From LipidomicsWiki
Sample preparation
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 %
Reference