Juan Manuel Marchante-Gayón

Quantitative speciation of selenium in human serum by affinity chromatography coupled to post-column isotope dilution analysis ICP-MS

A post-column isotope dilution analysis (IDA) methodology has been applied to carry out the quantitative speciation of selenium in human serum by affinity chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) with an octapole reaction system (ORS). The interfering argon dimers on the 78Se and 80Se isotopes were suppressed by pressurizing the octapole chamber with hydrogen. The separation of the selenium-containing proteins was evaluated both by anion exchange chromatography (Mono Q HR 5/5) and affinity chromatography (Hi-Trap Heparin- and Hi-Trap Blue-Sepharose columns). Quantification of selenium was performed by post-column isotope dilution analysis by continuous mixing of an enriched 77Se spike solution with the eluent from the column. Finally, the 78Se/77Se or 80Se/77Se isotope ratios were monitored and the amount of selenium bound to proteins was evaluated. The chromatographic separation of selenium-containing proteins in human serum by anion exchange chromatography was not satisfactory. On the other hand, the use of affinity chromatography allowed for a rapid, precise and convenient fractionation of those proteins. Three main selenium fractions could be separated and quantified: selenoprotein P, albumin and glutathione peroxidase. Mass balance performed under different experimental conditions showed quantitative selenium recovery. The proposed methodology was used to study qualitative and quantitative speciation of selenium in human serum samples from healthy volunteers and patients on haemodialysis. The distribution of selenium between plasma glutathione peroxidase (∼20%), selenoprotein P (∼55%) and albumin (∼20%) was similar in both populations.

Total determination and quantitative speciation analysis of selenium in yeast and wheat flour by isotope dilution analysis ICP-MS

Isotope dilution analysis (IDA) has been used to carry out the determination of total Se as well as Se speciation analysis in samples of yeast and wheat flour using an ICP-(Q)MS with an octapole reaction system (ORS). Total selenium was determined after microwave digestion of the samples, whereas enzymatic hydrolysis with protease and lipase was used for the speciation studies. Speciation analysis was performed by using an anion exchange column in order to separate the Se compounds. The separated Se species, eluting from the column, were quantified on-line by post-column IDA using an enriched 77Se spike solution, after adequate conversion of intensity chromatograms into mass flow chromatograms. To do this, the 78Se/77Se ratio was monitored, avoiding the 38Ar40Ar polyatomic interference on the 78Se isotope by pressurizing the octapole chamber with hydrogen. Under such conditions significant formation of SeH+ ions occurred which had to be mathematically corrected. Results showed that the main seleno-compound found in both matrices was selenomethionine, with concentration levels of approximately 59% of total Se for wheat flour and of 68% for yeast. Se(IV) and Se(VI) were detected in yeast samples, but only Se(IV) was present at a level to be quantified. Conversely, those two inorganic species of Se were not detected in wheat flour. The obtained results were included in an inter-laboratory exercise, organised by the University of Plymouth Enterprise Ltd. (UK), with excellent agreement between our results and the average obtained from the rest of the participants.

High-resolution ICP-MS determination of Ti, V, Cr, Co, Ni, and Mo in human blood and urine of patients implanted with a hip or knee prosthesis.

The main components (Ti, V, Cr, Co, Ni, and Mo) of metallic alloys currently used in hip and knee articular prostheses have been simultaneously determined in human whole blood and urine of implanted people by a (HR)-ICP–MS method previously developed in our laboratory. The determination of those elements has been carried out in patients with knee and hip prosthesis and in a group of pre-operation patients without any metallic device in their bodies, used as controls, demonstrating the usefulness of this technique to perform multielement analysis at ppt levels in complex matrices. The concentrations of V, Cr, Co, Ni, and Mo in urine and blood of implanted people turned out to be very similar to those obtained in control patients. However, raised Ti levels could be found both in urine and blood of patients with articular prostheses made or coated with a titanium alloy (Ti6Al4V).

Multi-elemental trace analysis of human serum by double-focusing ICP-MS

A method is described for the determination of a selected range of trace and ultratrace elements in human serum, using a double-focusing ICP-MS instrument (ELEMENT, Finnigan-MAT). Serum samples were diluted 1+4 with ultrapure water and addition of Sc, Ga, Y and Tl as internal standards, to a final concentration of 10 µg L –1 , was used for matrix interference correction. Low and medium resolution scans were performed on the instrument for the analyses, the first scan, at m/Δm=300, for the determination of Rb, Sr, Mo, Cd, Pb and U and the second scan, at m/Δm=3000, for the determination of those elements subject to interference at low resolution: Al, Ca, Cr, Mn, Fe, Co, Cu, Zn and Se. A SRM, 1598 Inorganic Constituents in Bovine Serum (NIST), was analysed for validation of the multi-element analysis method. Good agreement was achieved between the found levels and the certified values. The method was also applied to the multi-analysis of trace elements in serum samples from healthy subjects and from uraemic patients. Preliminary experiments carried out with both quadrupole and double-focusing ICP-MS instruments proved to be invaluable for the study of spectral and non-spectral interferences in ICP-MS detection.

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.

