Ceramide - ESI-MS/MS - Liebisch et al.

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Line 1: Line 1:
== Sample preparation ==
== Sample preparation ==
-
* lipid extraction according to [[Bligh and Dyer]]
+
* [[Lipid extraction according to Bligh and Dyer]]
* internal standards are added prior lipid extraction
* internal standards are added prior lipid extraction
* to avoid the loss of lipids, extraction was performed in glassware
* to avoid the loss of lipids, extraction was performed in glassware
Line 22: Line 22:
* Type: binary high pressure gradient (Agilent 1100)
* Type: binary high pressure gradient (Agilent 1100)
* Mode: isocratic flow gradient
* Mode: isocratic flow gradient
-
* Solvent(s): 5 mM ammonium formate/chloroform (3/1 = v/v)
+
* Solvent(s): Methanol containing 5 mM ammonium formate/chloroform (3/1 = v/v)
-
* Gradient:  
+
* Flow gradient:  
{| border="1" class="wikitable" style="text-align:center;"
{| border="1" class="wikitable" style="text-align:center;"
! Time [min]!! Flow [ml/min]  !! % Solvent A !! % Solvent B
! Time [min]!! Flow [ml/min]  !! % Solvent A !! % Solvent B
Line 48: Line 48:
* Type: Triple quadrupole (Micromass, Quattro Ultima)
* Type: Triple quadrupole (Micromass, Quattro Ultima)
* Ionization mode: ESI positive
* Ionization mode: ESI positive
-
* Ionization voltage:
+
* Ionization voltage: 3500 V
-
* Source temperature:
+
* Source temperature: 250°C
* Collision gas: Argon
* Collision gas: Argon
-
* Collision gas pressure:  
+
* Collision gas pressure: 1.0 10-3 Torr
* MS/MS-conditions:
* MS/MS-conditions:
** precursor ion scan of m/z 264.2
** precursor ion scan of m/z 264.2
Line 58: Line 58:
! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV]  
! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV]  
|-
|-
-
! Cer 14:0 - H2O
+
! Cer 14:0 - H2O (IS)
-
| 492.5|| 264.2 ||x
+
| 492.5|| 264.2 ||25 V
|-
|-
-
! Cer 14:0  
+
! Cer 14:0 (IS)
-
| 510.5|| 264.2 ||x
+
| 510.5|| 264.2 ||25 V
 +
|-
 +
! Cer 16:0 - H2O
 +
| 520.5|| 264.2 ||25 V
 +
|-
 +
! Cer 16:0
 +
| 538.5|| 264.2 ||25 V
 +
|-
 +
! Cer 17:0 - H2O (IS)
 +
| 534.5|| 264.2 ||25 V
 +
|-
 +
! Cer 17:0 (IS)
 +
| 552.5|| 264.2 ||25 V
 +
|-
 +
! Cer 18:0 - H2O
 +
| 548.5|| 264.2 ||25 V
 +
|-
 +
! Cer 18:0
 +
| 566.6|| 264.2 ||25 V
 +
|-
 +
! Cer 20:0 - H2O
 +
| 576.6|| 264.2 ||25 V
 +
|-
 +
! Cer 20:0
 +
| 594.6|| 264.2 ||25 V
 +
|-
 +
! Cer 22:1 - H2O
 +
| 602.6|| 264.2 ||25 V
 +
|-
 +
! Cer 22:1
 +
| 620.6|| 264.2 ||25 V
 +
|-
 +
! Cer 22:0 - H2O
 +
| 604.6|| 264.2 ||25 V
 +
|-
 +
! Cer 22:0
 +
| 622.6|| 264.2 ||25 V
 +
|-
 +
! Cer 23:0 - H2O
 +
| 618.6|| 264.2 ||25 V
 +
|-
 +
! Cer 23:0
 +
| 636.6|| 264.2 ||25 V
 +
|-
 +
! Cer 24:1 - H2O
 +
| 630.6|| 264.2 ||25 V
 +
|-
 +
! Cer 24:1
 +
| 648.6|| 264.2 ||25 V
 +
|-
 +
! Cer 24:0 - H2O
 +
| 632.6|| 264.2 ||25 V
 +
|-
 +
! Cer 24:0
 +
| 650.6|| 264.2 ||25 V
|-
|-
|}
|}
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=== Calibration and quantification ===
=== Calibration and quantification ===
-
* calibration by standard addition to sample matrix
+
* calibration type: matrix calibration - addition of naturally occurring species
* both [M+H]+ and [M+H-H2O]+ are used
* both [M+H]+ and [M+H-H2O]+ are used
* species used for calibration
* species used for calibration
Line 99: Line 153:
| 0-35pmol || 0-50pmol
| 0-35pmol || 0-50pmol
|}
|}
-
 
