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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).)

With the aim to distinguish between routes of exposition to mercury (Hg) in artisanal and small-scale gold mining (ASGM) communities and to distinguish between Hg contamination sources, Hg species composition should be performed in human biomarkers. In this work, Hg species-specific determination were determined in human hair samples (N = 96), mostly non-directly occupied in ASGM tasks, from the six most relevant gold mining Colombian regions. Therefore, MeHg, Hg(II) and THg concentrations were simultaneously determined by double spiking species-specific isotope dilution mass spectrometry (IDMS) and GC-ICP-MS. Only 16.67% of participants were involved at some point in AGSM works and fish consumption ranged from 3 to 7 times/week, which is between medium and high intake levels. The median concentration of THg obtained from all samples is higher than the reference dose weekly acceptable of MeHg intake established by the EPA (1 ppm), whereas a 25% were more than 4 times higher than the WHO level (2.2 μg Hg g -1 ). Median THg value of individuals consuming fish 5-7 times per week was significantly higher (p < 0.05) than those of the other consuming groups (12.5 μg Hg g -1 ). Most of the samples presented a % of MeHg relative to THg higher than 80%. The average % of Hg(II)/THg was 11% and only 10 individuals presented a Hg(II) content over 30%. No significant differences (p > 0.05) were found when the amount of Hg(II) was compared between people involved in AGSM task and people not involved. Interestingly, significant differences among the evaluated groups where found when the percentage of the Hg(II)/THg ratio of these groups were compared. In fact, people involved in AGSM tasks showed 1.7 times higher Hg(II)/THg vs. inhabitants uninvolved. This suggest that Hg(II) determination by IDMS-GC-ICP-MS could be a good proxy for evaluating Hg(II) adsorption by direct exposure to mercury vapors onto hair., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15 N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.

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.

We evaluate here the combination of two-dimensional liquid chromatography (2D-LC) in the multiple heart cutting mode and isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples. As a proof of concept, we attempt the quantification of proteotypic peptides of Apolipoprotein AIV (APOA4), Complement C3 (C3) and Vitronectin (VTN) which have been previously identified as potential candidate biomarkers of glaucoma. Using this 2D-LC strategy, analyte enrichment steps are avoided and the sample preparation involved after enzymatic digestion amounted to a simple centrifugation, evaporation of the supernatant and reconstitution in the 1D mobile phase. A mobile phase not compatible with the ESI source (10 mM KH 2 PO 4 at pH 2.7) was used in the first dimension as it provided a satisfactory chromatographic resolution of the peptides and a high buffering capacity avoiding changes in retention times when analyzing complex matrices like human serum. We also demonstrate that using coeluting labelled analogues of the target peptides, protein concentrations were not affected by slight retention time shifts affecting the amount of target peptides transferred to the second dimension. Satisfactory results were obtained when analyzing fortified serum samples (recoveries from 98 to 113%). Precisions in the range of 1-9% RSD were obtained when replicating the analysis of a pooled serum sample. The comparative analysis of serum samples from n = 94 control subjects and n = 91 patients diagnosed with primary open-angle glaucoma did not show significant differences in the APOA4, VTN and C3 concentrations in contrast with previous studies using immunoassays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

We have developed an analytical procedure to measure the carbon isotopic composition of multiple compounds even when there is a partial overlap in the chromatographic profiles and applied this procedure to measure the carbon isotopic composition of different metabolites in human urine and exhaled breath. Method development and validation was performed with CRM IAEA-600 caffeine after calibration of the reference CO 2 gas using a mixture of certified undecane, pentadecane and eicosane δ( 13 C) standards. The alternative data treatment procedure included the correction of time-lag between Faraday cup amplifiers (44 ms at mass 45 and -160 ms at mass 46), the calculation and correction of chromatographic isotope effects on each peak (isotope shifts) and the calculation of the isotope ratio for each compound using the linear regression slope procedure with data only at the top of the chromatographic peak. In that way, partial chromatographic overlap between different metabolites can be tolerated (resolution equal or higher than 1). The reproducibility (SD) of the carbon isotope composition of 93 metabolites in human urine (n = 8) from one volunteer was typically better than 0.5 δ( 13 C) (range 0.1-2.0 δ( 13 C), median 0.4 δ( 13 C)). The method was applied to follow the carbon isotope composition of different metabolites in human urine and exhaled breath after the oral administration of 100 mg of universally labelled 13 C-glucose to another human volunteer. It was demonstrated that isotopically labelled compounds could be detected in both samples even 2 h after administration. So, the developed methodology can be applied to multiple types of samples containing a large number of partially overlapping analytes including environmental applications, anti-doping control or metabolomics studies, including the use of enriched isotope tracers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Between November 19th, 2012 and December 3rd, 2012, 50 workers were intoxicated with gaseous Hg in San Juan de Nieva (Asturias, Spain) during the maintenance of a heat exchanger of a zinc manufacturer. We have quantified the concentration of methylmercury (MeHg), ethylmercury (EtHg) and Hg(II) in blood, hair and urine samples of those individuals taken three years after the accident. Blood, hair and urine of their closest relatives were also analyzed to assess whether the mercury burden present in the intoxicated individuals was due to the occupational exposure or to environmental or lifestyle-related factors. The determination of the mercury species in the samples was carried out applying multiple spiking Isotope Dilution GC-ICP-MS. This procedure corrects for possible interconversion reactions between the Hg species during the sample preparation procedure. Linear correlations were observed for both groups when plotting MeHg in blood vs MeHg in hair, and MeHg in hair vs Hg (II) in urine. The concentrations of Hg species in the intoxicated individuals were not significantly different from those obtained in the control group except for MeHg in blood. Significantly higher levels of MeHg in blood were obtained in some of the intoxicated individuals who had not consumed fish or seafood since the accident. A different correlation between MeHg in hair and MeHg in blood was obtained for these individuals compared to the control group who showed a hair-to-blood ratio consistent with the reported value for people exposed to Hg via fish consumption. Our results suggest that ingested MeHg followed the same pathway of deposition in hair in exposed and non-exposed individuals. However, the exposed individuals with high MeHg levels in blood showed a significantly different extent of MeHg deposition in hair compared to the control group., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mass spectrometry-based techniques, such as inductively coupled plasma mass spectrometry (ICPMS) and laser ablation (LA) ICPMS, combined with an isotope pattern deconvolution mathematical tool are proposed for a better understanding of supplementation studies in cultured cells. An in vitro model of human retinal pigment epithelium (HRPEsv) cells was treated with different concentrations (0-150 μm Zn, 1 mL) of enriched stable isotope tracers of Zn in the form of sulfate and/or gluconate. Supplementations with t68 ZnSO 4 or t70 Zn-gluconate alone and in combination (1:1 molar ratio) were investigated to evaluate the exogenous contribution and distribution of Zn in the treated cells. In order to obtain not only the Zn concentration for a cell population (mineralized cells) but also single cell information about the contribution of exogenous Zn and their distribution within micrometer cells structures, LA-ICPMS was employed to directly analyze cryopreserved cells. nat Zn, t68 Zn, and t70 Zn molar fraction images obtained from cells and cell aggregates allowed confirming the uptake of exogenous Zn by HRPEsv cells, being t68 Zn and t70 Zn molar fractions close to 1 in the cell nuclei. Under the selected experimental conditions tested (24 h treatments), no significant differences were obtained in the Zn distribution depending on its chemical form.

