From LipidomicsWiki
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 10 mM ammonium acetate/chloroform (3/1 = v/v)
Material | Material used | Internal Standard(s) | Internal Standard(s) added
|
Cultured cells
| 100µg protein | D7-FC, CE 17:0, CE 22:0 | 50ng each
|
Human plasma
| 20µl | D7-FC, CE 17:0, CE 22:0 | 1500ng each
|
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: isocratic flow gradient
- Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
- Flow 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: 3500 V
- Source temperature: 220°C
- Collision gas: Argon
- Collision gas pressure: 1.0 10-3 Torr
- Collision energy: 13 V
- MS/MS-mode: precursor ion scan of m/z 369.3
scan modi: selected reaction monitoring (SRM) for FC m/z 446,4 -> 369,3 (CE 2:0); m/z 453,4 -> 376,3 (D7-CE 2:0)
Data analysis and quantification
Data handling
- combine spectra above half peak height
- smooth combined spectrum (if necessary)
- centroid combined spectrum
- pick peak intensities
Isotope correction
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Cultured cells | Human plasma
|
LPC 16:0
| 0 - 100 pmol | 0 - 1000 pmol
|
LPC 18:0
| 0 - 100 pmol | 0 - 1000 pmol
|
LPC 18:1
| 0 - 100 pmol | 0 - 1000 pmol
|
Method validation
Precision
- CV within-run: 3 %(major), 5-10 % (minor)
- CV total: 10 % (major), 15-20 % (minor)
LOD
Sample data
Mass spectra
Reference