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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.

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).

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 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 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 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.

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.

Isotope dilution analysis (IDA) has been used to quantify total selenium, total solubilized selenium, and the selenomethionine (SeMet) amount in yeast and yeast-based nutritional supplements after acid microwave digestion and different enzymatic extraction procedures. For this purpose, both a (77)Se-enriched SeMet spike, previously synthesized and characterized in our laboratory, and a (77)Se(VI) spike were used. In the analysis of the nutritional supplements, the SeMet spike was added to the sample and extracted under different conditions, and the (78)Se/(77)Se and (80)Se/(77)Se isotope ratios were measured as peak area ratios after high-performance liquid chromatography (HPLC) separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The formation of SeH(+) and mass discrimination were corrected using a natural SeMet standard injected every three samples. Similarly, total solubilized selenium was measured in the extracts after enzymatic hydrolysis using the (77)Se-enriched SeMet as a spike by direct nebulization without a chromatographic separation. To establish a mass balance, total selenium was also determined by IDA-ICP-MS on the yeast tablets after microwave digestion using (77)Se(VI) as a spike. Results showed that all enzymatic procedures tested were able to solubilize total selenium quantitatively from the solid. However, the recovery for the species SeMet, the major selenium compound detected, was seriously affected by the enzymatic procedure employed and also by the matrix composition of the supplement evaluated. For the yeast sample, SeMet recovery increased from 68 to 76% by the combined use of driselase and protease. For the nutritional supplements, the two most effective procedures appeared to be protease and driselase/protease, with a SeMet recovery ranging from 49 to 63%, depending upon the supplement evaluated. In the case of in vitro gastrointestinal enzymolysis, the results obtained showed 26-37% SeMet recovery, while the rest of selenium was solubilized as other unknown compounds (probably Se-containing peptides).

Different liquid-solid extraction techniques, including room-temperature leaching with mechanical shaking, ultrasonic, and microwave-assisted extractions, have been evaluated for the quantitative speciation of tin for mono-, di-, and tributyltin (MBT, DBT, and TBT, respectively) in PACS-2 and BCR-646 certified reference materials. A methanol-acetic acid mixture was used as the extractant reagent in all cases. For this purpose, a mixed spike containing 119Sn-enriched MBT (79.7 At%), 118Sn-enriched DBT (86.7 At%), and 119Sn-enriched TBT (83.1 At%), was synthesized, characterized, and used for isotope dilution analysis. The isotopic composition of the mixed spike was determined by gas chromatography/ICPMS after aqueous ethylation using sodium tetraethylborate, and the determination of the concentration of the different species in the spike was performed by means of reverse isotope-dilution analysis using natural MBT, DBT, and TBT standards. In the analysis of the certified sediments, the sample was spiked with the mixed spike, extracted under different conditions, derivatized with sodium tetraethylborate, and extracted into hexane, and the isotope ratios 120/118 and 120/119 were measured as peak area ratios for all butyltin species after GC/ICPMS. Mass bias was corrected using a derivatized natural standard every three sample injections. Sequential degradation reactions during extraction (from TBT to DBT, from DBT to MBT, and from MBT to inorganic tin) were assumed, and mathematical equations were developed that allowed the determination of the correct species concentration and the decomposition factor for each of the transformation reactions. For ultrasonic extraction and mechanical shaking, negligible degradation reactions were observed. However, for microwave assisted extractions, degradation factors up to 7% (TBT to DBT) and 16% (DBT to MBT) were obtained for both reference materials when high-MW energy was applied in the extraction step. For the three extraction techniques tested, the DBT and TBT concentration values obtained for PACS-2 closely matched the certified values. However, for MBT the concentrations found by microwave and ultrasonic extraction were much higher than the certified value. This was not the case for mechanical shaking. The results obtained for BCR-646 using microwave assisted extraction were in good agreement with the certified values for all tin species.