Sphingosine-1-phosphate - LC-MS/MS - Schmidt et al.
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== Sample preparation == | == Sample preparation == | ||
* S1P and related compounds were extracted with methanol precipitation | * S1P and related compounds were extracted with methanol precipitation | ||
* internal standards were added prior lipid extraction | * internal standards were added prior lipid extraction | ||
- | * | + | * supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system |
{| class="wikitable" style="text-align:center" | {| class="wikitable" style="text-align:center" | ||
! Material !! Material used !! Internal Standard(s) !! Internal Standard(s) added | ! Material !! Material used !! Internal Standard(s) !! Internal Standard(s) added | ||
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|- | |- | ||
! 0 | ! 0 | ||
- | | 0.3 || | + | | 0.3 || 100 ||0 |
|- | |- | ||
! 0.6 | ! 0.6 | ||
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* Injection volume: 5µl | * Injection volume: 5µl | ||
* Wash solvent: n.d. | * 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 === | === Mass spectrometer === | ||
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|- | |- | ||
|} | |} | ||
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- | |||
== Method Validation == | == Method Validation == | ||
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=== LOD === | === LOD === | ||
- | + | <10.2 ng/mL for S1P | |
=== Recovery === | === Recovery === | ||
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=== Chromatogram === | === Chromatogram === | ||
- | == Reference == | + | == Reference == |
- | * H. Schmidt, R. Schmidt, G. Geisslinger ''Prostaglandins & other Lipid Mediators'' '''2006''', ''81'', 162-170. | + | |
- | [ | + | *[http://www.ncbi.nlm.nih.gov/pubmed/17085324?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum H. Schmidt, R. Schmidt, G. Geisslinger ''Prostaglandins & other Lipid Mediators'' '''2006''', ''81'', 162-170.] |
+ | |||
+ | [[Category:LC-MS/MS]] [[Category:Triple_quadrupole]] [[Category:Sphingolipids_(SP)]] [[Category:Sphingoid_bases_(SP01)]] [[Category:ESI]] [[Category:HPLC]] |
Current revision
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 | 100 | 0 |
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
<10.2 ng/mL for S1P
Recovery
84.3 ± 7.3 (S1P)