Sphingosine/Sphinganine - LC-MS/MS - Lieser et al.
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Revision as of 11:37, 5 August 2008
Contents |
Sample preparation
- lipid extraction according to Bligh&Dyer
- internal standards were added prior lipid extraction
- supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Fibroblasts | SPH 17:0 | 175 ng/mg cellular protein |
Analysed Matrices
fibroblasts
Analytes
C17-SPH (IS); SPH; SPA
Sample Preparation
- rinsing cells two times with ice-cold phosphate-buffered saline (PBS)
- centrifugation (240 g, 7 min)
- homogenization of pellets in dest. H2O by sonication
- protein determination of an aliquot of cell homogenate
- addition of IS (C17-SPH): 175 ng/ mg cellular protein
- lipid extraction according to Bligh and Dyer (1959)
- drying and resolvation of lipid extract in the mobile phase
Analytical Method
HPLC-MS
HPLC
- constant flow: 300 μl/ min (Waters Alliance 2790)
- mobile phase: MeOH/CHCl3 (3/1) containing 0,1 % HCOOH
- column: Thermo Hypersil-Keystone Beta Basic CYANO (3 μm, 50 mm * 2 mm)
- injection volume: 5 μl
- T (column): 30 °C
MS
- triple quadrupole MS (Quattro Ultima, Micromass)
- capillary voltage: 3000 V
- cone voltage: 35 V
- collision energy: 14 V
- collision gas: Ar (1.75*10^-3 torr)
- mass resolution above unit resolution
- scan modus: multiple reaction monitoring (MRM)
m/z 300 ->282, 300 -> 264, 300 -> 252 for SPH
m/z 302 ->284, 302 -> 266, 302 -> 254 for SPA
m/z 286 -> 268, 286 -> 250, 286 -> 238 for C17-SPH
- data analysis: MassLynx software coupled to self-programmed Exel macros
- analysis time: 3,5 min
Method Validation
Accuracy
Precision
8 % (SPH); 13 % (SPA)
LOD
9 fmol (SPH); 21 fmol (SPA)
LOQ
Recovery
92,0 ± 6,4 %
Internal Standard
C17-SPH
Reference
- B. Lieser, G.Liebisch, W. Drobnik, G. Schmitz Journal of Lipid Research 2003, 44, 2209-2216.
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