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

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**multiple reaction monitoring table of species observed frequently
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! HexCer d18:1<br>
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Revision as of 12:55, 27 April 2010

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

  • Analyst 1.4.2.

Calibration and quantification

  • calibration type: matrix calibration - addition of naturally occurring species
  • species used for calibration
Species Human plasma
S1P 0.352-3.518µmol/L
SA1P 0.175-1.75µmol/L
LPA 16:0 0.162-1.62µmol/L
LPA 18:0 0.0304-0.304µmol/L
LPA 18:1 0.0917-0.917µmol/L
LPA 18:2 0.308-3.08µmol/L
LPA 20:4 0.291-2.91µmol/L

Method Validation

Accuracy

90 - 106 %

Precision

CV: 3 % - 10 %

LOD

6 nmol/L for S1P, SA1P and <2nmol/l

Recovery

85% (S1P, SA1P); 80 % LPA 

Sample data

Mass spectra

Chromatogram 


Reference

Scherer et al. Clin Chem. 2009 Jun;55(6):1218-22.

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