Automated isolation of high-purity plasma albumin for isotope ratio measurements

Measurement of the incorporation of labeled amino acids in plasma albumin, isolated from plasma sampled at different time points after infusion start is a well-known technique to study human albumin synthesis. Unfortunately, no chromatographic method has been described yet, enabling the automated is olation of high-purity albumin from large numbers of plasma samples as is r equired to study the kinetics of this process. Therefore, we developed a fa st protein liquid chromatographic method, capable of processing 200 mu l am ounts of plasma in 74 min (injection to injection). The system can run unat tended as the FPLC system is connected to a sample processor equipped with a polyether ether ketone (PEEK) sample loop and a cooled sample tray. Album in isolation was divided into three steps. First, plasma samples were injec ted onto a I-mi Blue Sepharose HiTrap affinity column, equilibrated with 50 mmol/l phosphate buffer (pH 7.0). After elution of non-binding protein, sw itching the solvent to phosphate buffer with 1.5 mol/l sodium chloride elut ed albumin. The resulting albumin fraction was desalted on-line by directin g it through two consecutive HiTrap 5-ml desalting columns, whereafter it w as retained in the system within a 5-ml PTFE loop, connected to a motor val ve. After switching this valve, thus bypassing the sample loop, the phospha te buffers were changed automatically to Tris buffers. Final purification i nvolved elution of the captured fraction over a 1-ml ion-exchange Resource Q column, using a sodium chloride gradient, ranging from 0 to 0.5 mol/l in Tris buffer (20 mmol/l, pH 7.5). A more than 99% purity of the final albumi n fraction was confirmed by capillary electrophoresis. (C) 1999 Elsevier Sc ience B.V. All rights reserved.