Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection.

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 microg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100:1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm(-3) NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min(-1). The separated Zn species were determined in 1.0 cm3 eluate fractions "off line" by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm(-3) NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min(-1) and determination of the separated Zn species in 1.0 cm3 eluate fractions "off line" by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn-citrate was separated from both Zn-oxalate and from Zn-EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2-4%), tested for six parallel determinations (2 microg cm(-3) Zn) of Zn-EDTA, Zn-citrate and Zn-oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm(-3). The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.

Speciation of low molecular weight Al complexes in serum of CAPD patients.

Speciation of LMW--Al complexes was performed in human serum of six continuous ambulatory peritoneal dialysis (CAPD) patients in order to investigate the individual variability in the percentage and the composition of LMW--Al species. The total concentration of Al in serum ranged from 10 to 120 ng ml(-1). The samples with high total concentration of Al were analysed directly, while those of low total Al concentration were spiked with Al(3+). Spiked and non-spiked samples (100--120 ng ml(-1) of total Al) were microultrafiltered through a membrane filter (cut-off 30,000 Da) to separate Al-transferrin from LMW-Al complexes. On an anion-exchange fast protein liquid chromatography (FPLC) column, 0.2 ml of filtrate was injected. An aqueous -- 4 mol l(-1) NH(4)NO(3) linear gradient elution was applied for 10 min to separate LMW--Al complexes. Fractions of 0.2 ml collected throughout the chromatographic run were diluted 1:1 with water and Al determined 'off line' by electrothermal atomic absorption spectrometry (ETAAS). The characterisation of LMW-Al species eluted under the chromatographic peaks was performed also by electrospray tandem mass spectrometric (ES-MS-MS) analysis. It was found experimentally that the percentage of LMW--Al species in spiked and non-spiked serum ranged from 25 to 50% (in one non-spiked sample 100%). The following LMW--Al species were separated and identified during the chromatographic run: Al-phosphate and a mixture of Al-citrate and ternary Al-citrate--phosphate complexes. It was found experimentally that the distribution of these species varied among particular patients. Similar distribution of LMW--Al species was found in spiked serum of healthy volunteers.

Environmentally safe sewage sludge disposal: the impact of liming on the behaviour of Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn.

Dewatered sewage sludge containing relatively high total concentrations of Cr (945 micrograms ml-1), Cu (523 micrograms ml-1), Ni (1186 micrograms ml-1) and Zn (2950 micrograms ml-1) was treated with quicklime and sawdust for sludge disinfection and post-stabilisation. The mobility of the heavy metals in the sludge samples was assessed by applying a modified five-step Tessier sequential extraction procedure. Water was added as a first step for estimation of the proportion of the easily soluble metal fractions. To check the precision of the analytical work the concentrations of heavy metals in steps 1-6 of the extraction procedure were summed and compared to the total metal concentrations. The mass balance agreed within +/- 3% for Cd, Cu, Cr, and Zn and within +/- 5% for Ni, Pb, Fe and Mn. Data from the partitioning study indicate that in the lime-treated sludge at a pH of 12 the mobility of Cu and Ni notably increased with the solubilisation of these metals from their organic and/or carbonate and Fe and Mn oxide and hydroxide fractions, respectively. Liming slightly decreased the proportion of other heavy metals in the easily soluble fractions while its impact on the partitioning between other sludge phases was almost insignificant. Due to the increased solubility of Ni and Cu as well as potential Cr oxidation at high pH, liming cannot be recommended for sludge disinfection. Addition of sawdust did not change the heavy metal partitioning.

The uptake and speciation of various Al species in the Brassica rapa pekinensis.

An investigation was carried out on the uptake and speciation of Al species in Al tolerant Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plants were exposed to 10 microg cm(-3) of Al in the chemical forms of Al3+, Al-citrate and Al-malate in a time span from 1 up to 24 h. In each experiment the nutrient solution and stem sap were analysed by a combination of FPLC ICP AES and ES MS MS techniques. Speciation analysis enabled determination of particular chemical forms of Al present in the nutrient solution or in stem sap. The results indicate that Al3+ added to the nutrient solution remained as Al3+ in the solution during the experiments, but in the roots transformation to Al-malate occurred. Al was transported from roots to the upper parts of the plant as Al-malate (70%) and Al3+ (30%). Al-citrate or Al-malate added to the nutrient solution were transferred to the upper parts of the plant without transformation of their chemical forms.

