Sphingosine/Sphinganine - LC-MS/MS - Lieser et al.

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Contents

Sample preparation

  • lipid extraction according to Bligh&Dyer
  • internal standards were added prior lipid extraction
  • supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system
Material Internal Standard(s) Internal Standard(s) added
Fibroblasts SPH 17:0 175 ng/mg cellular protein

Analysed Matrices

fibroblasts

Analytes

C17-SPH (IS); SPH; SPA

Sample Preparation

  • rinsing cells two times with ice-cold phosphate-buffered saline (PBS)
  • centrifugation (240 g, 7 min)
  • homogenization of pellets in dest. H2O by sonication
  • protein determination of an aliquot of cell homogenate
  • addition of IS (C17-SPH): 175 ng/ mg cellular protein
  • lipid extraction according to Bligh and Dyer (1959)
  • drying and resolvation of lipid extract in the mobile phase

Analytical Method

HPLC-MS

HPLC
  • constant flow: 300 μl/ min (Waters Alliance 2790)
  • mobile phase: MeOH/CHCl3 (3/1) containing 0,1 % HCOOH
  • column: Thermo Hypersil-Keystone Beta Basic CYANO (3 μm, 50 mm * 2 mm)
  • injection volume: 5 μl
  • T (column): 30 °C
MS
  • triple quadrupole MS (Quattro Ultima, Micromass)
  • capillary voltage: 3000 V
  • cone voltage: 35 V
  • collision energy: 14 V
  • collision gas: Ar (1.75*10^-3 torr)
  • mass resolution above unit resolution
  • scan modus: multiple reaction monitoring (MRM)

m/z 300 ->282, 300 -> 264, 300 -> 252 for SPH

m/z 302 ->284, 302 -> 266, 302 -> 254 for SPA

m/z 286 -> 268, 286 -> 250, 286 -> 238 for C17-SPH

  • data analysis: MassLynx software coupled to self-programmed Exel macros
  • analysis time: 3,5 min

Method Validation

Accuracy

Precision

8 % (SPH); 13 % (SPA)

LOD

9 fmol (SPH); 21 fmol (SPA)

LOQ

Recovery

92,0 ± 6,4 %

Internal Standard

C17-SPH

Reference

  • B. Lieser, G.Liebisch, W. Drobnik, G. Schmitz Journal of Lipid Research 2003, 44, 2209-2216.

Institut für Klinische Chemie und Laboratoriumsmedizin; Klinikum der Universität Regensburg; Franz-Josef-Strauss-Allee 11; D-93042 Regensburg; Germany.

Fax: 49-941-944-6202; e-mail: gerd.schmitz@klinik.uni-regensburg.de

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