Electron Capture Negative Chemical Ionization Research Articles

Temporal trends of lipophilic organic contaminants in blue mussel (1994–2017) and eelpout (1994–2017) from the southern Baltic Sea

A time-trend study was carried out for two important Baltic Sea species, blue mussel (1994–2017, 11 samples) and eelpout (1994–2017, 11 samples), to track the changes in levels of regulated persistent organic pollutants (POPs) and show potential increases in the levels of the contaminants of emerging concern (CECs). It was carried out utilizing gas chromatography–high-resolution mass spectrometry (GC-HRMS) based non-target screening (NTS). Data were acquired in two modes – electron ionization (EI) and electron capture negative ion chemical ionization (ECNI) – to widen the contaminant coverage, and treated using a fast semi-automated NTS data processing workflow. The study revealed that >250 tentatively identified compounds show statistically significant temporal trends in Baltic blue mussel and eelpout. A large number of regulated substances, including but not limited to PCBs, DDTs and other organochlorine pesticides (OCPs), chlorobenzenes, and many polybrominated diphenyl ethers (PBDEs), showed significant declining trends, as was expected. Their rates of decline were in good agreement with previously reported data. In contrast, increasing trends were observed for many CECs, some polycyclic aromatic compounds (PAHs), and hydrocarbons. The CEC group included, among others, four compounds, namely, one personal care product ingredient, 2-ethylhexyl stearate, one brominated compound 1,2,3,5-tetrabromobenzene and two intermediates 4-isopropoxyaniline and bilobol dimethyl ether, that were reported in marine biota for the first time to the best of our knowledge. Several compounds, including four CECs and two unknown brominated compounds, showed levels considerably higher than the common legacy pollutants (CB-153 and BDE-99), which might be taken into consideration for future monitoring and risk assessment. In addition, this work revealed the presence of a plethora of organoiodinated compounds that exhibited statistically significant temporal trends in the samples under study, which could be of future interest.

Comprehensive non-target screening of biomagnifying organic contaminants in the Baltic Sea food web

High-resolution mass spectrometry (HRMS) based non-target screening (NTS) is a powerful approach for the simultaneous determination of multiple environmental contaminant classes in complex biota samples. In this study, trophic biomagnification factor (TMF) directed NTS was performed to find and (tentatively) identify known, emerging, and new chemical contaminants that are persistent and biomagnify in Baltic Sea biota. The investigated food web included seven species: one filter feeder (blue mussel, Mytilus edulis), two fish (eelpout, Zoarces viviparous; herring, Clupea harengus), two marine mammals (harbor porpoise, Phocoena phocoena; grey seal, Halichoerus grypus) and two birds (guillemot, Uria aalge; white-tailed sea eagle, Haliaeetus albicilla). The NTS procedure included extraction with organic solvent mixtures, two-step high-resolution gel permeation chromatography clean-up, Florisil® fractionation, gas chromatography (GC) HRMS analysis in electron ionization (EI) and electron capture negative ion chemical ionization (ECNI) modes, and NTS data processing. The latter was performed differently for the EI and ECNI data: the EI data were treated using a flexible and highly automated TMF-directed NTS workflow, whereas the ECNI data were treated with a simpler and less automated workflow that specifically screened for brominated compounds. The two workflows collectively revealed biomagnification (statistically significant TMF values) of >250 tentatively identified compounds, including legacy persistent organic pollutants (POPs), such as PCBs and PCB-related compounds, DDT and its metabolites, and organochlorine pesticides (OCPs), contaminants of emerging concern (CECs), and halogenated natural products (HNPs). Among the tentatively identified CECs, nine have not previously been reported in environmental biota samples. These included four polymer additives (used as antioxidants, rubber additives or plasticizers) and two cosmetic product additives (ethyl myristate and isopropyl palmitate). The CECs should be prioritized for future structure verification and quantification using reference standards.