Partitioning of Zn, Pb and Cd in river sediments from a lead and zinc mining area using the BCR three-step sequential extraction procedure.

The extent of pollution was investigated in sediments collected along the course of the river Meza and its tributaries in the lead and zinc mining area of the Mezica valley (Slovenia). In order to evaluate the heavy metal burden, total concentrations of Zn, Pb and Cd were determined and the partitioning of these metals between the easily and sparingly soluble sediment fractions was performed by the use of the slightly modified BCR sequential extraction procedure. The quality of analytical data was checked with Certified Reference Material CRM 601. Good agreement between the determined and certified or indicative metal concentrations was obtained. In order to estimate the natural and anthropogenic inputs of Zn, Pb and Cd in the sediments, normalisation to Al was applied. The results of the partitioning study indicate that Zn prevails in the most sparingly soluble fraction and is distributed between organic matter and sulfides, while a smaller proportion is found in the easily soluble fraction. Pb is distributed mainly between organic matter and sulfides, whereas Cd is predominantly associated with the most sparingly soluble fraction. Data from the normalisation procedure indicate that the anthropogenic inputs of Zn, Pb and Cd correlate with the very high total metal concentrations determined in sediments. The highest total Zn, Pb and Cd concentrations (16.3. 9.3 and 0.13 g kg(-1), respectively) were found in the sediment of the Helena rivulet, arising from former mining activities. In spite of the relatively low easily soluble metal fractions in the sediment, the concentrations of metals in these fractions are high, owing to the extremely high total metal concentrations. These data indicate severe pollution of the terrestrial and aquatic environment.

Speciation of aluminium in forest soil extracts by size exclusion chromatography with UV and ICP-AES detection and cation exchange fast protein liquid chromatography with ETAAS detection.

Aluminium speciation was studied in forest soil extracts by size exclusion chromatography (SE) with UV and inductively coupled plasma-atomic emission spectrometric (ICP-AES) detection and cation exchange fast protein liquid chromatography (FPLC) with ETAAS detection. Size exclusion chromatography was performed on a Superdex HR75 10/30 column. Isocratic clution with 0.15 mol dm(-3) NaCl in TRIS-HCl buffer (pH = 5.5) was applied over 100 min at a flow rate of 0.35 cm(3) min(-1). The chromatographic run was followed at 278 nm and separated Al species also determined 'off line' in 0.875 cm3 fractions by ICP-AES. The analytical procedure enabled speciation of high molecular weight Al complexes. Cation exchange FPLC was performed on a Mono S HR 5/5 column. Aqueous 8 mol dm(-3) NH4NO3 linear gradient elution was applied over 10 min at a flow rate of 1 cm3 min(-1). Separated Al species were collected in 0.5 cm3 fractions and Al determined 'off line' by ETAAS. The analytical procedure enabled speciation of some positively charged monomeric Al species. Negatively charged species were eluted with the solvent front. The combination of the two analytical techniques was successfully employed in speciation of Al in forest soil extracts. Water was used as an extracting solution. It was found experimentally that 80-95% of Al in aqueous extracts of forest soils exists in monomeric Al forms. Water soluble Al (30-40%) is bound to high molecular weight complexes with humic substances. The remaining monomeric Al in the low molecular weight fraction exists as AIF2+, Al-oxalate and Al-citrate species.

Use of nitric acid in sample pretreatment for determination of trace elements in various biological samples by ETAAS.

Trace elements in liquid biological samples may be determined by direct electrothermal atomic absorption spectrometry (ETAAS). In our previous work it was found that samples containing proteins or DNA may leak out of the graphite tube before the drying step, despite the addition of various modifiers. In order to keep the sample to the graphite tube, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid, or 5 microl of 32% v/v nitric acid was added to the graphite tube before ETAAS determination. Applying the proposed procedure, the concentrations of lead in eluted fractions after gel chromatographic separation of human cerebellar nucleus dentatus supernatant and platinum in isolated DNA samples were determined. The use of nitric acid in sample pretreatment prevent sample leakage out of the graphite tube, provided for even drying and considerably reduced nonspecific absorption in lead determination. The repeatability of measurements was better than + 6%. The accuracy of the procedure was checked by spiking samples. The recoveries for both elements lay between 93--104%. Nitric acid was found to be a better modifier than TRITON X-100.