A current remaining challenge in nanotechnology is the fast and reliable determination of the ratios between engineered nanoparticles and the species attached to their surface after chemical functionalization. The approach proposed herein based on the online coupling of asymmetric flow field-flow fractionation (AF4) with inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) allows for the first time the direct determination of such ratios in CdSe/ZnS core-shell quantum dot:rat monoclonal IgG2a antibody (QD:Ab) conjugate mixtures in a single run without any previous sample preparation (i.e., derivatization). AF4 provides full recovery and adequate resolution of the resulting bioconjugate from the excess of nanoparticles and proteins used in the different bioconjugation mixtures (1:1, 2:1, and 3:1 QD:Ab molar ratios were assessed). The point-by-point determination by ICP-MS/MS of the metal to sulfur ratios along the bioconjugate fractographic peak allowed disclosing the mixture of the different species in the bioconjugated sample, providing not only the limits of the range of QD:Ab ratios in the different bioconjugate species resulting after functionalization but also a good estimation of their individual relative abundance in the mixture. Interestingly, a wide variety of compositions were observed for the different bioconjugate mixtures studied (QD:Ab molar ratios ranging from 0.27 to 4.6). The resulting weighted QD:Ab ratio computed in this way for each bioconjugate peak matches well with both the global (average) QD:Ab ratio experimentally obtained by the simpler peak area ratio computation and the theoretical QD:Ab molar ratios assayed, which internally validates the procedure developed.

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13 C 2 -testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g -1 . The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Dissolved inorganic carbon (DIC) is one of the most important parameters to be measured in seawaters for climate change studies. Its quantitative assessment requires analytical methodologies with overall uncertainties around 0.05% RSD for clear evaluation of temporal trends. Herein, two alternative isotope dilution mass spectrometry (IDMS) methodologies (online and species-specific) using an isotope ratio mass spectrometer (IRMS) and two calculation procedures for each methodology have been compared. As a result, a new method for the determination of DIC in seawaters, based on species-specific IDMS with isotope pattern deconvolution calculation, was developed and validated. A 13 C-enriched bicarbonate tracer was added to the sample and, after equilibration and acidification, the isotope abundances at CO 2 masses 44, 45, and 46 were measured on an IRMS instrument. Notably, early spiking allows correcting for evaporations and/or adsorptions during sample preparation and storage and could be carried out immediately after sampling. Full uncertainty budgets were calculated taking into account all the factors involved in the determination (initial weights, concentration and isotope abundances of standards, and final IRMS measurements). The average DIC value obtained for CRM seawater agreed very well with the certified value. Propagated precision obtained ranged from 0.035 to 0.050% RSD for individual sample triplicates. Reproducibility, assessed by three independent experiments carried out in different working days, was excellent as well (-0.01% and 0.057%, error and full combined uncertainty, respectively). Additionally, the approach proposed improves on established methods by simplicity, higher throughput (15 min per sample), and lower volume requirements (10 mL).

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We report the first common methodology for the simultaneous determination of methylmercury (MeHg), ethylmercury (EtHg), and inorganic mercury (Hg(II)) in human blood hair and urine. With the exception of the initial sample mass (0.15 g for blood, 0.5 g for urine, and 0.1 g for hair), the same sample preparation and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS) measurement conditions are employed for the three matrixes providing experimental values in agreement with the certified values in the analysis of NIST SRM 955c (Caprine Blood) Level 3 and the certified human hairs IAEA 085 and IAEA 086. Also, the method provides quantitative recoveries for the three Hg species in the analysis of fortified human urine samples at 1, 2, and 5 ng Hg g -1 . Mercury species concentrations for levels 2 and 4 of SRM 955c are reported here for the first time. A systematic interconversion of EtHg into Hg(II) was obtained for all matrixes reaching values up to 95% in blood, 29% in hair, and 11% in urine. MeHg dealkylation was also observed in a lesser extent in blood and hair analyses, but it was not observed when analyzing urine samples. Hg methylation was not observed in any matrix. The amount of NaBPr 4 added for derivatization has been found to be the main factor responsible for Hg species interconversion. This work demonstrates for the first time that experimental conditions optimized for SRM 955c (caprine blood) are not valid for human blood samples as the optimum initial sample amount for a real sample is more than 3 times lower than that for SRM 955c.

