Simultaneous quantitation and identification of intact Nandrolone phase II oxo-metabolites based on derivatization and inject LC-MS/(HRMS) methodology

Alpha sensitive and selective derivatization and inject method for the quantification of intact nandrolone phase II oxo-metabolites was developed and validated using liquid chromatography - (tandem high resolution) mass spectrometry (LC-MS/(HRMS)). For the derivatization, Girard's reagent T (GRT) was used directly in natural urine samples and the analysis of the metabolites of interest was performed by direct injection into LC-MS/(HRMS) system operating in positive ionization mode. Derivatization enabled the efficient detection of nandrolone oxo-metabolites, while at the same time producing intense product ions under collision-induced dissociation (CID) conditions that are related to metabolites of the steroid backbone and not to the conjugated moieties. Glucuronide and sulfate metabolites of nandrolone were chromatographically resolved and quantified in the same run in the range of 1-100 ng mL-1, while at the same time structure identification could be performed for each metabolite. Full validation of the method was performed according to the World Anti-Doping Agency (WADA) International Standard for Laboratories (ISL). Nandrolone oxo-metabolites were quantified in two sets of urine samples, the first set consisted of real urine samples previously detected as negative and the second set consisted of urine samples collected from two excretion studies after nandrolone decanoate administration. The results for 19-norandrosterone glucuronide (19-NAG) and 19-noretiocholanolone glucuronide (19-NEG) were compared with those obtained by traditional gas chromatography - (tandem) mass spectrometry (GC-MS/[MS]) method. Accurate and convenient analysis of the intact phase II oxo-metabolites of nandrolone under the provisions of WADA's TD2023IDCR was accomplished with a dilute derivatization reagent (Girard's reagent T) and inject strategy. image