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

From LipidomicsWiki

Revision as of 13:51, 17 September 2008 by Max Scherer (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Sample preparation

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
  • 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

Method Validation

Precision

8 % (SPH); 13 % (SPA)

LOD

9 fmol (SPH); 21 fmol (SPA)

Recovery

92.0 ± 6,4 %

Reference

Personal tools
Create a book