Comparison of sub-2-μm particle columns for fast metabolite ID

The use of sub-2-mu m particle columns for fast high throughput metabolite ID applications was investigated. Three LC-MS methods based on different sub-2-mu m particle size columns using the same analytical 3 min gradient were developed (Methods A, B, and C). Method A was comprised of a 1.8 mu m particle column coupled to an MS, methods B and C utilized a 1.7 mu m particle column (BEH 50 x 2.1 mm(2) id) and 1.8 pin particle column coupled to a Q-TOF MS. The precision and the separation efficiency of the methods was compared with repeated standard injections (N = 10) of reference compounds verapamil (VP), propranolol, and fluoxetine. Separation efficiency and MS/MS spectral quality were also evaluated for separation and detection of VP and its two major metabolites norverapamil (NVP) and O-demethylverapamil (ODMVP) in human-liver microsomal incubates. Results show that 1.8 mu m particle columns show similar performance for separation of VP and its major metabolites and comparable spectral quality in MS' mode of the Q-TOF instrument compared to 1.7 mu m particle columns. Additionally, the study also confirmed that sub-2-mu m particle size columns can be operated with standard analytical HPLC but that performance is maximized by integrating column in UPLC method with reduced void volumes. All the methods are suitable for the determination of major metabolites for compounds with high metabolic turnover. The high throughput metabolite profile analysis using 384-well plate format of up to 48 compounds in incubates of humanliver microsomes was discussed.