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

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Contents

Sample preparation

  • S1P and related compounds were extracted with methanol precipitation
  • 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 50µl S1P 17:0 7.5 ng

Instrumentation and method

Pump

  • Type: binary high pressure gradient (Agilent 1100)
  • Mode: gradient elution
  • Solvent(s): Solvent A water/formic acid (100/0.1 v/v); Solvent B acetonitril/ tetrahydrofuran/formic acid (50/50/0.1 v/v)
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.3 100 0
0.6 0.3 57.5 42.5
5 0.3 0 100
10 0.3 0 100
10.5 0.3 57.5 42.5
14 0.3 57.5 42.5

Autosampler

  • Type: CTC Pal
  • Injection volume: 5µl
  • Wash solvent: n.d.

Column

  • Type: Luna RP C 18 with a C18 guard column (Phenomenex, Aschaffenburg, Germany)
  • length: 150 x 2 mm i.d., guard column 4 x 2 mm i.d.
  • Particle size: 5 µm

Mass spectrometer

  • Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
  • Ionization mode: ESI positive
  • Ionization voltage: 5400V
  • Source temperature: 400°C
  • Collision gas: Nitrogen
  • Collision gas pressure:
  • 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]
S1P 380.1 264.2 23V
S1P 17:0 366.2 250.1 23V

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 50-1000ng/mL

Method Validation

Accuracy

100.6 ± 6.7 (S1P)

Precision

intraday 96.2 - 104.4 % and interday precision 96.3 - 104.3 %

LOD

<10.2 ng/mL for S1P

Recovery

84.3 ± 7.3 (S1P)

Sample data

Mass spectra

Chromatogram

Reference

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