Cholesterol / Cholesterylester - ESI-MS/MS - Liebisch et al.

From LipidomicsWiki

Revision as of 18:07, 1 April 2009 by Gerhard Liebisch (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Sample preparation

  • Lipid extraction according to Bligh and Dyer
  • internal standards are added prior lipid extraction
  • to avoid the loss of lipids, extraction is performed in glassware
  • dried samples are derivatized by addition of 200 μl of acetyl chloride/chloroform = 1/5 (v/v) for 60 min at room temperature
  • derivatization reagents are removed by vacuum centrifugation
  • 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 D7-FC, CE 17:0, CE 22:0 3000ng D7-FC; 500ng each CE 17:0, CE 22:0
Human plasma 20µl D7-FC, CE 17:0, CE 22:0 10µg each D7-FC, CE 17:0, CE 22:0

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: 220°C
  • Collision gas: Argon
  • Collision gas pressure: 1.0 10-3 Torr
  • Collision energy: 13 V
  • MS/MS-mode:
    • selected reaction monitoring (SRM) for acetylated free cholesterol (FC) and D7-FC
    • precursor ion scan m/z 369.3 for cholesteryl ester (CE species)
Analyte Precursor [m/z] Precursor [m/z] Collision energy [eV]
CE 2:0 (acetylated FC) 446.4 369.3 13 V
D7-CE 2:0 (acetylated D7-FC) (IS) 453.4 376.3 13 V
CE species Scan 610 - 730 369.3 13 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
  • species used for calibration
Species Cultured cells Human plasma
FC 0 - 3 nmol 0 - 10 nmol
CE 16:0 0 - 500 pmol 0 - 10 nmol
CE 18:0 0 - 500 pmol 0 - 10 nmol
CE 18:1 0 - 500 pmol 0 - 10 nmol
CE 18:2 0 - 500 pmol 0 - 10 nmol

Method validation

Precision

  • CV within-run: 3 %(major), 5-10 % (minor)
  • CV total: 10 % (major), 15-20 % (minor)

LOD

  • 0.5 µM in plasma

Sample data

Mass spectra

Reference

Personal tools
Create a book