ABSTRACT
COVID-19 emergency required the development of specific therapeutic choices, especially for patient at higher risk to have a worst prognosis. Among the first and most widely used antivirals are remdesivir (RDV) and molnupiravir (MPV): these are both nucleoside analogs prodrugs, capable to inhibit viral RNA-dependent RNA polymerase by strand termination and a "mutational catastrophe", respectively. While RDV is only available for intravenous use in hospital, MPV is an oral drug, allowing domestic use. The circulating active metabolites of these drugs, GS-441524 and H-hydroxycitidine (NHC), respectively, are considered for the description of their pharmacokinetics (PK) and are related to their antiviral effect. Nevertheless, PK characteristics of RDV, MPV and their metabolites in the real-life use are still poorly explored, particularly due to the lack of validated methods for their quantification in human matrices. Therefore, in this work, we aimed at validating a fast, reliable and rugged ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/ MS) method for the simultaneous quantification of these prodrugs and their active metabolites in human plasma, following FDA and EMA guidelines. Sample preparation consisted in a protein precipitation protocol: 50 muL of plasma are added with 50 muL of a solution of isotope-labeled internal standards (IS) in water:methanol (50:50 v:v) and, then, with 400 muL of a mixture of acetonitrile:methanol (50:50 v:v), vortex mixed and centrifuged. After drying at 40 degreeC in vacuum centrifuge (1 h), the extracts are reconstituted with water 0.2% formic acid.Then, 5 muL of the extracts undergo UHPLC separation with a gradient run of water (Phase A) and acetonitrile:methanol 50:50 (v:v, Phase B), both with 0.2% of formic acid at 40degreeC in a core-shell reverse-phase column (Kinetex polar C18, 2.1x100 mm, 2.6 mum). The total runtime is 5 minutes and drug detection is performed by MRM, with 2 specific transitions for each compound and IS. The method fulfilled the requirements from FDA and EMA guidelines in terms of accuracy, precision, recovery, matrix effect, stability and it was applied to samples from a small cohort of patients treated with these drugs, confirming its eligibility for research and clinical use.