A time-trend guided non-target screening study of organic contaminants in Baltic Sea harbor porpoise (1988–2019), guillemot (1986–2019), and white-tailed sea eagle (1965–2017) using gas chromatography–high-resolution mass spectrometry

The rate of decline in regulated persistent organic pollutant (POP) concentrations in Baltic Sea biota has leveled off in recent years, with new contaminants frequently being discovered. There is, therefore, a need for comprehensive approaches to study occurrence and temporal trends of a wide range of environmental contaminants, including legacy POPs, contaminants of emerging concern (CECs), and new contaminants. In the current work, non-target screening (NTS) workflows were developed and used for, to the best of our knowledge, the first time-trend directed NTS of biota using gas chromatography–high-resolution mass spectrometry (GC-HRMS). To maximize contaminant coverage, both electron ionization (EI) and electron capture negative ion chemical ionization (ECNI) were used. The EI data were treated using highly automated workflows to find, prioritize, and tentatively identify contaminants with statistically significant temporal trends. The ECNI data were manually processed and reviewed prior to time-trend analysis. Altogether, more than 300 tentatively identified contaminants were found to have significant temporal trends in samples of Baltic guillemot, harbor porpoise, or white-tailed sea eagle. Significant decreases were found for many regulated chemicals, as could be expected, such as PCBs, polychlorinated terphenyls, chlorobenzenes, toxaphenes, DDT, other organochlorine pesticides, and tri- and tetra- bromodiphenyl ethers (BDEs). The rate of decline of legacy POPs agreed well with data reported from targeted analyses. Significant increases were observed for small polycyclic aromatic hydrocarbons, heptaBDEs, CECs, and terpenes and related compounds. The CECs included, among others, one plasticizer tributyl acetylcitrate (ATBC), two antioxidants 2,6-bis(1,1-dimethylethyl)phenol and 2,6-bis(tert-butyl)-4-(4-morpholinyl-methyl)phenol, and two compounds used in polymer production, trimethyl isocyanurate and 2-mercaptobenzothiazole, which had not previously been reported in biota. Their increased concentrations in biota indicate increased use and release. The increase in ATBC may be linked to increased use of it as a substitute for di-2-ethylhexyl phthalate (DEHP), which has been phased out over the last decade.

Non-targeted screening workflows for gas chromatography\u2013high-resolution mass spectrometry analysis and identification of biomagnifying contaminants in biota samples

The health of key species in the Baltic region has been impaired by exposure to anthropogenic hazardous substances (AHSs), which accumulate in organisms and are transferred through food chains. There is, thus, a need for comprehensive characterization of the occurrence and accumulation of AHSs in the ecosystem. In this study, we use a non-target screening (NTS) approach for this purpose. A major challenge in NTS of biological samples is the removal of matrix components such as lipids that may interfere with the detection and identification of compounds of interest. Here, we combine gel permeation chromatography with Florisil® column fractionation to achieve sufficient lipid removal for gas chromatography–high-resolution mass spectrometry analysis using electron ionization (EI) and electron capture negative ion chemical ionization (ECNI). In addition, we present new data processing workflows designed to systematically find and identify frequently occurring and biomagnifying AHSs, including known, emerging, and new contaminants. Using these workflows, we discovered a wide range of contaminants in tissue samples from blue mussels, fish, and marine mammals, and calculated their biomagnification factors (BMFs). Compounds with BMFs above 1 for herring and at least one marine mammal included legacy chlorinated pollutants (polychlorinated biphenyls, DDTs, chloro-benzenes/cyclohexanes, chlordanes, toxaphenes, dieldrin), polybrominated diphenyl ethers (PBDEs), and brominated biphenyls. However, there were also several halogenated natural products (halogenated methoxylated brominated diphenyl ethers, 1′-methyl-1,2′-bipyrroles, 1,1′-dimethyl-2,2′-bipyrroles, and the halogenated monoterpene mixed halogenated compound 1) as well as the novel flame retardant Dechlorane 602 and several polycyclic aromatic hydrocarbons, terpenoids, and steroids. The legacy pollutants exhibited the expected biomagnification behavior, demonstrating the utility of the unguided data processing workflow.Graphical abstract

