Ceramide - ESI-MS/MS - Liebisch et al.

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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 5 mM ammonium formate/chloroform (3/1 = v/v)
Material Material used Internal Standard(s) Internal Standard(s) added
Cultured cells 100µg protein Cer 14:0, Cer 17:0 50ng each
Human plasma 20µl Cer 14:0, Cer 17:0 50ng each

Instrumentation and method

Pump

  • Type: binary high pressure gradient (Agilent 1100)
  • Mode: isocratic flow gradient
  • Solvent(s): Methanol containing 5 mM ammonium formate/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: 250°C
  • Collision gas: Argon
  • Collision gas pressure: 1.0 10-3 Torr
  • MS/MS-conditions:
    • precursor ion scan of m/z 264.2
    • multiple reaction monitoring table of species observed frequently
Analyte Precursor [m/z] Precursor [m/z] Collision energy [eV]
Cer 14:0 - H2O (IS) 492.5 264.2 25 V
Cer 14:0 (IS) 510.5 264.2 25 V
Cer 16:0 - H2O 520.5 264.2 25 V
Cer 16:0 538.5 264.2 25 V
Cer 17:0 - H2O (IS) 534.5 264.2 25 V
Cer 17:0 (IS) 552.5 264.2 25 V
Cer 18:0 - H2O 548.5 264.2 25 V
Cer 18:0 566.6 264.2 25 V
Cer 20:0 - H2O 576.6 264.2 25 V
Cer 20:0 594.6 264.2 25 V
Cer 22:1 - H2O 602.6 264.2 25 V
Cer 22:1 620.6 264.2 25 V
Cer 22:0 - H2O 604.6 264.2 25 V
Cer 22:0 622.6 264.2 25 V
Cer 23:0 - H2O 618.6 264.2 25 V
Cer 23:0 636.6 264.2 25 V
Cer 24:1 - H2O 630.6 264.2 25 V
Cer 24:1 648.6 264.2 25 V
Cer 24:0 - H2O 632.6 264.2 25 V
Cer 24:0 650.6 264.2 25 V

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
  • both [M+H]+ and [M+H-H2O]+ are used
  • species used for calibration
Species Cultured cells Human plasma
Cer 16:0 0-35pmol 0-50pmol
Cer 18:0 0-35pmol 0-50pmol
Cer 20:0 0-35pmol 0-50pmol
Cer 24:1 0-35pmol 0-50pmol
Cer 24:0 0-35pmol 0-50pmol

Method validation

Precision

  • CV over all: below 10% in plasma samples

LOD

  • 0.3 pmol injected amount (in fibroblast lipid extract)

Recovery

  • recovery of Cer 6:0 above 90%

Sample data

Mass spectra

Reference

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