Sphingosine/Sphinganine - LC-MS/MS - Lieser et al.
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== Sample preparation == | == Sample preparation == | ||
- | * lipid extraction according to Bligh | + | * [[lipid extraction according to Bligh and Dyer]] |
* 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 | * 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 !! Internal Standard(s) !! Internal Standard(s) added |
|- | |- | ||
! Fibroblasts | ! Fibroblasts | ||
- | + | | SPH 17:0 || 175 ng/mg cellular protein | |
|} | |} | ||
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* Type: Waters Agilent Alliance 2790 | * Type: Waters Agilent Alliance 2790 | ||
* Mode: isocratic | * Mode: isocratic | ||
- | * Solvent(s): Solvent MeOH/CHCl3 (3/1) containing 0 | + | * Solvent(s): Solvent MeOH/CHCl3 (3/1) containing 0.1 % HCOOH |
* Flow: 300 µL/min | * Flow: 300 µL/min | ||
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* Ionization mode: ESI positive | * Ionization mode: ESI positive | ||
* Ionization voltage: 3000V | * Ionization voltage: 3000V | ||
- | |||
* Collision gas: Argon | * Collision gas: Argon | ||
* Collision gas pressure: 1.75*10^-3 torr | * Collision gas pressure: 1.75*10^-3 torr | ||
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== Method Validation == | == Method Validation == | ||
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=== Precision === | === Precision === | ||
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=== LOD === | === LOD === | ||
9 fmol (SPH); 21 fmol (SPA) | 9 fmol (SPH); 21 fmol (SPA) | ||
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=== Recovery === | === Recovery === | ||
- | 92 | + | 92.0 ± 6,4 % |
- | + | ||
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- | == Reference == | + | == Reference == |
- | + | ||
- | + | *[http://www.ncbi.nlm.nih.gov/pubmed/12897185?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum B. Lieser, G. Liebisch, W. Drobnik, G. Schmitz ''Journal Lipid research'' '''2003''', ''44(11)'', 2209-16.] | |
- | + | [[Category:LC-MS/MS]] [[Category:Triple_quadrupole]] [[Category:Sphingolipids_(SP)]] [[Category:Sphingoid_bases_(SP01)]] [[Category:ESI]] [[Category:HPLC]] |
Current revision
Contents |
Sample preparation
- lipid extraction according to Bligh and Dyer
- internal standards were added prior lipid extraction
- supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system
Material | Internal Standard(s) | Internal Standard(s) added |
---|---|---|
Fibroblasts | SPH 17:0 | 175 ng/mg cellular protein |
Instrumentation and method
Pump
- Type: Waters Agilent Alliance 2790
- Mode: isocratic
- Solvent(s): Solvent MeOH/CHCl3 (3/1) containing 0.1 % HCOOH
- Flow: 300 µL/min
Autosampler
- Type: CTC Pal
- Injection volume: 5µl
- Wash solvent: n.d.
Column
- Type: Thermo Hypersil-Keystone Beta Basic CYANO (Thermo scientific)
- length: 50 x 2mm i.d.
- Particle size: 3µm
- Temperature: 30°C
Mass spectrometer
- Type: Triple quadrupole (Quattro Ultima, Micromass)
- Ionization mode: ESI positive
- Ionization voltage: 3000V
- Collision gas: Argon
- Collision gas pressure: 1.75*10^-3 torr
- MS/MS-conditions:
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
---|---|---|---|
SPH | 300 | 282 | 14V |
SPH | 300 | 264 | 14V |
SPH | 300 | 252 | 14V |
SPA | 302 | 284 | 14V |
SPA | 302 | 266 | 14V |
SPA | 300 | 254 | 14V |
SPH C17 | 286 | 268 | 14V |
SPH C17 | 286 | 250 | 14V |
SPH C17 | 286 | 238 | 14V |
Data analysis and quantification
Software
- MassLynx coupled to self programmed Excel macros
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Fibroblasts |
---|---|
SPH, SPA | 0-60ng/175µg cellular protein |
Method Validation
Precision
8 % (SPH); 13 % (SPA)
LOD
9 fmol (SPH); 21 fmol (SPA)
Recovery
92.0 ± 6,4 %