Determination of O‐(chloroacetylcarbamoyl)fumagillol (TNP‐470; AGM‐1470) and two metabolites in plasma by high‐performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization

Abstract High performance liquid chromatography/mass spectrometry using a heat nebulizer atmospheric pressure chemical ionization interface was investigated as a sensitive and selective technique for the chromatographic separation and quantitation of the anti‐angiogenic drug TNP‐470 and its polar metabolites MII and MIV. Upon extraction from plasma, the analytes were measured using selected‐ion monitoring in the positive ionization mode and single quadrapole mass spectrometric detection. A trideuterated 13C‐labeled analog of TNP‐470 was used as an internal standard. Two plasma extraction procedures were evaluated. An acetonitrile precipitation method resulted in a recovery of 65.9% and 44.4% for MIV and TNP‐470, respectively, and a limit of quantitation of 2.5 ng ml−1 with a limit of detection of 0.63 ng ml−1 for both analytes. In contrast, a solid‐phase extraction method led to a higher recovery of 92.9% and 111.0% for MIV and TNP‐470, respectively, and a limit of quantitation of 0.63 ng ml−1 with a limit of detection of 0.16 ng ml−1 for both analytes. Intra‐day and inter‐day precisions were <10% and <18% for both analytes. The solid‐phase extraction, by decreasing plasma interferences present within the column capacity, also allowed the detection and quantitation of MII, a highly polar metabolite of TNP‐470. The recovery of MII in plasma was 77.4% with a limit of quantitation and detection of 2.5 and 1.25 ng ml−1, respectively. Intra‐day and inter‐day precisions were 6.19% and 2.99%. Quantitative analysis of plasma samples from a patient receiving 10 mg m−2 of TNP‐470 by a 1 h intravenous infusion is reported.