Comparison between one and two-dimensional liquid chromatographic approaches for the determination of plasmatic stroke biomarkers by isotope dilution and tandem mass spectrometry.

This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.

Determination of methylmercury and inorganic mercury in human hair samples of individuals from Colombian gold mining regions by double spiking isotope dilution and GC-ICP-MS.

With the aim to distinguish between routes of exposition to mercury (Hg) in artisanal and small-scale gold mining (ASGM) communities and to distinguish between Hg contamination sources, Hg species composition should be performed in human biomarkers. In this work, Hg species-specific determination were determined in human hair samples (N = 96), mostly non-directly occupied in ASGM tasks, from the six most relevant gold mining Colombian regions. Therefore, MeHg, Hg(II) and THg concentrations were simultaneously determined by double spiking species-specific isotope dilution mass spectrometry (IDMS) and GC-ICP-MS. Only 16.67% of participants were involved at some point in AGSM works and fish consumption ranged from 3 to 7 times/week, which is between medium and high intake levels. The median concentration of THg obtained from all samples is higher than the reference dose weekly acceptable of MeHg intake established by the EPA (1 ppm), whereas a 25% were more than 4 times higher than the WHO level (2.2 µg Hg g-1). Median THg value of individuals consuming fish 5-7 times per week was significantly higher (p < 0.05) than those of the other consuming groups (12.5 µg Hg g-1). Most of the samples presented a % of MeHg relative to THg higher than 80%. The average % of Hg(II)/THg was 11% and only 10 individuals presented a Hg(II) content over 30%. No significant differences (p > 0.05) were found when the amount of Hg(II) was compared between people involved in AGSM task and people not involved. Interestingly, significant differences among the evaluated groups where found when the percentage of the Hg(II)/THg ratio of these groups were compared. In fact, people involved in AGSM tasks showed 1.7 times higher Hg(II)/THg vs. inhabitants uninvolved. This suggest that Hg(II) determination by IDMS-GC-ICP-MS could be a good proxy for evaluating Hg(II) adsorption by direct exposure to mercury vapors onto hair.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Isotope Dilution Mass Spectrometry for Highly Precise Determination of Dissolved Inorganic Carbon in Seawater Aiming at Climate Change Studies.

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).

Instrumental Setup for Simultaneous Total and Speciation Analysis of Volatile Arsenic Compounds in Gas and Liquefied Gas Samples.

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.

Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

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.

Detection of transgenerational barium dual-isotope marks in salmon otoliths by means of LA-ICP-MS.

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.

Development and evaluation of an electrochemical biosensor for creatinine quantification in a drop of whole human blood.

An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor.

Fast and accurate procedure for the determination of Cr(VI) in solid samples by isotope dilution mass spectrometry.

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.

Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS.

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

Determination of ultra-trace levels of priority PBDEs in water samples by isotope dilution GC(ECNI)MS using 81Br-labelled standards.

A gas chromatography electron capture negative ionization mass spectrometry (GC(ECNI)MS) procedure for the determination of priority polybrominated diphenyl ethers (PBDEs; congeners 28, 47, 99, 100, 153 and 154) in water samples at regulatory EU levels has been developed. The method is based on the use of (81)Br-labelled PBDEs for isotope dilution analysis and the measurement of (79)Br/(81)Br isotope ratios in gas chromatography peaks with the electron capture negative ionization technique. The suitability of this ion source for the precise and accurate measurement of bromine isotope ratios has been demonstrated. The general ECNI-IDMS procedure was evaluated by the analysis of NIST SRM 1947 (Lake Michigan fish tissue) with satisfactory results. For the analysis of water samples, 500 mL of the samples were spiked with the labelled PBDEs and extracted with 10 mL isooctane for 30 min. The extract was evaporated down to ca. 100 µL and injected in the GC(ECNI)MS. Detection limits ranged from 0.014 (-1) to 0.089 pg mL(-1) depending on the congener. Recoveries from real water samples, spiked at a level of 0.5 pg mL(-1), ranged from 77% to 102%.

Use of the stable isotope (57) Fe to track the efficacy of the foliar application of lignosulfonate/Fe(3+) complexes to correct Fe deficiencies in cucumber plants.

