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We evaluate here different analytical strategies for the chromatographic separation and determination of N-acetyl-5-methoxytryptamine (MEL) and its oxidative metabolites N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine (AMK) and cyclic 3-hydroxymelatonin (c3OHM) in cell culture samples. Two dimensional liquid chromatography (2D-LC) in the multiple heart-cutting mode was compared with regular 1D chromatographic separations of MEL and its oxidative metabolites. Our results showed that the use of trifluoroacetic acid (TFA) as mobile phase modifier was required to obtain a satisfactory resolution and peak shapes particularly for c3OHM. As TFA is not compatible with ESI ionization the application of the MHC mode was mandatory for a proper chromatographic separation. We evaluate also different internal standardization approaches based on the combined use of a surrogate standard (5-methoxytryptophol) and an internal standard (6-methoxytryptamine) for MEL quantification in cell culture samples obtaining unsatisfactory results both by 1D- and 2D-LC-ESI-MS/MS (from 9 ± 2 to 186 ± 38%). We demonstrate that only the application of isotope dilution Mass Spectrometry through the use of an in house synthesized

Spanish Ministry of Science and Innovation through Project [PGC2018-097961-B-I00]

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 KH2PO4 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.

A procedure for the determination of Pb isotope ratios by coupling liquid chromatography to a multicollector ICP-MS has been developed. The procedure allows the direct injection of samples after dissolution without resorting to time-consuming off-line separation procedures. The separation of Pb from concomitant elements is carried out by anionic exchange as ethylenediamine tetraacetic acid (EDTA) chelates using EDTA and ammonium nitrate as a mobile phase. A flow injection system allows the injection of NIST 981 Pb isotopic standard, before and after the Pb peak from the sample, and the on-line addition of Tl for mass bias correction and bracketing. The procedure was validated by injecting NIST 981 into the chromatographic system and by comparing the results for real samples with the classical off-line separation procedure using Pb spec resins. The optimised procedure was applied to archaeological samples containing different concentrations of Pb. It was observed that the only limitation to the accuracy of the procedure was the concentration of Pb in the samples as no preconcentration is performed. Solid archaeological samples containing at least 500 μg g−1 of Pb can be studied using the proposed procedure.

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 13C-enriched bicarbonate tracer was added to the sample and, after equilibration and acidification, the isotope abundances at CO2 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).

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 11 cm long depending on the individual, were taken ca. three months after exposure. Single point laser ablation samples of 50 μm diameter were taken at 1 mm intervals starting from the root of the hairs. Sulfur-34 was used as internal standard. The ratio 202Hg/34S 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 42 mm 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, 9–15 mm/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.

This work presents the development of a methodology for the accurate and precise quantification of the renal biomarker Cystatin C in human urine by Isotope Dilution Mass Spectrometry (IDMS). The procedure is based on the addition of a known quantity of the proteotypic peptide ALDFAVG*EYNK labelled with 13C2-glycine to the urine sample followed by protein hydrolysis using trypsin. Then, preconcentration and purification of the isotope diluted peptide was carried out by a selective monoclonal antibody bound to magnetic beads and final measurement was done after injection of the sample in a HPLC-MS/MS triple quadrupole instrument. The isotopic distribution of the isotope diluted proteotypic peptide was measured by low resolution selected reaction monitoring. Using this aquisition mode, the bandpass of the first quadrupole was widened (FWHM =13 u) so the whole isotopic clusters for both the natural abundance and the labelled peptides entered the collision cell. The proposed acquisition mode provided similar accuracy and precision than the regular SRM mode (FWHM =0.7 u) but a higher sensitivity was observed. The purification of the sample by antibody based enrichment of the target peptide was shown to remove interfering compounds more efficiently in comparison with a sample purification based on semipreparative liquid chromatography. Using 5 ng of the labelled peptide it was possible to quantify Cystatin C in human urine in patients with normal and impaired renal function. Recoveries from 100 to 104% were obtained in samples containing from 90 to 700 μg L−1 of Cystatin C with relative standard deviations from 0.5 to 6%. The stability of Cystatin C in urine samples was evaluated under different storage conditions showing that only when the urine samples were stored at room temperature during more than 10 days, a significant degradation of Cystatin C was observed.

