Sphingosine-1-phosphate, Lysophosphatidic acid - LC-MS/MS - Scherer et al.

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Contents

Sample preparation

  • S1P and LPA were extracted with butanol
  • internal standards were added prior lipid extraction
  • supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system
Material Material used Internal Standard(s) Internal Standard(s) added
Human plasma 75µl 13C2D2-S1P, LPA 17:0 20 ng

Instrumentation and method

Pump

  • Type: binary and isocratic pump (Agilent 1200)
  • Mode: gradient elution
  • Solvent(s): Solvent A 90mM ammonium formate/formic acid (100/0.2 v/v); Solvent B acetonitril/formic acid (100/0.2 v/v)
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.8 5 95
0.5 0.8 5 95
1.5 0.8 50 50
1.7 0.8 50 50
1.9 0.8 5 95
2.2 0.8 5 95

Autosampler

  • Type: CTC Pal
  • Injection volume: 10µl
  • 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., pre-column filter
  • Particle size: 2.2 µm

Mass spectrometer

  • Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
  • Ionization mode: ESI negative
  • Ionization voltage: -4500V
  • Source temperature: 300°C
  • Collision gas: Nitrogen
  • Collision gas pressure: medium
  • MS/MS-conditions:
    • precursor ion scan of m/z 153 for LPA species screening
    • multiple reaction monitoring table of species observed frequently
Analyte Precursor [m/z] Precursor [m/z] Collision energy [eV]
S1P 378.2 79 58V
SA1P 380.2 79 58V
13C2D2-S1P 382.2 79 58V
LPA 16:0 409.2 153 30V
LPA 18:0 437.2 153 30V
LPA 18:1 435.2 153 30V
LPA 18:2 433.2 153 30V
LPA 20:4 457.2 153 30V
LPA 17:0 423.2 153 30V

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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