Sphingosine-1-phosphate - LC-MS/MS - Berdyshev et al.
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Contents |
Sample preparation
- S1P and related compounds were extracted with modified Bligh and Dyer using 0.1 N HCl for phase separation
- internal standards were added prior lipid extraction
- for acetylation the lipid extracts were treated with acetic anhydrid
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Human plasma | 100µl | S1P 17:0 | 40 pmol |
HPAECs | 1 x e6 | S1P 17:0 | 40 pmol |
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: gradient elution
- Solvent(s): Solvent A water/methanol/formic acid (20/80/0.5 v/v), 5 mM ammonium formate; Solvent B methanol/acetonitril/formic acid (59/40/0.5 v/v), 5 mM ammonium formate
- Gradient:
Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
---|---|---|---|
0 | 0.5 | 100 | 0 |
4 | 0.5 | 100 | 0 |
7 | 0.5 | 0 | 100 |
10 | 0.5 | 0 | 100 |
12 | 0.5 | 100 | 0 |
Autosampler
- Type: Agilent
- Injection volume: 10µl
- Wash solvent: n.d.
Column
- Type: Agilent Zorbax Eclipse XDB-C8 column
- length: 150 x 4.6 mm i.d.
- Particle size: 5 µm
- Retention time: 8.15 min (S1P), 7.9 min (C17-S1P); 8.4 min (DHS1P)
Mass spectrometer
- Type: Triple quadrupole (Applied Biosystems, API 4000)
- Ionization mode: ESI negative
- Ionization voltage: -4500V
- Source temperature: 520°C
- Collision gas: Nitrogen
- 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 | 462 | 402 | -28V |
S1P 17:0 | 448 | 388 | -26V |
DH-S1P | 464 | 404 | -28V |
Data analysis and quantification
Software
- Analyst 1.4.2.
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 | Plasma | HPAEC |
---|---|---|
S1P | 0-150pmol | 0-350pmol |
DHS1P | 0-120pmol | 0-350 pmol |
Method Validation
Accuracy
n.d.
Precision
n.d.
LOD
< 50fmol on column
Recovery
n.d.