23V
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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