The Open Chemical and Biomedical Methods Journal

2009, 2 : 1-12
Published online 2009 January 27. DOI: 10.2174/1875038900902010001
Publisher ID: TOCBMJ-2-1

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

Liia D. Vainchtein , Hilde Rosing , Dorla Mirejovsky , Van Huynh , Luigi Lenaz , Jan H.M. Schellens and Jos H. Beijnen
Astellas Pharma Europe B.V., Exploratory Development Department, Elisabethhof 1, 2353 EW Leiderdorp, P.O. Box 108, 2350 AC Leiderdorp, The Netherlands.

ABSTRACT

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 µL 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 µg/mL to 50 µg/mL for EO9, from 0.2 µg/mL to 50 µg/mL for EO5a and 0.1 µg/mL to 4 µg/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 pharmacologic studies with intravesically administered EO9.

Keywords:

Indoloquinones, bioreductive anticancer drugs, intravesical route, bladder cancer, DNA adducts..