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 human serum from eight healthy volunteers in order to investigate the individual variability in the percentage and composition of LMW-Al species. Spiked samples (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-exchange fast protein liquid chromatography (FPLC) column and aqueous 4 mol dm(-) (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 chromatographic run and Al was determined 'off-line' by electrothermal atomic absorption spectrometry (ETAAS). The characterisation of LMW-Al species in spiked serum was performed not only on the basis of the retention time (ETAAS detection), but also by electrospray (ES)-MS-MS analysis. A tandem quadrupole mass spectrometer equipped with a Z spray ion source as LC-MS interface was used for the identification of LMW ligands eluted under the chromatographic 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-MS-MS analysis, it was found that the main LMW-Al species present in serum were Al-citrate, Al-phosphate and ternary Al-citrate-phosphate complexes. The distribution of these species varied among particular individuals. In some 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 complexes 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 be 8%.