Simultaneous sample preparation and species-specific isotope dilution mass spectrometry analysis of monomethylmercury and tributyltin in a certified oyster tissue

A rapid, accurate, sensitive, and simple method for simultaneous speciation analysis of mercury and tin in biological samples has been developed. Integrated simultaneous sample preparation for tin and mercury species includes open focused microwave extraction and derivatization via ethylation. Capillary gas chromatography-inductively plasma mass spectrometry (CGC-ICPMS) conditions and parameters affecting the analytical performance were carefully optimized both for species-specific isotope dilution analysis of MMHg and TBT and for conventional analysis of MBT and DBT201Hg-enriched monomethylmercury and 117Sn-enriched tributyltin were used for species-specific isotope dilution mass spectrometry (SIDMS) analysis. As important, accurate isotope dilution analysis requires equilibration between the spike and the analyte to achieve successful analytical procedures. Since the spike stabilization and solubilization are the most critical and time-consuming steps in isotope dilution analysis, different spiking procedures were tested. Simultaneous microwave-assisted spike stabilization and solubilization can be achieved within less than 5 min. This study originally introduces a method for the simultaneous speciation and isotope dilution of mercury and tin in biological tissues. The sample throughput of the procedure was drastically reduced by fastening sample preparation and GC separation steps. The accuracy of the method was tested by both external calibration analysis and species-specific isotope dilution analysis using the first biological reference material certified for multielemental speciation (oyster tissue, CRM 710, IRMM). The results obtained demonstrate that isotope dilution analysis is a powerful method allowing the simultaneous speciation of TBT and MMHg with high precision and excellent accuracy. Analytical problems related to low recovery during sample preparation are thus minimized by SIDMS. In addition, a rapid procedure allows us to establish a performant routine method using CGC-ICPMS technique.     

Application of speciated isotope dilution mass spectrometry to evaluate extraction methods for determining mercury speciation in soils and sediments

Extraction techniques commonly used to extract methylmercury or mercury species from various matrixes have been evaluated regarding their potential to transform inorganic mercury to methylmercury, or vice versa, during sample preparation steps by applying speciated isotope dilution mass spectrometry. Two of the five tested methods were highly prone to form inorganic mercury from methylmercury. Some published methods converted methylmercury to inorganic mercury approximately 100% (including the spiked CH(3)(201)Hg(+)). In other methods, as much as 45% of methylmercury was converted to inorganic mercury during extraction. The methods evaluated included cold acid extraction and sonication. Other methods, such as the proposed EPA RCRA Draft Method 3200, microwave-assisted extraction, and another sonication-based methods induced very little or no methylmercury transformation to inorganic mercury. Among these three methods, the proposed Draft EPA Method 3200 was found to be the most efficient.     

Development of isotope dilution cold vapor inductively coupled plasma mass spectrometry and its application to the certification of mercury in NIST standard reference materials

An isotope dilution cold vapor inductively coupled plasma mass spectrometry (ID-CV-ICPMS) method featuring gaseous introduction of mercury via tin chloride reduction has been developed and applied to the quantification and certification of mercury in various NIST standard reference materials: SRM 966 Toxic Metals in Bovine Blood (30 ng x mL(-1)); SRM 1641d Mercury in Water (1.6 microg x mL(-1)); and SRM 1946 Lake Superior Fish Tissue (436 ng x g(-1)). Complementary mercury data were generated for SRMs and NIST quality control standards using cold vapor atomic absorption spectroscopy (CVAAS). Certification results for the determination of mercury in SRM 1641d using two independent methods (ID-CV-ICPMS and CVAAS) showed a degree of agreement of 0.3% between the methods. Gaseous introduction of mercury into the ICPMS resulted in a single isotope sensitivity of 2 x 10(6) counts x s(-1)/ng x g(-1) for 201Hg and significantly reduced the memory and washout effects traditionally encountered in solution nebulization ICPMS. Figures of merit for isotope ratio accuracy and precision were evaluated at dwell times of 10, 20, 40, 80, and 160 ms using SRM 3133 Mercury Spectrometric Solution. The optimum dwell time of 80 ms yielded a measured 201Hg/202Hg isotope ratio within 0.13% of the theoretical natural value and a measurement precision of 0.34%, on the basis of three replicate injections of SRM 3133.     

