Ceramide - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
Contents |
Sample Preparation
Material | Material used | Internal Standard(s) | Internal Standard(s) added | |
---|---|---|---|---|
Cultured cells | 100µg protein | Cer 14:0, Cer 17:0 | x ng | |
Human plasma | 20µl | Cer 14:0, Cer 17:0 | x ng |
- 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