Automated simultaneous isolation and quantitation of labeled amino acid fractions from plasma and tissue by ion-exchange chromatography

In order to trace metabolic pathways of amino acids in the body, a known labeled amount of an amino acid is infused. Dilution in the body pool is measured, using the specific activity and calculated by dividing the labeled amount of an amino acid (tracer) by its total pool (tracer + tracee). This paper describes a method, which combines fractionation and quantitation of multiple amino acids in one chromatographic run. To achieve this, we performed a classical amino acid ion-exchange separation on standard HPLC equipment. The column effluent was divided continuously into two solvent streams using a rapidly switching, pump controlled “split-valve”. The main part (90%) was directed to a computer controlled fraction collector, while the remaining 10% was mixed with o-phthaldialdehyde reagent after which fluorescence was measured. Using this system, 10–1000 μl of deproteinized plasma, representing a maximum of 50 nmol of each amino acid, could be fractionated and quantitated in the same chromatographic run. In addition to optimal counting efficiency of an off-line radioactivity counter, it enabled easy measurement of the specific activity of multiple amino acid tracers.