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Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations., (© 2023 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13 C 1 -labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13 C 1 -labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13 C 1 -labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments., (© 2024. The Author(s).)

High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of 15 N-monoiodotyrosine and 13 C 2 -diiodotyrosine through the iodination of 15 N-tyrosine and 13 C 2 -tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPy 2 BF 4 ). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g -1 (limits of quantification 0.06 and 0.15 ng g -1 ) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g -1 (limits of quantification 0.23 and 0.17 ng g -1 ) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mg -1 creatinine levels.

Background: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described., Results: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL., Conclusions: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13 C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15 N-labelled amino acids could be obtained.

Sulfur isotopic enrichment of urine metabolites in healthy and prostate cancer mice using 34 S enriched yeast and High Performance Liquid Chromatography coupled to Multicollector Inductively Coupled Plasma Mass Spectrometry (HPLC-MC-ICP-MS) has been evaluated. A 30 weeks experiment (since the eleventh to the fortieth week of life) was carried out collecting the urine of three healthy mice and three transgenic mice with prostate cancer during 24h after a single oral administration of a 34 S enriched yeast slurry. The isotopic enrichment of different sulphur metabolites was monitored by coupling a C18 reverse phase HPLC column with a multicollector ICP-MS using a membrane desolvating system. Quantification of sulfur in the chromatographic peaks was carried out by post-column isotope dilution using a 33 S enriched spike. Differences between the 34 S enrichment in the urine metabolites of healthy and prostate cancer mice were found from the beginning of the disease. Both populations could be differentiated using a principal component analysis (PCA). Finally, 7 unknown mice were correctly classified in each population using a linear discriminant analysis., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

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.

A gas chromatography electron capture negative ionization mass spectrometry (GC(ECNI)MS) procedure for the determination of priority polybrominated diphenyl ethers (PBDEs; congeners 28, 47, 99, 100, 153 and 154) in water samples at regulatory EU levels has been developed. The method is based on the use of (81)Br-labelled PBDEs for isotope dilution analysis and the measurement of (79)Br/(81)Br isotope ratios in gas chromatography peaks with the electron capture negative ionization technique. The suitability of this ion source for the precise and accurate measurement of bromine isotope ratios has been demonstrated. The general ECNI-IDMS procedure was evaluated by the analysis of NIST SRM 1947 (Lake Michigan fish tissue) with satisfactory results. For the analysis of water samples, 500 mL of the samples were spiked with the labelled PBDEs and extracted with 10 mL isooctane for 30 min. The extract was evaporated down to ca. 100 μL and injected in the GC(ECNI)MS. Detection limits ranged from 0.014 (-1) to 0.089 pg mL(-1) depending on the congener. Recoveries from real water samples, spiked at a level of 0.5 pg mL(-1), ranged from 77% to 102%.

A mixture of (81)Br-labeled polybrominated diphenyl ethers (PBDEs), previously synthesized in our laboratory, was separated by liquid chromatography for the individual isolation of different (81)Br-labeled PBDEs containing from 3 to 6 bromine atoms. The different fractions were collected, and a mixed labeled standard was then prepared adequate for the determination of priority PBDEs (congeners 28, 47, 99, 100, 153, and 154) in environmental samples. The spike mixture was then characterized using gas chromatography(electron ionization)MS (GC(EI)MS) both in isotope composition and concentration in combination with multiple least-squares. Contamination from natural abundance BDEs 153 and 154 was detected in the spike mixture, and a new isotope dilution equation was developed to take into account the natural abundance contribution from the spike. The spike mixture was shown to be stable during at least 4 months, and no isotope exchange between natural abundance and labeled PBDEs was detected during this period of time. Finally, the (81)Br-labeled PBDEs standard was used for the determination of congeners 28 (+33), 47, 49, 99, 100, 153, and 154 in a standard reference material (Lake Michigan fish tissue SRM 1947) using three different sample to spike ratios. No methodological calibration needed to be prepared, as no isotopic effects were detected using this labeling mode. Concentrations found were in agreement with the certified concentrations (recoveries between 89% and 116%), and reproducibility was always below 7% RSD. Kragten procedure was used to calculate expanded uncertainties. Very low limits of detection were obtained for all compounds (between 0.02 and 0.9 ng·g(-1)) using the procedure developed here., (© 2011 American Chemical Society)

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.