Aluminum inhibits the lysosomal proton pump from rat liver.

Lysosomes are cytoplasmatic organelles, delimitated by a single lipoprotein membrane, that contain several enzymes mostly belonging to the hydrolases in that they function mainly for intracellular digestion. Lysosomal internal pH is characteristically acidic and it is maintained around pH 4.5 by a proton pump, an ATPase, that uses energy from ATP hydrolysis to translocate H+ ions into lysosomes. In the presence of Al3+ the proton pump activity is markedly reduced compromising acidic vesicles functionality. Among different species utilized, Al2(SO4)3 and AlF3 were the most effective. Aluminum effect was not observed when the delta pH was produced artificially by nigericin.

Total metal concentrations and partitioning of Cd, Cr, Cu, Fe, Ni and Zn in sewage sludge.

Application of the BCR three-step sequential extraction procedure to sewage sludge samples collected at an urban wastewater treatment plant (Domzale, Slovenia) is reported. The total concentrations of Cd, Cr, Cu, Fe, Ni and Zn and their concentrations in fractions after extraction were determined by flame or electrothermal atomic absorption spectrometry (FAAS, ETAAS) under optimised measurement conditions. Total acid digestion including hydrofluoric acid (HF) treatment and aqua regia extraction were compared in order to estimate the efficiency of aqua regia extraction for determination of total metal concentrations in sewage sludge. It was found experimentally that aqua regia quantitatively leached these heavy metals from the sewage sludge and could therefore be applied in analysis of total heavy metal concentrations. The total concentrations of 856 mg kg(-1) Cr, 621 mg kg(-1) Ni and 2032 mg kg(-1) Zn were higher than those set by Slovenian legislation for sludge to be used in agriculture. Total concentrations of 2.78 mg kg(-1) Cd, 433 mg kg(-1) Cu and 126 mg kg(-1) Pb were below those permitted in the relevant legislation. CRM 146R reference material was used to follow the quality of the analytical process. The results of the BCR three-step sequential extraction procedure indicate high Ni and Zn mobility in the sludge analysed. The other heavy metals were primarily in sparingly soluble fractions and hence poorly mobile. Due to the high total Ni concentration and its high mobility the investigated sewage sludge could not be used in agriculture.

Determination of trace elements and calcium in bone of the human iliac crest by atomic absorption spectrometry.

A rapid and reliable analytical method for the determination of trace elements in human bone by atomic absorption spectrometry is reported. Calcium was determined to estimate the homogeneity of samples. Human bone from the iliac crest was obtained at autopsy of adult subjects. Before analysis samples were decomposed by microwave digestion and acid digestion in a Parr bomb. Zinc, rubidium, strontium, calcium and iron were determined by flame atomic absorption spectrometry (FAAS) and aluminium, copper and lead by electrothermal atomic absorption spectrometry (ETAAS) at optimum measurement conditions. The results for the two digestion procedures agreed for zinc, rubidium and calcium within +/-5%, for copper within +/-7% and for strontium, iron, aluminium and lead within +/-10%. The repeatability of measurement (R.S.D.) for determination of calcium and trace elements after microwave digestion and acid digestion in a Parr bomb was tested in one representative autopsy bone sample by six parallel determinations. It was found to be better than +/-5% either for microwave digested samples or samples digested in a Parr bomb, for all elements determined by FAAS and ETAAS techniques. The accuracy of the applied digestion procedures was checked by analysis of trace elements in NIST SRM 1486 Bone Meal reference material. Good agreement of the results with certified values was obtained for both digestion procedures. The microwave procedure developed for digestion of small amounts of sample was applied in trace elements analysis of bone biopsy samples from dialysis patients.

Problems related to determination of trace elements in spent continuous ambulatory peritoneal dialysis fluids by electrothermal atomic absorption spectrometry.

Critical parameters which influence the direct determination of trace elements in spent continuous ambulatory peritoneal dialysis (CAPD) fluids by electrothermal atomic absorption spectrometry (ETAAS) were investigated. Samples were collected after CAPD fluid exchange in 5.0 ml polyethylene cups and stored at -20 degrees C. Daily spent CAPD fluids were frozen immediately, while nightly spent CAPD fluids were frozen 2-3 h after the morning exchange. Before analysis samples were equilibrated to room temperature and analysed within 8 h. It was found experimentally that some samples which were not frozen immediately leaked out of the pyrolytically coated graphite tube before the drying step. In order to keep the sample in the graphite tube and to obtain reproducible measurements of copper, aluminium and iron, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid. The standard addition method was used in the calibration procedure. The results of direct ETAAS determinations for copper agreed well (+/-5-10%) with those obtained after acid digestion of CAPD fluids in Parr bomb. The procedure for direct determination of all three elements was validated with spiked samples.

