Identification of Nifurtimox metabolites in urine of pediatric Chagas disease patients by UHPLC-MS/MS

Abstract

Background Nifurtimox (NFX) is one of the only 2 available drugs for the treatment of Chagas disease, a parasitic disease endemic to Latin America. In spite of widespread use of this medication, little is known regarding its metabolism, particularly in children.The objective of this study was to develop a method to identify NFX metabolites in human samples, and apply it to the discovery of NFX metabolites in urine from pediatric patients undergoing treatment for Chagas disease.Methods Urine was collected from 12 pediatric patients and 8 healthy volunteers (controls), and anonymized before analysis. Informed consent was obtained from all participants. Samples were aliquoted, deproteinized with ACN (BNZ as internal standard) and centrifuged in cold. 10% of supernatant in water was injected into a 1.8 µm C18 column and chromatographed in 3.5 min under a water/ACN gradient at 0.4 mL/min in a Shimadzu Nexera X2 UHPLC equipment. Species were positively ionized by a Turbo IonSpray source. Metabolites were identified and characterized by an ABSciex 6500 QTRAP spectrometer through Enhanced-Mass-Screening (EMS), Neutral-Loss (NL), Precursor-Ion (PREC), Enhanced-Product-Ion (EPI) and MS3 experiments. For chromatographic monitoring, parameters were optimized and the three most intense Multiple-Reaction-Monitoring (MRM) transitions were selected.Results Denitrated NFX conjugated with cysteine (M1) and N-acetyl-cysteine (M2), as well as other phase I metabolites like saturated nitrile (M3), hydroxyamide (M4), carboxylic acid (M5) or aldehyde (M6) were identified in most samples. The final MS/MS detection method was high reproducible and sensitive for all metabolites.Conclusions We found the main NFX metabolites in pediatric urine using a fast MS/MS method that can allow us to efficiently study the role of NFX and its metabolites in pediatric treatment response and the adverse drug reactions, and in combination with PK/PD experiments will facilitate future clinical trials, and possibly develop new therapeutic drug monitoring strategies.