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Abstract

The toxicity, biological availability and mobility of aluminium (Al) depend on its chemical form. In order
to understand toxicity and transport of Al in human body it is important to identify and quantify Al species
present in human serum. This led to the development of sensitive and selective analytical methods for
reliable speciation of Al in serum. The aim of my PhD research work was the development of analytical
methods for the identification and quantification of high molecular mass (HMM-Al) and low molecular
mass (LMM-Al) Al compounds in human serum. For the separation of above-mentioned Al complexes
different anion-exchange monolithic and particled packed supports and size exclusion columns were used.
Concentration of Al in separated species was determined on-line by inductively coupled plasma mass
spectrometry (ICPMS). The characterisation of ligands which bind Al was performed by electrospray mass
spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Speciation of Al in spiked human serum (up to 300 ng mL-1 of Al) was performed using anion-exchange
monolithic separation support based on Convective interaction media (CIM®) in combination with size
exclusion chromatography (SEC) and ICPMS. The separation of proteins was performed on a weak anionexchange
CIM® diethylamine (DEAE) monolithic disk. It was experimentally proven that 90 ± 3 % of Al in
spiked serum investigated was eluted under the transferrin (Tf) peak. The proposed speciation procedure
removes LMM-Al species and enables reliable determination of the concentration and composition of Al
bound to proteins when the concentration of Al in serum is higher than 5 ng mL-1. In comparison to
chromatographic columns, the CIM® disks enable faster separation and simpler manipulation during
cleaning procedure. The developed analytical procedure enables reliable determination of the concentration
and the composition of HMM-Al species in spiked human serum.
A new analytical procedure using a CIM® monolithic column was developed for speciation of Al in
human serum at normal concentration levels. The separation of proteins was performed on a weak anionexchange
CIM® DEAE monolithic column. Separated Al species were detected on-line by ICPMS. It was
experimentally found that 91 ± 7 % of Al in human serum was bound Tf. Tf was identified not only on the
basis of the retention volume but also by acquity ultra performance liquid chromatography - electrospray
ionization mass spectrometry (UPLC-ESI-MS) and SDS-PAGE. The problem of extraneous contamination
with Al was successfully overcome by using efficient cleaning procedures of eluents and chromatographic
supports. The efficient cleaning was of paramount importance to perform Al speciation at extremely low
concentration levels. The repeatability of measurement tested for six consecutive separations of unspiked
serum was ± 8.6 %. The limits of detection and quantification (based on 3s and 10s of the blank) were 0.15
and 0.49 ng mL-1 of Al bound to Tf, respectively. To the best of our knowledge, this is the first report on
quantitative and reliable speciation of Al in human serum at normal concentration levels.
A new analytical procedure for the efficient, reliable and fast separation of proteins from LMM
compounds in serum was developed. A HiTrap desalting SEC column enables separation of HMM from
LMM species present in serum in 10 min. The Tris-hydrochloric acid buffer (pH= 7.4) was used as eluent.
In the first 5 min HMM-Al compounds were eluted followed by the elution LMM-Al species from 5 to 10
min. HMM and LMM species were collected in 5 mL fractions. The speciation of protein peak was
performed by anion-exchange monolithic disk coupled to ICPMS, while the LMM fraction was injected
onto the anion-exchange fast protein liquid chromatography column (FPLC) connected to ICPMS. It was
experimentally confirmed that 93 ± 4 % of Al is bound to Tf and that the remaining Al corresponded to
LMM-Al compounds. Citrate, one of the major LMM-Al species in serum was identified in the LMM
fraction by UPLC-ESI-MS. The rapidness and reliability of the developed analytical procedure represent a
promising tools for investigations of kinetics of Al binding to Tf.
The new-developed analytical procedures for speciation of HMM and LMM Al species in serum in
combination with other experimental and theoretical techniques, could help to explain the role, distribution
and toxicity of Al present in the human body.