Biosynthesis of isotopically enriched selenomethionine: application to its accurate determination in selenium-enriched yeast by isotope dilution analysis-HPLC-ICP-MS

Isotope dilution analysis is proposed for the determination of selenomethionine (SeMet) in Se-enriched yeast material by high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) using a 77Se-enriched selenomethionine spike obtained by yeast growth on a 77Se-rich culture medium. Different methods of yeast enrichment with selenium were evaluated to determine the most effective one. The use of two different culture media, amount of selenium added, amount of carbon source (glucose) and harvest time were evaluated in order to characterize the total selenium incorporated by the yeast cells. The yeast proteins were hydrolysed with protease XIV, and the isotopically enriched SeMet isolated by anion exchange chromatography. The isotopic composition of the SeMet in the spike solution was determined by on-line HPLC-ICP-MS and reverse isotope dilution was used for the characterization of the spike by means of a natural SeMet standard. In the analysis of the Se-enriched yeast material, the sample was spiked with 77Se-enriched SeMet, and hydrolyzed with a non-specific enzyme (protease XIV) at 37 °C for 20 hours or using an ultrasonic probe for 1 min. Both 78Se/77Se and 80Se/77Se isotope ratios were measured by ICP-MS with collision and reaction cell after the separation of selenomethionine by HPLC. The method was applied to the determination of selenomethionine in a candidate yeast reference material (SEAS project), and the results obtained were in agreement with those reported by other laboratories.

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

AbstractA 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-μm 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-μm-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. 34S/32S 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 34S/32S isotope amount ratios and δSV-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 δ34SV-CDT values are plotted for comparison. Slight variations of <1.2‰ were detected in the hair strands from UK residents whereas the traveler presented a variation of >5‰. 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. FigureThe measurement of longitudinal sulfur isotopic variations by LA-MC-ICP-MS in single human hair strands could play an important role in human identification providing information regarding geographical origin, recent movements and lifestyle of an individual

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.

HPLC-ICPMS and Stable Isotope-Labeled Approaches To Assess Quantitatively Ti(IV) Uptake by Transferrin in Human Blood Serum

Little is known about the effects of titanium found in patients wearing prostheses or about the biochemical pathways of this metal when used as an anticancer drug (e.g., titanocene dichloride). In this work, transferrin has been confirmed as the only carrier protein binding Ti in human blood serum samples by making use of different HPLC protein separations followed by element-specific Ti detection by ICPMS. Besides, isotope dilution analysis has been applied to the quantitative speciation of Ti-Tf in standards and human blood serum samples. Species-unspecific and species-specific isotope dilution modes have been explored. In the first case, very low Ti-Tf results were obtained even using two different chromatographic mechanisms, anion exchange (20-24%) and size exclusion (33-36%). Surprisingly, no major Ti species except Ti-Tf were observed in the chromatograms, suggesting that Ti(IV) hydrolysis and precipitation as inactive titanium oxide species could take place inside the chromatographic columns. These results demonstrate that chemical degradation of metalloproteins during analytical separations could ruin the sought speciation quantitative results. The isotope dilution species-specific mode, much more accurate in such cases, has been instrumental in demonstrating the possibility of gross errors in final metalloprotein quantification. For this purpose, an isotopically enriched standard of (49)Ti-Tf was synthesized and applied to the quantitative speciation of Ti-Tf again. Using this species-specific spike, Ti-Tf dissociation inside the chromatographic columns used could be corrected, and thus, quantitative Ti-Tf binding in serum (92-102%) was observed. In other words, the usefulness and potential of a species-specific isotope dilution analysis approach to investigate quantitatively metal-protein associations, which can be dissociated at certain experimental conditions, is demonstrated here for the first time.

Comparison of electrothermal atomic absorption spectrometry, quadrupole inductively coupled plasma mass spectrometry and double-focusing sector field inductively coupled plasma mass spectrometry for the determination of aluminium in human serum

A detailed examination of the performance of ICP-MS, using both a quadrupole-based and a double-focusing sector field instrument, for the determination of Al in human serum was carried out and the results were compared with those obtained by ETAAS as the reference technique for this type of analysis. Both ICP-MS techniques were compared in terms of observed matrix effects, spectral interferences and overall analytical performance. The results showed that ICP-MS was superior to ETAAS in terms of detection power [detection limits of 0.35 µg l–1 using sector field ICP-MS and 0.85 µg l–1 with quadrupole ICP-MS as compared with 2 µg l–1 for ETAAS determinations of Al in serum (3σ)]. Negligible matrix effects were observed using the quadrupole instrument for serum diluted 1+4 with water. However, the double-focusing instrument showed matrix effects even for a 1+9 dilution of the serum. In any case, such matrix effects could be eliminated by the use of Be and Sc as internal standards. Spectral interferences at m/z=27 in the serum matrix were observed by using the sector field instrument at a resolution of 3000. The analytical methods developed were applied to the determination of Al in reference serum samples with satisfactory results. However, Al serum determination in healthy subjects (n=20) showed that typical basal Al levels in serum required high resolution and, as a rule, were below the detection limit of sector field ICP-MS (i.e., less than 0.35 µg l–1).