From LipidomicsWiki
Sample preparation
- Lipid extracts according to Bligh and Dyer are spiked with internal standards, and get diluted 1:100 in acetonitrile/2-propanol 5:2, 1% ammonia acetate, 0.1% formic acid.
| Material
| Material used
| Internal Standards
| Internal Standard added
|
| Tissue extracts
| 5 - 100 mg tissue
| 24:0 PC, 24:0 PE, 24:0 PS
| 10uM each
|
Instrumentation and method
Pump
- Type: Thermo Accela
- Mode: Gradient
- Solvent A: Water with 1% ammonia acetate and 0.1% formic acid
- Solvent B: Acetonitrile/2-propanol 5:2 with 1% ammonia acetate and 0.1% formic acid
- Gradient:
| Time [min]
| Flow [ml/min]
| % Solvent A
| % Solvent B
|
| 0
| 0.25
| 65
| 35
|
| 4.0
| 0.25
| 30
| 70
|
| 20.0
| 0.25
| 0
| 100
|
| 30.0
| 0.25
| 0
| 100
|
Column
- Type: Thermo Hypersil GOLD C18, 100x1 mm, 1.9µm
- Temperature: 50°C
Autosampler
- Type: Accela
- Injection volume: 5ul
- Wash solvent: Solvent B
Mass spectrometer
- Type: Thermo LTQ-FT Ultra
- Ionization mode: positive ESI
- Ionization voltage: 4500V
- Capillary temperature: 250°C
- Tube Lens: 120V
- Capillary voltage: 35V
- Damping gas: Helium
- Mass calibration: <2ppm external
- MS-conditions:
| Analyte
| Scan range [m/z]
|
| Phosphatidylcholines
| 300 - 1000
|
| Phosphatidylethanolamines
| 300 - 1000
|
| Phosphatidylserines
| 300 - 1000
|
| Plasmalogens
| 300 - 1000
|
| Lysophosphatidylcholines
| 300 - 1000
|
| Lysophosphatidylethanolamines
| 300 - 1000
|
| Sphingomyelines
| 300 - 1000
|
Data analysis and quantitation
Data handling
- Peak areas for all analytes are calculated by QuanBrowser. Peak identification is based on exact mass (<2ppm) and retention time.
Calibration and quantitation
- calibration type: relative quantitation
- The internal standard is set to 100% and all other peaks are expressed as % relative to internal standards.
Method validation
Precision
Method precision for 5 injections is better than 15%. Inter day retention time stability is better than 1%.
