Phosphatidylcholine / Sphingomyelin - ESI-MS/MS - Liebisch et al.

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Revision as of 11:02, 17 September 2008 by Gerhard Liebisch (Talk | contribs)
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Contents

Sample preparation

  • Lipid extraction according to Bligh and Dyer
  • internal standards are added prior lipid extraction
  • to avoid the loss of lipids, extraction was performed in glassware
  • samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
Material Material used Internal Standard(s) Internal Standard(s) added
Cultured cells 100µg protein PC 28:0, PC 44:0 1250ng each
Human plasma 20µl PC 28:0, PC 44:0 3500ng PC 28:0, 5000ng PC 44:0

Instrumentation and method

Pump

  • Type: binary high pressure gradient (Agilent 1100)
  • Mode: isocratic flow gradient
  • Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
  • Flow gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.05 100 0
0.1 0.03 100 0
1.1 0.2 100 0
1.3 0.05 100 0

Autosampler

  • Type: CTC Pal
  • Injection volume: 20µl
  • Wash solvent: methanol/chloroform = 1/1

Mass spectrometer

  • Type: Triple quadrupole (Micromass, Quattro Ultima)
  • Ionization mode: ESI positive
  • Ionization voltage: 3500 V
  • Source temperature: 300°C
  • Collision gas: Argon
  • Collision gas pressure: 1.0 10-3 Torr
  • Collision energy: 30 V
  • MS/MS-mode: precursor ion scan of m/z 184.1

Data analysis and quantification

Data handling

  • combine spectra above half peak height
  • smooth combined spectrum (if necessary)
  • centroid combined spectrum
  • pick peak intensities

Isotope correction

Calibration and quantification

  • calibration type: matrix calibration - addition of naturally occurring species
  • species used for calibration
Species Cultured cells Human plasma
PC 34:1 0 - 1000 pmol 0 - 3000 pmol
PC 36:2 0 - 1000 pmol 0 - 3000 pmol
PC 38:4 0 - 1000 pmol 0 - 3000 pmol
PC 40:0 0 - 1000 pmol 0 - 3000 pmol
PC O 16:0/20:4 0 - 1000 pmol 0 - 3000 pmol
SM 16:0 0 - 1000 pmol 0 - 3000 pmol
SM 18:1 0 - 1000 pmol 0 - 3000 pmol
SM 18:0 0 - 1000 pmol 0 - 3000 pmol

Method validation

Precision

  • CV within-run: 4 % (major), 5-10 % (minor)
  • CV total: 10 % (major), 15 % (minor)

LOD

  • 0.6 μM in plasma

Sample data

Mass spectra

Reference

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