Determination of safingol, a sphingosine kinase inhibitor, in human plasma by liquid chromatography-electrospray quadrupole linear ion trap mass spectrometry

3961 Safingol, a saturated synthetic enantiomer of dihydrosphingosine, is a potential sphingosine kinase (SK) inhibitor that could decrease the formation of sphingosine-1 phosphaste (S1P) from degraded ceramide due to its stereoisomeric structure. Safingol has been reported to synergistically increase cytotoxicity of chemotherapeutic agents in tumor cell lines. An analytical method was developed for the quantitative determination of safingol in human plasma. Calibration curves were constructed in the range of 1-100 ng/ml, and were analyzed using a weight factor (1/x) proportional to the nominal concentration. Sample pretreatment involved a liquid-liquid extraction with ethyl acetate using 0.1 ml aliquots of plasma. The analyte was separated on a column (20mm × 2.1mm i.d.) packed with 3μm C18 material using a gradient elution with methanol (mobile phase B) and water containing 0.1% formic acid at a flow rate of 0.3 mL/min. The gradient profile was: hold at 60% mobile phase B for 0.5 min, linear gradient to 100% mobile phase B in 1 min, and hold at 100% mobile phase B for 1 min followed by 1 min re-equilibration. The mean retention time for safingol and C17 D-erythrosphinganine (internal standard) during the method validation was approximately 1.6 min and the overall chromatographic run time was established at 3 min. Detection was performed using a Q TRAPTM LC/MS/MS system by monitoring the ion transitions (MRM) from m/z 302.4 →59.9 (safingol) and m/z 288.3 →60.0 (C17 D-erythrosphinganine). Electrospray ionization was performed in the positive mode with an ionspray voltage of 5000eV. The other MS conditions were as follows: curtain gas, 25 psi; temperature 600 oC; ion source gas 1, 40 psi; ion source gas 2, 50 psi; CAD gas, medium; collision energy, 43 eV; declusting potential, 71 eV; entrance potential, 10 eV; collision cell exit potential 10 eV. The values for precision and accuracy were