Determination of selenomethionine and selenocysteine in human serum using speciated isotope dilution-capillary HPLC-inductively coupled plasma collision cell mass spectrometry

A method for the accurate determination of selenoamino acids in human serum by HPLC-ICPMS was developed using the species-specific isotope dilution analysis principle. A serum sample was enzymatically digested with a mixture of lipase and protease after derivatization of the selenocysteine residues with iodoacetamide. The selenoamino acid fraction was isolated by size exclusion LC followed by the separation of selenomethionine and the carboxymethylated selenocysteine by capillary HPLC. The isotope-specific determination of 77Se and 80Se was achieved on-line by ICP collision cell MS allowing the removal of polyatomic interferences. Quantification was carried out by isotope dilution using a 77Se-labeled selenomethionine spike and the determination of the 77Se/80Se ratio in the cHPLC selenomethionine peak. The accurately determined selenomethionine was used as an internal standard for the selenocysteine determination from the same chromatogram. The modification of the previously developed cHPLC-ICPMS interface allowed the reduction of the absolute detection limits twice (down to the 75-fg level), which resulted in the lowest ever reported procedural detection limits (below 0.5 ng g(-1) for a 450-mg serum sample). The precision was less than 5% RSD. The method was validated by the mass balance of selenium (amino acid incorporated vs total).     

Determination of methylmercury, ethylmercury, and inorganic mercury in mouse tissues, following administration of thimerosal, by species-specific isotope dilution GC-inductively coupled plasma-MS

Isotopically enriched HgO standards were used to synthesize CH3(200)Hg+ and C2H5(199)Hg+ using Grignard reagents. These species were employed for isotope dilution GC-ICPMS to study uptake and biotransformation of ethylmercury in mice treated with thimerosal, (sodium ethylmercurithiosalicylate) 10 mg L(-1) in drinking water ad libitum for 1, 2.5, 6, or 14 days. Prior to analysis, samples were spiked with aqueous solutions of CH3(200)Hg+, C2H5(199)Hg+, and 201Hg2+ and then digested in 20% tetramethylammonium hydroxide and extracted at pH 9 with DDTC/toluene. Extracted mercury species were reacted with butylmagnesium chloride to form butylated derivatives. Absolute detection limits for CH3Hg+, C2H5Hg+, and Hg2+ were 0.4, 0.2, and 0.6 pg on the basis of 3sigma of five separate blanks. Up to 9% of the C2H5Hg+ was decomposed to Hg2+ during sample preparation, and it is therefore crucial to use a species-specific internal standard when determining ethylmercury. No demethylation, methylation, or ethylation during sample preparation was detected. The ethylmercury component of thimerosal was rapidly taken up in the organs of the mice (kidney, liver, and mesenterial lymph nodes), and concentrations of C2H5Hg+ as well as Hg2+ increased over the 14 days of thimerosal treatment. This shows that C2H5Hg+ in mice to a large degree is degraded to Hg2+. Increased concentrations of CH3Hg+ were also observed, which was found to be due to impurities in the thimerosal.     

