Elucidation of Neu-P11 metabolism in urine of volunteers by liquid chromatography–tandem mass spectrometry

The novel melatonin agonist Neu-P11 is used in treatment of physiological insomnia. In animal studies Neu-P11 showed sleep-promoting effects. In a phase 1 study Neu-P11 was administered to cohorts of healthy young male volunteers in an ascending single dose study. Up to now the metabolism of this new drug in humans has not been investigated. The first aim of this study was to identify possible metabolites in pooled urine samples of the first collecting period (0–8 h) of volunteers with the highest Neu-P11 oral dosage (200 mg). The objective of the second part of the study was to estimate the concentrations of the main metabolites of Neu-P11 – in this urine sample. The analyte Neu-P11 and metabolites were separated from human urine using dilution and precipitation with acetonitrile. Samples were analyzed for formation of both phase I and phase II metabolites using LC–MS/MS in precursor ion mode, product ion mode, neutral loss mode and the multi-reaction monitoring mode (MRM). Urine samples were analyzed before and after addition of beta-glucuronidase and sulfatase. In the pooled urine sample eight metabolites could be proved. The parent drug, the sulfated demethylated Neu-P11, the sulfated 6OH-Neu-P11 and the di-oxygenated product gave the highest signals in these urine samples and probably had the highest concentration. But quantification without reference substances is not possible. So in the second part of the study the urine samples were additionally analyzed with UV-detection for a better estimation of the metabolite concentrations. The concentration of the sulfated metabolites was more than ten times higher than the concentration of the unchanged drug in urine. Other metabolites were not measurable with UV-detection. The di-oxygenated Neu-P11 and an additional mono-oxygenated Neu-P11 showed relatively high signals in MS/MS. Probably the other metabolites, namely glucuronides, unconjugated demethylated Neu-P11 and unconjugated 6OH-Neu-P11, were formed at a lesser extent.