Drug-intercalated nanoparticles are one pharmaceutical delivery modality being developed to target the release of small-molecule treatments to tumor tissues while minimizing the concentrations of free drug in the plasma. Characterizing the in vivo pharmacokinetics of these nanoparticles requires measurement of the released drug that is circulating in the plasma and the drug that remains bound within the particle. The development of bioanalytical assays is quite challenging as the drug plasma concentrations, by design, are very low while the concentrations of nanoparticles are orders of magnitude greater. Any disruption of the nanoparticles during sample processing will release the bound drug and completely mask the free drug concentrations. Therefore, assays must be able to quantitatively remove the nanoparticles to accurately measure free drug concentrations. In the system we present, quantitative removal of nanoparticles from the free drug is accomplished by binding the free drug to a Waters Oasis MCX SPE sorbent bed followed by extensive washing under mild conditions. Free drug concentrations are determined by elution of the captured drug from the SPE bed. Total drug in the system is determined from a second plasma aliquot by the disruption of the nanoparticles and precipitation of plasma proteins with acetonitrile. All concentrations are determined using liquid-chromatography/mass-spectrometry. Assays were validated according to FDA guidances. A linear calibration range of 10-1000 ng/mL was achieved when processing 25-microliters of plasma sample. The released drug in the plasma is free to bind to the SPE cation-exchange bed while the bound drug is shielded within the nanoparticle and removed in the bed washes. The released small molecule method meets the regulatory agency’s criteria for accuracy and precision. Analysis of spiked quality control samples produced intra-day precisions (%CV) and accuracies (%RE) in the ranges of 1.6-10.3% and -2.7-11.5%, respectively; and inter-day precisions and accuracies in the ranges of 3.8-7.4% and -0.3-8.5%, respectively. The validated assay was successfully applied for the quantitation of released drug in plasma following the dosing of nanoparticles to monkeys in a GLP toxicity study. The reproducibility of the analysis was demonstrated by the reanalysis of 238, (approximately 40%) of the study samples where 76.5% met an acceptance criterion of ±30% of the concentration determined in the original analysis.

Development, validation and application of a cation-exchange, solid-phase extraction for the determination of nanoparticle-released drug concentrations in plasma

256th National Meeting (2018)

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