## 4 Discussion  
In this study, we developed and validated a rapid HPLC-MS/MS to quantify Fingolimod and its active metabolite Fingolimod-P in human plasma. The method involved the use of Fingolimod-D4 and Fingolimod-P-D4 as internal standards, plasma protein precipitation with acetonitrile and chromatographic separation with tandem mass spectrometry detection. The method was validated following the criteria set in the \"EMA guidelines on bioanalytical method validation\" [13]. This method was successfully applied to plasma samples obtained from 15 RRMS patients under chronic treatment with Fingolimod ad  
Figure 2: Plasma levels of Fingolimod (light blue) and Fingolimod-P (red) measured in 15 patients after 6 (\(n=15\)), 12 (\(n=11\)) and 24 (\(n=5\)) months of treatment (Fingolimod 0.5 mg/day orally). Box plots show median, quartile range and min-max range. Absolute levels of Fingolimod and Fingolimod-P were analyzed by one-way ANOVA followed by Tukey multiple comparison post-hoc test. ministered orally at a dose of 0.5 mg once daily for up to 24 months. To our knowledge, this is the first analysis reporting plasmatic levels of Fingolimod and Fingolimod-P after such long-term treatments with the dosage recommended in the clinical practice. No significant differences were observed in the levels of Fingolimod between samples collected after 6, 12 and 24 months of treatment, in agreement with literature data showing that steady-state concentrations are reached in less than 2 months [14]. Importantly, the concentration profile at steady state is remarkably flat, with peak-to-trough fluctuations after daily administrations ranging approximately 20% [9] and thus, the time of blood collection is likely not so critical. The similar plasmatic levels for Fingolimod at the different time-points suggests that 24 months of daily treatment does not affect the drug's bioavailability. Our data also indicate similar plasmatic levels of the active metabolite Fingolimod-P along the 24-month treatment, suggesting that Fingolimod metabolism is unaffected by multiple daily doses. In addition, the levels of the active metabolite Fingolimod-P were consistently higher than the levels of the parent compound, by 2.2-fold on average. Contradictory results are reported in the literature regarding this latter observation. Thus, data obtained in single healthy subject receiving 5 mg/day of Fingolimod for 7 days showed higher levels of the metabolite after the first dose and similar levels of drug and metabolite after the 7 th dose [15]. On the contrary, higher levels of the drug in comparison to the metabolite were found in three other studies [9; 16] in which Fingolimod was administered at daily doses ranging 0.5-5 mg for 7-28 days. No data are available in the literature at the longer time-points (\(\geq\)6 months) considered in the present study. The analytical method described in this work can be useful to evaluate if the inter-individual variability in clinical responses might be explained by inter-individual differences in drug bioavailability, which can underlie either no or poor efficacy (insufficient blood concentrations) or adverse events (too high blood concentrations). Conceptualization, C.F., L.B., R.M., S.R., M.G. and J.L. ; methodology, C.F., A.P. and J.L. ; formal analysis, C.F., L.B., M.G. and J.L. ; investigations, C.F., L.B., S.R., R.M., M.G. and J.L. ; writing--original draft preparation, C.F., M.G. and J.L. ; writing--review and editing, C.F., L.B., R.M., M.G. and J.L. All authors have read and agreed to the published version of the manuscript. This research was funded by the Patient-Centered Outcomes Research Institute, grant number MS 1511-33406. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Foundation IRCCS Neurological Institute [PERSON] (protocol code N. 43, 10 August 2017). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.