Although analysis of metals and metalloids, such as arsenic, is widely spread in many different fields, their analysis in gas and liquefied gas samples is still a challenge. A new GC-ICP-MS set up has been developed for their simultaneous total and speciation analysis in gas and liquefied gas samples without the need of a preconcentration step. An arsine in nitrogen standard was used for optimization and evaluation of the system. Good linearity and detection limits in the very low ppt level for both total and speciation analyses were found. Liquefied butane pressurized under nitrogen and doped with arsine and a propylene real sample from a cracker plant were analyzed using both external calibration and standard additions methods. The good match between both quantifying approaches demonstrated almost negligible matrix effects, even for the total analysis. Application of the approach to check repartition of volatile elements or species between gas and liquid phases was performed in the real propylene sample. Finally, its potential applicability for the simultaneous total and speciation analysis of other elements, such as Hg, was also proved.

This work describes a methodology based on multiple linear regression and GC-MS for the determination of molar fractions of isotopically-labeled intracellular metabolites in cell cultures. Novel aspects of this work are: i) the calculation of theoretical isotopic distributions of the different isotopologues from an experimentally measured value of % 13C enrichment of the labeled precursor ii) the calculation of the contribution of lack of mass resolution of the mass spectrometer and different fragmentation mechanism such as the loss or gain of hydrogen atoms in the EI source to measure the purity of the selected cluster for each metabolite and iii) the validation of the methodology not only by the analysis of gravimetrically prepared mixtures of isotopologues but also by the comparison of the obtained molar fractions with experimental values obtained by GC-Combustion-IRMS based on 13 C/ 12 C isotope ratio measurements. The method is able to measure molar fractions for twenty-eight intracellular metabolites derived from glucose metabolism in cell cultures grown in the presence of 13 C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of 13 C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of 13 C atoms of the labeled precursor. The proposed procedure was able to accurately quantify gravimetrically prepared mixtures of natural and labeled glucose molar fractions of 0.07% and mixtures of natural and labeled glycine at molar fractions down to 0.7%. The method was applied to initial studies of glucose metabolism of different prostate cancer cell lines., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 developed a novel, rapid and easy calculation procedure for Mass Isotopomer Distribution Analysis based on multiple linear regression which allows the simultaneous calculation of the precursor pool enrichment and the fraction of newly synthesized labelled proteins (fractional synthesis) using linear algebra. To test this approach, we used the peptide RGGGLK as a model tryptic peptide containing three subunits of glycine. We selected glycine labelled in two 13 C atoms ( 13 C 2 -glycine) as labelled amino acid to demonstrate that spectral overlap is not a problem in the proposed methodology. The developed methodology was tested first in vitro by changing the precursor pool enrichment from 10 to 40% of 13 C 2 -glycine. Secondly, a simulated in vivo synthesis of proteins was designed by combining the natural abundance RGGGLK peptide and 10 or 20% 13 C 2 -glycine at 1 : 1, 1 : 3 and 3 : 1 ratios. Precursor pool enrichments and fractional synthesis values were calculated with satisfactory precision and accuracy using a simple spreadsheet. This novel approach can provide a relatively rapid and easy means to measure protein turnover based on stable isotope tracers. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

We have found clear evidence of direct adsorption of mercury in human hair after the occupational exposure to mercury vapour. We have performed both longitudinal analysis of human hair by laser ablation ICP-MS and speciation analysis by gas chromatography ICP-MS in single hair strands of 5 individuals which were occupationally exposed to high levels of mercury vapour and showed acute mercury poisoning symptoms. Hair samples, between 3.5 and 11cm long depending on the individual, were taken ca. three months after exposure. Single point laser ablation samples of 50μm diameter were taken at 1mm intervals starting from the root of the hairs. Sulfur-34 was used as internal standard. The ratio (202)Hg/(34)S showed a distinct pattern of mercury concentration with much lower levels of mercury near the root of the hair and high levels of mercury near the end of the hair. In all cases a big jump in the concentration of mercury in hair occurred at a given distance from the root, between 32 and 42mm depending on the individual, with a high and almost constant concentration of mercury for longer distances to the root. When we took into account the rate of hair growth in humans, 9-15mm/month, the jump in mercury concentration agreed approximately with the dates when the contamination occurred with the new growing hair showing much lower mercury concentration. In some cases the concentration of mercury at the tip of the hair was ca. 1000 times higher than that near the root. Additionally, speciation studies confirmed that mercury in all hair samples was present as inorganic mercury. The only explanation for these results was the direct adsorption of mercury vapour in hair at the time of exposure., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The degradation of butyltin compounds in surface water samples under different storage conditions has been studied. A triple spike solution, containing monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) labelled with a different tin isotope, was added to the sample to calculate the extent of the interconversion reactions among butyltin compounds. Real surface water samples (river water) were collected and stored in glass, polypropylene or polytetrafluoroethylene (PTFE) containers. The presence of light, addition of acetic acid, storage temperature (22, 4 or -18 °C), and the influence of a filtration step were evaluated. Moreover, Milli-Q water with and without the addition of a high concentration of humic acids was prepared in parallel and the results compared to those obtained from the real samples. The water samples were analysed by gas chromatography-tandem mass spectrometry (GC-MS/MS) in selected reaction monitoring (SRM) mode at two different storage times (2 weeks and 4 months after its preparation) to carry out both a short- and a long-term stability study. The lowest butyltin degradation was obtained when the samples were stored at -18 °C in the dark. Under these conditions, both TBT and DBT showed negligible dealkylation factors after 2 weeks. After 4 months, DBT dealkylation to MBT increased up to 19 % but TBT degradation was not observed.

