A strategy for absolute quantitation of isomers using high performance liquid chromatography-ion mobility mass spectrometry and material balance principle

Accurate characterization of isomers is a quite challenge and time-consuming work. A major bottleneck is the lack of relevant reference standards. In this work, we conducted a proof-of-concept study to develop a standard-free method for absolute quantitation of isomers by using high performance liquid chromatography-ion mobility mass spectrometry and material balance principle. The isomer structures were characterized by matching the rank order of experimental collision cross sections (CCSs) to that of theoretical CCSs. Then a time-dependent hydrolysis protocol derived from material balance equation was used for the calculation of the relative correction factor (F). Finally, the multi-level normalized matrix approach was developed to absolutely and precisely quantitate isomeric compounds. To assess this method, quercetin-liver microsome reaction pool was applied. With the developed method, four isomeric metabolites of quercetin were accurately analyzed, and metabolic profiling of quercetin isomeric metabolites was firstly reported with the absolute quantitation results. This is an accurate yet simple method for the absolute quantitation of positional isomers of interest.