High-throughput sample preparation procedures for the quantitation of a new bone integrin ανβ3 antagonist in human plasma and urine using liquid chromatography–tandem mass spectrometry

High throughput LC–MS/MS assays to quantitate a new ανβ3 bone integrin antagonist (I) in human plasma and urine have been developed using instruments programmed to automate sample preparation procedures. Packard liquid handling system-MultiPROBE® II EX was programmed for preparing calibration standards in control plasma and urine, acidifying all standards, quality control (QC), and clinical samples with necessary dilutions, and adding the internal standard to the acidified samples. TOMTEC Quadra 96™ was programmed to perform the solid phase extraction (SPE) process on a 3 M 96-well mixed phase cation standard density (MPC-SD) plate to isolate the analytes from the sample matrix. The extract collected from both types of matrices was directly injected into reversed-phase LC–MS/MS system with a Turbo Ion Spray (TIS) interface in the positive ionization mode. The plasma and urine assays have the calibration range of 0.5–1500 and 2–6000 ng/mL, respectively. Validation of the automated and the manual plasma assays showed that application of MultiPROBE II to sample preparation gave comparable accuracy and precision. Overall, the automated approaches with minimum manual intervention enhanced the throughput of sample preparation.