Pablo Rodríguez-González

Species-Specific Stable Isotope Fractionation of Mercury during Hg(II) Methylation by an Anaerobic Bacteria (Desulfobulbus propionicus) under Dark Conditions

This work reports the first results on the stable isotope fractionation of Hg during methylation by anaerobic bacteria under dark conditions. The GC-MC-ICPMS methodology employed is capable of simultaneously measuring the species-specific isotopic composition of different Hg species within the same sample. We have studied Hg isotopic fractionation caused by methylation of Hg(II) standard reference material NIST-3133 in the presence of the pure bacterial strain Desulfobulbus propionicus MUD10 (DSM 6523) under fermentative conditions. We have measured the isotopic composition of Hg(II) and monomethyl mercury (MMHg) in these cultures as a function of time and calculated delta-values for both species versus the starting material (NIST-3133) as a delta-zero standard. Two different strategies for the incubation were applied: single sampling cultures and a continuous sampling culture. The results obtained have shown that under the conditions employed in this work the methylation of Hg(II) causes mass-dependent fractionation of the Hg isotopes for both Hg(II) substrate and produced MMHg. Such a process occurred under the exponential growth of the bacteria which preferentially methylate the lighter isotopes of Hg. After 96 h for the continuous culture and 140 h for the single sampling cultures, we observed a change in the fractionation trend in the samples at a similar cell density value (ca. 6.0 x 10(7) cells mL(-1)) which suggests the increasing contribution of a simultaneous process balancing methylation extent such as demethylation. Assuming that Rayleigh type fractionation conditions are met before such suppression, we have obtained a alpha(202/198) fractionation factor of 1.0026 +/- 0.0004 for the single sampling cultures.

Simultaneous Determination of Species-Specific Isotopic Composition of Hg by Gas Chromatography Coupled to Multicollector ICPMS

This work presents the simultaneous online determination of the isotopic composition of different Hg species in a single sample by the hyphenation of gas chromatography (GC) with multicollector-inductively coupled plasma mass spectrometry (MC-ICPMS). With the use of commercially available instrumentation, precise and accurate species-specific Hg isotope delta values (per mil deviation of the Hg isotope ratio in the sample relative to a reference standard) have been obtained online from consecutive GC transient signals. The use of isothermal temperature programs to extend the elution of the Hg species, the proper selection of the peak integration window, as well as the preconcentration of real samples are critical to provide optimal counting statistics. Also, isotope ratio drift during transient signal elution was overcome by introducing a mixed Hg(II) and MeHg standard bracketing scheme and expressing all results using the delta-notation relative to SRM NIST-3133. Using the proposed methodology, we have obtained an external 2SD precision of 0.56 per thousand for delta (202)Hg that is more than 10 times smaller than the overall Hg stable isotope variation thus far observed in terrestrial samples. The measurement of species-specific Hg isotopic composition relative to SRM NIST-3133 has been validated versus two other analytical techniques, i.e., conventional nebulization (CN) of Hg(II) solution and cold vapor (CV) generation of Hg (0) vapor. A good agreement between the species-specific delta values obtained by the different techniques has been obtained in secondary fractionated reference standard (UM-Almaden) and environmental matrixes, i.e., BCR-CRM 464 (tuna fish) and IAEA-085 (human hair). The results show mass-dependent and mass-independent fractionation in environmental samples, i.e., mass-independent fractionation of odd isotopes (199)Hg and (201)Hg in tuna fish was observed. This methodology provides new possibilities for the future study of species-specific stable isotope geochemistry of Hg and other trace metals.

Development of a triple spike methodology for validation of butyltin compounds speciation analysis by isotope dilution mass spectrometry

The triple spike GC-ICP-MS methodology developed in Part 1 of this paper has been applied here to study and validate different extraction procedures previously reported in the analytical literature for speciation analysis of biological materials in the determination of butyltin compounds. Microwave assisted extraction, mechanical shaking, alkaline hydrolysis with tetramethylammonium hydroxide (TMAH) and enzymatic digestion have been evaluated under different conditions for the determination of these species in mussel tissue CRM-477. The results obtained showed extensive degradation of the species using alkaline hydrolysis with TMAH and also a lack of isotope equilibration when using enzymatic digestion. The use of microwave assisted extraction with methanol–acetic acid showed the best results in terms of low degradation, rapid isotope equilibration and quantitative recoveries. Quantitative extraction was also obtained, however, by mechanical shaking at 37 °C using methanol–acetic acid as extractant, with no degradation of the species. The developed triple spike methodology was able to correct for as much as 50% degradation of DBT to form MBT during sample pre-treatment. Moreover, isotope equilibration studies performed using different extraction procedures have shown that the required complete isotope equilibration is achieved only after the naturally occurring organotin compounds are completely released to the solution from the solid matrix.

