Sphingosine/Sphinganine - LC-MS/MS - Lieser et al.
From LipidomicsWiki
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* Ionization mode: ESI positive | * Ionization mode: ESI positive | ||
* Ionization voltage: 3000V | * Ionization voltage: 3000V | ||
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* Collision gas: Argon | * Collision gas: Argon | ||
* Collision gas pressure: 1.75*10^-3 torr | * Collision gas pressure: 1.75*10^-3 torr |
Revision as of 12:08, 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 | Internal Standard(s) | Internal Standard(s) added | |
---|---|---|---|
Fibroblasts | SPH 17:0 | 175 ng/mg cellular protein |
Instrumentation and method
Pump
- Type: Waters Agilent Alliance 2790
- Mode: isocratic
- Solvent(s): Solvent MeOH/CHCl3 (3/1) containing 0,1 % HCOOH
- Flow: 300 µL/min
Autosampler
- Type: CTC Pal
- Injection volume: 5µl
- Wash solvent: n.d.
Column
- Type: Thermo Hypersil-Keystone Beta Basic CYANO (Thermo scientific)
- length: 50 x 2mm i.d.
- Particle size: 3µm
- Temperature: 30°C
Mass spectrometer
- Type: Triple quadrupole (Quattro Ultima, Micromass)
- Ionization mode: ESI positive
- Ionization voltage: 3000V
- Collision gas: Argon
- Collision gas pressure: 1.75*10^-3 torr
- MS/MS-conditions:
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
---|---|---|---|
SPH | 300 | 282 | 14V |
SPH | 300 | 264 | 14V |
SPH | 300 | 252 | 14V |
SPA | 302 | 284 | 14V |
SPA | 302 | 266 | 14V |
SPA | 300 | 254 | 14V |
SPH C17 | 286 | 268 | 14V |
SPH C17 | 286 | 250 | 14V |
SPH C17 | 286 | 238 | 14V |
Data analysis and quantification
Software
- MassLynx coupled to self programmed Excel macros
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Fibroblasts |
---|---|
SPH, SPA | 0-60ng/175µg cellular protein |
Method Validation
Precision
8 % (SPH); 13 % (SPA)
LOD
9 fmol (SPH); 21 fmol (SPA)
Recovery
92,0 ± 6,4 %