Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 microL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.