Application of acoustic ejection mass spectrometry for plasma protein binding assay using flux dialysis

Flux dialysis, a superior method for plasma protein binding (PPB) measurement of compounds with challenging properties, has limitations in early-stage drug discovery due to multi-timepoint sampling and prolonged testing cycles. This study combines flux dialysis with acoustic ejection mass spectrometry (AEMS) to develop an innovative method that accelerates analytical throughput in PPB assays during drug discovery and demonstrates its application for the rapid and precise determination of the unbound fraction (f_u) in plasma. Herein, we validated this approach using 10 commercially available compounds with known f_u values-imipramine, indomethacin, itraconazole, lapatinib, nicardipine, warfarin, chlorpromazine, rivastigmine, zonisamide, and ritonavir-with a wide f_u range covering from very high binding (f_u ≤ 0.01) to low binding (f_u > 0.10) in human plasma. By leveraging the advantages of chromatography-free analysis and nanoliter droplet ejection mode, AEMS achieves a speed of 3 seconds per sample using only 30 nL of sample volume. Our results showed that the f_u values measured correlate strongly (R² > 0.96) with those measured by liquid chromatography-tandem mass spectrometry. Additionally, f_u values by AEMS correlate highly (R² > 0.95) with those reported in the literature. In conclusion, this method presents a high-throughput, accurate, and efficient solution for PPB assays, improving speed by 25-fold compared to the liquid chromatography-tandem mass spectrometry method. SIGNIFICANCE STATEMENT: This study bridges the gap between flux dialysis and acoustic ejection mass spectrometry by creating a synergistic analytical framework for plasma protein binding assays, addressing limitations of both methods and enabling high-throughput applications with improved accuracy and efficiency. The combination of flux dialysis and acoustic ejection mass spectrometry will make a positive contribution to the development of high-throughput in vitro absorption, distribution, metabolism and excretion assays in drug discovery.