Phosphatidylcholine / Sphingomyelin - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
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 10 mM ammonium acetate/chloroform (3/1 = v/v)
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Cultured cells | 100µg protein | PC 28:0, PC 44:0 | 1250ng each |
Human plasma | 20µl | PC 28:0, PC 44:0 | 3500ng PC 28:0, 5000ng PC 44:0 |
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: 300°C
- Collision gas: Argon
- Collision gas pressure: 1.0 10-3 Torr
- Collision energy: 30 V
- MS/MS-mode: precursor ion scan of m/z 184.1
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
- using Excel Macros correcting the peak intensities in a sequential algorithm starting from low mass species
- five isotope peaks including the monoisotopic were used
- the detailed algorithm is described in the appendix of Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Cultured cells | Human plasma |
---|---|---|
PC 34:1 | 0 - 1000 pmol | 0 - 3000 pmol |
PC 36:2 | 0 - 1000 pmol | 0 - 3000 pmol |
PC 38:4 | 0 - 1000 pmol | 0 - 3000 pmol |
PC 40:0 | 0 - 1000 pmol | 0 - 3000 pmol |
PC O 16:0/20:4 | 0 - 1000 pmol | 0 - 3000 pmol |
SM 16:0 | 0 - 1000 pmol | 0 - 3000 pmol |
SM 18:1 | 0 - 1000 pmol | 0 - 3000 pmol |
SM 18:0 | 0 - 1000 pmol | 0 - 3000 pmol |
Method validation
Precision
- CV within-run: 4 % (major), 5-10 % (minor)
- CV total: 10 % (major), 15 % (minor)
LOD
- 0.6 μM in plasma