Design and development of a two-dimensional system based on hydrophilic and reversed-phase liquid chromatography with on-line sample treatment for the simultaneous separation of excreted xenobiotics and endogenous metabolites in urine

Abstract

In the present work we describe a two-dimensional liquid chromatographic system (2D-LC) with detection by mass spectrometry (MS) for the simultaneous separation of endogenous metabolites of clinical interest and excreted xenobiotics deriving from exposure to toxic compounds. The 2D-LC system involves two orthogonal chromatographic modes, hydrophilic interaction liquid chromatography (HILIC) to separate polar endogenous metabolites and reversed-phase (RP) chromatography to separate excreted xenobiotics of low and intermediate polarity. Additionally, the present proposal has the novelty of incorporating an on-line sample treatment based on the use of restricted access materials (RAMs), which permits the direct injection of urine samples into the system. The work is focused on the instrumental coupling, studying all possible options and attempting to circumvent the problems of solvent incompatibility between the RAM device and the two chromatographic columns, HILIC and RP. The instrumental configuration developed, RAM-HILIC-RPLC-MS/MS, allows the simultaneous assessment of urinary metabolites of clinical interest and excreted compounds derived from exposure to toxic agents with minimal sample manipulation. Thus, it may be of interest in areas such as occupational and environmental toxicology in order to explore the possible relationship between the two types of compounds.

Keywords: 2D liquid chromatography; HILIC; biomarkers; on-line treatment; urine.