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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).

Current procedures for the evaluation of spectral accuracy of mass spectrometers are limited by the lack of certified isotopic reference materials and the high uncertainty in the isotopic composition of natural abundance molecules. The calculated uncertainties in the ratio M + 1/M for natural abundance molecules containing any number of C, H, N and/or O atoms are close to 5% relative because of the natural variability of the isotopic composition of carbon. So, we have developed two alternative measurement procedures with much lower theoretical uncertainties for a better evaluation of spectral accuracy in both single and triple quadrupole analysers. The first method is based on the measurement of the M + 2/M, M + 4/M + 2, etc. ratios for halogenated organic compounds containing either Cl or Br. The theoretical uncertainties for these ratios because of natural variability are in the order of 0.3 to 1.0% making them suitable for the evaluation of spectral accuracy with the additional advantage that there is no need to take into account other limitations such as cluster purity or poor mass resolution. This procedure was applied to the evaluation of a single quadrupole GC-MS instruments using natural abundance PCB and PBDE standards with satisfactory results. The second method can be applied to tandem instruments and takes advantage of the loss of two halogen atoms when PCB and PBDE standards are fragmented by Collision Induced Dissociation. Theoretical SRM transition ratios can be calculated as a pure combinatorial probability with theoretical uncertainties lower than 0.1%. By combining PCBs and PBDEs with different number of halogen atoms, a mass range from 100 to 700 u and abundance ratios from 0.1 to 10 can be evaluated. The use of penta-chlorinated PCBs and/or penta-brominated PBDEs is finally recommended for the evaluation of spectral accuracy of mass spectrometers with the EI source. Copyright © 2016 John Wiley & Sons, Ltd.

GC-FID has been effectively used as a universal quantification technique for volatile organic compounds for a long time. In most cases, the use of the ECN allows for quantification by GC-FID without external calibration using only the response of a single internal standard. In this paper we compare the performance characteristics of GC-FID with those of post-column (13)C Isotope Dilution GC-Combustion-MS for the absolute quantification of organic compounds without the need for individual standards. For this comparison we have selected the quantification of FAMEs in biodiesel. The selection of the right internal standard was critical for GC-FID even when ECN were considered. On the other hand, the nature of the internal standard was not relevant when GC-Combustion-MS was employed. The proposed method was validated with the analysis of the certified reference material SRM 2772 and comparative data was obtained on real biodiesel samples. The analysis of the SRM 2772 biodiesel provided recoveries in the range 100.6-103.5% and 96.4-103.6% for GC-combustion-MS and GC-FID, respectively. The detection limit for GC-combustion-MS was found to be 4.2ng compound/g of injected sample. In conclusion, the quantitative performance of GC-Combustion-MS compared satisfactorily with that of GC-FID constituting a viable alternative for the quantification of organic compounds without the need for individual standards.

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

This work describes the first application of 37Cl-labeled compounds to isotope dilution mass spectrometry (IDMS). The synthesis of 12 37Cl-labeled polychlorinated biphenyls (PCBs) was carried out by the chlorination of biphenyl with isotopically enriched chlorine gas, generated by the direct oxidation of Na37Cl with potassium peroxymonosulfate. After an exhaustive purification due to the presence of other congeners, the concentration and the isotopic enrichment of all 37Cl-labeled PCBs in the mixture was determined. The proposed procedure allows the simultaneous quantification of every isotope diluted PCB congener in a single gas chromatography-tandem mass spectrometry (GC-MS/MS) injection without resorting to a methodological calibration graph. The results obtained here demonstrate that the use of 37Cl-labeled analogues provides results in agreement with the certified values of three different Certified Reference Materials (marine sediment SRM 1944, fish tissue 1947, and loamy soil CRM 962-50) and analyt...

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

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.

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

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

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 β 2 -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 β 2 -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 β 2 -agonists in human urine and bovine liver, even at the sub ng g −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 C 1 -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.

We have developed a novel, rapid and easy calculation procedure for Mass Isotopomer Distribution Analysis based on multiple linear regression which allows the simultaneous calculation of the precursor pool enrichment and the fraction of newly synthesized labelled proteins (fractional synthesis) using linear algebra. To test this approach, we used the peptide RGGGLK as a model tryptic peptide containing three subunits of glycine. We selected glycine labelled in two

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 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.