BACKGROUND: During the last decade, environmental concerns regarding the use of recalcitrant synthetic chelates to overcome iron chlorosis has increased and new ligands such as lignosulfonates (LS) have been evaluated. However, the efficacy of these products is variable. In this work a hardwood (eucalyptus) and softwood (spruce) LS were compared to try to relate their physico-chemical characteristics and their efficacy. Also two more products derived from the eucalyptus lignosulfonate were tested. RESULTS: All the LS tested presented a good ability to complex Fe, but only the spruce LS was capable to maintain significant amounts of soluble Fe above pH 8. According to the FTIR data, structural changes related to the Fe source (Fe(2+) or Fe(3+) ) used to form the complex occurred in the LS molecule and might influence their efficacy. Cucumber (Cucumis sativus L. cv Ashley) chlorotic plants were used to test lignosulfonate efficacy when applied through foliar sprays in comparison with FeSO(4) and EDTA/(57) Fe(3+) . The (57) Fe content of plants sprayed with LS was very low in respect to the EDTA treatment, but this was not reflected in the biomass and re-greening rates. Eucalyptus LS modifications improve its efficacy for iron chlorosis recovery to levels similar to those found for the spruce LS. Two applications of the LS are recommended. CONCLUSIONS: Lignosulfonates did not require surfactants for their application; they did not burn the leaves, and had a stimulating effect on the vegetative growth of the plants. So these by-products could be a good alternative when applied through foliar sprays for cucumber plants.

Comprehensive Isotope Ratio Metabolomics: Gas chromatography Isotope Ratio Mass Spectrometry of urinary metabolites and exhaled breath.

We have developed an analytical procedure to measure the carbon isotopic composition of multiple compounds even when there is a partial overlap in the chromatographic profiles and applied this procedure to measure the carbon isotopic composition of different metabolites in human urine and exhaled breath. Method development and validation was performed with CRM IAEA-600 caffeine after calibration of the reference CO2 gas using a mixture of certified undecane, pentadecane and eicosane δ(13C) standards. The alternative data treatment procedure included the correction of time-lag between Faraday cup amplifiers (44 ms at mass 45 and -160 ms at mass 46), the calculation and correction of chromatographic isotope effects on each peak (isotope shifts) and the calculation of the isotope ratio for each compound using the linear regression slope procedure with data only at the top of the chromatographic peak. In that way, partial chromatographic overlap between different metabolites can be tolerated (resolution equal or higher than 1). The reproducibility (SD) of the carbon isotope composition of 93 metabolites in human urine (n = 8) from one volunteer was typically better than 0.5 δ(13C) (range 0.1-2.0 δ(13C), median 0.4 δ(13C)). The method was applied to follow the carbon isotope composition of different metabolites in human urine and exhaled breath after the oral administration of 100 mg of universally labelled 13C-glucose to another human volunteer. It was demonstrated that isotopically labelled compounds could be detected in both samples even 2 h after administration. So, the developed methodology can be applied to multiple types of samples containing a large number of partially overlapping analytes including environmental applications, anti-doping control or metabolomics studies, including the use of enriched isotope tracers.

Multiple heart-cutting two dimensional liquid chromatography and isotope dilution tandem mass spectrometry for the absolute quantification of proteins in human serum.

We evaluate here the combination of two-dimensional liquid chromatography (2D-LC) in the multiple heart cutting mode and isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples. As a proof of concept, we attempt the quantification of proteotypic peptides of Apolipoprotein AIV (APOA4), Complement C3 (C3) and Vitronectin (VTN) which have been previously identified as potential candidate biomarkers of glaucoma. Using this 2D-LC strategy, analyte enrichment steps are avoided and the sample preparation involved after enzymatic digestion amounted to a simple centrifugation, evaporation of the supernatant and reconstitution in the 1D mobile phase. A mobile phase not compatible with the ESI source (10 mM KH2PO4 at pH 2.7) was used in the first dimension as it provided a satisfactory chromatographic resolution of the peptides and a high buffering capacity avoiding changes in retention times when analyzing complex matrices like human serum. We also demonstrate that using coeluting labelled analogues of the target peptides, protein concentrations were not affected by slight retention time shifts affecting the amount of target peptides transferred to the second dimension. Satisfactory results were obtained when analyzing fortified serum samples (recoveries from 98 to 113%). Precisions in the range of 1-9% RSD were obtained when replicating the analysis of a pooled serum sample. The comparative analysis of serum samples from n = 94 control subjects and n = 91 patients diagnosed with primary open-angle glaucoma did not show significant differences in the APOA4, VTN and C3 concentrations in contrast with previous studies using immunoassays.

