A rapid UPLC-MS/MS method for simultaneous determination of flunitrazepam, 7-aminoflunitrazepam, methadone and EDDP in human, rat and rabbit plasma

► Methadone (MET) and Flunitrazepam (FNZ) are frequently co-abused drugs. ► Rapid and sensitive UPLC-MS/MS assay for simultaneous quantification of FNZ, MET and metabolites. ► The assay could be used for pharmacokinetic and forensic applications. ► Full validation was performed over a wide linearity range (LOQ=0.5 ng/mL, ULOQ=2000 ng/mL). ► The method was applied on real human samples and in a pharmacokinetic interaction study in animals.