Enhanced Characterization of Metabolites using Microflow UPLC-MS and Collision Cross Section Measurements

Metabolites of Midazolam were produced from incubations in mouse, rat and human hepatocytes. The assay is based on the direct analysis of the extracts produced by protein precipitation and subsequent analysis using a new microfluidic device combining an ultra high pressure liquid chromatography (UPLC) analytical column with a nano-flow electrospray source. The ceramic device contains a 5 cm analytical column of 150 µm internal diameter along with the ionization emitter in one interchangeable unit. All microfluidic, gas and electrical connections are automatically engaged when the ceramic microfluidic device is inserted into the source enclosure. All experiments were performed on a Waters Synapt G2-Si Q-tof mass spectrometer operating in MSE data acquisition mode. Detected metabolites of interest were subject to separation by ion mobility spectrometry and the CCS calculated for all the isomeric forms detected.