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

From LipidomicsWiki

(Difference between revisions)
Jump to: navigation, search
(Method Validation)
(Method Validation)
Line 103: Line 103:
=== Recovery ===
=== Recovery ===
-
84.3 ± 7.3 (S1P); 87,9 ± 8.2 (SPH); 85.7 ± 7.7 (SAPH);
+
84.3 ± 7.3 (S1P)
-
77.5 ± 10.5 (SA1P); 82.4 ± 11.9 (C17 S1P); 77.5 ± 10.5 (C17 SPH)
+
== Internal Standard ==
== Internal Standard ==

Revision as of 12:58, 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: 5µl
  • Wash solvent: n.d.

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

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

Precision

interday and intraday precision


LOD

LOD: <10.2 ng/mL for S1P

Recovery

84.3 ± 7.3 (S1P)

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)

Personal tools
Create a book