Determination of mercury in coal by isotope dilution cold-vapor generation inductively coupled plasma mass spectrometry

A method based on isotope dilution cold-vapor inductively coupled plasma mass spectrometry (ID-CV-ICPMS) has been developed for high-accuracy determinations of mercury in bituminous and sub-bituminous coals. A closed-system digestion process employing a Carius tube is used to completely oxidize the coal matrix and chemically equilibrate the mercury in the sample with a 201Hg isotopic spike. The digestates are diluted with high-purity quartz-distilled water, and the mercury is released as a vapor by reduction with tin(II) chloride. Measurements of 201Hg/202Hg isotope ratios are made using a quadrupole ICPMS system in time-resolved analysis mode. The new method has some significant advantages over existing methods. The instrument detection limit is less than 1 pg/mL. The average blank (n = 17) is 30 pg, which is roughly 1 order of magnitude lower than the equivalent microwave digestion procedure. The detection limit in coal is blank limited and is approximately 40 pg/g. Memory effects are very low. The relative reproducibility of the analytical measurements is approximately 0.5% for mercury concentrations in the range 10-150 ng/g. The method has been used to measure mercury concentrations in six coal reference materials, SRM 1632b (77.4 ng/g), SRM 1632c (94.3 ng/g), BCR 40 (433.2 ng/g), BCR 180 (125.0 ng/g), BCR 181 (135.8 ng/g), and SARM 20 (252.6 ng/g), as well as a coal fly ash, SRM 1633b (143.1 ng/g). The method is equally applicable to other types of fossil fuels including both crude and refined oils.     

Potential for using isotopically altered metalloproteins in species-specific isotope dilution analysis of proteins by HPLC coupled to inductively coupled plasma mass spectrometry

The production and evaluation of an isotopically enriched metalloprotein standard for use as a calibrant in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is described. Using a model system involving the copper-containing protein rusticyanin (Rc) from the bacterium Acido-thiobacillus ferrooxidans, it was possible to demonstrate the analytical conditions that could be used for the measurement of metalloproteins by on-line IDMS analysis. Rc was chosen because it is a well-characterized protein with an established amino acid sequence and can be produced in suitable quantities using a bacterial recombinant system. Three different forms of the protein were studied by organic and inorganic mass spectrometry: the native form of the protein containing a natural isotopic profile for copper, an isotopically enriched species containing virtually all of its copper as the 65Cu isotope, and the nonmetalated apo form. Incorporation of the copper isotopes into the apo form of the protein was determined using a UV-vis spectrophotometric assay and shown to be complete for each of the copper-containing species. The experimental conditions required to maintain the conformational form of the protein with a nonexchangeable copper center were established using +ve electrospray mass spectrometry. A pH 7.0 buffer was found to afford the most appropriate conditions, and this was then used with HPLC-ICP-MS to verify the stability of the copper center by analysis of mixtures of different isotopic solutions. No exchange of the enriched copper isotope from Rc with an added naturally abundant inorganic copper cation was observed under a neutral pH environment, indicating that species-specific ID-MS analysis of metalloproteins is possible.     

Development and evaluation of an isotope dilution LC/MS method for the determination of total homocysteine in human plasma

Elevated plasma homocysteine has been identified as a strong and independent risk factor for cardiovascular diseases, and recently, it has been associated with the development of dementia in older adults. Selected ion-monitoring isotope-dilution LC/MS (electrospray) has been developed and evaluated as a reference method for the accurate determination of total homocysteine in human plasma. Homocysteine is quantitatively isolated from plasma via the use of anion-exchange resins and then detected and quantified in stabilized plasma extracts with selected ion-monitoring LC/MS. This method is shown to be highly comparable to LC/MS/MS determinations in terms of its analytical accuracy and precision, yet this alternative measurement approach does not necessitate the enhanced instrumentation or added expense required of tandem MS/MS determinations. LC/MS detection of homocysteine was linear (standard error of the estimate for the regression line was 0.0323) over 3 orders of magnitude, and the calculated limits of detection and quantification were 0.06 micromol/L (0.12 ng on column) and 0.6 micromol/L (1.2 ng on column), respectively. Independent calibration curves showed excellent linearity (r2 > or = 0.996) between 0 and 25 micromol/L homocysteine over a 3-day period. The accuracy and precision of total homocysteine measurements for patient samples and quality control pools using LC/MS were compared to total homocysteine measurements using LC/MS/MS, GC/MS, FPIA, and LC-FD. LC/MS performed well in relation to the other homocysteine methods in terms of its capability to accurately quantify plasma homocysteine over the normal range (5-15 micromol/L).     