Determination of trace elements in a large series of spent peritoneal dialysis fluids by atomic absorption spectrometry.

An analytical procedure is reported for the determination of six elements in a large series of spent dialysis fluid samples. Determinations of aluminium, chromium, copper, manganese and iron were made by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman background correction, while zinc was analysed by flame atomic absorption spectrometry (FAAS). Because of the complex matrix with high salt content and a high content of proteins, the measurement parameters were optimised for each particular element determined by ETAAS. The samples were collected in polyethylene eppendorf cups and stored in a freezer at -20 degrees C. When the elements were determined by ETAAS the standard addition method was applied in the calibration procedure. The sample (10 microl) was injected into a cuvette and careful drying and long ashing of samples at temperatures between 850 and 1000 degrees C performed. Triton X-100 was added before each determination to reduce the matrix effects of the proteins. Zinc was determined by FAAS in an air acetylene flame under the usual recommended procedure, calibrating with aqueous standards. The limits of detection (3sigma basis) were 1.0 microg x l(-1) for aluminium, 0.20 microg x l(-1) for chromium, 0.40 microg x l(-1) for copper, 0.20 microg x l(-1) for manganese, 0.50 microg x l(-1) for iron and 5.0 microg x l(-1) for zinc. The reproducibility of the measurements for aluminium, copper, iron and zinc was better than +/-3.0%. It was worse for manganese and chromium (+/-6.0 and +/-12.0%, respectively), since these two elements were present in very low concentrations in all the samples analysed.

Optimisation of parameters for determination of rubidium in spent CAPD fluids by flame and electrothermal atomic absorption spectrometry.

An analytical procedure is reported for the determination of rubidium in spent continuous ambulatory peritoneal dialysis (CAPD) fluids by flame and electrothermal atomic absorption spectrometry (FAAS, ETAAS). Samples of spent CAPD fluids were collected as 5 ml aliquots in polyethylene tubes and stored in a freezer at -20 degrees C. Before analysis, samples were equilibrated to room temperature and analysed within 8 h. A total of 2 mg ml(-1) of caesium was added to each sample and standard solution to overcome interferences from ionisation. An air-acetylene flame was applied in FAAS determinations. Analysis was performed against aqueous standards. The calibration graph was linear from 30.0 up to 5000 microg l(-1) Rb, while the limit of detection (3 s) was found to be 20.0 microg l(-1) rubidium. Good repeatability of measurement (RSD 1%) was obtained. Parameters were also optimised for determination of rubidium in spent CAPD fluids by ETAAS. Ten-fold diluted samples (3.5% nitric acid) were analysed applying standard addition calibration. The calibration graph was linear from 2.0 up to 30.0 microg l(-1) rubidium, while the limit of detection (3 s) was found to be 1.0 microg l(-1) rubidium (sample volume 10 microl). Good repeatability of measurement (RSD 5%) was obtained. The results of direct determination by FAAS and ETAAS were compared to those obtained after acid digestion of samples in Parr bombs. The accuracy of the procedure for direct determination was checked by spiking samples. In 73% of samples analysed, the differences between the results obtained by the two techniques, either for direct determinations of samples or for samples digested in a Parr bomb did not exceed +/-10%.

Intratumoral cisplatin administration in electrochemotherapy: antitumor effectiveness, sequence dependence and platinum content.

Electrochemotherapy using intratumoral cisplatin administration was tested on EAT tumors in mice. Mice were treated with eight electric pulses (100 micros, 1 Hz, 1040 V) and/or cisplatin (1, 2, 4 and 8 mg/kg). Cisplatin treatment resulted in up to 20 days of tumor growth delay. Electrochemotherapy resulted in tumor cures; local tumor control reached a plateau at 4 mg/kg in 67% of tumor cures. The maximal effect of electrochemotherapy was achieved when cisplatin was injected 5 min before or simultaneously with electric pulses application. Approximately two times more platinum was bound to DNA in electrochemotherapy than in cisplatin treated tumors at all time points tested. Our study shows that electrochemotherapy with intratumoral cisplatin administration is a very effective local treatment of EAT tumors with high curability rate.