A mixture of different (81)Br-labeled polybrominated diphenyl ethers (PBDEs) was prepared and characterized for its future use as spike for the isotope dilution analysis of PBDEs. The synthesis was carried out by direct bromination of diphenyl ether using (81)Br enriched Br(2) obtained after aqueous oxidation of bromide with potassium peroxymonosulfate and extraction into dichloromethane. The number of bromine atoms introduced in the diphenyl ether molecule depended on the molar ratio between bromine and diphenyl ether. The final mixture prepared contained a mixture of tri-, tetra-, penta-, and hexabrominated PBDEs with a larger concentration of the tetrabrominated congener BDE-47. The isotopic composition of bromine in the resulting PBDEs mixture was determined by GC(ICP)MS and resulted in a 99.53% enrichment of the isotope 81 of bromine. The concentration of three of the PBDE congeners (28, 47, and 99) in the mixture was determined by reverse isotope dilution analysis using a certified, natural abundance, PBDEs mixture and both GC(ICP)MS and GC(EI)MS. For this purpose, the fragmentation and isotope distribution patterns of the different PBDE cogeners in the positive electron ionization source were studied in detail both for natural abundance and labeled compounds. A procedure based on isotope pattern deconvolution was developed which allowed the direct determination of the concentration of the labeled PBDEs in the spike mixture by GC(EI)MS. Finally, the GC(EI)MS isotope pattern deconvolution procedure was applied for the determination of natural abundance congeners 28, 47, and 99 in spiked waters at ng L(-1) levels. Detection limits below 0.5 ng L(-1) could be obtained for all compounds using only 100 mL of sample and liquid-liquid extraction with isooctane.

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.

Titanium (Ti) has long been regarded as an inert and biocompatible metal, ideal for biomedical applications such as dental implants or joint replacements. However, concerns about the biocompatibility of Ti have lately arisen. Unfortunately, information on reliable Ti baseline physiological levels in blood and organ tissues is still pending and the real effects of physiological corrosion as opposed to wear processes of Ti or Ti alloys implants is controversial so far. In this work a previously developed and validated methodology, based on using double-focusing inductively coupled plasma mass spectrometry (DF-ICP-MS) has been used to establish Ti basal levels in blood and organs (heart, liver, spleen, kidneys, and lungs) of Wistar rats. These data were compared with the levels found in three Wistar rats implanted with a Ti wire embedded in their femur for 18 months, in order to assign possible Ti released purely due to non-wear physiological mechanisms. Results showed that Ti content in all the selected organ tissues and blood was higher than previously determined Ti basal levels, clearly showing both corrosion of the Ti implant and systemic Ti accumulation in target tissues. These results indicate that Ti metal corrosion occurs. This seems to be the only mechanism responsible in the long term for the observed passive dissolution of Ti of the implant in the absence of wear. A comparative study of the systemic distribution of the soluble and particulate Ti potentially released from Ti implants was also carried out by intraperitoneally injection of soluble Ti(citrate)(3) and insoluble TiO(2) particles, respectively. Different systemic Ti storage was observed. Whereas soluble Ti was rapidly transported to all distal organs under study, TiO(2) particles were only accumulated in lung tissue.

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.

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 (Ti(6)Al(4)V).