The current EU legislation lays down the Environmental Quality Standards (EQS) of 45 priority substances in surface water bodies. In particular, the concentration of tributyltin (TBT) must not exceed 0.2ngL(-1) and analytical methodologies with a Limit of Quantification (LOQ) equal or below 0.06ngL(-1) are urged to be developed. This work presents a procedure for the determination of ultratrace levels of TBT in water samples by Isotope Dilution and GC-MS/MS operating in Selected Reaction Monitoring (SRM) mode which meets current EU requirements. The method requires the monitorization of five consecutive transitions (287>175 to 291>179) for the sensitive and selective detection of TBT. The measured isotopic distribution of TBT fragment ions was in agreement with the theoretical values computed by a polynomial expansion algorithm. The combined use of Tandem Mass Spectrometry, a sample volume of 250mL, the preconcentration of 1mL of organic phase to 30μL and an injection volume of 25μL by Programmed Temperature Vaporization provided a LOQ of 0.0426ngL(-1) for TBT (calculated as ten times the standard deviation of nine independent blanks). The recovery for TBT calculated in Milli-Q water at the EQS level was 106.3±4%. A similar procedure was also developed for the quantification of dibutyltin (DBT) and monobutyltin (MBT) in water samples showing satisfactory results. The method was finally implemented in a routine testing laboratory to demonstrate its applicability to real samples obtaining quantitative recoveries for TBT at the EQS level in mineral water, river water and seawater., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We describe the instrumental modification of a commercial gas chromatography isotope ratio mass spectrometer (GC-IRMS) and its application for on-line carbon isotope dilution. The main modification consisted in the addition of a constant flow of enriched (13)CO2 diluted in helium after the chromatographic column through the splitter holder located inside the chromatographic oven of the instrument. In addition, and in contrast to the conventional mode of operation of GC-IRMS instruments where the signal at m/z 45 is amplified 100-fold with respect to the signal at m/z 44, the same signal amplification was used in both Faraday cups at m/z 44 and 45. Under these conditions isotope ratio precision for the ratio 44/45 was around 0.05% RSD (n=50). The evaluation of the instrument was performed with mixtures of organic compounds including 11 n-alkanes, 16 PAHs, 12 PCBs and 3 benzothiophenes. It was observed that compounds of very different boiling points could be analysed without discrimination in the injector when a Programmable Temperature Vaporizer (PTV) injector was employed. Moreover, the presence of heteroatoms (Cl or S) in the structure of the organic compounds did not affect their combustion efficiency and therefore the trueness of the results. Quantitative results obtained for all the analytes assayed were excellent in terms of precision (<3% RSD) and accuracy (average relative error≤4%) and what is more important using a single and simple generic internal standard for quantification., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aims of the work were to detect and quantify hexavalent chromium in 14 soil samples from an area in Lombardia (northern Italy) contaminated by two polluted water plumes. Cr(VI) was extracted from the solid samples by applying focused microwaves in an alkaline medium after Cr(III) complexation with EDTA. Cr(VI) was reduced to Cr(III) when previously reported extraction conditions for the analysis of certified reference materials were used, and Cr(VI) could not be reliably quantified in the soil samples. The influence of organic matter and iron contents in the samples on the reduction of Cr(VI) was subsequently studied using a new set of soil samples with different iron and organic matter concentrations. Isotope dilution mass spectrometry (IDMS) measured two different enriched stable isotopes of Cr (54 and 53) to evaluate the reduction extent of hexavalent chromium during the analytical procedure. The extraction conditions were optimized to obtain the lowest amount of Cr(VI) reduction and quantify Cr(VI) in the polluted soil samples from Lombardia.

