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

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Flux dialysis, a superior method for plasma protein binding 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 plasma protein binding 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 ten 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 (R 2 >0.96) with those measured by LC-MS/MS. Additionally, f u values by AEMS correlate highly (R 2 >0.95) with those reported in literatures. In conclusion, this method presents a high-throughput, accurate, and efficient solution for plasma protein binding assays, improving speed by 25-fold compared to LC-MS/MS method.