Gas chromatography-combustion-mass spectrometry with postcolumn isotope dilution for compound-independent quantification: its potential to assess HS-SPME procedures.

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.

Synthesis of 81Br-labeled polybrominated diphenyl ethers and their characterization using GC(EI)MS and GC(ICP)MS.

A mixture of different (81)Br-labeled polybrominated diphenyl ethers (PBDEs) was prepared and characterized for its future use as spike for the isotope dilution analysis of PBDEs. The synthesis was carried out by direct bromination of diphenyl ether using (81)Br enriched Br(2) obtained after aqueous oxidation of bromide with potassium peroxymonosulfate and extraction into dichloromethane. The number of bromine atoms introduced in the diphenyl ether molecule depended on the molar ratio between bromine and diphenyl ether. The final mixture prepared contained a mixture of tri-, tetra-, penta-, and hexabrominated PBDEs with a larger concentration of the tetrabrominated congener BDE-47. The isotopic composition of bromine in the resulting PBDEs mixture was determined by GC(ICP)MS and resulted in a 99.53% enrichment of the isotope 81 of bromine. The concentration of three of the PBDE congeners (28, 47, and 99) in the mixture was determined by reverse isotope dilution analysis using a certified, natural abundance, PBDEs mixture and both GC(ICP)MS and GC(EI)MS. For this purpose, the fragmentation and isotope distribution patterns of the different PBDE cogeners in the positive electron ionization source were studied in detail both for natural abundance and labeled compounds. A procedure based on isotope pattern deconvolution was developed which allowed the direct determination of the concentration of the labeled PBDEs in the spike mixture by GC(EI)MS. Finally, the GC(EI)MS isotope pattern deconvolution procedure was applied for the determination of natural abundance congeners 28, 47, and 99 in spiked waters at ng L(-1) levels. Detection limits below 0.5 ng L(-1) could be obtained for all compounds using only 100 mL of sample and liquid-liquid extraction with isooctane.

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

This work demonstrates, for the first time, the applicability of multiple spiking isotope dilution analysis to molecular mass spectrometry exemplified by the speciation analysis of mercury using GC(EI)MS instrumentation. A double spike isotope dilution approach using isotopically enriched mercury isotopes has been applied for the determination of inorganic mercury Hg(II) and methylmercury (MeHg) in fish reference materials. The method is based on the application of isotope pattern deconvolution for the simultaneous determination of degradation-corrected concentrations of methylmercury and inorganic mercury. Mass isotopomer distributions are employed instead of isotope ratios to calculate the corrected concentrations of the Hg species as well as the extent of species degradation reactions. The isotope pattern deconvolution equations developed here allow the calculation of the different molar fractions directly from the GC(EI)MS mass isotopomer distribution pattern and take into account possible impurities present in the spike solutions employed. The procedure has been successfully validated with the analysis of two different certified reference materials (BCR-464 and DOLT-4) and with the comparison of the results obtained by GC(ICP)MS. For the tuna fish matrix (BCR-464), no interconversion reactions were observed at the optimized conditions of open focused microwave extraction at 70 degrees C during 8 min. However, significant demethylation was found under the same conditions in the case of the certified dogfish liver DOLT-4. Methylation and demethylation factors were confirmed by GC(ICP)MS. Transformation reactions have been found to depend on the sample matrix and on the derivatization reagent employed. Thus, it is not possible to recommend optimum extraction conditions suitable for all types of matrices demonstrating the need to apply multiple spiking methodologies for the determination of MeHg and Hg(II) in biological samples. Double spike isotope dilution analysis methodologies using widespread GC(EI)MS instrumentation are proposed here for the routine analysis of inorganic mercury and methylmercury in fish samples. The estimated method detection limits were below 10 ng g(-1) for both mercury species. Precision was evaluated for the concentrations present in the certified reference materials (CRMs) which vary from 0.1 to 5 microg g(-1), achieving values of coefficients of variation ranging from 7% to 2%. The concentrations obtained in both CRMs analyzed were in excellent agreement with the certified values, demonstrating the accuracy of the method at these concentration levels.

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

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

Novel HPLC-ICP-MS strategy for the determination of beta2-transferrin, the biomarker of cerebrospinal fluid (CSF) leakage.

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.

Internal correction of hafnium oxide spectral interferences and mass bias in the determination of platinum in environmental samples using isotope dilution analysis.

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