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

Instrumentation and method

Pump

  • Type: Waters Agilent Alliance 2790
  • Mode: isocratic
  • Solvent(s): Solvent MeOH/CHCl3 (3/1) containing 0,1 % HCOOH
  • Flow: 300 µL/min

Autosampler

  • Type: CTC Pal
  • Injection volume: 5µl
  • Wash solvent: n.d.

Column

  • Type: Thermo Hypersil-Keystone Beta Basic CYANO (Thermo scientific)
  • length: 50 x 2mm i.d.
  • Particle size: 3µm
  • Temperature: 30°C

Mass spectrometer

  • Type: Triple quadrupole (Quattro Ultima, Micromass)
  • Ionization mode: ESI positive
  • Ionization voltage: 3000V
  • Source temperature:
  • Collision gas: Argon
  • Collision gas pressure: 1.75*10^-3 torr
  • MS/MS-conditions:
    • multiple reaction monitoring table of species observed frequently
Analyte Precursor [m/z] Precursor [m/z] Collision energy [eV]
SPH 300 282 14V
SPH 300 264 14V
SPH 300 252 14V
SPA 302 284 14V
SPA 302 266 14V
SPA 300 254 14V
SPH C17 286 268 14V
SPH C17 286 250 14V
SPH C17 286 238 14V

Data analysis and quantification

Software

  • MassLynx coupled to self programmed Excel macros

Calibration and quantification

  • calibration type: matrix calibration - addition of naturally occurring species
  • species used for calibration
Species Fibroblasts
SPH, SPA 0-60ng/175µg cellular protein

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

Precision

8 % (SPH); 13 % (SPA)

LOD

9 fmol (SPH); 21 fmol (SPA)

Recovery

92,0 ± 6,4 %

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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