## 1 Introduction  
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neuroinflammation that lead to extensive astrogliosis, demyelination, and neurons lost [1]; MS represents the leading cause of neurological disability in young people and is commonly diagnosed between 20 and 40 years old [2]. Relapsing-remitting MS (RRMS) accounts for about 80% of patients with MS and is characterized by recurrent acute exacerbations (relapses) of neurological symptoms, followed by periods of partial or complete recovery (remission) [3].  
Disease-modifying therapies (DMTs) represent the standard care for RRMS, aiming to reduce the frequency and severity of relapses, extending the time intervals between relapses and slowing the progression to permanent disability [4]. Among the most prescribed first-line DMTs, interferon beta and glatiramer acetate are administered by injections and associated with injection-site reactions, poor patient adherence and moderate efficacy [5]. Novel oral agents with different mechanisms of action have been approved in recent years for the treatment of MS, both as first- and second-line therapies [6]. Among them, Fingolimod (FTY720, Gilenya; Novartis International AG, Basel, Switzerland) was the first oral therapy approved for the treatment of RRMS; Fingolimod is a sphingosine 1 phosphate-receptor modulator that selectively and reversibly retains naive and central memory T-lymphocytes within lymph node avoiding penetration in other tissues such as the central nervous system [7,8]. After oral administration, Fingolimod is slowly absorbed (t\({}_{\text{max}}\): 12-16 h) and is transformed by reversible phosphorylation to Fingolimod-Phosphate (Fingolimod-P), the active metabolite [9,10]. Based on two large clinical trials [11,12], the use of Fingolimod was approved in the USA (September 2010) as a first-line treatment and recommended in the EU (by March 2011) for patients with high disease activity despite previous treatment with at least one other DMT and individuals with rapidly evolving severe RRMS. Efficacy data of Fingolimod have been successively updated by long-term observational studies on some of those patients enrolled in the original trials [8].  
Here, we validated a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of Fingolimod and its active metabolite Fingolimod-P in human plasma. The method was set up for its application within a multicentric randomized pragmatic trial whose primary objective was to compare the effectiveness of Fingolimod versus dimethyl-fumarate in RRMS patients (PRAG-MS, EURDACT number 2017-000559-26, ClinicalTrials.gov Identifier: NCT03345940). In fact, the project also aimed to investigate, as an exploratory endpoint, the association between blood drug concentrations and clinical responses over 6, 12 and 24 months of treatment. Unfortunately, the project was terminated due to the slowness of the recruitment activity. Here, we present the data obtained measuring Fingolimod and Fingolimod-P in a subset of the recruited patients, as a complement of the method development and as a proof of its applicability to clinical samples.