Louis Mees

Arsenic speciation in serum of uraemic patients based on liquid chromatography with hydride generation atomic absorption spectrometry and on-line UV photo-oxidation digestion

Abstract An on-line method was developed for the speciation of arsenic species in human serum, including monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC). The method is based on cation-exchange liquid chromatography (LC) separation, UV-photo-oxidation for sample digestion and continuous hydride generation (HG) atomic absorption spectrometry (AAS) for the measurement of arsenic in the LC eluent. By developing the technique of argon segmented flow in the post-column eluent, an substantial improvement in Chromatographic resolution for the separation of these four arsenic species was obtained. The LC separation, photo-oxidation, hydride generation and AAS measurement could be completed on-line within 10 min. The detection limits were 1.0, 1.3, 1.5 and 1.4 μg/l of arsenic for MMA, DMA, AsB and AsC, in serum respectively. The concentration of the 4 species was determined in serum samples of patients with chronic renal insufficiency. Only AsB and DMA were significantly detected by the present method. The main part of arsenic in human serum is AsB. No MMA, AsC and inorganic arsenic were detected in these 6 samples.

Speciation of antimony(III) and antimony(V) species by using high-performance liquid chromatography coupled to hydride generation atomic absorption spectrometry

A miniaturized HPLC column (Supelcosil LC-SAX 1, 2 cm×4.6 mm id) was coupled to HGAAS for the speciation of inorganic SbIII and SbV species in spiked water samples. The baseline separation of these two Sb species was achieved by using aqueous 50 mmol l–1 tartrate solution at pH 5.5 as eluent. The retention times were 0.5 and 2.8 min for SbV and SbIII, respectively. The hydrides were generated with 3% NaBH4 and 1.8 mol l–1 HCl solutions. The absorbance was linearly related to the concentration of SbV species in the range 2.5–100 µg l–1 (r=0.9996) and of SbIII species in the range 5.0–100 µg l–1 (r=0.9990) with detection limits of 1.0 and 2.0 µg l–1, for SbV and SbIII, respectively. The precision, evaluated by using the relative standard deviation (RSD), was 3.3% and 1.5% (n=10) for SbV and SbIII at concentrations of 50 µg l–1, and 4.1% and 3.9% (n=10) at concentrations of 10 µg l–1, respectively. The detection limits achieved in the present work are acceptable for screening samples with respect to the EU drinking and surface water limits (10 µg l–1), but are not sufficiently sensitive to measure real levels in natural waters. The accuracy of the method was evaluated by the analysis of Sb species in several spiked water samples, as there is yet no certified reference material available.

Identification of some arsenic species in human urine and blood after ingestion of Chinese seaweed Laminaria

Algae contain high amounts of arsenic in the form of arsenosugars. The metabolism and toxicology of these arsenic species are not yet fully understood. Three sets of experiments have been conducted in which the alga Laminaria was ingested by 2 to 5 healthy volunteers. Total arsenic concentrations in urine and in blood, packed blood cells and serum have been determined using ICP-MS and HGAFS, respectively. Neutron activation analysis was used for the determination of the total arsenic content in algae samples. Speciation analysis of urine and serum samples has been carried out using HPLC-ICP-MS. HPLC-ES-MS/MS has been used for structural confirmation. The stability of the arsenosugars in simulated gastric fluid was studied for both boiled and unboiled seaweed. A maximum level of arsenic in urine appears within 15 to 25 h after ingestion. Total arsenic and speciation analysis revealed no marked increase in arsenic blood, serum and packed cells levels up to 7 h after ingestion. Dimethylarsinic acid (DMA), methylarsonic acid (MA) and dimethylarsinoylethanol (DMAE) have been positively identified in urine sampled after algae intake. Another 5 species remain unknown. In simulated gastric fluid incubated with algae, the larger share of the arsenosugars degrade within a short time span into a compound with a mass of 254 Da.

Speciation of toxicologically important arsenic species in human serum by liquid chromatography–hydride generation atomic absorption spectrometry

An on-line method was developed for the speciation of trivalent and pentavalent inorganic arsenic (As), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in serum. The method is based on HPLC separation of As species and continuous HGAAS for the measurement of As in the HPLC eluate. Three types of HPLC system were evaluated in order to find a method that was applicable to serum samples: reversed-phase ion-pair chromatography, polymer-based anion-exchange chromatography and silica-based anion-exchange chromatography. As the last-named appeared more suitable, the analytical conditions were optimized, and a procedure is proposed for the speciation of As species in serum. Phosphate buffer of pH 4.5 at a concentration of 30 mmol l–1 was used as the mobile phase. Serum samples were adjusted to the same pH as that of the mobile phase before injecting them onto the column. The detection limits were 0.49, 0.44, 0.92 and 0.40 µg l–1 for AsIII, AsV, MMA and DMA in serum, respectively. The HPLC separation, hydride generation and AAS measurement could be completed on-line within 10 min. The method was applied to the speciation of As in the serum of patients with chronic renal disease. The only As species that could be measured in the serum of the patients was DMA.

More than Tenfold Increase of Arsenic in Serum and Packed Cells of Chronic Hemodialysis Patients

Arsenic concentrations were determined in serum and packed cells of 7 chronic hemodialysis patients, in fresh dialysate and in a heparin solution. The analytical technique was radiochemical neutron activation analysis. The accuracy of the method was tested by the analysis of As in certified reference materials. Patients showed elevated serum and packed cell arsenic concentrations compared with controls (serum, range: 2.3-79.8 ng As/ml, median: 11.5 ng As/ml; versus range: 0.132-4.783 ng As/ml, median: 0.38 ng As/ml; packed cells, range: 2.1-68.4 ng As/g, median: 9.5 ng As/g; versus range: 0.51-14.44 ng As/g, median: 3.17 ng As/g). Arsenic concentrations remained unaltered, before versus after a single hemodialysis treatment. The arsenic contents of serum and packed cells were significantly correlated (n = 7, r = 0.96, p < 0.05). No arsenic could be detected in the heparin solution or in the dialysate.

