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This work represents the first reporting of a comprehensive bioanalytical GLP methodology detailing the mass spectrometric quantitation of PF-05212384 dosed as a targeted polymeric encapsulated nanoparticle (PF-07034663) to monkeys. Polymeric nanoparticles are a type of drug formulation that enables the sustained release of an active therapeutic agent (payload) for targeted delivery to specific sites of action such as cancer cells. Through the careful design and engineering of the nanoparticle formulation, it is possible to improve the biodistribution and safety of a given therapeutic payload in circulation. However, the bioanalysis of nanoparticles is challenging due to the complexity of the nanoparticle drug formulation itself and the number of pharmacokinetic end points needed to characterize the in vivo exposure of the nanoparticles. Gedatolisib, also known as PF-05212384, was reformulated as an encapsulated targeted polymeric nanoparticle. The bioanalytical assays were validated to quantitate both total and released PF-05212384 derived from the encapsulated nanoparticle (PF-07034663). Assay performance calculated from quality control samples in three batch runs demonstrated intraday precision and accuracy within 10.3 and 12.2%, respectively, and interday precision and accuracy within 9.1 and 8.5%, respectively. This method leveraged automation to ease the burden of a laborious and complicated sample pretreatment and extraction procedure. The automated method was used to support a preclinical safety study in monkeys in which both released and total PF-05212384 concentrations were determined in over 1600 monkey plasma study samples via LC-MS/MS.

Hydrophilic retention coefficients for 17 peptides were calculated based on retention coefficients previously published for TSKgel silica‐60 and were compared with the experimental elution profile on a Waters Atlantis HILIC silica column using TFA and methanesulfonic acid (MSA) as ion‐pairing reagents. Relative peptide retention could be accurately determined with both counter‐ions. Peptide retention and chromatographic behavior were influenced by the percent acid modifier used with increases in both retention and peak symmetry observed at increasing modifier concentrations. The enhancement of net peptide polarity through MSA pairing shifted retention out by nearly five‐fold for the earliest eluting peptide, compared with TFA. Despite improvements in retention and efficiency (Neff) for MSA over TFA, a consistent reduction in calculated selectivity (α) was observed. This result is believed to be attributed to the stronger polar contribution of MSA masking and diminishing the underlying influence of the amino acid residues of each associated peptide. Finally, post‐column infusion of propionic acid and acetic acid was evaluated for their potential to recover signal intensity for TFA and MSA counter‐ions for LC‐ESI‐MS applications. Acetic acid generally yielded more substantial signal improvements over propionic acid on the TFA system while minimal benefits and some further reductions were noted with MSA.