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

Cheap and cheerful: Postcolumn carbon-isotope dilution with (13)CO(2) in combination with the coupling of gas chromatography, combustion, and EIMS (valve in position B in the picture) provides a generic quantitative approach for every organic compound without the need for specific standards. A GC-MS instrument can be upgraded by a simple low-cost modification without any loss of the structural information provided by electron ionization sources.

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.

In order to evaluate alternative analytical methodologies to study the geographical origin of ciders, both multi-elemental analysis and Sr isotope abundance ratios in combination with multivariate statistical analysis were estimated in 67 samples from England, Switzerland, France and two Spanish regions (Asturias and the Basque Country). A methodology for the precise and accurate determination of the (87)Sr/(86)Sr isotope abundance ratio in ciders by multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) was developed. Major elements (Na, K, Ca and Mg) were measured by ICP-AES and minor and trace elements (Li, Be, B, Al, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Y, Mo, Cd, Sn, Sb, Cs, Ba, La, Ce, W, Tl, Pb, Bi, Th and U) were measured by ICP-MS using a collision cell instrument operated in multitune mode. An analysis of variance (ANOVA test) indicated that group means for B, Cr, Fe, Ni, Cu, Se, Cd, Cs, Ce, W, Pb, Bi and U did not show any significant differences at the 95% confidence level, so these elements were rejected for further statistical analysis. Another group of elements (Li, Be, Sc, Co, Ga, Y, Sn, Sb, La, Tl, Th) was removed from the data set because concentrations were close to the limits of detection for many samples. Therefore, the remaining elements (Na, Mg, Al, K, Ca, Ti, V, Mn, Zn, As, Rb, Sr, Mo, Ba) together with (87)Sr/(86)Sr isotope abundance ratio were considered for principal component analysis (PCA) and linear discriminant analysis (LDA). Finally, LDA was able to classify correctly 100% of cider samples coming from different Spanish regions, France, England and Switzerland when considering Na, Mg, Al, K, Ca, Ti, V, Mn, Zn, As, Rb, Sr, Mo, Ba and (87)Sr/(86)Sr isotope abundance ratio as original variables.

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

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.

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.

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.

The development of a rapid, precise and accurate speciation method for the simultaneous determination of mono-, di- and tributyltin in environmental samples is described. The method is based on using isotope dilution gas chromatography/mass spectrometry (GC/MS) with electron ionization, a widely used technique in routine testing laboratories. A mixed spike containing 119Sn-enriched monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) was used for the isotope dilution of the samples. Five molecular ions were monitored for each analyte, corresponding to the 116Sn, 117Sn, 118Sn, 119Sn and 120Sn isotopes. The detection at masses corresponding to 116Sn and 117Sn were used to correct for m + 1 and m + 2 contributions of 13C from the organic groups attached to the tin atom on the 118Sn, 119Sn and 120Sn masses with simple mathematical equations and the concentrations of the butyltin compounds were calculated based on the corrected 118Sn/119Sn and 120Sn/119Sn isotope ratios. The 119Sn-enriched multispecies spike was applied with satisfactory results to the simultaneous determination of MBT, DBT and TBT in three certified reference materials: two sediments, PACS-2 and BCR 646, and the mussel tissue CRM 477. The method was compared with a previously published GC/inductively coupled plasma MS isotope dilution procedure, developed in our laboratory, by injecting the same samples into both instruments. Comparable analytical results in terms of precision and accuracy are demonstrated for both atomic and molecular mass spectrometric detectors. Thus, reliable quantitative organotin speciation analysis can be achieved using the more widespread and inexpensive GC/MS instrument. Copyright © 2004 John Wiley & Sons, Ltd.

