Sphingolipid profiling - LC-MS/MS - Scherer et al.

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Contents

Sample preparation

  • sphingolipids were extracted with butanol
  • internal standards were added prior lipid extraction
  • supernatant was transferred to a glass vial and injected
Material Material used Internal Standard(s) Internal Standard(s) added
Cell homogenate 100µg SPH d17:1 20 ng


SPC d17:1 2 ng


GluCer 12:0 20 ng


LacCer 12:0 20 ng


Cer1P 12:0 20 ng

Instrumentation and method

Pump

  • Type: binary and isocratic pump (Agilent 1200)
  • Mode: gradient elution
  • Solvent(s): Solvent A water containing 0.2% formic acid and 200 mM ammonium formate ; Solvent B acetonitrile containing 0.2% formic acid
  • column flow was directed only from 1.0 to 3.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.8 0
100
0.1 0.8 0 100
0.11 0.8 10 90
2.5 0.8 50 50
3.5 0.8 50 50
3.51 0.8 0 100
4.5
0.8
0
100

Autosampler

  • Type: CTC Pal
  • Injection volume: 2µl
  • Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH

Column

  • Type: Interchim HILIC column (Interchim, Montlucan, France)
  • length: 50 x 2 mm i.d., 0.5 µm pre-column filter
  • Particle size: 1.8 µm
  • Column temperature: 50°C

Mass spectrometer

  • Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
  • Ionization mode: ESI positive
  • Ionization voltage: 5500V
  • Source temperature: 400°C
  • Collision gas: Nitrogen
  • Collision gas pressure: medium
  • MS/MS-conditions:
    • precursor ion scan of m/z 264 for HexCer, LacCer, Cer1P d18:1 species screening
    • neutral loss scan of m/z 98 for Cer1P d18:0 species screening
    • multiple reaction monitoring table of species observed frequently
Analyte Precursor [m/z] Product [m/z] Collision energy [eV]
SPH d18:1 300.3 282.2 17

300.3 252.2 25
SPH d18:0 302.3 284.2 21

302.3 254.2 29
SPH t18:0 318.3 282.2 23
Dimethyl-SPH d18:1 328.3 280.3 29
Trimethyl-SPH d18:1
342.3 60.1 49
SPC d18:1
465.3
184
31
HexCer d18:1
[M+H]+
264.3
55
LacCer d18:1
[M+H]+
264.3
65
Cer1P d18:1
[M+H]+
264.3
39
Cer1P d18:0
[M+H]+ neutral loss scan of 98
29

Data analysis and quantification

Software

Calibration and quantification

  • calibration type: matrix calibration - addition of naturally occurring species
  • species used for calibration
Species Cultured cells [pmol] 
SPH d18:1 30 – 300
SPH d18:0 15 - 150
SPH t18:0 10 - 100
Dimethyl-SPH d18:1 0.3 - 3
Trimethyl-SPH d18:1 0.3 - 3
SPC d18:1 10 - 100
GluCer d18:1/16:0 25 - 250
GlaCer d18:1/24:1 25 - 250
LacCer d18:1/16:0
25 - 250
LacCer d18:1/24:0
25 - 250
Cer1P d18:1/16:0
15 - 150
Cer1P d18:1/24:0
15 - 150

Method Validation

Accuracy

90 - 110 %

Precision

CV: below 10 %

LOD

below 10 fmol on column, except PhytoSPH 25 fmol and dhCer-1P 50 fmol on column

Recovery

60 - 70%

Sample data

Mass spectra

Chromatogram 


Reference

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