Increasing high-throughput Discovery bioanalysis using automated selected reaction monitoring compound optimization, ultra-high-pressure liquid chromatography, and single-step sample preparation workflows

摘要

QuickQuan is an integrated software package for Thermo Scientific triple quadrupole mass spec-trometers that allows users to automate routine operations ranging from method development to dataprocessing. QuickQuan automated optimization of compound-selected reaction monitoring (SRM)transitions by evaluating both positive and negative polarities during an infusion. Whichever modeproduces the most intense Q1 scan is then carried to product ion spectra. QuickQuan then writesthese SRM methods to a shared network database. The total volume of compound needed is 100 μLinfused over approximately 1.6 min. The auto-optimization is carried out in 96-well plates and doesnot require an operator present. The SRM database was shared between two identical TSQ Quantummass spectrometers. For data acquisition, QuickQuan automatically created a sequence file completewith a data processing method pre-populated with compound IDs and corresponding SRM tran-sitions. To increase throughput we coupled each Finnigan Quantum with ultra-high-pressure liquidchromatography (uHPLC) accomplished using 4× Ultra Flux quaternary pumps that were designedto handle pressures up to 15 000 psi. The total run time for all analyses was 1.2 min using BEH 1.7 p.mparticle C18 columns. Further time reductions were realized with sample preparation accomplishedusing Strata Impact protein precipitation plates which provided an in-well protein crash and 0.20micron filtering in a one-step process. Pharmacokinetic data turnaround time was significantlyimproved by combining these three techniques of automated method development with the speedefficiency of uHPLC and a single step in well sample preparation.