Different spike solutions available for the determination of butyltin compounds by isotope dilution analysis are described and applied for the determination of butyltin compounds in PACS-2 certified reference material. Additionally, those spike solutions were evaluated during the course of an interlaboratory exercise organised by the National Research Council of Canada and the Laboratory of the Government Chemist (UK) in order to quantify tributyltin in a pilot sediment. The aim of this project was to evaluate the capabilities of isotope dilution mass spectrometry to reduce the uncertainty in the certification of Reference Materials for the speciation of organotin compounds. All participants were supplied with a 17Sn-enriched TBT solution from the Laboratory of the Government Chemist (UK). In our case, we performed the analysis of the pilot sediment also using a 119Sn enriched spike (mixed mono-, di- and tributyltin) and a 118Sn-119Sn double spike. The use of these additional spike solutions not only allowed the determination of monobutyltin and dibutyltin in the pilot sediment but also the evaluation and correction of possible extraction-derived rearrangement reactions. An excellent agreement amongst our results and between the participants was obtained with a precision of 8.4% RSD at a level of ca. 80 ng TBT g(-1) (as Sn).

Pressurized liquid extraction using the accelerated solvent extractor (Dionex ASE 200) has been evaluated for the determination of mono-, di- and tributyltin (MBT, DBT, and TBT, respectively) in PACS-2 certified reference material. A double-enriched spike containing 119Sn-enriched MBT and TBT and 118Sn-enriched DBT allowed for the simultaneous determination of the three butyltin species and the factors governing species interconversion. The stability of the spike was evaluated by reverse isotope dilution experiments covering more than one year with satisfactory results. Quantitative recoveries using ASE for TBT and DBT were obtained at temperatures above 110 degrees C. The effect of the extraction time and number of static cycles was evaluated. Results suggest that extraction efficiency was quantitative with extraction times as low as 10 min for all butyltin species at 110 degrees C. Decomposition reactions were only detected at the higher temperatures assayed (140 and 175 degrees C) and that was only for the degradation of DBT to MBT (approximately 4%). The results found for MBT were approximately 25% higher than the certified value for the PACS-2 sediment reference material in agreement with previous results obtained by ultrasonic and microwave assisted extraction.

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

The extraction/pre-concentration capabilities of the liquid ion exchanger 7-(4-ethyl-1-methyloctyl)-8-hydroxyquinoline (Kelex-100) immobilized on octadecyl silica have been evaluated for the analysis of trace metals in sea-water by quadruple ICP–MS (ICP–QMS) and the results are compared with those directly obtained by double focusing ICP–MS (ICP–SMS), after a simple 1+4 dilution of the sea-water sample. The retention capabilities of the different materials (Kelex-100 retained on Amberlite XAD-7 and Bondapack C-18 (BC-18)) were evaluated by using copper as test element and ICP–QMS for detection. Kelex-100 supported onto BC-18 provided the best performance and was evaluated, both on-line and off-line, for the pre-concentration of different trace elements present in sea-water. Using the selected “off-line” procedure, satisfactory recoveries (95–104%) and adequate detection limits in the ng l −1 range (V, 10; Ni, 30; Mn, 70; Cu, 170; Zn, 150; Cd, 7; Pb, 10 and U, 1 ng l −1 ) were obtained. Such multi-elemental trace metal determinations in sea-water were also attempted directly, after a simple 1+4 dilution of the sample and calibration with internal standards (IS) using different mass resolutions to avoid polyatomic interferences when necessary. The analytical performance characteristics and limitations of both approaches for multi-elemental determinations in coastal sea-water are compared.

The measurement of isotope ratios (within individual species), for a range of organolead compounds, was carried out by coupling a capillary gas chromatograph (HP 6890) to an inductively coupled plasma mass spectrometer (HP 4500). Optimum conditions for the separation and detection of the derivatised lead organometallic compounds were studied focusing on the precise and accurate measurement of isotope ratios on each chromatographic peak. Three lead isotopes were monitored, 206, 207 and 208 using 66 ms integration time per isotope. Under optimum conditions adequate chromatographic peak profiles could be measured. Based on peak area ratios, isotope ratios were determined for five butylated lead compounds (trimethyllead, dimethyllead, triethyllead, diethyllead and inorganic lead) and mixed methyl-ethyl tetraalkyllead compounds from leaded gasoline. Mass bias was corrected using both ethylated and butylated NIST 981 isotopic standard (Common Lead) and a certified enriched 204Pb solution. No isotope fractionation effects were observed during derivatisation of the different organometallic compounds. Applications of this methodology include the differentiation between Pb sources of the different standards used, the measurement of Pb isotope ratios in the organolead compounds present in airborne particulate matter of Oviedo to ascertain their origin and the comparison with isotope ratios in the BCR CRM 605 urban dust reference material. Significant differences between lead isotope ratios in the different species of organolead compounds and in total lead (after acid digestion of the samples) were observed. These differences can be used to trace back sources of lead contamination, by resorting to the speciation data, in cases where the isotope ratios of total lead in the samples are inconclusive in identifying Pb contamination sources.