The spectral interference of 87Rb on 87Sr for the measurement of 87Sr/86Sr isotope ratios in solid samples by laser ablation multicollector (MC)-ICP-MS could be corrected by combining the transient signal of the sample with a Rb pulse from the nebulisation of a pure Rb standard. This dual sample introduction system, combined with a multiple linear regression data treatment procedure, allowed the interference free measurement of the 87Sr/86Sr isotope ratio in solid samples. Additionally, when the signals for Rb and Sr could be separated in time, even only partially, the application of the multiple linear regression procedure allowed us to compute also the 87Sr/86Sr isotope ratio free of spectral interference. The fact that Sr was concentrated in the seed coat of beans, whereas Rb was mainly present in the cotyledon, allowed us to develop a laser ablation multicollector ICP-MS procedure for the authentication of Asturian beans which are subject to fraud with the import of South American beans. By combining the 87Sr/86Sr isotope ratio with the 88Sr/85Rb elemental ratio we were able to distinguish between Asturian and South American beans in spite of the large 87Sr/86Sr isotopic variability of Asturian beans.

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 13C/12C 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 13C-labeled Glucose. The validation strategies demonstrate a satisfactory accuracy and precision of the proposed procedure. Also, our results show that the minimum value of 13C incorporation that can be accurately quantified is significantly influenced by the calculation of the spectral purity of the measured cluster and the number of 13C 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.

The monastery of San Andres de Arroyo (established in 1181) is one the best examples of Cistercian architecture in the Spanish region of Castilla y Leon. In this study, strontium isotope ratios were used to study the population of the recently excavated graveyard of this monastery. Twenty-nine individuals (of which 13 had preserved teeth) found in burials from the 13th and 14th centuries were subjected to analysis. Enamel 87Sr/86Sr ratio values obtained by using a multicollector ICP-MS instrument were used to distinguish between local and non-local individuals. Additionally, an anthropological study based on entheseal changes and other paleopathological conditions was carried out in all the exhumed individuals. This combined data allowed us to define the chemical and osteological diversity of a medieval Spanish monastery and its surrounding village. Among the usual profiles for a rural society, two individuals, both showing strong indications of being foreign to the area, were found to exhibit evidence of physical activities compatible with military training and activity. The present study can be considered as the first experimental indication that during the 13th and 14th centuries Spanish monasteries served as a last refuge for soldiers, which is compatible with historical records and previous indirect evidence.

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.

The analytical methodology for the in vivo study of selenium metabolism using two enriched selenium isotopes has been modified, allowing for the internal correction of spectral interferences and mass bias both for total selenium and speciation analysis. The method is based on the combination of an already described dual-isotope procedure with a new data treatment strategy based on multiple linear regression. A metabolic enriched isotope ((77)Se) is given orally to the test subject and a second isotope ((74)Se) is employed for quantification. In our approach, all possible polyatomic interferences occurring in the measurement of the isotope composition of selenium by collision cell quadrupole ICP-MS are taken into account and their relative contribution calculated by multiple linear regression after minimisation of the residuals. As a result, all spectral interferences and mass bias are corrected internally allowing the fast and independent quantification of natural abundance selenium ((nat)Se) and enriched (77)Se. In this sense, the calculation of the tracer/tracee ratio in each sample is straightforward. The method has been applied to study the time-related tissue incorporation of (77)Se in male Wistar rats while maintaining the (nat)Se steady-state conditions. Additionally, metabolically relevant information such as selenoprotein synthesis and selenium elimination in urine could be studied using the proposed methodology. In this case, serum proteins were separated by affinity chromatography while reverse phase was employed for urine metabolites. In both cases, (74)Se was used as a post-column isotope dilution spike. The application of multiple linear regression to the whole chromatogram allowed us to calculate the contribution of bromine hydride, selenium hydride, argon polyatomics and mass bias on the observed selenium isotope patterns. By minimising the square sum of residuals for the whole chromatogram, internal correction of spectral interferences and mass bias could be accomplished. As a result, the tracer/tracee ratio could be calculated for each selenium-containing species and a time relationship for synthesis and degradation established. Both selenite and selenized yeast labelled with (77)Se were employed for comparative purposes.

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 method has been developed for the accurate determination of platinum by isotope dilution analysis, using enriched (194)Pt, in environmental samples containing comparatively high levels of hafnium without any chemical separation. The method is based on the computation of the contribution of hafnium oxide as an independent factor in the observed isotope pattern of platinum in the spiked sample. Under these conditions, the ratio of molar fractions between natural abundance and isotopically enriched platinum was independent of the amount of hafnium present in the sample. Additionally, mass bias was corrected by an internal procedure in which the regression variance was minimised. This was possible as the mass bias factor for hafnium oxide was very close to that of platinum. The final procedure required the measurement of three platinum isotope ratios (192/194, 195/194 and 196/194) to calculate the concentration of platinum in the sample. The methodology has been validated using the reference material "BCR-723 road dust" and has been applied to different environmental matrices (road dust, air particles, bulk wet deposition and epiphytic lichens) collected in the Aspe Valley (Pyrenees Mountains). A full uncertainty budget, using Kragten's spreadsheet method, showed that the total uncertainty was limited only by the uncertainty in the measured isotope ratios and not by the uncertainties of the isotopic composition of platinum and hafnium.

Isotope pattern deconvolution is a mathematical technique for isolating distinct isotope signatures from mixtures of natural abundance and enriched tracers. In iron metabolism studies measurement of all four isotopes of the element by high-resolution multicollector or collision cell ICP-MS allows the determination of the tracer/tracee ratio with simultaneous internal mass bias correction and lower uncertainties. This technique was applied here for the first time to study iron uptake by cucumber plants using 57Fe-enriched iron chelates of the o,o and o,p isomers of ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA) and ethylenediamine tetraacetic acid (EDTA). Samples of root, stem, leaves, and xylem sap, after exposure of the cucumber plants to the mentioned 57Fe chelates, were collected, dried, and digested using nitric acid. The isotopic composition of iron in the samples was measured by ICP-MS using a high-resolution multicollector instrument. Mass bias correction was computed using both a natural abundance iron standard and by internal correction using isotope pattern deconvolution. It was observed that, for plants with low 57Fe enrichment, isotope pattern deconvolution provided lower tracer/tracee ratio uncertainties than the traditional method applying external mass bias correction. The total amount of the element in the plants was determined by isotope dilution analysis, using a collision cell quadrupole ICP-MS instrument, after addition of 57Fe or natural abundance Fe in a known amount which depended on the isotopic composition of the sample.

This work presents the comparison of four different mass spectrometric techniques coupled to gas chromatography (single quadrupole ICP-MS, triple quadrupole ICP-MS/MS, single quadrupole NCI-MS and triple quadrupole EI-MS/MS) for the detection of polychlorinated biphenyls (PCBs) in environmental samples and their determination by a new isotope dilution mass spectrometry (IDMS) approach. A mixture of twelve priority PCBs labelled with 37Cl was employed as the species-specific isotopically labelled internal standard. The 37Cl-labelled PCBs enable the use of both molecular and elemental ionization sources, such as ICP or NCI, as the isotopic label is in the heteroatom. First, the comparison was carried out by assessing the capabilities of all instruments to measure chlorine isotope ratios and calculating the isotopic enrichment of the labelled analogues. Finally, the analysis of the Certified Reference Material SRM 1941b (organics in marine sediment) containing PCBs in the low ng g−1 range was carried out for method comparison. Elemental ionization sources such as ICP and NCI combined with quadrupole mass spectrometry provided chlorine specific detection and high sensitivity for higher chlorinated compounds but suffered from high background signals from other chlorine containing, co-eluting compounds in the sample which prevented the accurate measurement of PCB-specific chlorine isotope ratios. On the other hand, the use of GC-MS/MS in the selected reaction monitoring mode (SRM) provided selective and accurate measurements but suffered from lower sensitivity for higher chlorinated compounds.

The development of a rapid, precise and accurate analytical method for the determination of methylmercury in tuna fish samples is described. The method is based on the use of isotope dilution GC/MS with electron impact ionization, a widespread technique in routine testing laboratories. A certified spike containing 202Hg-enriched methylmercury was used for the isotope dilution of the samples. After extraction of the methylmercury from the sample, methylmercury was propylated using sodium tetrapropyl borate in SPME vials and the analytes were sampled from the headspace for 15 min. For isotope measurements, the molecular ion (MePrHg+) was used in the SIM mode. Five molecular ions were monitored, corresponding to the 198Hg, 199Hg, 200Hg, 201Hg and 202Hg isotopes. The detection at masses corresponding to 198Hg was used to correct for m + 1 contributions of 13C from the organic groups attached to the mercury atom on the 199Hg, 200Hg, 201Hg and 202Hg masses with simple mathematical equations, and the concentration of methylmercury was calculated on the basis of the corrected 200Hg/202Hg isotope ratio. The 202Hg-enriched methylmercury spike was applied, with satisfactory results, to the determination of methylmercury in the certified reference material BCR 464. The method was successfully applied to the determination of methylmercury in tuna fish samples, and the obtained results were included in the CCQM-P39 interlaboratory exercise, organized by the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) with excellent agreement between our results and the average obtained by the other participants. Copyright © 2005 John Wiley & Sons, Ltd.

An in vitro gastrointestinal digestion approach in combination with species-specific isotope dilution analysis has been employed for the first time to study the transformation reactions as well as the solubilisation of butyltin species throughout a simulated human digestion. Different sample preparation procedures were assayed in order to avoid problems derived from lack of isotope equilibration between the endogenous and the isotopically-enriched added species. A "triple spike" approach, which can be used to calculate the corrected concentrations of mono-, di-, and tributyltin (MBT, DBT and TBT, respectively), as well as six interconversions, was employed throughout this work. In order to calculate and compare the species degradation factors, a triple spike solution containing each butyltin species enriched in a different isotope was added to the simulated gastric and intestinal fluids before the digestion procedures in the presence and in the absence of a solid biological matrix (commercial mussel tissue). Additionally, by analysing the soluble and insoluble fractions resulting from the simulated digestion of a commercial mussel tissue (gastric and gastric plus intestinal digestion), total mass balances for each butyltin compound could be derived. For this purpose, the isotopically-enriched species were added after the enzymatic digestions in order to avoid problems derived from lack of isotope equilibration. The mass balances provided information not only about the solubilisation but also about the degradation of the butyltin species during the digestion procedures. Good agreement between the degradation factors calculated under all experiments performed in this work and between those reported in previous works were obtained. The most serious degradation observed was that of DBT to produce MBT, whereas slight degradations of TBT and MBT were detected. Moreover, a worrying 61% of the original total butyltin content present in a commercial mussel tissue was found to be solubilised after complete simulated gastrointestinal digestion, with minimal degradation of TBT.

Five common placolith-bearing coccolithophorid algae—Gephyrocapsa oceanica, Coccolithus pelagicus, Calcidiscus leptoporus, Umbilicosphaera sibogae (var. sibogae and var. foliosa), and Emiliania huxleyi—were cultured to investigate controls on Sr partitioning in coccolith calcite. For identical temperature and media composition, Sr partitioning varies by more than 30% in exponential phase cultures of the five species and is linearly related to rates of calcite production/cell (ρ=0.91). Exponential phase culture experiments with three strains of C. leptoporus and six strains of G. oceanica at varying temperatures show variations in Sr partitioning of 20% and 30%, respectively. With C. leptoporus, Sr partitioning is equally correlated with temperature and calcification rate (ρ=0.8), which themselves are highly correlated; the slope of the relationship between DSr and calcification rate is comparable to that observed in all species at constant temperature. However, in G. oceanica, increased temperature appears to enhance Sr incorporation by up to 2% to 1.6% °C−1 in the range of 15 to 30 °C. The strong influence of calcification rate on Sr partitioning may be useful for inferring past variations in coccolithophorid productivity from Sr partitioning in coccolith sediments if the influence of temperature on Sr partitioning can be resolved. Because the relationship between calcite production and Sr partitioning is linear, a proportional change in calcification should be expressed much more strongly in the Sr/Ca ratio of large species with rapid calcite production than in smaller species, which produce calcite more slowly. Consequently, it may be possible to separate temperature and calcification influences on coccolith Sr/Ca by separately analyzing Sr/Ca in species that produce calcite rapidly and those that produce calcite slowly, if both undergo comparable relative changes in calcification rates.