This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

We propose a peptide-based isotope dilution mass spectrometry approach for Cystatin C determination in human serum samples, a clinical marker for renal status for which backup by a mass spectrometry based primary method has been missing so far. In contrast to common protocols, the isotope labelled version of the proteotypic signature peptide is designed such as keeping the isotopic difference as little as possible with respect to the peptide released from the protein. Peptides labelled in two (13)C atoms are added to the serum samples just before proteolysis. After two steps of chromatographic purification the sample is measured by selected reaction monitoring using a LC-MS/MS. Resolution of the first quadrupole is reduced to transmit the whole parent ion cluster to the collision cell for monitoring accurate isotopic distributions of the molecular fragments. Molar fractions of labelled and natural abundance peptides are directly obtained from the experimental mass spectra of the in-cell fragment ions. Thus, the natural abundance protein concentration is obtained from the fragment-ion spectrum of the sample without resorting to extra calibration runs. Applicability of the approach is demonstrated by the measurement of the serum concentration of Cystatin C in Reference Material ERM R-DA471/IFCC and real samples., Biological Significance: Cystatin C is used as an alternative marker instead of, or in combination with creatinine for non-invasive determination of glomerular filtration rates. Advantages advocating in favour of Cystatin C in diagnosis of chronic kidney diseases are the lower variability of its serum level and, particularly, virtual independence on sex, age and muscle mass. However, in order to capitalize, accuracy of measurement has to be in proportion with the predictive power of the marker. Though there are label-free methods available for screening purposes or high-throughput analysis, achieving high levels of reliability and accuracy in quantitative proteomics takes reference to isotope labelled materials. Present routine assays (mainly nephelometry, turbidimetry and ligand-binding assays) are known to leave improvement to be desired in that respect. Absolute quantification based on enzymatic signature-peptides provides a method principle establishing traceability to the International System of Units on the level of primary methods. The kind of technique is capable, by this way, of high accuracy value-assignment to matrix materials needed for calibration of present routine assays, where not completely replacing them. Cystatin C measurement by isotope dilution mass spectrometry is developed in this study with the aim of making available this tool to support diagnostics of kidney function in the same way., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Seven β 2 -agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC-ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally 13 C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg -1 levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg -1 ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg -1 for all compounds, except to salbutamol in bovine urine which was of 0.029ngg -1 . The use of compound-specific isotopically labelled analogues minimally labelled in 13 C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of β 2 -agonists in human and bovine urine., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report on the determination of trace elements in solid samples by the combination of on-line double isotope dilution and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The proposed method requires the sequential analysis of the sample and a certified natural abundance standard by on-line IDMS using the same isotopically-enriched spike solution. In this way, the mass fraction of the analyte in the sample can be directly referred to the certified standard so the previous characterization of the spike solution is not required. To validate the procedure, Sr, Rb and Pb were determined in certified reference materials with different matrices, including silicate glasses (SRM 610, 612 and 614) and powdered samples (PACS-2, SRM 2710a, SRM 1944, SRM 2702 and SRM 2780). The analysis of powdered samples was carried out both by the preparation of pressed pellets and by lithium borate fusion. Experimental results for the analysis of powdered samples were in agreement with the certified values for all materials. Relative standard deviations in the range of 6-21% for pressed pellets and 3-21% for fused solids were obtained from n=3 independent measurements. Minimal sample preparation, data treatment and consumption of the isotopically-enriched isotopes are the main advantages of the method over previously reported approaches., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We present a novel method for the purity assessment of peptide standards which is applicable to any water soluble peptide. The method is based on the online (13)C isotope dilution approach in which the peptide is separated from its related impurities by liquid chromatography (LC) and the eluent is mixed post-column with a continuous flow of (13)C-enriched sodium bicarbonate. An online oxidation step using sodium persulfate in acidic media at 99°C provides quantitative oxidation to (12)CO2 and (13)CO2 respectively which is extracted to a gaseous phase with the help of a gas permeable membrane. The measurement of the isotope ratio 44/45 in the mass spectrometer allows the construction of the mass flow chromatogram. As the only species that is finally measured in the mass spectrometer is CO2, the peptide content in the standard can be quantified, on the base of its carbon content, using a generic primary standard such as potassium hydrogen phthalate. The approach was validated by the analysis of a reference material (NIST 8327), and applied to the quantification of two commercial synthetic peptide standards. In that case, the results obtained were compared with those obtained using alternative methods, such as amino acid analysis and ICP-MS. The results obtained proved the value of the method for the fast, accurate and precise mass purity assignment of synthetic peptide standards., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A general method for the determination of the enrichment of isotopically labelled molecules by mass spectrometry (MS) is described. In contrast to other published procedures, the method described here takes into account and corrects for measurement errors such as the contribution at M - 1 due to loss of hydrogen or lack of spectral resolution and provides an uncertainty value for the determined enrichment. The general procedure requires the following steps: (1) evaluation of linearity in the mass spectrometer by injecting the natural abundance compound at different concentration levels, (2) determination of the purity of the mass cluster using the natural abundance analogue, (3) calculation of the theoretical isotope composition of the labelled compound using different tentative isotope enrichments, (4) calculation of 'convoluted' isotope distributions for the labelled compound taking into account the purity of the mass cluster determined with the natural abundance analogue and (5) comparison of the isotope distributions measured for the labelled compound with those calculated for different isotope enrichments using linear regression. The method was applied to a series of commercially available (13)C- and (2)H-labelled compounds and to a suite of singly (13)C-labelled β2-agonist prepared in-house both by gas chromatography (GC)-MS, GC-tandem MS (MS/MS) and liquid chromatography-MS/MS with satisfactory results. It was observed that the main uncertainty source for the isotope enrichment was the uncertainty in the purity of the measured cluster as determined with the natural abundance compound., (Copyright © 2014 John Wiley & Sons, Ltd.)

