Investigation of low molecular weight Al complexes in human serum by fast protein liquid chromatography (FPLC)-ETAAS and electrospray (ES)-MS-MS techniques

Speciation of low molecular weight (LMW) Al complexes was performed in huma n serum from eight healthy volunteers in order to investigate the individua l variability in the percentage and composition of LMW-Al species. Spiked s amples (100-120 ng cm(-3) Al3+) were microultrafiltered through a membrane filter (cut-off 30 000 Da) to separate Al bound to transferrin from LMW-Al complexes. A 0.5 cm(3) volume of the filtrate was injected onto an anion-ex change fast protein liquid chromatography (FPLC) column and aqueous 4 mol d m(-) (3) NH4NO3 linear gradient elution was applied for 10 min to separate LMW-Al complexes. Fractions of 0.2 cm(3) were collected throughout the chro matographic run and Al was determined 'off-line' by electrothermal atomic a bsorption spectrometry (ETAAS). The characterisation of LMW-Al species in s piked serum was performed not only on the basis of the retention time (ETAA S detection), but also by electrospray (ES)-MS-MS analysis. A tandem quadru pole mass spectrometer equipped with a Z spray ion source as LC-MS interfac e was used for the identification of LMW ligands eluted under the chromatog raphic peaks. It was found experimentally that the amount of LMW-Al species in spiked serum ranged from 14 to 55%. On the basis of FPLC-ETAAS and ES-M S-MS analysis, it was found that the main LMW-Al species present in serum w ere Al-citrate, Al-phosphate and ternary Al-citrate-phosphate complexes. Th e distribution of these species varied among particular individuals. In som e of them Al-citrate and Al-phosphate were the main LMW-Al species in serum , while in others the ternary Al-citrate-phosphate complex was also present . The serum of some other individuals did not contain Al-phosphate and the main LMW-Al species were either Al-citrate and Al-citrate-phosphate complex es or Al-citrate species alone. The limit of detection for the separated Al species on the FPLC column was 5.0 ng cm(-3), while the RSD was found to b e 8%.