Investigation of high-throughput ultrafiltration for the determination of an unbound compound in human plasma using liquid chromatography and tandem mass spectrometry with electrospray ionization

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an αvβ3 bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen® filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1–500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.