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

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== Reference ==
== Reference ==
* [http://www.jlr.org/cgi/content/full/40/8/1539 G.Liebisch, W. Drobnik, M. Reil, B. Trümbach, R. Arnecke, B. Olgemöller, A. Roscher, G. Schmitz Journal of Lipid Research 1999, 40, 1539-1546.]
* [http://www.jlr.org/cgi/content/full/40/8/1539 G.Liebisch, W. Drobnik, M. Reil, B. Trümbach, R. Arnecke, B. Olgemöller, A. Roscher, G. Schmitz Journal of Lipid Research 1999, 40, 1539-1546.]
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10428992
[[Category:Flow injection analysis]]
[[Category:Flow injection analysis]]
[[Category:Triple quadrupole]]
[[Category:Triple quadrupole]]

Revision as of 12:47, 22 July 2008

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): 5 mM ammonium formate/chloroform (3/1 = v/v)
  • 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:
  • Source temperature:
  • Collision gas: Argon
  • Collision gas pressure:
  • 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 492.5 264.2 x
Cer 14:0 510.5 264.2 x

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

Accuracy

Precision

LOD

LOQ

Recovery

Sample data

Mass spectra

Chromatogram

Reference

10428992

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