Determination of high molecular mass Al species in serum and spent CAPD fluids of dialysis patients combining SEC and anion-exchange FPLC with ETAAS detection

Abstract

Speciation of high molecular mass Al compounds (HMM-Al) in human serum is commonly performed by size-exclusion or ion-exchange chromatography. In general, size-exclusion chromatographic (SEC) columns are not able to completely separate transferrin from albumin. Fast protein liquid chromatography (FPLC), using an anion-exchange column, enables separation of transferrin from albumin. However, the possibility exists of coelution of LMM–Al species (e.g., citrate, phosphate) with HMM–Al species. In this case the concentration of HMM–Al species could be overestimated. It is therefore important to remove LMM–Al species before anion-exchange FPLC separation. For this purpose a novel analytical approach was developed for speciation of HMM–Al species in human serum by combination of SEC (Superdex 75 HR 10/30) and anion-exchange FPLC (Mono Q HR 5/5 anion-exchange FPLC) with UV detection at 278 nm. 1 cm³ of serum was injected onto the SEC column. Isocratic elution using 0.05 mol dm⁻³ TRIS-HCl + 0.03 mol dm⁻³ NaHCO₃ was applied. It was experimentally proved that proteins were eluted in a 5 cm³ peak that was collected into a polyethylene cup. A 0.5 cm³ aliquot of the sample was then injected onto the anion-exchange FPLC column. The separation of serum proteins was obtained by applying linear gradient elution from 100% buffer A (0.05 mol dm⁻³ TRIS-HCl + 0.03 mol dm⁻³ NaHCO₃) to 100% buffer B (A + 0.25 mol dm⁻³ NaCl). Well-resolved protein peaks were obtained. 0.25 cm³ fractions were collected during the chromatographic run and Al determined by ETAAS. It was experimentally proved that 90 ± 5% of Al in spiked serum from a dialysis patient was eluted under the transferrin peak which was identified, not only on the basis of the retention volume, but also by the SDS-PAGE electrophoresis. The same procedure may be applied for speciation of Al in spent CAPD fluids of dialysis patients if the concentration of Al is higher than10 ng cm⁻³. The proposed speciation procedure removes LMM–Al species and enables reliable determination of the concentration and composition of Al bound to proteins by anion-exchange FPLC-ETAAS.