Selective drug metabolite trace analysis by very high-volume injections and heartcut two-dimensional (2D)-ultrahigh performance liquid chromatography (UHPLC).

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.