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

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Revision as of 12:34, 21 July 2008


Contents

Sample preparation

  • S1P and related compounds were extracted with methanol precipitation
  • internal standards were added prior lipid extraction
  • supernetants were 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 57.5 42.5
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: 20µl
  • Wash solvent: methanol/chloroform = 1/1

Mass spectrometer

  • Type: Triple quadrupole (Micromass, Quattro Ultima)
  • Ionization mode: ESI positive
  • Ionization voltage:
  • Source temperature:
  • Collision gas: Argon
  • 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]
Cer 14:0 - H2O 492.5 264.2 x
Cer 14:0 510.5 264.2 x

Sample Preparation

S1P and related compounds were extracted with methanol precipitation. Therefore, the prepared standard mixture were centrifuged at 10,000 x g for 5 min, and supernatants were transferred to glass vials prior to injection into the ESI-LC-MS/MS system.

Analytical Method

HPLC-MS-MS

HPLC

HPLC: Agilent 1100 Series binary pump (G1312A); Agilent degasser (G13791A); HTC Pal autosampler (Chromtech)

columns: Luna RP (150 mm L x 2 mm i.d., 5 µm particle size, 100 Å pore size); C18 guard column (4 mm Lx 2 mm i.d.)

eluent A: water/formic acid (100/0.1 v/v); eluent B: acetonitril/ tetrahydrofuran/formic acid (50/50/0.1 v/v)

linear gradient: A/B = 57.5/42.5 (0 - 0.6 min) A/B = 0/100 (5 -10 min) A/B = 57.5/42.5 (10.5 - 14 min)

flow rate: 0.3 ml/min; injection volume: 5 μl

MS 4000 Q-Trap (Applied Biosystems)

triple quadrupole; Turbo V source ion spray; positive ion mode;

electrospray voltage: 5400 V; source temperature: 400 °C; auxiliary gas: 30 psi; nebulizer gas: 30 psi; curtain gas: 10 psi; collision gas: 4 psi; dwell time: 100 ms

All quadrupoles were working at unit resolution linear ion trap. Quantitation was performed with Analyst Software V1.4 (Applied Biosystems).

Method Validation

Accuracy

100.6 ± 6.7 (S1P); 100.8 ± 4.8 (SPH); 100.6 ± 3.6 (SAPH); 99.7 ± 3.1 (SA1P)

Precision

interday and intraday precision

LOD

n.d.

LOQ

LLOQ: <10.2 ng/mL for S1P; <4.6 ng/mL for SPH; <1.9 ng/mL for SAPH; 0.57 ng/mL for SA1P

Recovery

84.3 ± 7.3 (S1P); 87,9 ± 8.2 (SPH); 85.7 ± 7.7 (SAPH); 77.5 ± 10.5 (SA1P); 82.4 ± 11.9 (C17 S1P); 77.5 ± 10.5 (C17 SPH)

Internal Standard

C17 S1P for quantitation of S1P and SA1P; C17 SPH for quantitation of SPH and SAPH

Reference

  • H. Schmidt, R. Schmidt, G. Geisslinger Prostaglandins & other Lipid Mediators 2006, 81, 162-170.

Pharmazentrum Frankfurt/ZAFES; Institut für Klinische Pharmakologie; Klinikum der Johann Wolfgang Goethe-Universität; Theodor-Stern-Kai 7; 60590 Frankfurt am Main; Germany.

Tel: +49-69-6301-7618; Fax: +49-69-6301-7636; e-mail: Helmut.Schmidt@em.uni-frankfurt.de (H. Schmidt)

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