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.

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.

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.

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.

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.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone.

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g-1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.

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.

Accurate and sensitive determination of molar fractions of 13C-Labeled intracellular metabolites in cell cultures grown in the presence of isotopically-labeled glucose.

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.

Evaluation of the spectral accuracy of mass spectrometers using compounds containing Cl or Br atoms.

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.

A simplified calculation procedure for mass isotopomer distribution analysis (MIDA) based on multiple linear regression.

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 13 C atoms (13 C2 -glycine) as labelled amino acid to demonstrate that spectral overlap is not a problem in the proposed methodology. The developed methodology was tested first in vitro by changing the precursor pool enrichment from 10 to 40% of 13 C2 -glycine. Secondly, a simulated in vivo synthesis of proteins was designed by combining the natural abundance RGGGLK peptide and 10 or 20% 13 C2 -glycine at 1 : 1, 1 : 3 and 3 : 1 ratios. Precursor pool enrichments and fractional synthesis values were calculated with satisfactory precision and accuracy using a simple spreadsheet. This novel approach can provide a relatively rapid and easy means to measure protein turnover based on stable isotope tracers. Copyright © 2016 John Wiley & Sons, Ltd.

Evidence of the direct adsorption of mercury in human hair during occupational exposure to mercury vapour.

We have found clear evidence of direct adsorption of mercury in human hair after the occupational exposure to mercury vapour. We have performed both longitudinal analysis of human hair by laser ablation ICP-MS and speciation analysis by gas chromatography ICP-MS in single hair strands of 5 individuals which were occupationally exposed to high levels of mercury vapour and showed acute mercury poisoning symptoms. Hair samples, between 3.5 and 11cm long depending on the individual, were taken ca. three months after exposure. Single point laser ablation samples of 50µm diameter were taken at 1mm intervals starting from the root of the hairs. Sulfur-34 was used as internal standard. The ratio (202)Hg/(34)S showed a distinct pattern of mercury concentration with much lower levels of mercury near the root of the hair and high levels of mercury near the end of the hair. In all cases a big jump in the concentration of mercury in hair occurred at a given distance from the root, between 32 and 42mm depending on the individual, with a high and almost constant concentration of mercury for longer distances to the root. When we took into account the rate of hair growth in humans, 9-15mm/month, the jump in mercury concentration agreed approximately with the dates when the contamination occurred with the new growing hair showing much lower mercury concentration. In some cases the concentration of mercury at the tip of the hair was ca. 1000 times higher than that near the root. Additionally, speciation studies confirmed that mercury in all hair samples was present as inorganic mercury. The only explanation for these results was the direct adsorption of mercury vapour in hair at the time of exposure.

Simultaneous determination of α-, ß- and γ-hexabromocyclododecane diastereoisomers in water samples by isotope dilution mass spectrometry using (81)Br-labeled analogs.

This work describes the synthesis, characterization and application of three (81)Br-labeled diastereosiomers of hexabromocyclododecane (HBCD) for the accurate and precise determination of α-, ß- and γ-HBCD in water samples by isotope dilution mass spectrometry. The synthesis of the labeled analogs was carried out by bromination of cis, trans, trans-1,5,9-cyclododecatriene with (81)Br-enriched bromine. After isolation and purification by semipreparative HPLC, each diastereoisomer was characterized in terms of concentration and isotopic enrichment. Then, they were added to the samples to simultaneously quantify the three HBCD diastereoisomers in a single LC-MS/MS injection without resorting to a methodological calibration graph. The results obtained here demonstrate that the use of (81)Br-labeled analogs provides accurate and precise determinations of α-, ß- and γ-HBCD in real water samples. The limits of quantification obtained in real samples for α-, ß- and γ-HBCD were 0.022, 0.073 and 0.015 ng L(-1), respectively, significantly lower than those required by the European Directive 2013/39/EC.

Study of the degradation of butyltin compounds in surface water samples under different storage conditions using multiple isotope tracers and GC-MS/MS.

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.

Determination of ultratrace levels of tributyltin in waters by isotope dilution and gas chromatography coupled to tandem mass spectrometry.

The current EU legislation lays down the Environmental Quality Standards (EQS) of 45 priority substances in surface water bodies. In particular, the concentration of tributyltin (TBT) must not exceed 0.2ngL(-1) and analytical methodologies with a Limit of Quantification (LOQ) equal or below 0.06ngL(-1) are urged to be developed. This work presents a procedure for the determination of ultratrace levels of TBT in water samples by Isotope Dilution and GC-MS/MS operating in Selected Reaction Monitoring (SRM) mode which meets current EU requirements. The method requires the monitorization of five consecutive transitions (287>175 to 291>179) for the sensitive and selective detection of TBT. The measured isotopic distribution of TBT fragment ions was in agreement with the theoretical values computed by a polynomial expansion algorithm. The combined use of Tandem Mass Spectrometry, a sample volume of 250mL, the preconcentration of 1mL of organic phase to 30µL and an injection volume of 25µL by Programmed Temperature Vaporization provided a LOQ of 0.0426ngL(-1) for TBT (calculated as ten times the standard deviation of nine independent blanks). The recovery for TBT calculated in Milli-Q water at the EQS level was 106.3±4%. A similar procedure was also developed for the quantification of dibutyltin (DBT) and monobutyltin (MBT) in water samples showing satisfactory results. The method was finally implemented in a routine testing laboratory to demonstrate its applicability to real samples obtaining quantitative recoveries for TBT at the EQS level in mineral water, river water and seawater.

Modification of a commercial gas chromatography isotope ratio mass spectrometer for on-line carbon isotope dilution: Evaluation of its analytical characteristics for the quantification of organic compounds.

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 (<3% RSD) and accuracy (average relative error≤4%) and what is more important using a single and simple generic internal standard for quantification.

Quantification of Cr(VI) in soil samples from a contaminated area in northern Italy by isotope dilution mass spectrometry.

The aims of the work were to detect and quantify hexavalent chromium in 14 soil samples from an area in Lombardia (northern Italy) contaminated by two polluted water plumes. Cr(VI) was extracted from the solid samples by applying focused microwaves in an alkaline medium after Cr(III) complexation with EDTA. Cr(VI) was reduced to Cr(III) when previously reported extraction conditions for the analysis of certified reference materials were used, and Cr(VI) could not be reliably quantified in the soil samples. The influence of organic matter and iron contents in the samples on the reduction of Cr(VI) was subsequently studied using a new set of soil samples with different iron and organic matter concentrations. Isotope dilution mass spectrometry (IDMS) measured two different enriched stable isotopes of Cr (54 and 53) to evaluate the reduction extent of hexavalent chromium during the analytical procedure. The extraction conditions were optimized to obtain the lowest amount of Cr(VI) reduction and quantify Cr(VI) in the polluted soil samples from Lombardia.

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.

Determination of Cystatin C in human serum by isotope dilution mass spectrometry using mass overlapping peptides.

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 (13)C 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.