The accurate determination of creatinine in serum is essential for the diagnosis and treatment of kidney diseases. The determination of serum creatinine in clinical laboratories is routinely carried out by the Jaffe method or by enzymatic methods that may suffer from interferences. So, the development of reliable, fast and interference-free routine methods for complex serum samples is required. A fast method using isotope dilution mass spectrometry (IDMS) and gas chromatography mass spectrometry (GC-MS) was developed using minimally (13)C labeled creatinine analogs, multiple linear regression and rapid derivatization of creatinine with acetylacetone in 2 min by using focused microwave technology. The proposed method was validated with the analyses of two Certified Reference Materials (ERM-DA252a and ERM-DA253a) and compared with the Jaffe and enzymatic methods when analyzing real serum samples containing variable levels of bilirubin The proposed method is capable of providing accurate serum creatinine concentrations in less than 45 min from sample arrival to full data treatment and can be an alternative routine procedure for creatinine determinations in complex serum samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of β2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different β2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC-ESI-MS in complex samples. In this procedure singly (13)C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of β2-agonists in human urine and bovine liver, even at the sub ngg(-1) and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97-114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of (13)C1-labeled standards and IPD is a reliable approach for accurate LC-MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study evaluates the use of an individual-specific transgenerational barium dual-isotope procedure and its application to salmon specimens from the Sella River (Asturias, Spain). For such a purpose, the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in combination with multiple linear regression for the determination of the isotopic mark in the otoliths of the specimens is presented. In this sense, a solution in which two barium-enriched isotopes ((137)Ba and (135)Ba) were mixed at a molar ratio of ca. 1:3 (N Ba137/N Ba135) was administered to eight returning females caught during the spawning period. After injection, these females, as well as their offspring, were reared in a governmental hatchery located in the council of Cangas de Onís (Asturias, Spain). For comparison purposes, as well as for a time-monitoring control, egg and larva data obtained by solution analysis ICP-MS are also given. Otoliths (9-month-old juveniles) of marked offspring were analysed by LA-ICP-MS demonstrating a 100 % marking efficacy of this methodology. The capabilities of the molar fraction approach for 2D imaging of fish otoliths are also addressed.

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.

Isotope dilution mass spectrometry (IDMS) based on isotope pattern deconvolution (IPD) has been applied here to MS/MS (QqQ) in order to carry out the quantification and confirmation of organic compounds in complex matrix water samples without the use of a methodological IDMS calibration graph. In this alternative approach, the isotope composition of the spiked sample is measured after fragmentation by SRM and deconvoluted into its constituting components (molar fractions of natural abundance and labeled compound) by multiple linear regression (IPD). The procedure has been evaluated for the determination of the pharmaceutical diclofenac in effluent and influent urban wastewaters and fortified surface waters by UHPLC (ESI) MS/MS using diclofenac-d(4) as labeled compound. Calculations were performed acquiring a part and the whole fragment cluster ion, achieving in all cases recoveries within 90-110% and coefficients of variation below 5% for all water samples tested. In addition, potential false negatives arising from the presence of diclofenac-d(2) impurities in the labeled compound were avoided when the proposed approach was used instead of the most usual IDMS calibration procedure. The number of SRM transitions measured was minimized to three to make possible the application of this alternative technique in routine multi-residue analysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We present here a new environmental measurement method for the rapid extraction and accurate quantification of Cr(VI) in solid samples. The quantitative extraction of Cr(VI) is achieved in 10 minutes by means of focused microwave assisted extraction using 50 mmol/L Ethylendiamintetraacetic acid (EDTA) at pH 10 as extractant. In addition, it enables the separation of Cr species by anion exchange chromatography using a mobile phase which is a 1:10 dilution of the extracting solution. Thus, neutralization or acidification steps which are prone to cause interconversion of Cr species are not needed. Another benefit of using EDTA is that it allows to measure Cr(III)-EDTA complex and Cr(VI) simultaneously in an alkaline extraction solution. The application of a 10 minutes focused microwave assisted extraction (5 min at 90 °C plus 5 min at 110 °C) has been shown to quantitatively extract all forms of hexavalent chromium from the standard reference materials (SRM) candidate NIST 2700 and NIST 2701. A double spike isotope dilution mass spectrometry (IDMS) procedure was employed to study chromium interconversion reactions. It was observed that the formation of a Cr(III)-EDTA complex avoided Cr(III) oxidation for these two reference materials. Thus, the use of a double spiking strategy for quantification is not required and a single spike IDMS procedure using isotopically enriched Cr(VI) provided accurate results.

A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single (13)C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d(9)-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g(-1)) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized (13)C(1)-clenbuterol and a commercially available d(9)-clenbuterol. The detection limit of the method in human urine was 0.050 ng g(-1) with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the (13)C(1)-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD.

Background: During the last decade, environmental concerns regarding the use of recalcitrant synthetic chelates to overcome iron chlorosis has increased and new ligands such as lignosulfonates (LS) have been evaluated. However, the efficacy of these products is variable. In this work a hardwood (eucalyptus) and softwood (spruce) LS were compared to try to relate their physico-chemical characteristics and their efficacy. Also two more products derived from the eucalyptus lignosulfonate were tested., Results: All the LS tested presented a good ability to complex Fe, but only the spruce LS was capable to maintain significant amounts of soluble Fe above pH 8. According to the FTIR data, structural changes related to the Fe source (Fe(2+) or Fe(3+) ) used to form the complex occurred in the LS molecule and might influence their efficacy. Cucumber (Cucumis sativus L. cv Ashley) chlorotic plants were used to test lignosulfonate efficacy when applied through foliar sprays in comparison with FeSO(4) and EDTA/(57) Fe(3+) . The (57) Fe content of plants sprayed with LS was very low in respect to the EDTA treatment, but this was not reflected in the biomass and re-greening rates. Eucalyptus LS modifications improve its efficacy for iron chlorosis recovery to levels similar to those found for the spruce LS. Two applications of the LS are recommended., Conclusions: Lignosulfonates did not require surfactants for their application; they did not burn the leaves, and had a stimulating effect on the vegetative growth of the plants. So these by-products could be a good alternative when applied through foliar sprays for cucumber plants., (2010 Society of Chemical Industry.)

