Stability assessment of different chelating moieties used for elemental labeling of bio-molecules

Abstract

Integrating elemental labeling in quantitative LC-ICP-MS based bio-analysis requires fundamental experiments concerning the stability of complexes during analysis. In a competitive approach complex stability of the chelating moieties 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraaceticacid (DOTA), 1,4,7-triazacyclononane-N,N′,N′′-triacetic acid (NOTA) and diethylenetriaminepentaacetic dianhydride (DTPA) in combination with 11 different lanthanides was investigated under typical chromatographic conditions. Measurements were carried out via LC-ICP-QMS using a novel mixed mode separation method. The influence of chromatographic separation, pH and temperature on complex stability constants was assessed regarding further applications of multiplexing in bio-analytical assays. The limit of detection (LOD) for LC-ICP-QMS was 0.03 nM for all investigated Tm complexes (0.15 fmol absolute). Quantification of the complexes was performed via external, flow injection based calibration. For all investigated complexes the stability was significantly decreased by the chromatographic conditions. Moreover, complexation by DOTA revealed two different signals suggesting the presence of a stable intermediate product. Ln³⁺–DOTA and Ln³⁺–NOTA complexes provided high stability at 5 °C and 37 °C over a time of 12 hours, whereas Ln³⁺–DTPA complexes showed significant degradation at 37 °C.