View source

== Sample preparation ==

* [[Lipid extraction according to Bligh and Dyer]]
* internal standards are added prior lipid extraction
* to avoid the loss of lipids, extraction was performed in glassware
* samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) 

{| class="wikitable" style="text-align:center"
! Material !! Material used  !! Internal Standard(s) !! Internal Standard(s) added
|-
! Cultured cells
| 100µg protein || PC 28:0, PC 44:0 || 1250ng each
|-
! Human plasma
| 20µl ||  PC 28:0, PC 44:0 || 3500ng PC 28:0, 5000ng PC 44:0
|}

== Instrumentation and  method ==


=== Pump ===
* Type: binary high pressure gradient (Agilent 1100)
* Mode: isocratic flow gradient
* Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
* Flow gradient: 
{| border="1" class="wikitable" style="text-align:center;"
! Time [min]!! Flow [ml/min]  !! % Solvent A !! % Solvent B
|-
! 0 
| 0.05 || 100 ||0
|-
! 0.1 
| 0.03 || 100 ||0
|-
! 1.1 
| 0.2 || 100 ||0
|-
! 1.3 
| 0.05 || 100 ||0
|}

=== Autosampler ===
* Type: CTC Pal
* Injection volume: 20µl
* Wash solvent: methanol/chloroform = 1/1

=== Mass spectrometer ===
* Type: Triple quadrupole (Micromass, Quattro Ultima)
* Ionization mode: ESI positive
* Ionization voltage: 3500 V
* Source temperature: 300°C
* Collision gas: Argon
* Collision gas pressure: 1.0 10-3 Torr
* Collision energy: 30 V
* MS/MS-mode: precursor ion scan of m/z 184.1

== Data analysis and quantification ==

=== Data handling === 
* combine spectra above half peak height
* smooth combined spectrum (if necessary)
* centroid combined spectrum
* pick peak intensities

=== Isotope correction === 
* using Excel Macros correcting the peak intensities in a sequential algorithm starting from low mass species
* five isotope peaks including the monoisotopic were used
* the detailed algorithm is described in the appendix of [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15522827 Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.]

=== Calibration and quantification ===
* calibration type: matrix calibration - addition of naturally occurring species
* species used for calibration
{| class="wikitable" style="text-align:center"
! Species!! Cultured cells !! Human plasma 
|-
! PC 34:1
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! PC 36:2
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! PC 38:4
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! PC 40:0
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! PC O 16:0/20:4
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! SM 16:0
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! SM 18:1
| 0 - 1000 pmol || 0 - 3000 pmol
|-
! SM 18:0
| 0 - 1000 pmol || 0 - 3000 pmol
|}

== Method validation ==

=== Precision ===
* CV within-run: 4 % (major), 5-10 % (minor)
* CV total: 10 % (major), 15 % (minor)

=== LOD ===
* 0.6 μM in plasma

== Sample data ==

=== Mass spectra ===

== Reference  ==

*[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15522827 Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.]

[[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Glycerophosphocholines_(GP01)]] [[Category:Phosphosphingolipids (SP03)]]