Gel electrophoresis coupled to inductively coupled plasma-mass spectrometry using species-specific isotope dilution for iodide and iodate determination in aerosols

In this paper, we present an online coupling of gel electrophoresis (GE) and inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of iodine species (iodide and iodate) in liquid (seawater) and aerosol samples. For the first time, this approach is applied to the analysis of small molecules, and initial systematic investigations revealed that the migration behavior as well as the detection sensitivity strongly depends on the matrix (e.g., high concentrations of chloride). These effects could consequently affect the accuracy of analytical results, so that they need to be considered for the analysis of real samples. The technique used for quantification is species-specific isotope dilution analysis (ssIDA), which is a matrix-independent calibration method under certain conditions. We demonstrate that the use of 129I-enriched iodide and iodate allows the correction of the impact of the matrix on both, the electrophoretic migration and the detection sensitivity of the ICP-MS. After optimization, this coupling offers a novel and alternative method in the analysis of iodine compounds in various matrices. Here, we demonstrate the analytical capability of the technique for the chemical characterization of marine aerosols. The results show the presence of iodide and iodate at the ng m(-3) and sub-ng m(-3) level in the investigated aerosol samples, which were taken at the coastal research station in Mace Head, Ireland. These results are in good agreement with other recent studies, which demonstrated that the iodine chemistry in the marine atmosphere is only poorly understood. In addition to iodide and iodate, another iodine compound could be separated and detected in certain samples with high total iodine concentrations and was identified as elemental iodine, probably in form of triiodide, by peak matching. However, it may arise from an artifact during sample preparation.     

Determination of methionine and selenomethionine in selenium-enriched yeast by species-specific isotope dilution with liquid chromatography-mass spectrometry and inductively coupled plasma mass spectrometry detection

Selenomethionine (SeMet) and methionine (Met), liberated by acid hydrolysis of selenium-enriched yeast, were quantified by liquid chromatography-mass spectrometry (LC/MS) using standard additions calibrations as well as isotope dilution (ID) based on species-specific (13)C-enriched spikes. LC inductively coupled plasma mass spectrometry (ICPMS) was also employed for the quantification of SeMet, and (74)Se-enriched SeMet was used for ID calibration. The results were evaluated to ascertain the feasibility of using these methods in a campaign to certify selenized yeast. Good agreement was found between the methods, which, when averaged, gave concentrations of 5482.2 +/- 101 and 3256.9 +/- 217.4 microg/g for Met and SeMet, respectively. This corresponds to a 1.68:1 Met-to-SeMet ratio in the yeast. Quantification by ID LC/MS and LC ICPMS yields the most precise sets of results with relative standard deviations in the range 0.5-1.3% (n = 6). A total selenium concentration of 2064.6 +/- 45.4 microg/g was obtained for this yeast material. The extraction efficiency and a mass balance budget were determined. Acid hydrolysis liberated 81.0% of the total selenium present. SeMet comprised 79.0% of the extracted selenium and 63.9% of the total selenium present in the yeast.     

Correction of species transformations in the analysis of Cr(VI) in solid environmental samples using speciated isotope dilution mass spectrometry

Speciation of Cr(VI) in solid environmental samples is challenging because of the transformations between Cr(VI) and Cr(III). EPA method 3060A completely extracts Cr(VI) in a hot alkaline solution and preserves the solublized Cr(VI). This procedure, however, can oxidize Cr(III) in some chemical forms. On the other hand, the reverse transformation may occur during neutralization and acidification following the extraction step. We developed a method that is capable of monitoring and correcting for such bidirectional species transformations to determine Cr(VI) in solid samples. In this method, we spike a sample with a 53Cr(VI) spike (enriched in 53Cr) and a isoCr(III) spike (enriched in 50Cr). The large quantity of isoCr(III) in an easily oxidizable form competes with sample Cr(III) in the oxidization, reducing the method-induced oxidation of sample Cr(III). This method also corrects for the reduction of Cr(VI). The theory is presented and is evaluated experimentally. The analysis of chromite ore processing residue, fly ash, and standard reference material SRM 1645 showed that the oxidation of sample Cr(III) could cause positive biases as high as 163% if no correction is performed.     

