Simultaneous Quantitative Analysis of EO9 (Apaziquone) and its Conversion Products EO5a and EO9-Cl in Human and Dog Urine by High-Performance Liquid Chromatography Coupled with Electrospray Tandem Mass Spectrometry

A sensitive and specific LC-MS/MS assay for the quantitative determination of anticancer agent EO9 and its conversion products EO5a and EO9-Cl in human and dog urine is presented. A 20-μL-urine aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and diluted with 180 �L 0.1 M ammonium acetate - methanol (7:3, v/v). Next, 25 μL-volumes were injected onto the HPLC system. Separation was achieved on a 150 � 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide - methanol (gradient system)). Detection was executed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range from 0.1 �g/mL to 50 � g/mL for EO9, from 0.2 � g/mL to 50 � g/mL for EO5a and 0.1 � g/mL to 4 � g/mL for EO9-Cl using 20 μL of stabilized urine samples. Validation results demonstrate that EO9, EO5a and EO9-Cl concentrations can be accurately and precisely quantified in human and dog urine. This assay is used now to support pre-clinical and clinical pharma- cologic studies with intravesically administered EO9.