Sphingosine/Sphinganine - LC-MS/MS - Lieser et al.
From LipidomicsWiki
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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 | |
|} | |} | ||
- | == | + | == 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 | ||
+ | {| border="1" class="wikitable" style="text-align:center" | ||
+ | ! 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 | |
- | * | + | {| class="wikitable" style="text-align:center" |
- | + | ! Species!! Fibroblasts | |
- | + | |- | |
- | + | ! SPH, SPA | |
- | + | | 0-60ng/175µg cellular protein | |
- | + | |- | |
- | + | |} | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
== Method Validation == | == Method Validation == | ||
- | |||
- | |||
=== Precision === | === Precision === | ||
Line 67: | Line 94: | ||
=== LOD === | === LOD === | ||
9 fmol (SPH); 21 fmol (SPA) | 9 fmol (SPH); 21 fmol (SPA) | ||
- | |||
- | |||
=== Recovery === | === Recovery === | ||
- | 92 | + | 92.0 ± 6,4 % |
- | + | ||
- | + | ||
- | + | ||
- | == 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 %