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

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Contents

Sample Preparation

  • rinsing cells two times with ice-cold phosphate-buffered saline (PBS)
  • centrifugation (800 g)
  • homogenization of pellets in dest. H2O by sonication or lysis in 0,2 % sodium dodecyl sulfonate (SDS)
  • protein determination of an aliquot of cell homogenate
  • addition of 0,5 μg C6-CER to cell lysate
  • lipid extraction according to Bligh and Dyer (1959)
  • addition of 0,5 μg C8-CER (IS)/ mg protein to the CHCl3-phase


Analysed Matrices

skin fibroblasts

Analytes

C6-CER; C16-CER; C18-CER; C8-CER; C2-CER; C24:1-CER


To avoid the loss of lipids, the whole cell procedure was performed in glassware.

Analytical Method

ESI-MS-MS: PE Sciex API 36

  • dissolving all standards and cell extracts in 5 mM ammonium formate in MeOH/CHCl3 (3/1) prior to analysis
  • injection of 35 μl calibrator and analytical sample by an autosampler
  • constant flow: 20 μl/ min
  • scan modus: positive precursor ion scan (PIS) of m/z = 264
  • ion spray voltage: 5900 V
  • orifice voltage: 52 V
  • collision energy: 35 V
  • collision gas N2: 5.0 (2.1*10^-5 torr)
  • step size: 1 amu
  • dwell time: 15 ms
  • collection window: 5 min
  • data analysis: PE Sciex software LC Tune 1.3 and Sample control 1.3

Method Validation

Accuracy

Precision

LOD

LOQ

Recovery

Internal Standard

C8-CER

Reference

  • G.Liebisch, W. Drobnik, M. Reil, B. Trümbach, R. Arnecke, B. Olgemöller, A. Roscher, G. Schmitz Journal of Lipid Research 1999, 40, 1539-1546.

Institut für Klinische Chemie und Laboratoriumsmedizin; Klinikum der Universität Regensburg; Franz-Josef-Strauss-Allee 11; D-93042 Regensburg; Germany.

Fax: 49-941-944-6202; e-mail: gerd.schmitz@klinik.uni-regensburg.de

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