Recent advances in isotope dilution analysis for elemental speciation

Following the concepts and applications included in our tutorial review published in 2005, this review presents and critically discusses new concepts, methodologies and trends that have appeared during the last five years of scientific development in the field of isotope dilution analysis for elemental speciation. Although general concepts have been detailed previously in several high quality reviews, we will highlight selected applications that, in our opinion, have significantly contributed to the improvement of the state of the art in this field. Methodological advances, such as the irruption of the Isotope Pattern Deconvolution theory, are commented in detail. Important metrological achievements, such as the organisation of international comparisons by the Comite consultatif pour la quantite de matiere (CCQM) or the certification of new reference materials using Isotope Dilution Analysis are also included. Particular attention has been paid to the developments in the field of bioanalytical chemistry. The application of enriched stable isotopes in metallomics, clinical chemistry and quantitative proteomics are covered in this review. Also, metabolic applications of enriched isotopes are included when speciation work is carried out. Finally, future trends are identified together with the extension of concepts and applications typical of elemental speciation to other scientific fields.

Single and multiple spike procedures for the determination of butyltin compounds in sediments using isotope dilution GC-ICP-MS

The combined use of single and multiple spike solutions for the determination of butyltin compounds in sediments by species-specific isotope dilution mass spectrometry has been evaluated in this work in order to validate an analytical procedure proposed for routine determinations. For this purpose, a test sediment sample was analysed during the course of an intercomparison exercise using both a single isotope spike solution (a mixture of mono-, di- and tributyltin enriched in 119Sn) and a triple spike solution containing each butyltin species enriched with a different tin isotope. The triple spike methodology (able to correct for interconversion reactions) was employed for the optimisation of the microwave assisted extraction procedure that was subsequently applied for the analysis of the test sediment. The determination of butyltin compounds in the test sediment was performed using both spike solutions under the same extraction conditions. An excellent agreement between the results obtained using both approaches and those provided by all laboratories participating in the intercomparison exercise was obtained, validating in this way the methodologies employed in this work. Uncertainty budgets were calculated for both spiking methodologies. The complexity of the triple spike methodology, in which the simultaneous measurement of nine tin isotope ratios must be carried out, provided additional sources of error in obtaining degradation-corrected concentrations of the three butyltin species. Additionally, uncertainty budgets were obtained also for the calculation of degradation factors F1 (degradation of TBT to DBT) and F2 (degradation of DBT to MBT). The results demonstrated that their uncertainty was independent of the uncertainty of the measurement of the concentration of the butyltin species, both in the spike solution and in the sample, when using a multiple spiking approach, and depended only on the measured isotope ratios and on the uncertainty in the isotopic composition of the spike.

Determination of butyltin compounds in coastal sea-water samples using isotope dilution GC-ICP-MS

A rapid, accurate, sensitive and simple method for the simultaneous determination of mono-, di- and tributyltin (MBT, DBT and TBT, respectively) in coastal sea-water samples was developed. The method is based on the application of isotope dilution methodologies using a 119Sn enriched spike containing all three butyltin compounds. Sample volumes of 100 ml were introduced in pre-cleaned glass volumetric flasks and spiked with the mixed enriched spike. After 15 min equilibration, sodium tetraethylborate, acetate buffer of pH 5.4 and 1 ml of hexane were added and the mixture shaken for 10 min. The hexane phase was recovered, evaporated to near dryness under argon, and finally 1 μl was injected in the GC-ICP-MS system. Detection was performed at masses 118, 119 and 120. The concentration of the butyltin compounds in the samples was calculated based on the average value from both the 118/119 ratio and the 120/119 ratio. In this way mass bias was compensated without resorting to external mass bias correction. Instrumental detection limits were ca. 50 fg for all butyltin species (as Sn) while method detection limits were blank limited and resulted to be 0.27, 0.09 and 0.04 ng l−1 (as Sn) for TBT, DBT and MBT, respectively. Recovery experiments both at 10 and 70 ng l−1 levels for TBT were quantitative and precision was about 5% RSD at levels between 2 and 20 ng l−1 for all three butyltin compounds. The method was applied to investigate the levels of butyltin compounds in coastal sea-water from Gijon (Asturias, Spain) where no previous data existed.

Development of a triple spike methodology for validation of butyltin compounds speciation analysis by isotope dilution mass spectrometry. Part I. Synthesis of the spike, characterisation and development of the mathematical equations

A methodology for the determination of butyltin compounds by isotope dilution mass spectrometry based on a triple spike approach is presented. A spike solution containing monobutyltin, dibutyltin and tributyltin, each compound enriched with different tin isotopes, has been synthesised, characterised and applied to the simultaneous determination of the three butyltin compounds by isotope dilution GC-ICP-MS. Mathematical equations correcting for all possible degradation reactions which could occur during sample pre-treatment were developed, allowing the accurate calculation of the three butyltin concentrations sought plus the values of six possible interconversion yieids, regardless of the extent of degradation. The triple tin spike solution has shown itself to be stable over more than 7 months, both in isotope composition and elemental concentration, provided that acetic acid is present in the solution. The proposed methodology has been finally evaluated by performing the determination of butyltin compounds in the certified reference material CRM-477 (Mussel Tissue), under different microwave assisted extraction conditions, with satisfactory results.

Overcoming matrix effects in electrospray: Quantitation of β-agonists in complex matrices by isotope dilution liquid chromatography–mass spectrometry using singly 13C-labeled analogues

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.

Multiple spiking species-specific isotope dilution analysis by molecular mass spectrometry: simultaneous determination of inorganic mercury and methylmercury in fish tissues.

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.

Evaluation of minimal 13C-labelling for stable isotope dilution in organic analysis

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.