Quantitative determination of trace amounts of Al-citrate by anion-exchange FPLC-ETAAS

An anion-exchange fast protein liquid chromatographic-electrothermal atomic absorption spectrometric procedure (FPLC-ETAAS) was developed for determination of trace amounts of negatively charged Al-citrate in the pH range 3.5-8.0. Aqueous-4 mol dm(-3) NH(4)NO(3) linear gradient elution at a flow rate of 1 cm(3) min(-1) was applied for 10 min to separate Al-citrate on a FPLC Mono Q HR 5/5 column. The separated aluminium species were determined 'off line' by ETAAS in 0.5 cm(3) fractions. After separation the column was regenerated for 5 min with 4 mol dm(-3) NH(4)NO(3) and equilibrated with water. All reagents used in the separation procedure were cleaned with a silica based LiChrosorb RP-18 HPLC column to remove traces of aluminium. The main advantage of NH(4)NO(3) as eluent lies in its ability to decompose quantitatively in the graphite tube during the ashing step, which enables reproducible analysis of aluminium in the separated fractions. Using the procedure developed reproducible (RSD+/-2.0%) and quantitative determination of negatively charged Al-citrate at a retention time of 4.5 min was obtained. The LOD was found to be 2.0 ng cm(-3) of Al-citrate. The technique was successfully applied for the determination of Al-citrate in human serum. Spiked samples (50-150 ng Al(3+) cm(-3)) were microultrafiltered through a membrane filter (cut-off 30 000 Da) to separate aluminium bound to transferrin from low molecular weight aluminium complexes. It was found that 15-19% of aluminium in spiked samples from healthy volunteers passed through the membrane. By applying FPLC separation it was proved that all the aluminium in the filtrate corresponded to Al-citrate. The analytical technique developed enabled quantitative and reproducible determination (RSD+/-3.0%) of Al-citrate in spiked human serum at levels which could be found in patients undergoing long term haemodialysis.