Isotope-dilution analysis in combination with GC-ICP-MS detection has been applied to the determination of butyltin species in environmental samples. Different spikes containing the isotopically labeled butyltin species have been synthesized in the laboratory after optimization of the reaction conditions. The isotopic compositions of the tin species in the different spike solutions were determined by GC-ICP-MS after derivatization by aqueous ethylation with sodium tetraethylborate. Reverse isotope-dilution analysis was used for quantitation of the spike solutions by means of natural MBT, DBT, and TBT standards. The mixed spikes were used for simultaneous analysis of MBT, DBT and TBT in the certified reference materials, PACS-2, CRM 462, and CRM 646, with satisfactory results. The excellent agreement of the different speciation results obtained by use of the different spikes is a good indicator of the precision, accuracy, and reliability which can be achieved by using isotope-dilution analysis for trace metal speciation. Application of a double spike containing (119)Sn-enriched MBT (79.7 At%), (118)Sn-enriched DBT (86.7 At%), and (119)Sn-enriched TBT (83.1 At%) also enabled evaluation of the conditions resulting in quantitative extraction of the species from the solid matrix, in combination with possible alterations depending on the different extraction procedures used (mechanical shaking, ultrasounds, and microwaves). Mathematical equations used for this purpose computed the correct species concentrations directly and, additionally, the decomposition factors (from TBT to DBT and from DBT to MBT) after precise measurement of the (119)Sn/(120)Sn and (118)Sn/(120)Sn ratios for all butyltin species by GC-ICP-MS.

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.

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.

A post-column Isotope Dilution Analysis (IDA) methodology has been used to carry out the quantitative speciation of selenium (Se) in industrial wastewaters by ion chromatography coupled to ICP-MS with an octapole reaction system (ORS). The interfering argon dimers on the 78Se and 80Se isotopes were suppressed by pressurizing the octapole chamber with 4 mL min-1 of hydrogen. The inorganic Se species selenite [Se (IV)], selenate [Se (VI)] and selenocyanate [SeCN-] were separated by ion chromatography (IC) using isocratic elution with 20 mmol L-1 Na2CO3 / 2 mmol L-1 NaHCO3 as eluent. After the separation, the eluent carbonates were converted to CO2 on-line by a membrane self-regenerating suppressor, in order to avoid clogging problems in the nebulizer or in the cones of the ICP-MS interface. Detection limits (as 3σ of the baseline noise) of 0.18, 0.13 and 1.0 ng g-1 were achieved for Se (IV), Se (VI) and SeCN- respectively. Quantification of selenium in each separated species was performed using post-column isotope dilution analysis by continuous mixing of an enriched 77Se spike solution with the effluent from the suppressor column. Finally, the 78Se/77Se or 80Se/77Se isotope ratios were monitored and the concentration of the corresponding selenium species was evaluated. The proposed methodology has been successfully applied to study the quantitative speciation of selenium in petroleum refinery wastewaters.

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 of1.2 per thousand were detected in the hair strands from UK residents whereas the traveler presented a variation of5 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.

International audience; A new double-spiking approach, based on a multiple-spiking numerical methodology, has been developed and applied for the accurate quantification of inorganic mercury (IHg) and methylmercury (MeHg) by GC-ICPMS in different environmental matrices such as water, sediments and a wide range of biological tissues. For this purpose, two enriched mercury species (201MeHg and 199IHg) were added to the samples before sample preparation in order to quantify the extents of the methylation and demethylation processes, and thereby correct the final species concentrations. A critical evaluation of the applicability of this methodology was performed for each type of matrix, highlighting its main advantages and limitations when correcting for the conversion reactions of the species throughout the whole sample preparation procedure. The double-spike isotope dilution (DSIDA) methodology was evaluated by comparing it with conventional species specific isotope dilution (IDA) when analysing both certified reference materials and environmental samples (water, biotissues and sediment). The results demonstrate that this methodology is able to provide both accurate and precise results for IHg and MeHg when their relative concentrations are not too different (ratio MeHg/IHg∈>∈0. 05), a condition that holds for most natural waters and biotissues. Significant limitations on the accurate and precise determination of the demethylation factor are however observed, especially for real sediment samples in which the relative concentrations of the species are substantially different (ratio MeHg/IHg∈

Enriched spikes of 53Cr, 57Fe, 67Zn, 95Mo, 111Cd, 143Nd, 150Sm, 199Hg and 207Pb were characterised on a multi-collector ICP-MS instrument both in terms of isotopic composition and concentration. The isotopic composition of the spikes was determined using either a natural abundance or certified standard for mass bias correction, except for lead where thallium internal correction was used. It was observed that the use of weighted regression lines for mass bias correction provided similar or, in most cases, lower uncertainties in the isotopic composition of the spikes in comparison with unweighted regression lines or single ratio measurements. For the establishment of the spike concentration by reverse isotope dilution analysis, isotope pattern deconvolution was evaluated and mass bias could be corrected internally in each blend by minimising the variance of the multiple linear regression model. It was also observed that isotope pattern deconvolution with internal mass bias correction provided lower uncertainties in the concentration of the spikes in comparison with the usual procedure based on external mass bias correction.

Two speciated isotope dilution (SID) approaches consisting of a single-spike (SS) method and a double-spike (DS) method including a reaction/transformation model for the correction of inadvertent transformations affecting mercury species were compared in terms of accuracy, method performance, and robustness for the simultaneous determination of methylmercury (MeHg), inorganic mercury (iHg), and total mercury (HgT) concentrations in five biological Standard Reference Materials (SRMs). The SRMs consisted of oyster and mussel tissue materials displaying different mercury species concentration levels and different textural/matrix properties including freeze-dried (FD) materials (SRMs 1566b, 2976, and 2977) and cryogenically prepared and stored fresh-frozen (FF) materials (SRMs 1974a, 1974b). Each sample was spiked with (201)iHg (Oak Ridge National Laboratory, ORNL) and Me(202)Hg (Institute for Reference Materials and Measurements. IRMM-670) solutions and analyzed using alkaline microwave digestion, ethylation, and gas chromatography inductively coupled plasma mass spectrometry (GC/ICP-MS). The results obtained by the SS-SID method suggested that FF and FD materials are not always commutable for the simultaneous determination of iHg, MeHg, and HgT, due to potential transformation reactions resulting probably from the methodology and/or from the textural/matrix properties of the materials. These transformations can occasionally significantly affect mercury species concentration results obtained by SS-SID, depending on the species investigated and the materials considered. The results obtained by the DS-SID method indicated that the two classes of materials were commutable. The simultaneous and corrected concentrations of iHg, MeHg, and HgT obtained by this technique were not found to be statistically different form the certified and reference concentration together with their expanded uncertainty budgets for the five SRMs investigated, exemplifying the robustness, the accuracy, and the improved commutability of this method compared to SS-SID measurements.

A standard GC-MS instrument with electron impact ionisation has been used to develop a fast, simple and reliable method for the simultaneous determination of monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) in water samples. Isotope dilution analysis (IDA) is used for the determination of species, taking advantage of a commercially available spike solution containing a mixture of MBT, DBT and TBT enriched in 119Sn. Method detection limits for 100-mL samples were between 0.18 and 0.25 ng L(-1) for the three butyltin compounds with typical RSD between 2 and 4% at levels between 100 and 10 ng L(-1), respectively. Recovery of tin species in spiked samples (natural water, wastewater and seawater) was quantitative. The stability of butyltin compounds in collected seawater samples was also studied. The addition of a 1% (v/v) glacial acetic acid preserved tin species in the samples for at least 5 days at room temperature. The IDA method was finally implemented in a routine testing laboratory and it was subsequently accredited by the Spanish National Accreditation Body according to the requirements of UNE-EN ISO/IEC 17025.

[1] Although coccolith calcite is abundant in carbonate sediments, it has not been previously utilized for Mg/Ca paleothermometry. Cleaning experiments with synthetic composite samples (reagent CaCO3 powder and organic matter from noncalcifying marine algae Chlorella) are used to evaluate which traditional and/or novel cleaning methods permit us to recover the known carbonate Mg/Ca ratio. The most effective cleaning treatment, causing the least effect on carbonate chemistry and most complete and rapid oxidation of algal organic matter, was oxidation in an equal volume mixture of sodium hypochlorite (2.8%) and hydrogen peroxide (30%). However, in some organic- rich samples this method may not remove sufficient noncarbonate Mg to permit precise determination of carbonate Mg/Ca. Mg/Ca ratios in small culture samples may be determined precisely (relative standard deviation of 0.65%) using sector field inductively coupled plasma-mass spectrometry. Measurements of Mg/Ca in coccoliths from several species suggest that temperature may be an important control over Mg partitioning in coccolith calcite, although cleaning issues are likely to be an important limitation on paleoceanographic application.