Imposex and butyltin burden were assessed in Nassarius nitidus, Bolinus brandaris and Hexaplex trunculus collected at five stations in the Bizerta lagoon. Biological analysis showed that imposex followed type (a) in N. nitidus (distal evolution), against type (d) in the two muricids (proximal evolution). Imposex indices were higher in sites located nearby sources of tributyltin and N. nitidus was the least affected species of the five sites, followed by B. brandaris and H. trunculus. Butyltin analysis showed lower accumulation in N. nitidus followed by H. trunculus and B. brandaris. This study has allowed the gathering of data on imposex in a snail studied for the first time in Tunisia (N. nitidus). It suggests the possibility of using such snail as a complementary species for organotin monitoring programs in the Mediterranean and further confirmed that H. trunculus is the most suitable species for such investigations.

A dual-stable isotope tracer experiment was carried out with Fe-deficient sugar beet plants grown hydroponically and resupplied with differentially Fe labeled racemic and meso Fe(iii)-chelates of the ethylendiamine di(o-hydroxyphenylacetic) acid (o,oEDDHA). No short-term Fe isotope exchange reactions occurred in the nutrient solution and plants did not discriminate between (54)Fe and (57)Fe. After 3-6 h, stable Fe isotopes, chelating agents and chelates were analyzed in roots, xylem sap and leaves by ICP-MS and HPLC-ESI/TOFMS. Ferric chelate reductase rates, xylem transport and total uptake were 2-fold higher with the meso isomer than with the racemic one. Both chelating agent isomers were incorporated and distributed by plants at similar rates, in amounts one order of magnitude lower than those of Fe. After 6 h of Fe resupply, most of the Fe acquired was localized in roots, whereas most of the chelating agent was in leaves. In a separate experiment, Fe-deficient sugar beet and tomato plants were treated with different concentrations of Fe(iii)-o,oEDDHA (with a meso/racemic ratio of 1). The xylem sap Fe concentration at 24 h was unaffected by the chelate concentration, with xylem Fe(iii)-o,oEDDHA accounting for 1-18% of total Fe and xylem meso/racemic ratio close to 1. Although most of the Fe coming from Fe(iii)-o,oEDDHA was taken up through a reductive dissociative mechanism, a small part of the Fe may be taken up via non-dissociative mechanisms.

A quadrupole GC-MS instrument with an electron ionization (EI) source has been modified to enable application of postcolumn isotope dilution analysis for the standardless quantification of organic compounds injected in the gas chromatograph. Instrumental modifications included the quantitative conversion of the separated compounds into CO(2), using a postcolumn combustion furnace, and the subsequent mixing of the gas with a constant flow of (13)CO(2) diluted in helium. The online measurement of the (12)CO(2)/(13)CO(2) (44/45) ratio in the EI-MS allowed us to obtain quantitative data without resorting to compound-specific standards. Validation of the procedure involved the analysis of standard solutions containing different families of organic compounds (C(9)-C(20) linear hydrocarbons, BTEX and esters) obtaining satisfactory results in all cases in terms of absolute errors (<6%) and precision (<4% RSD). The developed procedure showed excellent linearity over the range assayed (2 orders of magnitude) and adequate detection limits for carbon containing compounds (0.8 pg C s(-1)). The generic value of this compound-independent calibration approach was assessed by studying the quantitative performance of Head Space-Solid Phase Microextraction (HS-SPME). The proposed compound-independent quantification by EI-MS permits comparison of the performance of different fibers by assessing analyte recoveries with extreme robustness, simplicity, and precision.

Asialo-Transferrin (S(0)) or beta(2)-Transferrin is an accepted marker of cerebrospinal fluid (CSF) leakage from the subarachnoid space into the nasal or aural cavity as the result of a head trauma. At this time, the S(0) detection method of choice is isoelectric focusing on polyacrylamide gel with direct immunofixation of transferrin (Tf) and silverstaining which are time-consuming techniques (> 5 h). The aim of this study is to present an alternative methodology for determination of Tf sialoforms present in CSF samples, specifically S(0), based on the detection of the Fe associated with those forms. A double spiking methodology is developed by saturating the protein with isotopically enriched iron ((57)Fe) and conducting post-column Isotope Dilution Analysis (IDA) with (54)Fe after separation of the different sialoforms by anion exchange chromatography. The results obtained have been validated using a certified serum (ERM-DA470) and applied with satisfactory results to a pooled CSF sample.

A procedure for Stable Isotope Dilution Analysis in molecular Mass Spectrometry which does not require a methodological calibration graph and can be applied in combination with minimal labelling has been evaluated. This alternative approach is based on the determination of the molar fractions for each pure isotope pattern (natural abundance or labelled) contributing to the isotope pattern observed in the mixture of natural abundance and labelled molecules by multiple linear regression. Two labelled compounds, (13)C(1)-labelled or (13)C(6)-labelled phenol, were compared to study the influence of the number of (13)C atoms in the labelled molecule. The procedure was evaluated by comparing the results obtained for the determination of phenol in NIST 1584 CRM by GC-EI-MS using the classical isotope dilution calibration procedure and the new procedure based on multiple linear regression of isotope patterns without a calibration graph. The results obtained using the proposed procedure agreed well with both the certified values and those obtained using the classical Isotope Dilution Mass Spectrometry (IDMS) calibration procedures. For the evaluated procedure, a full uncertainty budget determination has been developed taking into account all uncertainty sources, including those derived from the uncertainties in the isotope patterns of the natural and labelled compounds. The measurements with the (13)C(1)-labelled phenol provided lower propagated uncertainties in comparison to the use of (13)C(6)-labelled phenol.

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.

