Quantitative HPLC/ETAAS hybrid method with an on-line metal scavenger for studying the protein binding and speciation of aluminum and iron

Abstract

A quantitative metal-free high-performance liquid chromatographic/electrothermal atomic absorption spectrometric (HPLC/ETAAS) technique allowing an adequate separation of the proteins and of the inorganic/organic metal species of interest was developed. A silica-based scavenger was placed proximal to the injection valve retaining any remaining Al and Fe originating from buffer solutions and recipients. ETAAS instrumental conditions and matrix modifiers were carefully selected to eliminate interferences occurring due to the salt gradient elution. The postelution Al recovery (143-1428 micrograms L-1; 100-microL sample volume) was found to be 100.1 +/- 15.3% (N = 50) whereas that of Fe was 97.3 +/- 2.0% (N = 20; 442-2653 micrograms L-1). The protein recovery varied between 95 and 105%. The HPLC/ETAAS intraassay CV (N = 3) was 8.0% for Al (eluted amount of Al bound to transferrin (Tf), 7.3 ng) whereas that of Fe was 1.4% (eluted amount of Fe bound to Tf, 124.8 ng). The intraassay CV of the HPLC Tf determination was 1.6%. The proposed method is sensitive and accurate, allowing for the first time quantitative evaluation of the competition between Al and Fe for protein binding at clinical relevant concentrations. The technique will also be used for studying the protein binding of elements other than Al and Fe.

Metal speciation in biological fluids — a review

“…In recent years, a lot of studies have been carried out in order to develop appropriate interfaces between chromatographic instruments and flame AAS [28][29][30], hydride generation or cold vapour atomization [31][32][33], electrothermal atomizers [34][35][36] and plasmas [-37-40].…”

“…HPLC with a totally metal-free system coupled to ETAAS allows an adequate separation of the proteins and speciation of aluminium and iron has been reported by Van Landeghem et al [36]. A silica-based scavenger was placed near to the injection valve retaining any residual aluminium and iron originating from buffer solutions and recipients.…”

“…In a pioneering study, for the determination of iron species in serum samples, an HPLC-ETAAS hyphenated method with an on-line metal scavenger for studying the protein binding has been recently reported [36]. Due to the currently introduced procedures for the treatment of anaemia in dialysis patients involving a relative iron deficiency in these subjects, the study on the competition between A1 and Fe for binding to transferrin got renewed interest.…”

Quantitative determination of transferrin in cerebrospinal fluid using an enzyme linked immunosorbent assay

“…However, besides the availability of accurate speciation techniques [2] methodologies are necessary allowing a reliable transferrin determination at ultra-low concentrations to calculate the aluminum-transferrin saturations in both human and animal CSF samples. Whereas the transferrin concentration in human serum is ± 3g/L, it is only ± 20mg/L in CSF.…”

“…Moreover, when transferrin binding of aluminum and other metals in CSF samples has to be observed by means of e.g. electrothermal atomic absorption spectrometry/high performance liquid chromatographic (ETAAS/HPLC) [2], the existing methods are not sensitive enough to detect the ultra low concentrations of the protein in the multi-fold diluted HPLC fractions. Therefore an ELISA based technique presenting a very low detection limit was developed to perform studies on the toxicity of aluminum at the brain level.…”