From LipidomicsWiki
Sample preparation
- 50µl of sample are spiked with 450 µl methanol containing internal standard. Following that samples are voretexed and centrifuged. 200µl of the supernatant are transferred into an autosampler vial.
- sample sovlent: Methanol / Water 9:1
| Material
| Material used
| Internal Standard(s)
| Internal Standard(s) added
|
| Serum
| 50µl
| C17 Sphingosine-1-phosphate
| 20ng/ml
|
Instrumentation and method
Pump
- Type: Binary Agilent 1100
- Mode: Gradient
- Solvent A: Water (0.1% formic acid)
- Solvent B: Methanol / THF 50:50 (0.1% formic acid)
- Gradient:
| Time [min]
| Flow [ml/min]
| % Solvent A
| % Solvent B
|
| 0
| 0.3
| 57.5
| 42.5
|
| 1.0
| 0.3
| 57.5
| 42.5
|
| 3.5
| 0.3
| 3
| 97
|
| 10.5
| 0.3
| 3
| 97
|
Column
- Type: Phenomenex Luna C18(2), 150x2mm, 5um
- Temperature: 20°C
Autosampler
- Type: Agilent 1100
- Injection volume: 10ul
- Wash solvent: Methanol
Mass spectrometer
- Type: 4000QTrap
- Ionization mode: negative ESI
- Ionization voltage: 4000V
- Source temperature: 400°C
- Gas 1: 30psi
- Gas 2: 30psi
- Collision gas: Nitrogen
- MS/MS-conditions:
- multiple reaction monitoring table
| Analyte
| Precursor [m/z]
| Fragment [m/z]
| Collision energy [eV]
|
| S1P
| 380
| 264
| 23
|
| C17-S1P
| 366
| 250
| 23
|
Data analysis and quantitation
Data handling
- Data processing is done automatically by InteliQuan
Calibration and quantitation
- calibration type: Standard curve
- Calibration range: 1 - 400 ng/ml
Method validation
Accuracy
Mean accuracy of 5 replicates is between 95% and 110%
Precision
Method precision for 5 replicates is better than 15%
Limit of detection
5 pg on coumn
Reference
