Rapid quantitative bioanalysis using LC-HRIM-QTOFMS with flexible sample delivery and variable traveling wave profiles

Incorporating an additional high-resolution ion mobility (HRIM) dimension can increase throughput and deliver results with high sensitivity and minimal ion suppression for bioanalytical LC-MS applications. However, the lengthy HRIM separation step conventionally comes at the cost of less data points per LC peak and a reduced working range, compromising quantification goals. This work addresses these challenges by using an alternative method for sample delivery and HRIM traveling wave profiles to establish methods for rapid quantification of small molecules and metabolites in complex samples. New HRIM-QTOFMS workflows for rapid, quantitative methods with sufficient sampling of LC peaks to harness high-resolution IM separation and obtain high quality analytical data were developed. Variable traveling wave profiles, which vary the applied amplitude and frequency over an HRIM scan, were used to increase the sampling of LC peaks to >15 points per peak. By optimizing LC sample introduction with feed injection, peak areas were improved and the total analysis times could be further reduced to < 5 min per sample using short LC columns (3 to 5 cm). This allowed riboflavin produced by three different engineered strains of the yeast Komagataella phaffii to be quantified in supernatant and cell lysates in 5 min per sample, along with other flavin nucleotides. Additionally, phytosiderophores exuded by graminaceous plants (barley and sorghum) were identified and quantified in agreement with a validated LC-MS/MS method, and with an analysis time of 3 min per sample. This study presents a new combination of rapid LC and HRIM separation dimensions with integrated feed injection mode for quantitative bioanalytical applications. We demonstrate that high-throughput methods for routine analysis of small molecules in biological samples can be realized by using HRIM traveling wave profiles to boost sampling across LC peaks and through post-HRIM fragmentation for the quantification of isomers. • Rapid, quantitative LC-HRIM-MS methods for two applications were developed • High IM resolving power (> 200) and feed injection for LC are key for fast methods • Variable traveling wave profiles for HRIM boosts LC sampling rates • HRIM-filtered EICs resolve selective fragments without isolation of precursors • Quantified flavins from bioproduction and eight phytosiderophores in plant samples