The speciation of organolead compounds in airborne particulate matter was determined by gas chromatography–inductively coupled plasma mass spectrometry. The analytes were extracted from the sample using 0.5 M acetic acid in methanol. The ionic organolead compounds were extracted into hexane using diethydithiocarbamate at pH 9 in the presence of EDTA to complex inorganic Pb. Derivatisation of the organolead species was performed with butylmagnesium chloride. Separation and detection of the butylated trimethyllead (TML), dimethyllead (DML), triethyllead (TEL) and diethyllead (DEL) was achieved. Detection limits, as Pb, were 3.2 fg for TML, 2.4 fg for DML, 1.8 fg for TEL and 9 fg for DEL for a 1 μl sample injection. Typical precision of repetitive injections was 1.5–4% using tetraethyllead (TrEL) as internal standard. The method proposed was validated by the determination of TML in the reference material CRM 605 and was applied to the analysis of airborne particulate matter in the atmosphere of the city of Oviedo. DML, DEL and methylethyllead (MEL) were detected at pg m−3 levels.

Two different approaches for the derivatisation of selenomethionine and detection by gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) are compared and applied for the determination of the amino acid in pharmaceutical preparations used as selenium nutritional supplements. The first derivatisation is accomplished in two steps, starting with the esterification of the carboxylic acid group of the seleno-amino acid using propan-2-ol, followed by the acylation of the amino group with trifluoroacetic acid anhydride. Alternatively, the ethyl chloroformate–ethanol method was carried out for the simultaneous esterification and acylation steps catalysed by a pyridine solution. The derivatives were finally extracted into chloroform and injected into the gas chromatograph. The different parameters affecting the efficiency of both derivatisation approaches were optimised, as well as the instrumental operating conditions. Standard GC-MS chromatograms and mass spectra for both derivatisation procedures were also obtained to confirm the structure of the corresponding derivatised amino acids. The propan-2-ol–trifluoroacetic acid anhydride procedure was finally selected and was used to determine selenomethionine in a parenteral solution by GC-ICP-MS. The advantages and disadvantages of both procedures are discussed.

The development of an ICP-MS method for the determination of three essential trace elements, Fe, Cu and Zn, in human serum by isotope dilution analysis is described. A double focusing ICP mass spectrometer (ELEMENT) working at a nominal resolution of 3000 allowed the complete resolution of polyatomic ions overlapping the main isotopes of these elements. The isotope ratios measured for the determination of Fe, Cu and Zn were 57 Fe/ 56 Fe, 65 Cu/ 63 Cu and 67 Zn/ 64 Zn, respectively. Instrumental parameters of the mass spectrometer were optimised for optimum precision, while keeping the total time of analysis under 6 min (2 min per element). The RSD values obtained for the isotope ratios of Fe (57/56), Cu (65/63) and Zn (67/64) were 0.7, 0.2 and 0.8%, respectively, for a natural standard aqueous solution of 50 µg l –1 . The measured isotope ratios were corrected for dead time on the detector and mass discrimination errors. The sample to spike ratio was optimised to minimise the relative error on the isotope ratio measurement. Serum samples were diluted 1+19 with ultrapure water after the addition of the 57 Fe, 65 Cu and 67 Zn spikes. The proposed method was validated by the analysis of NIST SRM 1598 Inorganic Constituents in Bovine Serum, the levels found being in agreement with the certified values. Finally, the method was applied to the analysis of 21 serum samples from healthy blood donors.

