Accelerator mass spectrometry in the attomolar concentration range for 14C-labeled biologically active compounds in complex matrixes

Abstract

Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical method suitable for detection of sub-nanomolar concentrations of labeled biological substances such as pharmaceutical drugs in body fluids. A limiting factor in extending the concentration measurements to the sub-picomolar range is the natural ¹⁴C content in living tissues. This can be circumvented by separating the labeled drug from the tissue matrix with, for example, liquid chromatography. The analysis of drugs and their metabolites or endogenous compounds in biological fluids by liquid chromatography is usually complicated and the sample preparation step remains the most serious problem both with regard to losses and degradation of the analyte, and also automation of the analysis. In this article a method for detection and quantification of extremely low concentrations of ¹⁴C-labeled biomolecules in biological fluids by AMS is described. The use of a column switched chromatographic system incorporating a restricted-access media (RAM) column allowed the direct injection of untreated human plasma samples, which reduces the total time of analysis and makes automation of the sample preparation step possible. As the separated total drug amount is in the attogram to femtogram region, it is not possible to use a standard AMS sample preparation method, where mg sizes are required. We have utilized a sensitive carbon carrier method where a ¹⁴C-deficient compound is added to the HPLC fractions and the composite sample is prepared and analysed by AMS. The method shows remarkable sensitivity, low background values and good linearity, allowing the detection and quantification of a pharmaceutical drug in human plasma in the low femtomolar and down to the attomolar concentration range.