Chemical speciation of arsenic in serum of uraemic patients.

Chemical speciation of arsenic was carried out in serum of a total of 51 uraemic patients: 19 non-dialysis (ND), 18 haemodialysis (HD) and 14 continuous ambulatory peritoneal dialysis (CAPD) patients. The low molecular mass As species were separated by ion-exchange liquid chromatography and measured on-line by hydride generation atomic absorption spectrometry (HGAAS). The high molecular mass As species were separated by fast protein liquid chromatography, either size-exclusion, ion-exchange or affinity chromatography, and the fractions were digested and measured off-line with HGAAS. The mean total As concentrations in the serum of the three groups of the uraemic patients were significantly higher than the reference value (6.47 +/- 4.28, 5.12 +/- 5.58 and 4.67 +/- 5.41 micrograms l-1 for HD, ND and CAPD patients, respectively, versus the reference value of 0.96 +/- 1.52 micrograms l-1. The major As species in serum of the patients were dimethylarsinic acid (DMA) and arsenobetaine. The HD patients showed a significantly higher mean DMA level than ND and CAPD patients. No selective removal of different As species in serum of HD patients was observed after 4 h of haemodialysis. The inorganic As species in serum were bound to proteins, mainly transferrin (about 5-6% of total As in serum). This binding may play an important role in arsenic detoxification.

Cr(III) and Cr(VI) speciation measurements in environmental reference materials

The production of reference materials for quality control of Cr(III) and Cr(VI) speciation in environmental samples is described. It concerns in the first place two lyophilized solutions containing Cr(III) and Cr(VI) at different concentrations, respectively representative for drinking water and filter leaching solutions, and in the second place filters loaded with welding dust. Twenty-four laboratories with experience in the field participated in an intercomparison exercise organized to validate the suitability of the reference materials and to gauge the state-of-the-art of Cr speciation throughout Europe. The outcome of this exercise is discussed.

Certification of the contents of the chromium(III) and chromium(VI) species and total chromium in a lyophilised solution (CRM 544)

The preparation is described of a batch of 1100 vials containing a lyophilised solution, with CrVI and CrIII contents as in natural or tap water and a total Cr content <50 µg l–1 following the European Community Directive 80/778/EEC, L229/20, D48 on the quality of drinking water. The homogeneity and stability studies and the analytical work performed for the certification of the contents of the CrIII and CrVI species and the total Cr content are also described. The paper contains all the results and gives the methods used to certify the measured concentrations of CrVI (22.79 µg l–1), CrIII (26.77 µg l–1) and total Cr (49.34 µg l–1) after reconstitution in 20 ml. A brief description of the preliminary feasibility study is also given.

Br, Rb, Zn, Fe, Se and K in Blood of Colorectal Patients by INAA and PIXE

A preliminary study was conducted on blood samples and blood fractions of 11 colorectal patients and 10 healthy subjects (controls) in Belgium, in order to determine the concentration of some vital trace elements. Two non destructive analytical methods were used for the determination: INAA and PIXE. The agreement between PIXE and INAA was within about +/- 10% for plasma, but for Rb, Se and Fe in whole blood and red cells a difference of +/- 20% was noted; part of the discrepancy may be due to self absorption problems in PIXE, and for Rb, spectral interferences also may have contributed. The precision of the INAA method for the elements studied was found to be +/- 3% for whole blood and red cells and +/- 5% for plasma; the accuracy for Br, Rb and Zn was better than +/- 10% and +/- 17% for Se. The ratios of the concentrations in whole blood to red cells and whole blood to plasma were not significantly different for normals and cancer cases and, therefore, in future studies analysis of whole blood only may be sufficient. The mean values for Br, Rb, Br/Rb ratio, K, Fe and Se were significantly lower for cancer cases than for healthy individuals, and this might be applicable as an additional parameter for differentiating normals from malignant cases.

Speciation measurements by HPLC-HGAAS of dimethylarsinic acid and arsenobetaine in three candidate lyophilized urine reference materials.

Speciation measurements of dimethylarsinic acid (DMA) and arsenobetaine (AsB) in three candidate lyophilized urine reference materials are described. The measurements were based on cation-exchange liquid chromatography coupled to hydride generation atomic absorption spectrometry with on-line digestion of the organic. As species by alkaline persulfate solution aided by ultraviolet radiation. Arsenic concentrations as DMA were significantly different in the three samples. The mean values for the three samples were 4.1 +/- 0.3, 55.3 +/- 1.2 and 134.1 +/- 1.5 micrograms l-1, respectively. No significant differences in AsB concentrations were observed among the three samples. The mean As concentrations as AsB in the three samples were 17.4 +/- 0.4, 17.7 +/- 0.2 and 17.5 +/- 0.3 micrograms l-1, respectively. By off-line digestion of the urine samples, total As concentrations in the three materials were also obtained. The mean values were 23.4 +/- 0.3, 76.6 +/- 1.6 and 151.3 +/- 1.8 micrograms l-1, respectively. These results correlated well with the results obtained by neutron activation analysis in our laboratory (r = 0.999; p < 0.0001).