Sphingosine-1-phosphate - LC-MS/MS - Berdyshev et al.

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Contents

Sample preparation

  • S1P and related compounds were extracted with modified Bligh and Dyer using 0.1 N HCl for phase separation
  • internal standards were added prior lipid extraction
  • for acetylation the lipid extracts were treated with acetic anhydrid
Material Material used Internal Standard(s) Internal Standard(s) added
Human plasma 100µl S1P 17:0 40 pmol
HPAECs 1 x e6 S1P 17:0 40 pmol

Instrumentation and method

Pump

  • Type: binary high pressure gradient (Agilent 1100)
  • Mode: gradient elution
  • Solvent(s): Solvent A water/methanol/formic acid (20/80/0.5 v/v), 5 mM ammonium formate; Solvent B methanol/acetonitril/formic acid (59/40/0.5 v/v), 5 mM ammonium formate
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.5 100 0
4 0.5 100 0
7 0.5 0 100
10 0.5 0 100
12 0.5 100 0

Autosampler

  • Type: Agilent
  • Injection volume: 10µl
  • Wash solvent: n.d.

Column

  • Type: Agilent Zorbax Eclipse XDB-C8 column
  • length: 150 x 4.6 mm i.d.
  • Particle size: 5 µm
  • Retention time: 8.15 min (S1P), 7.9 min (C17-S1P); 8.4 min (DHS1P)

Mass spectrometer

  • Type: Triple quadrupole (Applied Biosystems, API 4000)
  • Ionization mode: ESI negative
  • Ionization voltage: -4500V
  • Source temperature: 520°C
  • Collision gas: Nitrogen
  • Collision gas pressure:
  • MS/MS-conditions:
    • multiple reaction monitoring table of species observed frequently
Analyte Precursor [m/z] Precursor [m/z] Collision energy [eV]
S1P 462 402 -28V
S1P 17:0 448 388 -26V
DH-S1P 464 404 -28V

Data analysis and quantification

Software

  • Analyst 1.4.2.

Calibration and quantification

  • calibration type: standard addition without matrix for plasma and in the presence of total lipids extracted from HPAECs.
  • species used for calibration
Species Plasma HPAEC
S1P 0-150pmol 0-350pmol
DHS1P 0-120pmol 0-350 pmol



Method Validation

Accuracy

n.d.

Precision

n.d.

LOD

< 50fmol on column

Recovery

n.d.

Sample data

Mass spectra

Chromatogram

Reference

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