A liquid chromatographic/tandem mass spectroscopic method for quantification of the cyclic peptide melanotan-II. Plasma and brain tissue concentrations following administration in mice.

Melanotan-II (MT-II), a synthetic analogue of the natural melanocortin peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), is well known for the anorexic effects it elicits in rodents. These effects are, at least partly, associated with agonistic action on the centrally located melanocortin receptors, MC3R and MC4R. Whether MT-II exerts this effect via brain penetration still remains unclear. In order to address this question we administered MT-II in rodents at efficacious doses and then employed a sensitive methodology for the determination of MT-II in plasma and brain samples. MT-II was extracted from mouse plasma and brain tissue by acetonitrile precipitation followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The described assay improved significantly previously reported MT-II levels of quantification in rat plasma and brain. The lower limits of quantification (LLOQs) of 0.5 ng/mL and 2.5 ng/g were obtained in 50 microL plasma and 100 microL brain homogenate, respectively. The calibration curve was linear over the concentration range of 0.5-500 ng/mL for plasma and 2.5-250 ng/g for brain tissue. The method was successfully applied in measuring levels of MT-II in plasma and brain tissue following intraperitoneal (ip) administration of 1 mg/kg of peptide in mice. Following administration of MT-II, clearance from plasma was rapid. The sensitivity of the assay allowed the determination of low concentrations of MT-II (11.4 +/- 5.5 ng/g) in brain homogenate at 30 min after dosing. However, the brain concentrations when compared with the high plasma levels of MT-II at the same time point confirmed the low penetrability of the peptide in mouse brain.