Sphingosine-1-phosphate - LC-MS/MS - Bielawski et al.
From LipidomicsWiki
Contents |
Sample preparation
- Sphingolipids were extracted with iso-propanol:water:ethyl acetate (30:10:60)
- The water phase is re-etxtracted
- 50 µL IS were added prior lipid extraction
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Cells in culture | 1 x e6 - 10 e6 cells | S1P 17:0, C17 SPH, 17C16 Cer, 18C17 Cer, 18C6-SM, 18C17-SM | 1µM (S1P, SPH, Cer); 5µM SM |
tissues | 0.5 - 1 mg protein | S1P 17:0, C17 SPH, 17C16 Cer, 18C17 Cer, 18C6-SM, 18C17-SM | 1µM (S1P, SPH, Cer); 5µM SM |
serum | 50 - 100µL | S1P 17:0, C17 SPH, 17C16 Cer, 18C17 Cer, 18C6-SM, 18C17-SM | 1µM (S1P, SPH, Cer); 5µM SM |
Instrumentation and method
Pump
- Type: Surveyor quaternary HPLC pump
- Mode: gradient elution
- Solvent(s): Solvent A: 1 mM Ammonium formate in Methanol with 0.2% formic acid; solvent B: 2 mM Ammonium formate in water with 0.2% formic acid
- Gradient:
Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
---|---|---|---|
0 | 0.5 | 80 | 20 |
4.5 | 0.5 | 90 | 10 |
7 | 0.5 | 90 | 10 |
28 | 0.5 | 99 | 1 |
29 | 0.5 | 80 | 20 |
33 | 0.5 | 80 | 20 |
Autosampler
- Type: Surveyor
- Injection volume: 20µl
- Wash solvent: n.d.
Column
- Type: BDS Hypersil C8 (Phenomenex)
- length: 150 x 3.2 mm i.d.
- Particle size: 3 µm
- Retention time: 4.92 min (S1P), 4.3 min (C17-S1P); 2.16 min (SPH), 2.44 min (C17-SPH); 14-18 min (Cer), 3.5-14.5 min (SM)
Mass spectrometer
- Type: Triple quadrupole (Thermo Finnigan)
- Ionization mode: ESI positive
- Ionization voltage: n.d.
- Source temperature: n.d.
- Collision gas: n.d.
- Collision gas pressure:
- MS/MS-conditions:
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
---|---|---|---|
S1P | 380.1 | 264.2 | 23eV |
S1P 17:0 | 366.2 | 250 | 20eV |
SPH | 300.4 | 282.2 | 18eV |
C17 SPH | 286.1 | 268 | 17eV |
Data analysis and quantification
Software
- n.d.
Calibration and quantification
- calibration type: standard addition without matrix for plasma and in the presence of total lipids extracted from HPAECs.
- species used for calibration
Species | Serum, tissue, cells |
---|---|
SPH, S1P | 1-250pmol |
Cers | 2.5-400pmol |
SMs | 12.5-2000pmol |
Method Validation
Accuracy
n.d.
Precision
n.d.
LOD
n.d.
Recovery
n.d.