Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

Development of a direct procedure for the measurement of sulfur isotope variability in beers by MC-ICP-MS.

In this work, a multi-collector inductively coupled plasma mass spectrometer (MC-ICP-MS) was evaluated for the direct measurement of sulfur stable isotope ratios in beers as a first step toward a general study of the natural isotope variability of sulfur in foods and beverages. Sample preparation consisted of a simple dilution of the beers with 1% (v/v) HNO(3). It was observed that different sulfur isotope ratios were obtained for different dilutions of the same sample indicating that matrix effects affected differently the transmission of the sulfur ions at masses 32, 33, and 34 in the mass spectrometer. Correction for mass bias related matrix effects was evaluated using silicon internal standardization. For that purpose, silicon isotopes at masses 29 and 30 were included in the sulfur cup configuration and the natural silicon content in beers used for internal mass bias correction. It was observed that matrix effects on differential ion transmission could be corrected adequately using silicon internal standardization. The natural isotope variability of sulfur has been evaluated by measuring 26 different beer brands. Measured delta(34)S values ranged from -0.2 to 13.8 per thousand. Typical combined standard uncertainties of the measured delta(34)S values were < or = 2 per thousand. The method has therefore great potential to study sulfur isotope variability in foods and beverages.

Measurement of longitudinal sulfur isotopic variations by laser ablation MC-ICP-MS in single human hair strands.

A new method for the measurement of longitudinal variations of sulfur isotope amount ratios in single hair strands using a laser ablation system coupled to a multicollector inductively coupled plasma mass spectrometer (LA-MC-ICP-MS) is reported here for the first time. Ablation parameters have been optimized for the measurement of sulfur isotope ratios in scalp human hair strands of 80-120-microm thickness and different washing procedures have been evaluated. The repeatability of the method has been tested and the ability to measure sulfur isotopic variations in 1,000-microm-long hair segments has been evaluated. A horse hair sample previously characterized for carbon and nitrogen isotope ratios in an interlaboratory study has been characterized by LA-MC-ICP-MS to be used as an in-house standard for the bracketing of human hair strands. (34)S/(32)S isotope amount ratios have been measured and corrected for instrumental mass bias adopting the external standardization approach using National Institute of Standards and Technology (NIST) RM8553 and full uncertainty budgets have been calculated using the Kragten approach. Results are reported as both (34)S/(32)S isotope amount ratios and deltaS(V-CDT) values (sulfur isotopic differences relative to a reference sample expressed in the Vienna Canyon Diablo Troilite (V-CDT) scale) calculated using NIST RM8553, NIST RM8554, and NIST RM8556 to anchor results to the V-CDT scale. The main advantage of the new method versus conventional gas source isotope ratio mass spectrometry measurements is that longitudinal variations in sulfur isotope amount ratios can be resolved. Proof of concept is shown with human scalp hair strands from three individuals, two UK residents and one traveler (long periods of time abroad). The method enables monitoring of longitudinal isotope ratio variations in single hair strands. Absolute ratios are reported and delta(34)S(V-CDT) values are plotted for comparison. Slight variations of <1.2 per thousand were detected in the hair strands from UK residents whereas the traveler presented a variation of >5 per thousand. Thus, the measurement of sulfur isotopic variations in hair samples has potential to be an indicator of geographical origin and recent movements and could be used in combination with isotope ratio measurements in water/foodstuffs from different geographical locations to provide important information in nutritional and geographical studies.

Application of isotope dilution analysis for the evaluation of extraction conditions in the determination of total selenium and selenomethionine in yeast-based nutritional supplements.

Isotope dilution analysis (IDA) has been used to quantify total selenium, total solubilized selenium, and the selenomethionine (SeMet) amount in yeast and yeast-based nutritional supplements after acid microwave digestion and different enzymatic extraction procedures. For this purpose, both a (77)Se-enriched SeMet spike, previously synthesized and characterized in our laboratory, and a (77)Se(VI) spike were used. In the analysis of the nutritional supplements, the SeMet spike was added to the sample and extracted under different conditions, and the (78)Se/(77)Se and (80)Se/(77)Se isotope ratios were measured as peak area ratios after high-performance liquid chromatography (HPLC) separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The formation of SeH(+) and mass discrimination were corrected using a natural SeMet standard injected every three samples. Similarly, total solubilized selenium was measured in the extracts after enzymatic hydrolysis using the (77)Se-enriched SeMet as a spike by direct nebulization without a chromatographic separation. To establish a mass balance, total selenium was also determined by IDA-ICP-MS on the yeast tablets after microwave digestion using (77)Se(VI) as a spike. Results showed that all enzymatic procedures tested were able to solubilize total selenium quantitatively from the solid. However, the recovery for the species SeMet, the major selenium compound detected, was seriously affected by the enzymatic procedure employed and also by the matrix composition of the supplement evaluated. For the yeast sample, SeMet recovery increased from 68 to 76% by the combined use of driselase and protease. For the nutritional supplements, the two most effective procedures appeared to be protease and driselase/protease, with a SeMet recovery ranging from 49 to 63%, depending upon the supplement evaluated. In the case of in vitro gastrointestinal enzymolysis, the results obtained showed 26-37% SeMet recovery, while the rest of selenium was solubilized as other unknown compounds (probably Se-containing peptides).

Reference values for trace and ultratrace elements in human serum determined by double-focusing ICP-MS.