The design, construction and performance characteristics of an easily removable interface to couple a gas chromatograph (GC) to an inductively coupled plasma mass spectrometer (ICP-MS) for trace element speciation is described. The interface is based on the use of a heated metallic tube where the last part of the capillary GC column is inserted. This metallic tube is connected to a metallic T-piece where a flow of argon carrier gas is introduced perpendicularly and externally to the metallic tube, creating a high velocity flow of intermediate sheath gas, which prevents condensation of the analytes eluting from the column on the walls of the T-piece or on the connection tubing to the ICP-MS torch. A flexible non-heated PTFE tube is used to connect the exit of the T-piece to the ICP-MS torch. This new interface has been applied to the separation and detection of different organometallic compounds of Se, S, Pb, Hg and Sn.

The analytical potential of fast protein liquid chromatography (FPLC) coupled with a double focusing inductively coupled plasma mass spectrometer (DF-ICP-MS) was evaluated for the multielemental speciation of trace elements in human serum. The separation of the main serum proteins was performed in a MonoQ (HR5/5) anion exchange column using an ammonium acetate gradient (0-0.5 mol l –1 ) at the physiological pH of 7.4 (0.05 mol l –1 TRIS-HCl buffer) as the mobile phase. Proteins were first detected on-line spectrophotometrically at 295 nm and specific detection of metals was also carried out on-line by using a double focusing ICP-MS working at both low (m/Δm=300) and medium resolution (m/Δm=3000) in order to avoid possible polyatomic interferences. The use of variable resolution to carry out multielement speciation studies allowed the detection of Ca, Sr, Fe, Cu, Zn, Se, Mn, Cr, Pb, Al and Sn in different serum samples and introduces a new dimension to this area of investigation. From the multielemental study it was observed that detectable levels of Cr, Al, Pb and Sn were present in uraemic sera while those elements could not be detected in healthy subjects.

A method is described for the determination of a selected range of trace and ultratrace elements in human serum, using a double-focusing ICP-MS instrument (ELEMENT, Finnigan-MAT). Serum samples were diluted 1+4 with ultrapure water and addition of Sc, Ga, Y and Tl as internal standards, to a final concentration of 10 µg L –1 , was used for matrix interference correction. Low and medium resolution scans were performed on the instrument for the analyses, the first scan, at m/Δm=300, for the determination of Rb, Sr, Mo, Cd, Pb and U and the second scan, at m/Δm=3000, for the determination of those elements subject to interference at low resolution: Al, Ca, Cr, Mn, Fe, Co, Cu, Zn and Se. A SRM, 1598 Inorganic Constituents in Bovine Serum (NIST), was analysed for validation of the multi-element analysis method. Good agreement was achieved between the found levels and the certified values. The method was also applied to the multi-analysis of trace elements in serum samples from healthy subjects and from uraemic patients. Preliminary experiments carried out with both quadrupole and double-focusing ICP-MS instruments proved to be invaluable for the study of spectral and non-spectral interferences in ICP-MS detection.

A rapid and fairly accurate semiquantitative method for the determination of trace amounts of metals in water samples of low and high salinity was successfully applied to a double focusing inductively coupled plasma mass spectrometer. The method was used at both low (m/ Δm=300) and medium (m/Δm=3000) resolution, establishing the corresponding molar response curve in both modes. The ionisation conditions (Tionandne) were calculated through the Saha equation by adjusting the molar response of a multi-element solution to a third order polynomial equation by least squares. The validation of the proposed methodology was performed by elemental analysis of a Riverine Water reference material (SLRS-3) from the National Research Council of Canada. Results obtained were in good agreement with the certified values, working at low resolution when no polyatomic interferences were found and at medium resolution when necessary. Matrix effects were also evaluated using sea-water samples, after a 5-fold dilution. An increase in the analytical response for all the elements, used as internal standards, in comparison with the corresponding curve in Milli-Q water was observed. However, once the ionisation conditions had been calculated on the same saline matrix, recoveries of most of the elements under study were satisfactory for semiquantitative analysis.

