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

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== Reference ==
== Reference ==
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B. Lieser, G.Liebisch, W. Drobnik, G. Schmitz
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* B. Lieser, G.Liebisch, W. Drobnik, G. Schmitz ''Journal of Lipid Research'' '''2003''', ''44'', 2209-2216.
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''Journal of Lipid Research'' '''2003''', ''44'', 2209-2216.
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Institut für Klinische Chemie und Laboratoriumsmedizin; Klinikum der Universität Regensburg; Franz-Josef-Strauss-Allee 11; D-93042 Regensburg; Germany.
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Fax: 49-941-944-6202; e-mail: gerd.schmitz@klinik.uni-regensburg.de

Revision as of 12:03, 30 July 2007

Contents

Abstract

A fast and convenient methodology for the quantification of sphingosine (SPH) and the biosynthetic intermediate sphinganine (SPA) based on HPLC electrospray ionization tandem mass spectrometry was developed by Lieser et al. This method allows a high-throughput quantification of sphingoid bases from crude lipid extracts and thus constitutes a valuable tool for studies of cellular sphingolipid metabolism and signaling.

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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