the interconversion of these two opposing signaling molecules, it is essential to examine their levels simultaneously. In the present study, we developed a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify the levels of Sph and S1P in biological samples using C17-Sph and C17-S1P as internal standards. With one step of methanol-induced protein precipitation, each sample was subjected to LC-MS/MS analysis using positive electrospray ionization under selected reaction monitoring mode. The running time was within 4 min with a simple mobile phase consisting of methanol-0.1% formic acid (95:5, v/v) at a flow rate of 0.2 mL/min. Standard curves were linear over ranges of 1-100 ng/mL for Sph and 0.1-10 ng/mL for S1P with correlation coefficient (r²) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for S1P. The intra-batch and inter-batch precision was less than 15% for all quality control samples. The recoveries of the method were found to be 76.36-89.84%. The method was applied to simultaneously determine the Sph and S1P levels in mouse kidney, human plasma, and HEK 293 cells treated with tumor necrosis factor-α (TNF-α) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-α whereas decreased in cells treated with DMS. These results indicated that this new LC-MS/MS method was rapid, sensitive, specific and reliable to quantify Sph and S1P levels in biological samples simultaneously.
authors.affiliation: Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Wai Huan Dong Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, China.
authors.forename: Tian
authors.initials: T
authors.lastname: Lan
authors.forename: Huichang
authors.initials: H
authors.lastname: Bi
authors.forename: Weihua
authors.initials: W
authors.lastname: Liu
authors.forename: Xi
authors.initials: X
authors.lastname: Xie
authors.forename: Suowen
authors.initials: S
authors.lastname: Xu
authors.forename: Heqing
authors.initials: H
authors.lastname: Huang
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D016018:Least-Squares Analysis; D008246:Lysophospholipids / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D008246:Lysophospholipids; D014409:Tumor Necrosis Factor-alpha; C060506:sphingosine 1-phosphate; D013110:Sphingosine