A detailed examination of the performance of ICP-MS, using both a quadrupole-based and a double-focusing sector field instrument, for the determination of Al in human serum was carried out and the results were compared with those obtained by ETAAS as the reference technique for this type of analysis. Both ICP-MS techniques were compared in terms of observed matrix effects, spectral interferences and overall analytical performance. The results showed that ICP-MS was superior to ETAAS in terms of detection power [detection limits of 0.35 µg l–1 using sector field ICP-MS and 0.85 µg l–1 with quadrupole ICP-MS as compared with 2 µg l–1 for ETAAS determinations of Al in serum (3σ)]. Negligible matrix effects were observed using the quadrupole instrument for serum diluted 1+4 with water. However, the double-focusing instrument showed matrix effects even for a 1+9 dilution of the serum. In any case, such matrix effects could be eliminated by the use of Be and Sc as internal standards. Spectral interferences at m/z=27 in the serum matrix were observed by using the sector field instrument at a resolution of 3000. The analytical methods developed were applied to the determination of Al in reference serum samples with satisfactory results. However, Al serum determination in healthy subjects (n=20) showed that typical basal Al levels in serum required high resolution and, as a rule, were below the detection limit of sector field ICP-MS (i.e., less than 0.35 µg l–1).

A semiquantitative procedure for the determination of trace metals in natural waters and sediments by ICP-MS was developed. The method is based on the establishment of the instrumental molar response curveversus mass using a multi-elemental solution containing elements from Mg to Bi with different ionisation potentials. The ionisation conditions in the plasma (electron density, ne, and ionisation temperature, Tion) were determined by least-squares fitting, using the Saha equation, by adjusting the molar response curve to a third-order polynomial. The effect of matrix constituents, such as Na+, K+, Ca2+, Mg2+, Cl– and SO42–, on both the shape of the molar response curve and on the plasma ionisation conditions was evaluated. It was observed that the ionisation degrees calculated in the plasma did not change significantly in the presence of the matrix constituents. However, the shape of the molar response curve was affected by high concentrations of matrix components and this effect was corrected by adding internal standards to the samples. The semiquantitative procedure was applied to the analysis of local natural waters and the results were compared with those obtained by external calibration and standard additions. Validation was performed by analysis of riverine water (SLRS-3) and sediment (PACS-1 and MESS-2) reference materials from the National Research Council of Canada with satisfactory results.

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.

A procedure for the universal detection and quantification of polar organic compounds separated by liquid chromatography (LC) based on postcolumn carbon isotope dilution mass spectrometry (IDMS) was developed. The eluent from the LC column is mixed online with a continuous flow of (13)C-enriched sodium bicarbonate, and the sodium persulfate oxidation reaction in acidic media is employed to achieve isotope equilibration. All carbon-containing compounds eluting from the column are oxidized to (12)CO(2) and (13)CO(2), respectively, and the carbon dioxide is separated from the aqueous phase using a gas-permeable membrane. The gaseous carbon dioxide is then carried to the mass spectrometer for isotope ratio measurements. Different water-soluble organic compounds were evaluated using a flow injection configuration to assess the efficiency of the oxidation process. Most water-soluble organic compounds tested showed quantitative oxidation. However, chemical structures involving conjugated C═N double bounds and guanidinium-like structures were found to be resistant to the oxidation and were further studied. For this purpose, (13)C(1)-labeled creatine (with the isotopic label in the guanidinium group) was employed as model compound. Specific conditions for the quantitative oxidation of these compounds required lower flow rates and the addition of metallic catalysts. This novel approach was tested as a universal detection and quantification system for LC. A simple standard mixture of four amino acids was separated under 100% aqueous conditions and quantified without the need for specific standards with good accuracy and precision using potassium hydrogen phthalate as internal standard. The main field of application of the developed method is for the purity assessment of organic standards with direct traceability to the International System of Units (SI).

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

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

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

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

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.

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.

