Methods: 100 μL of standard or samples were placed in a tube and then methanol containing 0.6 μL of concentrated HCl, and 30 pmol of C17-S1P were added. Then CHCl3, 1M NaCl, and 3M NaOH were added. The tubes were vigorously vortexed and centrifuged and the alkaline aqueous phase containing S1P was transferred to a new tube. The aqueous fractions of the S1P extracts were then mixed with CHCl3 and 6 N HCl. The CHCl3 phase was transferred to a new tube and dried with a vacuum evaporator. Then dephosphorylation of S1P was carried out. 100 uL of HF was added to each sample and sealed with parafilm in an ice bath. After 1 h, the HF was evaporated under a stream of nitrogen. The reaction was quenched by the addition of 300uL 10N sodium hydroxide. Then it was extracted with CHCl3 and CH3OH. After the drying process, samples were resolved in methanol and injected into LC-MS/MS.

Results: The HF dephosphorylation reaction conditions were optimized in consideration of the reaction temperature and time. After adding HF the tube was incubated at 4°C, 25°C, and 40°C overnight to determine the optimal temperature. As a result of the experiment, the reaction at 25° C was optimal for removing the phosphate group of S1P. The dephosphorylation yield increased from 1 min to 60 min and became stable after 60 min. Under the optimized gradient conditions, the retention time of S1P in the chromatography system was 5.99 min and that of C17-S1P was 5.74 min. For validation conditions, ICH M10, Q2(R1), and MFDS guidelines were referenced. The calibration equation was y=0.0074x+0.0516 (n=3), and the correlation coefficient value (R2) was 0.9996 (n=3). The limit of quantitation is 10 nM (S/N ratio >10) and the limit of detection (S/N ratio >3) is 5 nM. Accuracy in recoveries ranged from 97.25 to 104.53% with 3 individuals on 30, 250, and 800 nM (pmol/mL) spike samples. Reproducibility was repeated 6 times and had an RSD (%) of 4.67%.

Conclusion: In conclusion, our findings demonstrate that HF dephosphorylation reaction is optimal at 25° C and 60 min incubation time for removing the phosphate group of S1P. The results indicated that the limit of quantitation and the limit of detection is 10 and 5 nM, respectively.

Acknowledgment: This research was supported by "Regional Innovation Strategy (RIS)" through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(MOE)(2021RIS-001)