Multielement trace-element speciation in metal-biomolecules by chromatography coupled with ICP-MS.

The state-of-the-art of multi-element speciation in biological materials by hybrid techniques with inductively coupled plasma-mass spectrometric (ICP-MS) detection is critically reviewed with special attention to multi-metal speciation in metallothioneins (MT) and metallothionein-like proteins (MLP). The most common separation techniques used for the purpose include conventional size-exclusion chromatography (SEC) (with off-line detection) and anion-exchange (AE) or reversed-phase (RP) HPLC coupled on-line to ICP-MS. Advantages and limitations of the different commercial element-selective ICP-MS detectors, including the quadrupole (Q), double focusing (DF), and time-of-flight (TOF) mass analysers, are discussed. Procedures reported for speciation analysis, by use of hybrid techniques, for multi-metal speciation in mussel MLP and rabbit liver MT are illustrated with special emphasis on work performed in the authors' laboratory.

Speciation of metallothionein-like proteins of the mussel Mytilus edulis at basal levels by chromatographic separations coupled to quadrupole and double-focusing magnetic sector ICPMS.

Characterization and partial purification of metallothionein-like proteins (MLPs) of the mussel Mytilus edulis from natural populations of three coastal regions in Spain were performed. Size exclusion chromatography (SEC) with quadrupole (Q-ICPMS) or double-focusing inductively coupled plasma mass spectrometry (DF-ICPMS) detection was used first for speciation of cadmium in such natural samples and those of mussels exposed to 500 mg x L(-1) Cd in an aquarium tank. SEC results showed always a single Cd-MLP peak (MLP fraction). The contents in Cd, Cu, and Zn of this MLP fraction, of the high molecular weight protein pool (HMW), and of the whole cytosol were then measured by DF-ICPMS. Then, a given aliquot (50 microL) of MLPs with the highest values for UV molecular absorption at 254 nm (also the maximum sulfur and Cd, Cu, or Zn contents) was used to further fractionation. Fast protein liquid chromatography "on line" with Q-ICPMS was used for the purpose. Two Cd-MLP isoforms (MLP-1, MLP-2), with retention times (tR) of 15.7 and 16.0 min, were then detected in cytosols of the mussel samples of aquarium tank and also of the industrial area and Galicia coast. Conversely, wild coast mussels did not show any Cd-MLP signals at all. Analysis of essential elements copper and zinc in such cytosols by FPLC-Q-ICPMS revealed that these two metals were associated just to MLP-1. These results tend to indicate a different role for the two MLP isoforms detected in mussels (i.e., essential metals' homeostasis role seems to be tied to the MLP-1 isoform only). They illustrate the fact that trace metal speciation of unknown species in biological materials is becoming a challenge and points to the use of several complementary analytical techniques to obtain the required speciation information.

Metal distribution patterns in the mussel Mytilus edulis cytosols using size-exclusion chromatography and double focusing ICP-MS detection.

In order to estimate metal distribution patterns in biomolecules of different sizes and their possible relationship with environmental heavy metal contamination, multi-elemental distributions in different fractions of the cytosols of mussels were studied. To do so, samples were collected from natural populations of two coastal regions in Spain: a wild (uncontaminated) coast and an industrialised (contaminated) area in Asturias. Moreover, some commercial mussels from the Galicia coast were also investigated for comparison. Aliquots of the mussel cytosol extracts from each sample were applied to a calibrated Sephadex G-75 column (100 x 1 cm) and forty 3 ml fractions were obtained. After suitable dilution, 18 trace metals were determined by double focusing inductively coupled plasma mass spectrometry (DF-ICP-MS). The use of DF-ICP-MS detection allowed the resolution of several spectral interferences that cannot be resolved by quadrupole ICP-MS. Accurate results for ultratrace elements at basal levels are possible even after sample dilution to prevent matrix effects. After biomolecule-metal association pattern has been established, quantitative analysis of mussel cytosols from the three coastal areas was carried out, using external aqueous calibration plus standard additions to correct for possible matrix effects. Results showed that total metal contents increased following the expected order: wild coast < Galicia coast < industrial area coast. Speciation of Cu, Zn, Ca, U, Ni, Mo, Mn, Cr, V, Cd, Al and Sb showed a similar distribution pattern among cytosolic ligands for all the studied samples. Conversely, Fe, Pb, Sn, Co, Hg and Ag were found to exhibit different speciation patterns when samples from industrialised (contaminated) and non-industrialised areas were compared.