This work demonstrates, for the first time, the applicability of multiple spiking isotope dilution analysis to molecular mass spectrometry exemplified by the speciation analysis of mercury using GC(EI)MS instrumentation. A double spike isotope dilution approach using isotopically enriched mercury isotopes has been applied for the determination of inorganic mercury Hg(II) and methylmercury (MeHg) in fish reference materials. The method is based on the application of isotope pattern deconvolution for the simultaneous determination of degradation-corrected concentrations of methylmercury and inorganic mercury. Mass isotopomer distributions are employed instead of isotope ratios to calculate the corrected concentrations of the Hg species as well as the extent of species degradation reactions. The isotope pattern deconvolution equations developed here allow the calculation of the different molar fractions directly from the GC(EI)MS mass isotopomer distribution pattern and take into account possible impurities present in the spike solutions employed. The procedure has been successfully validated with the analysis of two different certified reference materials (BCR-464 and DOLT-4) and with the comparison of the results obtained by GC(ICP)MS. For the tuna fish matrix (BCR-464), no interconversion reactions were observed at the optimized conditions of open focused microwave extraction at 70 degrees C during 8 min. However, significant demethylation was found under the same conditions in the case of the certified dogfish liver DOLT-4. Methylation and demethylation factors were confirmed by GC(ICP)MS. Transformation reactions have been found to depend on the sample matrix and on the derivatization reagent employed. Thus, it is not possible to recommend optimum extraction conditions suitable for all types of matrices demonstrating the need to apply multiple spiking methodologies for the determination of MeHg and Hg(II) in biological samples. Double spike isotope dilution analysis methodologies using widespread GC(EI)MS instrumentation are proposed here for the routine analysis of inorganic mercury and methylmercury in fish samples. The estimated method detection limits were below 10 ng g(-1) for both mercury species. Precision was evaluated for the concentrations present in the certified reference materials (CRMs) which vary from 0.1 to 5 microg g(-1), achieving values of coefficients of variation ranging from 7% to 2%. The concentrations obtained in both CRMs analyzed were in excellent agreement with the certified values, demonstrating the accuracy of the method at these concentration levels.

A procedure for the determination of the uncertainties in the theoretical mass isotopomer distribution of molecules due to natural variations in the isotope composition of their constituting elements is described here for the first time. For this purpose, a Visual Basic macro for Microsoft Excel was written by adapting the direct stepwise calculation algorithm published by Kubinyi (Anal. Chim. Acta 1991, 247, 107-119, Fig. 1). In our procedure no pruning threshold factors were used to eliminate round up errors for large molecules. Then, the Kragten procedure of uncertainty propagation (Analyst 1994, 119, 2161-2165) was applied taking into account the correlation coefficients between the isotope abundances of the corresponding atoms. For bi-isotopic elements (C, H, N, Cl, Br) the correlation coefficients were given the value of -1. For tri- and tetra-isotopic elements the correlation coefficients were calculated using the mass dependent fractionation law used in stable isotope geochemistry and values of +1 or -1 were obtained depending on the isotope system considered. It was observed that for small organic molecules of natural isotope abundances, such as phenol or polybrominated diphenylethers, the method provided relatively small propagated uncertainties similar in magnitude to those measured experimentally. For (13)C-labelled molecules the calculated uncertainties were mainly due to the uncertainties in the isotope enrichment of (13)C and were much larger than the experimental uncertainties. For large molecules of natural isotope abundances, such as peptide C(68)H(107)N(17)O(25) (NIST 8327 RM), the uncertainties in their mass isotopomer distributions were much larger and their source could be assigned mainly to the uncertainty of the natural isotope composition of carbon. When the size of the molecule was even larger, such as bovine insulin (C(254)H(377)N(65)O(75)S(6)), Kragten procedure provided a good estimate for the uncertainty when the most probable isotope composition of carbon in mammals was used in the calculations., (2010 Elsevier B.V. All rights reserved.)

The identification of Fe transport forms in plant xylem sap is crucial to the understanding of long-distance Fe transport processes in plants. Previous studies have proposed that Fe may be transported as an Fe-citrate complex in plant xylem sap, but such a complex has never been detected. In this study we report the first direct and unequivocal identification of a natural Fe complex in plant xylem sap. A tri-Fe(III), tri-citrate complex (Fe(3)Cit(3)) was found in the xylem sap of Fe-deficient tomato (Solanum lycopersicum Mill. cv. 'Tres Cantos') resupplied with Fe, by using an integrated mass spectrometry approach based on exact molecular mass, isotopic signature and Fe determination and retention time. This complex has been modeled as having an oxo-bridged tri-Fe core. A second complex, a di-Fe(III), di-citrate complex was also detected in Fe-citrate standards along with Fe(3)Cit(3), with the allocation of Fe between the two complexes depending on the Fe to citrate ratio. These results provide evidence for Fe-citrate complex xylem transport in plants. The consequences for the role of Fe to citrate ratio in long-distance transport of Fe in xylem are also discussed.

Levels of imposex (superimposition of male characters, upon females) and the presence of sterile females are assessed in the gastropod Nassarius nitidus (Jeffreys, 1867), at 22 locations in the Basque Country (northern Spain). At 18 of these localities, butyltin bioaccumulation (tributyltin (TBT); dibutyltin; monobutyltin) was analysed using isotope dilution and GC-ICP-MS. Higher imposex levels and TBT body burden were found in confined harbours, with a large vessel traffic or the presence of a fishing fleet or a shipyard. For the first time, four apparently sequential types of aborted capsules are described in this species. Another novelty is the interspecific comparison between imposex intensities in sympatrically living populations of N. nitidus and N. reticulatus. This showed that sensitivity to TBT pollution of both species is relatively similar (in terms of Relative Penis Length Index). Since N. nitidus has a restricted habitat distribution due to its low presence in wave-exposed habitats, its complementary use with other species is recommended for its use in TBT monitoring programmes.