== Method validation ==
== Method validation ==
-
 
-
=== Accuracy ===
 
=== Precision ===
=== Precision ===
 +
* CV over all: below 10% in plasma samples
=== LOD ===
=== LOD ===
-
 
+
* 0.3 pmol injected amount (in fibroblast lipid extract)
-
=== LOQ ===
+
=== Recovery ===
=== Recovery ===
 +
* recovery of Cer 6:0 above 90%
== Sample data ==
== Sample data ==
Line 117: Line 169:
=== Mass spectra ===
=== Mass spectra ===
-
=== Chromatogram ===
+
== Reference  ==
-
== Reference ==
+
*[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10428992 G.Liebisch et al. Journal of Lipid Research 1999, 40, 1539-1546.]
-
* [http://www.jlr.org/cgi/content/full/40/8/1539 G.Liebisch, W. Drobnik, M. Reil, B. Trümbach, R. Arnecke, B. Olgemöller, A. Roscher, G. Schmitz Journal of Lipid Research 1999, 40, 1539-1546.]
+
-
[[Category:Flow injection analysis]]
+
[[Category:Flow_injection_analysis]] [[Category:ESI]][[Category:Triple_quadrupole]] [[Category:Ceramides_(SP02)]]
-
[[Category:Triple quadrupole]]
+

Current revision

Contents

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 5 mM ammonium formate/chloroform (3/1 = v/v)
Material Material used Internal Standard(s) Internal Standard(s) added
Cultured cells 100µg protein Cer 14:0, Cer 17:0 50ng each
Human plasma 20µl Cer 14:0, Cer 17:0 50ng each

Instrumentation and method

Pump

  • Type: binary high pressure gradient (Agilent 1100)
  • Mode: isocratic flow gradient
  • Solvent(s): Methanol containing 5 mM ammonium formate/chloroform (3/1 = v/v)
  • Flow gradient:
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: 250°C
  • Collision gas: Argon
  • Collision gas pressure: 1.0 10-3 Torr
  • 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]
Cer 14:0 - H2O (IS) 492.5 264.2 25 V
Cer 14:0 (IS) 510.5 264.2 25 V
Cer 16:0 - H2O 520.5 264.2 25 V
Cer 16:0 538.5 264.2 25 V
Cer 17:0 - H2O (IS) 534.5 264.2 25 V
Cer 17:0 (IS) 552.5 264.2 25 V
Cer 18:0 - H2O 548.5 264.2 25 V
Cer 18:0 566.6 264.2 25 V
Cer 20:0 - H2O 576.6 264.2 25 V
Cer 20:0 594.6 264.2 25 V
Cer 22:1 - H2O 602.6 264.2 25 V
Cer 22:1 620.6 264.2 25 V
Cer 22:0 - H2O 604.6 264.2 25 V
Cer 22:0 622.6 264.2 25 V
Cer 23:0 - H2O 618.6 264.2 25 V
Cer 23:0 636.6 264.2 25 V
Cer 24:1 - H2O 630.6 264.2 25 V
Cer 24:1 648.6 264.2 25 V
Cer 24:0 - H2O 632.6 264.2 25 V
Cer 24:0 650.6 264.2 25 V

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

Calibration and quantification

  • calibration type: matrix calibration - addition of naturally occurring species
  • both [M+H]+ and [M+H-H2O]+ are used
  • species used for calibration
Species Cultured cells Human plasma
Cer 16:0 0-35pmol 0-50pmol
Cer 18:0 0-35pmol 0-50pmol
Cer 20:0 0-35pmol 0-50pmol
Cer 24:1 0-35pmol 0-50pmol
Cer 24:0 0-35pmol 0-50pmol

Method validation

Precision

  • CV over all: below 10% in plasma samples

LOD

  • 0.3 pmol injected amount (in fibroblast lipid extract)

Recovery

  • recovery of Cer 6:0 above 90%

Sample data

Mass spectra

Reference

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