Determination of methionine and selenomethionine in yeast by species-specific isotope dilution GC/MS

A method for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast using species-specific isotope dilution (ID) gas chromatography/mass spectrometry (GC/MS) is described. Samples were digested by refluxing for 16 h with 4 M methanesulfonic acid. Analytes were derivatized with methyl chloroformate and extracted into chloroform for GC/MS analysis. In addition to use of commercially available 13C-enriched Met and SeMet spikes for species specific ID analysis, a 74Se-enriched SeMet spike was also available for comparison of results. In selective ion monitoring mode, the intensities of ions at m/z 221, 222, 269, 270, and 263 were used to calculate the 221/222, 269/270, and 269/263 ion ratios for quantification of Met and SeMet. Concentrations of 5959 +/- 33 and 3404 +/- 12 microg g(-1) (one standard deviation, n = 6) with relative standard deviations of 0.55 and 0.36% for Met and SeMet, respectively, were obtained using 13C-enriched spikes. A concentration of 3417 +/- 8 microg g(-1) (one standard deviation, n = 6) was obtained using the 74Se-enriched SeMet spike. The concentration of SeMet measured in the yeast is equivalent to 66.43 +/- 0.24% of total Se and 30.31 +/- 0.11% of total Met is in the form of SeMet. Method detection limits (three times the standard deviation) of 3.4 and 1.0 microg g(-1) were estimated for Met and SeMet, respectively, based on a 0.25-g subsample of yeast with 1 mL of extract used for derivatization. A similar concentration of 5930 +/- 29 microg g(-1) (one standard deviation, n = 4) for Met and a lower concentration of 2787 +/- 49 microg g(-1) (one standard deviation, n = 4) for SeMet were obtained for this yeast sample using species-specific ID analysis based on GC/MS with 13C-enriched Met and SeMet spikes when a 2-h open microwave digestion approach using 8 M methanesulfonic acid was used.     

Quantitative analysis of proteins via sulfur determination by HPLC coupled to isotope dilution ICPMS with a hexapole collision cell

Quantitative analysis of proteins is an essential part and also constitutes a major challenge in modern proteomics. Quantification of proteins by inductively coupled plasma mass spectrometry (ICPMS) offers an alternative method for quantitative proteomics. In this study, we developed a method of absolute quantification of proteins via sulfur by size exclusion chromatography (SEC) coupled to ICPMS with a collision cell (ICP-CC-MS) and postcolumn isotope dilution. Bovine serum albumin (BSA), superoxide dismutase (SOD), and metallothionein-II (MT-II) served as model proteins. Enriched 34S, 65Cu, and 67Zn isotopic solutions were continuously mixed with the eluate from the SEC. Oxygen was added as a reactive gas into the collision cell where sulfur reacts with oxygen to form sulfur-oxygen ion, the ratio of 32S16O(+)/34S16O(+) thus representing 32S(+)/34S(+). The absolute quantity of proteins could be calculated by the isotopic dilution equation and the content of sulfur in the proteins. The detection limits for BSA, SOD, and MT-II are 8, 31, and 15 pmol, respectively. The relative standard deviations for the proteins are less than 3%. The ratios of S/Cu and S/Zn in the proteins were also determined. The quantitative method was validated by comparing with gravimetric results.     

