Sphingosine-1-phosphate - LC-MS/MS - Schmidt et al.
From LipidomicsWiki
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=== Column === | === Column === | ||
- | * Type: Luna RP C | + | * Type: Luna RP C 18 with a C18 guard column (Phenomenex, Aschaffenburg, Germany) |
* length: 150 x 2 mm i.d., guard column 4 x 2 mm i.d. | * length: 150 x 2 mm i.d., guard column 4 x 2 mm i.d. | ||
* Particle size: 5 µm | * Particle size: 5 µm |
Revision as of 15:55, 23 July 2008
Contents |
Sample preparation
- S1P and related compounds were extracted with methanol precipitation
- internal standards were added prior lipid extraction
- supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Human plasma | 50µl | S1P 17:0 | 7.5 ng |
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: gradient elution
- Solvent(s): Solvent A water/formic acid (100/0.1 v/v); Solvent B acetonitril/ tetrahydrofuran/formic acid (50/50/0.1 v/v)
- Gradient:
Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
---|---|---|---|
0 | 0.3 | 57.5 | 42.5 |
0.6 | 0.3 | 57.5 | 42.5 |
5 | 0.3 | 0 | 100 |
10 | 0.3 | 0 | 100 |
10.5 | 0.3 | 57.5 | 42.5 |
14 | 0.3 | 57.5 | 42.5 |
Autosampler
- Type: CTC Pal
- Injection volume: 5µl
- Wash solvent: n.d.
Column
- Type: Luna RP C 18 with a C18 guard column (Phenomenex, Aschaffenburg, Germany)
- length: 150 x 2 mm i.d., guard column 4 x 2 mm i.d.
- Particle size: 5 µm
Mass spectrometer
- Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
- Ionization mode: ESI positive
- Ionization voltage: 5400V
- Source temperature: 400°C
- Collision gas: Nitrogen
- Collision gas pressure:
- MS/MS-conditions:
- precursor ion scan of m/z 264.2
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
---|---|---|---|
S1P | 380.1 | 264.2 | 23V |
S1P 17:0 | 366.2 | 250.1 | 23V |
Data analysis and quantification
Software
- Analyst 1.4.2.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Human plasma |
---|---|
S1P | 50-1000ng/mL |
Method Validation
Accuracy
100.6 ± 6.7 (S1P)
Precision
intraday 96.2 - 104.4 % and interday precision 96.3 - 104.3 %
LOD
LOD: <10.2 ng/mL for S1P
Recovery
84.3 ± 7.3 (S1P)