Chemical derivatization as a novel strategy for selective and sensitive determination of intracellular di-and triphosphate anabolites in peripheral blood mononuclear cells.

Quantitation of intracellular tri-phosphate anabolite, GSK1-TP, was required to understand drug efficacy of a proprietary molecule, GSK1; while quantitation of the di-phosphate, GSK1-DP, provided an indicator of potential GSK1-TP instability during sample processing and storage. A novel derivatization approach with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hexylamine was developed to mitigate the challenges associated with the analysis of GSK1-DP and GSK1-TP, rendering them more amendable to reversed-phase LC-MS/MS detection. Extensive effort was spent to minimize the analyte loss and cell counts variability during peripheral blood mononuclear cells (PBMC) collection and best practices were thoroughly discussed. A solution of 30/70/2 (v/v/v) RPMI-1640/methanol/trichloroacetic acid proved to be an effective method of analyte stabilization and recovery. Methods were developed for simultaneous analysis of GSK1-DP and GSK1-TP with a limit of quantitation of 2.0 ng/mL in dog and rat PBMC lysate, with a subsequent improvement to 0.1 ng/mL for the analysis of GSK1-TP in human PBMC lysate. All three assays were found to be robust over the PBMC concentration range of 1-25 × 10 lysed cells/mL. This novel methodology could alleviate some challenges associated with the bioanalysis of intracellular phosphorylated anabolites such as PBMC collection variability, analyte instability, poor chromatographic retention, high carryover, matrix effect and insufficient assay sensitivity.