Comparative studies on the certification of reference materials by ICPMS and TIMS using isotope dilution procedures

A comparison of different isotope dilution mass spectrometric (IDMS) procedures using inductively coupled plasma mass spectrometry (ICPMS) and thermal ionization mass spectrometry (TIMS) was carried out to examine the degree of equivalence between the used procedures in terms of requirements for reference material certification. The comparison was based on the measurement results and their uncertainties. The sample used in this study is a pure zinc metal to be certified by the Bureau Communie de Référence (BCR) for amount contents of different trace elements. This study focuses on cadmium and thallium. The TIMS values contributed to the certified values. To guarantee identical conditions as far as possible for the procedures under investigation, the samples were split into subsamples after spiking and digestion took place. Thus, every IDMS procedure started with an identical set of samples. In total, four different IDMS procedures and one external calibration procedure using internal standardization as an example of routine analysis were applied. The IDMS procedures divide in a group with and a group without trace/matrix separation. Multicollector TIMS (TI-MC-MS) and multicollector ICPMS (ICP-MC-MS) were used in combination with trace/matrix separation, whereas quadrupole ICPMS (ICP-QMS) and ICP-MC-MS were also applied to nonseparated samples. All IDMS results agree well within their combined uncertainties, while some results from the external calibration procedure do not. IDMS results obtained by ICPMS without separation are comparable to those obtained by TI-MC-MS with separation regarding precision and accuracy. The smallest uncertainties were achieved using ICP-MC-MS in combination with trace/matrix separation.     

High-throughput assay of oleopentanedialdheydes in extra virgin olive oil by the UHPLC-ESI-MS/MS and isotope dilution methods

The quality of extra virgin olive oil is associated with the presence of microcomponents whose healing effects have been proved in some special cases. The enzymatic hydrolysis of oleuropein and ligstroside, and of their demethylated analogues, affords four different pentanedialdehydes, and for one of which, 2-(4-hydroxyphenyl)ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate, also known as oleocanthal, an anti-inflammatory effect was quite recently carefully assessed. Extra virgin olive oil is now worldwide considered as a functional food whose daily intake, as for the Mediterranean diet, helps consumers in keeping a constant level of nonsteroidal anti-inflammatory drug (NSAID) in the blood. The presence of these active principles provides, therefore, olive oil with an important added value. In the framework of the actions of the recently funded Agrifood Regional Center, which should coordinate the scientific research and production worlds, an absolute analytical method was developed for the mass spectrometric detection of the two most abundant NSAIDs, Tyr-OLPD and HTyr-OLPD (oleopentanedialdehydes (OLPDs) conjugated to p-hydroxyphenylethanol and 3,4-dihydroxyphenylethanol, respectively), by UHPLC-ESI-MS/MS.     

Comparison of isotope dilution mass spectrometry methods for the determination of total homocysteine in plasma and serum

Two independent methods have been critically evaluated and applied to the measurement of total homocysteine in serum and plasma: solid-phase anion extraction (SPAE) gas chromatography/mass spectrometry (GC/MS) and protein precipitation liquid chromatography/tandem mass spectrometry (LC/MS/MS). In addition, analysis of samples prepared by SPAE was accomplished by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS. These methods have been used to determine total homocysteine levels in several existing serum-based Standard Reference Materials (SRMs) from the National Institute of Standards and Technology and in patient plasma samples provided by the Centers for Disease Control and Prevention. The precision of the homocysteine measurements in serum and plasma was critically evaluated, and method comparisons were carried out using Bland-Altman plots and bias analysis. On the basis of the excellent precision and close agreement of the mass spectrometric (MS) methods, the MS-based methods will be used for certification of a serum-based SRM for homocysteine and folates.     

Screening for organic phosphorus compounds in aquatic sediments by liquid chromatography coupled to ICP-AES and ESI-MS/MS

The structures of organic phosphorous (P) compounds in aquatic sediments are to a large extent unknown although these compounds are considered to play an important role in regulating lake trophic status. To enhance identification of these compounds, a liquid chromatography (LC) method for their separation was developed. The stationary phase was porous graphitic carbon (PGC), and the mobile phases used in the gradient elution were compatible with both inductive coupled plasma atomic emission spectroscopy (ICP-AES) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). With LC-ICP-AES, eight different P containing peaks could be observed in the P chromatogram indicating that at least eight different P compounds were separated. With the setup of an information dependent acquisition (IDA) with ESI-MS/MS, the mass over charge ( m/ z) of compounds containing a phosphate group (H 2PO 3 (-), m/ z 97) could be measured and further fragmentation experiments gave additional information on the structure of almost 40 separated P compounds, several were verified to be nucleotides. ICP-AES was very suitable in the development of the LC method and allowed screening and quantification of P compounds. The presented LC-ESI-MS/MS technique was able to identify several sediment organic P compounds.     

