Quantitation of TNP-470 and its metabolites in human plasma: sample handling, assay performance and stability

A selective and sensitive assay for the determination of TNP-470 and two of its metabolites, AGM-1883 and M-II, in human plasma was developed. The assay involved liquid-liquid extraction followed by analysis using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Because TNP-470 is most stable in a pH of 4-5, an acidification procedure was utilized to prevent degradation of TNP-470 during sample collection which involved acidifying the whole blood sample collected with 5 mg of citric acid per ml of blood. Liquid-liquid extraction using an organic solvent mixture was chosen over solid-phase extraction to minimize the degradation of TNP-470 during solvent evaporation.