Determination of hexamethylene bisacetamide, an antineoplastic compound, in mouse and human plasma by LC-MS/MS

Hexamethylene bisacetamide (HMBA) is a polar compound which has recently been discovered to have antineoplastic activity by up-regulating the expression of an endogenous antiproliferative breast cancer protein, HEXIM1 (hexamethylene bisacetamide inducible protein 1) in vivo. HMBA has been shown in the past to induce terminal differentiation in multiple leukemia types at a concentration of 2-5 mM, but its phase I and II clinical trials were largely unsuccessful due to serious side effects (notably, thrombocytopenia) with dose escalation. In this work, a sensitive and simple LC-MS/MS method for direct determination of HMBA in mouse and human plasma is described. Plasma samples were prepared by deproteinization with acetonitrile. Separation was achieved on a Waters Atlantis® T3 (2.1 mm × 50 mm, 3 μm) column with retention times of 2.2 and 3.7 min for HMBA and 7MBA (internal standard), respectively. The quantitation was carried out by tandem mass spectrometry using positive MRM mode. The linear range of the method was 0.500-100 ng/mL in both mouse and human plasma with injection volume of 5 μL. This method has been validated in accordance with the US Food and Drug Administration (FDA) guidelines for bioanalytical method development and applied to the determination of HMBA concentrations in FVB mice over time after a single dose of HMBA in saline (0.9% NaCl) at 10 mg/kg.