Simultaneous determination of mono-, di-, and tributyltin in sediments by isotope dilution analysis using gas chromatography--ICPMS

A mixed spike containing 119Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) was prepared by direct butylation of 119Sn-enriched tin metal using a 1:3 molar excess of butyl chloride with iodide and triethylamine as catalysts. The isotopic composition of the different tin species in the spike solution was determined by gas chromatography- ICPMS after aqueous ethylation using sodium tetraethylborate. Reverse isotope dilution analysis was used for the characterization of the spike by means of natural MBT, DBT, and TBT standards. No species transformation was evident during derivatization from the reverse isotope dilution experiments based on the measured isotope ratios both before and after spiking. The mixed spike was applied to the simultaneous analysis of MBT, DBT, and TBT in certified reference materials, PACS-2 and CRM 646, with satisfactory results.     

Identification of selenium-containing glutathione S-conjugates in a yeast extract by two-dimensional liquid chromatography with inductively coupled plasma MS and nanoelectrospray MS/MS detection

An approach for the identification of unknown selenium-containing biomolecules was developed, enabling the identification of selenodiglutathione (GS-Se-SG) and the mixed selenotrisulfide of glutathione and cysteinylglycine (GS-Se-SCG) in aqueous yeast extracts. The method consists of two-dimensional liquid chromatography, inductively coupled plasma mass spectrometry (ICPMS) and nanoelectrospray tandem mass spectrometry. Analytes were separated by size-exclusion chromatography followed by preconcentration and separation on a porous graphitic carbon HPLC column. The HPLC effluent was monitored for selenium by ICPMS, and two selenium-containing fractions were isolated and analyzed by nanoelectrospray MS. The nanoelectrospray technique has a low sample consumption of approximately 80 nL/min, enabling a preconcentration of the sample to a few microliters. Mass spectra of the two fractions showed the characteristic Se isotopic pattern centered at m/z 693.1 and 564.0 for the [M + H]+ 80Se ions. MS/MS spectra of adjacent parent ions confirmed the presence of Se. The two selenium species were identified as GS-Se-SG and GS-Se-SCG by collision induced dissociation (CID). The accurately measured masses of the most abundant 691 and 693 u parent ions are in good agreement (differences = 3 ppm) with the theoretical masses. To our knowledge, this is the first identification of GS-Se-SG and GS-Se-SCG in biological matrixes by MS/MS.     

Negative chemical ionization GC/MS determination of nitrite and nitrate in seawater using exact matching double spike isotope dilution and derivatization with triethyloxonium tetrafluoroborate

The alkylation of nitrite and nitrate by triethyloxonium tetrafluoroborate allows determination of their ethyl esters by headspace gas chromatography/mass spectrometry (GC/MS). In the present study, significant improvement in analytical performance is achieved using negative chemical ionization providing detection limits of 150 ng/L for NO(2)(-) and 600 ng/L for NO(3)(-), an order of magnitude better than those achieved using electron impact ionization. The derivatization procedure was optimized and alkaline conditions adopted to minimize conversion of nitrite to nitrate (determined to be 0.07% at 100 mg/L NO(2)(-)) and to avoid the exchange of oxygen between the analytes and the solvent (water). Quantitation entails use of isotopically enriched standards (N(18)O(2)(-) and (15)NO(3)(-)), which also permits monitoring of potential conversion from nitrite to nitrate during the analysis (double spike isotope dilution).