Quantitative determination of nystatin in human plasma using LC–MS after inhalative administration in healthy subjects

The antifungal polyene antibiotics nystatin was tested in a clinical trial to describe pharmacokinetics and safety after repeated administration of Nystatin “Lederle” sterile powder in healthy volunteers. To monitor the nystatin concentration–time profile in plasma we developed a sensitive method in the range of 1–100 ng/ml based on liquid chromatography coupled with tandem mass spectrometry. The target substance was separated from the biological matrix on C18 solid-phase extraction cartridges with methanol. The Chromatography was performed isocratically using a reversed phase Caltrex Resorcinearene column. The mobile phase consisted of 5 mM ammonium formate buffer and acetonitrile (40:60, v/v). The mass spectrometer works with electrospray ionization in its positive selected ion monitoring (SIM) mode using the respective MH+ ions, m/z 926.6 for nystatin and m/z 924.4 for amphotericin B as internal standard. The method validation was performed according to the demands and international criteria for validation of bioanalytical methods and was successfully applied to the quantification of nystatin in human plasma in the pharmacokinetic trial.