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 < 8 % and < 7 % relative error at all times, respectively. Plasma samples were stable over at least three freeze-thaw cycles, but the variation of short-term stability after 24h at room temperature for lower limit of quantitation was relatively large (18%). Thus, we suggest that safingol plasma samples should be analyzed within 8 hour after the samples are thawed for the most accurate results. In conclusion, a simple and sensitive assay method using HPLC-MS/MS is developed to measure plasma concentrations of safingol in human. This method will be used to quantify safingol in human plasma samples in future clinical trial.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA