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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

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.

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.

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%.

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.

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.

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.

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.

cited By 16; International audience; The processes driving the inadvertent transformations of inorganic mercury (iHg) and methylmercury (MeHg) in cryogenically homogenized fresh-frozen (FF) and freeze-dried (FD) biological standard reference materials (SRM) were investigated using alkaline digestion, derivatization and gas chromatography inductively coupled plasma mass spectrometry (GC-ICP-MS). Labile inorganic mercury (201iHg) and methylmercury (Me202Hg) isotopic standards and their cysteine-complexed analogs (201Hg(Cys) 2 and Me202HgCys) were used in a double-spike speciated isotope dilution (SID) model to document the influence of complexing ligands on the equilibration, the reactivity and the transformation processes between isotopic mercury species and their endogenous analogs. Cysteine-complexed and labile Me202Hg spiking standards displayed similar equilibration processes in both classes of materials, leading to accurate MeHg determinations with negligible methylation transformations. Labile 201iHg standards provided accurate iHg concentration results in both material series, although an apparent demethylation reaction specific of FF materials was detected, with a negligible effect in FD materials. Cysteine-complexed 201iHg standards led to higher but inaccurate iHg concentrations and higher demethylation yields in both materials. This comparison illustrated a significant influence of complexing ligands on the equilibration processes between labile and/or complexed 201iHg species and their endogenous analogs. The derivatization step was found to catalyze these non-equilibrium conditions with different derivatization yields between labile and complexed iHg species, whereas no differences were observed for MeHg species. When complexation disparities existed in solution between 201iHg species and their endogenous analogs, this process specifically affected the determination of 200/201iHg ratios relative to 200/202iHg, 200/201MeHg and 200/202iHg ratios, which are all involved in the double-spike SID model to establish transformation yields and mercury species concentrations. This process and the influence of demethylating agents were presumably responsible for the apparent demethylation reaction only observed in FF materials. This result gave rise to several questions on the influence of freeze-drying procedures on the lability/complexation patterns of mercury species and/or on the activity of demethylating agents. A complementary analytical step consisting of maximizing the equilibrium conditions between isotopic and endogenous mercury species together with inhibiting the activity of demethylating agent is proposed. When applying these conditions, the two classes of materials displayed negligible transformation reactions, improving their commutability for the simultaneous determination of iHg and MeHg. The approach proposed in this work allowed for the first time documentation of the potential and limitations of speciated isotope dilution procedures to handle the problem of lability/complexation and ligand interactions in natural biological matrices when considering multiple-spike SID approaches. © The Royal Society of Chemistry 2008.

cited By 12; International audience; Six metrology institutes (NMIs) representing at the Comité International des Poids et Mesures (CIPM) 4 Member States of the Metre Convention and 2 international organisations, and 8 "expert" laboratories selected outside CIPM have compared their capabilities to quantitatively measure methylmercury (MeHg) in a prepared tuna material containing approximately 4.3 mg kg-1 Hg. This comparison was the object of the CIPM-Comité Consultatif pour la Quantité de Matière (CCQM) Pilot Study 39, organised by the Institute for Reference Materials and Measurements (IRMM), from the European Commission - Joint Research Centre. Beside the test material itself, a bottle of the BCR-464 tuna Certified Reference Material (CRM) and an ampoule of IRMM-670, a 202Hg isotope enriched MeHg candidate isotopic CRM, were distributed to all participants, who were free to apply the measurement strategy of their choice. Four, including 1 NMI, relied on external calibration or the method of standard additions, whereas the other 10 implemented an isotope dilution mass spectrometry (IDMS) approach and chose to use the IRMM-670 for their measurements. Alkaline digestion at room temperature (with manual shaking) or high temperature (under sonication, oven or hot plate conditions) was employed by most participants, with hydrochloric acid leaching the second most popular choice. Alkylation (4 phenylations, 4 ethylations and 3 propylations) in the aqueous phase was preferred by a large majority over butylation by the Grignard reaction. All participants were requested to estimate the uncertainty associated with their results and 9 out of 14 stated relative combined uncertainties below 6% (k = 2). Despite this apparent consensus, the perception of which factor caused the largest contribution to this estimation differed among participants because of the differences in the analytical methodologies deployed but also because of wide differences of the concepts of uncertainty estimation. The 'mixture mode' (MM) median, calculated also from the measurement uncertainties stated by the participants, was 1.967 ± 0.204 × 10-5 mol kg -1 (95% confidence). Twelve of the results were re-grouped within a range of less than 0.3 × 10-5 mol kg-1 (MM median = 1.967 ± 0.162 × 10-5 mol kg-1, 95% confidence): they nearly all (1 exception) overlapped with each other within k = 2 stated uncertainties. For the other 2 results the uncertainty seemed to have been particularly underestimated as they lay, respectively, at more than 20% above and less than -40% below the overall average. The relative standard deviation of the results of 9 laboratories out of the 10 that applied IDMS was about 2.6%. It can be assumed from the degree of equivalence shown by 12 out of 14 study participants that, at present, laboratories worldwide are potentially able to supply accurate results for MeHg in fish-type matrices (containing about 2 × 10-5 mol kg-1) within ± 10% uncertainty. This encouraging outcome permitted scheduling of a follow-up CCQM-K43 key comparison for a lower MeHg content level in salmon tissues. © The Royal Society of Chemistry 2005.