Possibilities for speciation of Al-citrate and other negatively charged Al complexes by anion-exchange FPLC-ICP-AES.

An anion-exchange fast protein liquid chromatographic-inductively coupled plasma atomic emission spectrometric procedure (FPLC-ICP-AES) was developed for speciation of Al-citrate and other negatively charged Al complexes. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange FPLC column over a pH range from 3.5 to 11.0. An aqueous-NaNO(3) (4 mol dm(-3)) linear gradient elution was applied over 10 min for separation of a particular Al species. The separated Al species were determined in 0.5 cm(3) eluate fractions ;off line' by ICP-AES. Under optimal analytical procedures Al-citrate was separated from Al-oxalate and Al-EDTA in a neutral pH range. Good reproducibility of the FPLC-ICP-AES procedure was obtained for determination of a particular Al species at optimal measurement conditions (RSD +/-2%). Al(3+) and neutral Al-citrate species were strongly adsorbed on the column resin and did not interfere with the separation of negatively charged Al complexes. Al(OH)(4)(-) species were separated from Al-citrate in an alkaline pH region, but quantitatively determined only at a pH of 11.0. The distribution of Al species over a pH range from 3.5 to 11.0 agreed with the reported calculated data. The limit of detection (3sigma basis) for separated Al species was 0.1 mug cm(-3).

Quantitative determination of trace amounts of Al-citrate by anion-exchange FPLC-ETAAS.

An anion-exchange fast protein liquid chromatographic-electrothermal atomic absorption spectrometric procedure (FPLC-ETAAS) was developed for determination of trace amounts of negatively charged Al-citrate in the pH range 3.5-8.0. Aqueous-4 mol dm(-3) NH(4)NO(3) linear gradient elution at a flow rate of 1 cm(3) min(-1) was applied for 10 min to separate Al-citrate on a FPLC Mono Q HR 5/5 column. The separated aluminium species were determined 'off line' by ETAAS in 0.5 cm(3) fractions. After separation the column was regenerated for 5 min with 4 mol dm(-3) NH(4)NO(3) and equilibrated with water. All reagents used in the separation procedure were cleaned with a silica based LiChrosorb RP-18 HPLC column to remove traces of aluminium. The main advantage of NH(4)NO(3) as eluent lies in its ability to decompose quantitatively in the graphite tube during the ashing step, which enables reproducible analysis of aluminium in the separated fractions. Using the procedure developed reproducible (RSD+/-2.0%) and quantitative determination of negatively charged Al-citrate at a retention time of 4.5 min was obtained. The LOD was found to be 2.0 ng cm(-3) of Al-citrate. The technique was successfully applied for the determination of Al-citrate in human serum. Spiked samples (50-150 ng Al(3+) cm(-3)) were microultrafiltered through a membrane filter (cut-off 30 000 Da) to separate aluminium bound to transferrin from low molecular weight aluminium complexes. It was found that 15-19% of aluminium in spiked samples from healthy volunteers passed through the membrane. By applying FPLC separation it was proved that all the aluminium in the filtrate corresponded to Al-citrate. The analytical technique developed enabled quantitative and reproducible determination (RSD+/-3.0%) of Al-citrate in spiked human serum at levels which could be found in patients undergoing long term haemodialysis.

Determination of platinum in tumour tissues after cisplatin therapy by electrothermal atomic absorption spectrometry.

A simple procedure is described for the determination of platinum in tumours after cisplatin therapy. Tumours were digested in 65% nitric acid by incubation at 37 degrees C for 2 days and platinum analysed under optimum conditions by electrothermal atomic absorption spectrometry with Zeeman background correction. The reproducibility of measurements in general was better than +/- 2%. The calibration graph was linear from 30.0 up to 1000 micrograms l-1 of platinum, while the limit of detection (3 sigma) was found to be 3.0 micrograms l-1 (sample volume 20 microliters). Aqueous standard solutions and the standard addition method were applied in the calibration procedure. Under the recommended analytical conditions, the sample matrix did not influence the determination of platinum significantly. In 72% of samples analysed the differences between results obtained by the two calibration procedures did not exceed +/- 5%.