High-throughput protein binding assay using cross-sample pooling in combination with high-resolution mass...

The fraction of unbound drugs (ƒ_u ) is a useful pharmacokinetic parameter in understanding drug disposition (Absorption, Distribution, Metabolism, Excretion), pharmacological activity and toxicity. Therefore, protein binding assays are frequently performed in drug development, creating a high demand for biological, experimental and analytical resources. Our work aims to increase binding assay throughput and comprehensiveness, while reducing biological and experimental consumption without compromising data quality by introducing cross-pooling and cassetting procedures, followed by a rapid and informative high-resolution mass spectrometry (HRMS) analysis.

Individual drugs were spiked into a test matrix and incubated in a rapid equilibrium dialysis device. After incubation, a cross-pooling procedure was performed, in which the samples of one drug were equalized with the complementary matrix provided from a different drug. The same drugs were also assayed with a conventional method, in which samples were equalized with the newly prepared complementary matrix. Cross-pooled samples were further cassetted to increase throughput. The samples were analyzed by high-performance liquid chromatography coupled with an Orbitrap HRMS, and the f_u values were calculated and compared between the cross-pooling and conventional sampling procedures.

Highly comparable human plasma f_u values of 27 drugs representing different chemical classes and wide-ranging f_u values were obtained by conventional and cross-pooling procedures, The tight correlation was further validated in other species (rat, mouse) and matrices (microsomes, brain). In addition, the cassetted samples showed highly consistent f_u values compared to their noncassetted counterparts. Moreover, HRMS analysis not only showed highly consistent and repeatable quantification results compared to the "gold standard" triple quadrupole (QqQ) analysis, but also demonstrated outstanding advantage over QqQ in enabling a high-throughput, informative and versatile analysis.

This work demonstrates that the cross-pooling procedure with further sample cassetting using HRMS is experimentally and analytically feasible to allow a higher throughput (increased by up to 8-fold), resource-effective (reducing matrix consumption by 50%, minimizing time spent on method development and platemap design), analytically dependable (accurate quantification), and versatile (metabolite elucidation and low recovery troubleshooting) analysis.