Comparison of liquid chromatography and supercritical luidchromatography coupled to compact single quadrupole massspectrometer for targeted in vitro metabolism assay

The goal of this study was to evaluate the combination of powerful chromatographic methods and com-pact single quadrupole MS device for simple in vitro cytochrome P450 (CYP) inhibition assay, insteadof more expensive triple quadrupole MS/MS detectors. For this purpose, two modern chromatographicapproaches (ultra-high pressure liquid chromatography (UHPLC) and ultra-high performance supercrit-ical fluid chromatography (UHPSFC)) were tested in combination with simple MS detector. To show theapplicability for an in vitro CYP-mediated metabolism assay using the cocktail approach, a method wasfirst developed in UHPLC–MS to separate a mixture of 8 probe substrates and 8 CYP-specific metabolites.A screening procedure was initially applied to determine the best combination of a column, an organicmodifier and a mobile-phase pH, followed by fine tuning of the conditions (i.e., gradient profile, temper-ature and pH) using HPLC modelling software. A similar sequential method development procedure wasalso evaluated for UHPSFC–MS. For method development, where peak tracking is necessary, the use ofsingle quadrupole MS was found to be extremely valuable for following the investigated analytes. Ulti-mately, a baseline separation of the 16 compounds was achieved in both UHPLC–MS and UHPSFC–MSwith an analysis time of less than 7 min. In a second series of experiments, sensitivity was evaluated,and LOQ values were between 2 and 100 ng/mL in UHPLC–MS, while they ranged from 2 to 200 ng/mLin UHPSFC–MS. Based on the concentrations employed for the current in vitro phase I metabolism assay,these LOQ values were appropriate for this type of application. Finally, the two analytical methods wereapplied to in vitro CYP-dependent metabolism testing. Two well-known phytochemical inhibitors, yohim-bine and resveratrol, were investigated, and reliable conclusions were drawn from these experimentswith both UHPLC–MS and UHPSFC–MS. At the end, the proposed strategy of optimized chromatographycombined with simple MS device has been shown to potentially compete with the widely used combi-nation of generic chromatography and highly selective MS/MS device for simple in vitro CYP inhibitionassays. In addition, our analytical method may be easier to use in a routine environment; the instrumentcost is significantly reduced and the two developed methods fit for purpose