In order to investigate the potentially bioavailable selenium-containing compounds in the selenized yeast candidate reference material SEAS 6, a two-dimensional (size exclusion-reversed phase) chromatography approach has been worked out. Electrospray tandem mass spectrometry (ESI Q-TOF MS) was then used for off-line identification of low molecular weigh selenocompounds generated during the gastrointestinal digestion. Selenomethionine (SeMet) was the major compound identified in the gastrointestinal extract while SeMet selenoxide was its main degradation product formed after medium and long-term sample storage, respectively. Total Se and SeMet were quantified in both the soluble extracts and the residue. Results showed that 89+/-3% of total Se was extracted after gastrointestinal digestion, but only 34+/-1% was surprisingly quantified as free SeMet. The rest of Se was present as many other low, medium and high molecular weight Se-species, which could be detected and further characterized by using the two-dimensional chromatography approach proposed here. Interestingly, most of Se-species seemed to be Se-peptides unspecifically produced by the gastrointestinal juice. These results show for the first time that while the efficiency of human gastrointestinal digestion to dissolve Se-containing proteins present in yeast may be high, its efficiency to convert them into free SeMet is much lower. Se-species present in the insoluble residue (not assimilated by the organism), accounting for 11+/-1% of the total Se in selenized yeast, were also studied. After treatment with SDS (denaturing agent) only 13+/-2% of this "insoluble" Se was solubilized, indicating that it was mainly non-protein bound and likely associated to other insoluble matrix components.

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).

Ultrasonic slurry sampling electrothermal vaporisation inductively coupled plasma mass spectrometry (USS-ETV-ICP-MS) was applied to the elemental analysis of silicate based minerals, such as talc or quartz, without any pre-treatment except the grinding of the sample. The electrothermal vaporisation device consists of a tungsten coil connected to a home-made power supply. The voltage program, carrier gas flow rate and sonication time were optimised in order to obtain the best sensitivity for elements determined. The relationship between the amount of sample in the slurry and the signal intensity was also evaluated. Unfortunately, in all cases, quantification had to be carried out by the standard additions method owing to the strong matrix interferences. The global precision of the proposed method was always better than 12%. The limits of detection, calculated as three times the standard deviation of the blank value divided by the slope of the calibration curve, were between 0.5 ng/g for As and 3.5 ng/g for Ba. The method was validated by comparing the concentrations found for Cu, Mn, Cr, V, Li, Pb, Sn, Mg, U, Ba, Sr, Zn, Sb, Rb and Ce using the proposed methodology with those obtained by conventional nebulisation ICP-MS after acid digestion of the samples in a microwave oven. The concentration range in the solid samples was between 0.2 microg/g for Cr and 60 microg/g for Ba. All results were statistically in agreement with those found by conventional nebulisation.

A sector field high-resolution (HR)-ICP-MS and an octapole reaction system (ORS)-ICP-MS have been compared for the simultaneous determination of traces of metals (Ti, V, Cr, Co, Ni, and Mo) released from dental implants and articular prostheses in human biological fluids. Optimum sample treatments were evaluated to minimize matrix effects in urine and whole blood. Urine samples were diluted tenfold with ultrapure water, whereas whole blood samples were digested with high-purity nitric acid and hydrogen peroxide and finally diluted tenfold with ultrapure water. In both matrices, internal standardization (Ga and Y) was employed to avoid potential matrix interferences and ICP-MS signal drift. Spectral interferences arising from the plasma gases or the major components of urine and whole blood were identified by (HR)-ICP-MS at 3,000 resolving power. The capabilities of (HR)-ICP-MS and (ORS)-ICP-MS for the removal of such spectral interferences were evaluated and compared. Results indicate that polyatomic interferences, which hamper the determination of such metallic elements in these biological samples, could be overcome by using a resolving power of 3,000. Using (ORS)-ICP-MS, all those elements could be quantified except Ti and V (due to the polyatomic ions 31P16O and 35Cl16O, respectively). The accuracy of the proposed methodologies by (HR)- and (ORS)-ICP-MS was checked against two reference materials. Good agreement between the given values and the concentrations obtained for all the analytes under scrutiny was found except for Ti and V when analyzed by (ORS)-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.

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.

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.

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).

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.