Enhanced resolution triple-quadrupole mass spectrometry for ultra-sensitive and quantitative analysis of the investigational anticancer agent EO9 (apaziquone) and its metabolite EO5a in human and dog plasma to support (pre)-clinical studies of EOquin given intravesically

A highly sensitive and selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to quantify the experimental anticancer agent EO9 and its metabolite EO5a in biological matrices. A 200-microL aliquot of human/dog plasma was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 mL of ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate/methanol (7:3, v/v) and 20-microL volumes were injected onto the LC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide/methanol (gradient system)). The detection was performed by a Finnigan TSQ Quantum Ultra equipped with an electrospray ionization source operated in positive mode and enhanced mass resolution capability. It demonstrated improved sensitivity with a factor 10-20 for EO9 and EO5a over a 3-decades dynamic range, with acceptable accuracy and precision, when compared with the previously described assay for EO9 and EO5a, developed by our group, using an API 2000. The assay quantifies a range from 0.5 to 500 ng/mL for EO9 and EO5a using 200-microL human plasma and dog samples. The described mass resolution method was successfully applied for the evaluation of the pharmacokinetic profile of EO9 and its metabolite EO5a in human and dog plasma.