Ceramide - ESI-MS/MS - Liebisch et al.
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== Data analysis and quantification == | == Data analysis and quantification == | ||
Revision as of 12:59, 29 April 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 | x ng |
| Human plasma | 20µl | Cer 14:0, Cer 17:0 | x ng |
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
| Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
|---|---|---|---|
| Cer 14:0 | 534 | x | x |