Large volume injection, up to 5 mL, was evaluated and optimised for the on-line ion chromatographic separation of Rb and Sr before ICP-MS measurement of Sr isotope ratios. Flat-topped chromatographic peaks, ideally suited for multicollector ICP-MS isotope ratio measurements, could be obtained when the composition of the mobile phase (nitric acid and 18-crown-6 ether) was identical to the matrix of the sample. Under those conditions rubidium eluted at the dead volume of the column while strontium produced a flat-topped transient signal with several minutes of stable plateau. On-line data acquisition during several minutes at the plateau of Sr signal allowed high precision Sr isotope ratio measurement. The developed procedure was evaluated for Sr isotope ratio measurements on different types of samples, including cider, apples, apple leaves, and soil extracts, in the frame of a long-term project aiming at origin authentication using strontium isotope ratio measurements. It was observed that sample matrix caused broadening of the strontium chromatographic peak and loss of flat-topped peak profile. Under those circumstances the addition of the complexing crown-ether 18-crown-6 both to samples and chromatographic eluent provided two distinct advantages. First, a drastic increase in the retention of strontium was observed which could be modulated by increasing the concentration of nitric acid in the eluent up to 900 mM. This increase in the eluent HNO(3) concentration allowed the application of the method to acid soil digests and other high acidity samples. Second, the matrix of the sample did not affect any more the chromatographic peak profile and similar chromatographic separations could be obtained for samples and standards maintaining the flat-topped Sr peak profile. Sample preparation consisted of a simple 1:10 dilution of the cider or pre-treated solid samples by adding HNO(3) (900 mM) and 18-crown-6 ether (5mM) to obtain similar composition in the sample solution and the HPLC eluent.

A methodology for the study of the absorption and metabolism of butyltin compounds in laboratory animals using isotopically enriched species was developed. The method is based on the oral administration of 119Sn-labeled monobutyltin (MBT), 118Sn-labeled dibutyltin (DBT), and 117Sn-labeled tributyltin (TBT) to the animals and the measurement of both the concentration and isotopic composition of these compounds in the different tissues by GC-ICPMS. The degradation of butyltin compounds during their metabolism was computed using least-squares isotope pattern deconvolution, and their concentration was measured by reverse isotope dilution analysis using natural-abundance MBT, DBT, and TBT standards. Male Wistar rats were used as models to evaluate the proposed methodology. Preliminary toxicological results obtained with one rat indicate that TBT is highly absorbed (64.4%), and it is found in all organs with relatively high levels in stomach and intestines. The apparent absorption of DBT was 27.3% and was mainly found in liver, kidney, and intestines. However, a large proportion of the found DBT is formed from the degradation of TBT (approximately 40% of the found DBT in liver is degraded TBT). The apparent absorption of MBT was found to be 12.5%, and the originally administered MBT was mainly recovered in the feces. However, MBT was clearly detected in liver, kidney, stomach, intestines, and urine as degradation products of DBT and TBT. Although a significant variability from rat to rat is expected to be obtained, the analytical variability provided by this methodology is small enough to yield meaningful biological results. The results obtained demonstrate that the developed methodology is able to follow qualitatively, quantitatively, and simultaneously the specific metabolic pathways of different species of a given element.

The fragmentation patterns of butyltin compounds (mono-, di-, and tributyltin) in an electron impact ion source were studied using an isotope pattern reconstruction algorithm with emphasis on isotope ratio measurements from molecular clusters. For this purpose, standards of natural tin isotope abundance and a (119)Sn-enriched mixture of the three compounds were both ethylated and propylated using sodium tetraalkylborates. The corresponding mass spectra of the various tetraalkyltin compounds prepared were obtained by GC/MS after their extraction with hexane. The results showed that pure interference-free molecular clusters were obtained only for certain R(3)Sn(+) ions where no isobaric overlap with R(2)SnH(+) ions occurred (e.g. BuEt(2)Sn(+) overlaps with Bu(2)SnH(+)). These ions are ideal candidates for accurate Sn isotope ratio measurements, while isotope pattern perturbing interferences are observed for other molecular fragments down to Sn(.)(+). Isotope pattern reconstruction algorithm thus can be used as an analytical tool to ensure the absence of molecular interferences--a requirement for accurate isotope ratio measurements from molecular clusters. The relevance of these studies for the determination of butyltin compounds in environmental samples by isotope dilution GC/MS is also discussed.