Reference values for trace and ultratrace elements concentrations in healthy human serum, measured by double-focusing inductively coupled plasma-mass spectrometry (ICP-MS), are presented. Blood donors from Asturias (Spain) were selected as the reference population (n=59). Blood samples were collected, after donation, taking the necessary precautions to avoid contamination. All subjects analyzed had normal renal function and nutritional status, as shown from their creatinine and albumin levels. A total number of 14 elements (Al, Ca, Cr, Mn, Fe, Co, Cu, Zn, Rb, Sr, Mo, Cd, Pb, and U) were monitored almost simultaneously. Serum samples were diluted 1+4 with ultrapure water and matrix interferences were corrected using Sc, Ga, Y, and Tl as internal standards. Fe, Cu, and Zn were also determined by isotope dilution analysis (IDA). Reference trace element concentrations intervals observed containing 95% of the reference distribution after excluding outliers are presented. Fourteen serum samples from hemodialysis patients were also analyzed for comparison. High levels of Al, Cr, Sr, Mo, Mn, Pb, U, Co, and Cu and low levels of Fe, Zn, and Rb were found in the serum samples from hemodialysis patients compared to the corresponding reference values observed in this work.

Urinary selenium speciation by high-performance liquid chromatography-inductively coupled plasma mass spectrometry: advantages of detection with a double-focusing mass analyser with a hydride generation interface.

A detailed comparison of the performance of inductively coupled plasma mass spectrometry (ICP-MS), with quadrupole and double-focusing instruments for the speciation of selenium in urine has been carried out. Selenium sensitivity about 23-59 times higher with double-focusing ICP-MS detection was observed, but limits of detection were only 1-8.7 times better because of background noise. Selenium species separation has been carried out by both reversed-phase and vesicle-mediated high-performance liquid chromatography (HPLC), coupled on-line with the detector via conventional nebulization and via on-line focused microwave digestion-hydride generation. A remarkable improvement in sensitivity (28-110 times better for (77)Se depending on the chromatographic system) and elimination of interference problems from the urinary matrix or the components of the mobile phases were achieved when an on-line microwave digestion-hydride generation interface was used, but the background noise was much higher than with conventional nebulization. Therefore, the limits of detection were not as low as expected from such improvement in the sensitivity. More selenocompounds can be separated, and a slight improvement in the sensitivity and limits of detection was obtained when the vesicle-mediated HPLC system was used as compared with reverse-phase chromatography. However, the use of several complementary chromatographic systems, such as reverse-phase HPLC, is recommended to bring some light on the selenocompounds present in basal human urine. Comparative data of rat urine speciation are also given.

Different quantification approaches for the analysis of biological and environmental samples using inductively coupled plasma mass spectrometry.

For the analysis of biological and environmental materials by inductively coupled plasma mass spectrometry (ICP-MS), several quantification procedures can be used depending on the precision and accuracy required. Semi-quantitative methods based on the molar response curve were compared with conventional external calibration and standard additions for the analysis of waters and sewage sludges. For the analysis of biological materials, where higher quality data were required, isotope dilution analysis using enriched isotopes was applied. It was observed that the molar sensitivity for different elements in ICP-MS was a simple function of the mass of the isotopes measured after normalization for ionization efficiency which could be fitted to a third-order polynomial equation. Element ionization adjustments for the third-order polynomial, using the Saha equation, allowed the calculation of the plasma ionization temperature and electron density. For the determination of trace metals in waters and sewage sludges, the samples were spiked with different internal standards, ionization corrections were performed and the results obtained agreed with those obtained by external calibration and standard addition within a factor of 2 but, on average, the agreement was within 20%. The determination of molybdenum in biological reference materials was performed by isotope dilution analysis taking into account possible sources of error in the measurements by ICP-MS such as mass discrimination, detector dead time, isobaric interferences and random error propagation.

Serum and tissue selenium contents related to renal disease and colon cancer as determined by electrothermal atomic absorption spectrometry.

Microwave digestion with nitric acid and hydrogen peroxide was applied to the determination of selenium in biological tissues by Electrothermal Atomic Absorption Spectrometry (ETAAS). Validation of this method is presented in terms of adequate recovery of selenium from standard reference materials and the method is applied to carcinogen human colon tissue. Ultramicrofiltration was used to study selenium protein binding and its fractionation and speciation in blood serum. These studies showed that 95% of the total selenium in serum seems to be bonded to high-molecular-weight proteins. Experiments with renal failure patients showed lower selenium levels than in the health population (0.57 +/- 0.23 mM versus 0.81 +/- 0.11 mM). A wider distribution pattern of total serum selenium concentration (from 0.1 to 1 mM) was clearly observed in renal failure patients. However, the ultramicrofiltrable selenium fraction was always constant, even in the presence of desferrioxamine (DFO).

Selenium speciation by coupling vesicle mediated HPLC with off-line ETAAS and on-line focused microwave digestion HG-AAS detection.

A novel High Performance Liquid Chromatography (HPLC) method for the separation of selenium species with specific detection by off-line Electrothermal Atomization Atomic Absorption Spectrometry (ETAAS) or on-line focused microwave digestion (MW) Hydride Generation Atomic Absorption Spectrometry (HG-AAS) is described. Vesicular mobile phases of the cationic surfactant didodecyldimethylammonium bromide (DDAB) have been evaluated for the liquid chromatographic separation of inorganic selenium (selenite and selenate) and different selenoaminoacids (selenocystine, selenomethionine and selenoethionine) on a C(18) reversed-phase column modified by DDAB molecules. The effects of different parameters (pH, buffer and vesicle concentrations) of the mobile phase on the retention times have been determined. The detection limit for selenium with the proposed off-line HPLC-ETAAS method has been found to be 5 microg/L of Se. The detection limit using HPLC-"on line" focused microwave digestion-HG-AAS has been found to be 1 microg/L of Se, with a precision (repeatability) better than +/- 5%. The latter proved to be an exceptional on-line real-time chromatographic detector for selenium speciation purposes.