Glycerophospholipid Profiling - FT-MS
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*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. | *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. | ||
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Current revision
Contents |
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%.