Solid-phase Extraction for Analysis of Short-Chain Chlorinated Paraffins in Water Samples

Abstract A method for analysis of short-chain chlorinated paraffins (SCCPs) in water based on solid-phase extraction (SPE) was established using electron capture negative chemical ionization mass spectrometry (GC-ENCI-MS). The SPE parameters, including the sorbent, eluent and elution volume were optimized. The Agilent Bond Elut-C18 was precleaned with 3 mL of hexane and 3 mL of methanol, and subsequently conditioned with 3 mL H2O prior to use. After the water samples were enriched by the cartridge, the sample was washed with 3 mL of 10% (V/V) methanol and eluted with 3 mL of hexane-dichloromethane (1:1, V/V) solution. The results indicated that the limits of detection (LOD) for SCCPs were 18 ng L−1, while the corresponding limits of quantitation (LOQ) were 60 ng L−1. The spiked recovery range was 90%–135%, with a relative standard deviation of less than 10%. Finally, 20 surficial water samples around Beijing were collected and SCCPs contents were analyzed using the proposed method. The SCCPs concentrations ranged from less than LOD to 682 ng L−1 with a mean concentration of 216 ng L−1.

Elevated Concentrations of 4-Bromobiphenyl and 1,3,5-Tribromobenzene Found in Deep Water of Lake Geneva Based on GC×GC-ENCI-TOFMS and GC×GC-μECD.

We quantified the concentrations of two little-studied brominated pollutants, 1,3,5-tribromobenzene (TBB) and 4-bromobiphenyl (4BBP), in the deep water column and sediments of Lake Geneva. We found aqueous concentrations of 625 ± 68 pg L–1 for TBB and 668 ± 86 pg L–1 for 4BBP over a depth range of 70–191.5 m (near-bottom depth), based on duplicate measurements taken at five depths during three separate 1 month sampling periods at our sampling site near Vidy Bay. These levels of TBB and 4BBP were 1 or 2 orders of magnitude higher than the quantified aqueous concentrations of the components of the pentabrominated biphenyl ether technical mixture, which is a flame retardant product that had a high production volume in Europe before 2001. We observed statistically significant vertical concentration trends for both TBB and 2,2′,4,4′,6-pentabromobiphenyl ether in the deep water column, which indicates that transport and/or degradation processes affect these compounds. These measurements were enabled by application of a comprehensive two-dimensional gas chromatograph coupled to an electron capture negative chemical ionization time-of-flight mass spectrometer (GC×GC-ENCI-TOFMS) and to a micro-electron capture detector (GC×GC-μECD). GC×GC-ENCI-TOFMS and GC×GC-μECD were found to be >10× more sensitive toward brominated pollutants than conventional GC×GC-EI-TOFMS (with an electron impact (EI) ionization source), the latter of which had insufficient sensitivity to detect these emerging brominated pollutants in the analyzed samples. GC×GC also enabled the estimation of several environmentally relevant partitioning properties of TBB and 4BBP, further confirming previous evidence that these pollutants are bioaccumulative and have long-range transport potential.

Isobutane Made Practical as a Reagent Gas for Chemical Ionization Mass Spectrometry.

As a reagent gas for positive- and negative-mode chemical ionization mass spectrometry (CI-MS), isobutane (i-C4H10) produces superior analyte signal abundance to methane. Isobutane has never been widely adopted for CI-MS because it fouls the ion source more rapidly and produces positive CI spectra that are more strongly dependent on reagent gas pressure compared with methane. Isobutane was diluted to various concentrations in argon for use as a reagent gas with an unmodified commercial gas chromatograph-mass spectrometer. Analyte spectra were directly compared using methane, isobutane, and isobutane/argon mixtures. A mixture of 10% i-C4H10 in argon produced twice the positive-mode analyte signal of methane, equal to pure isobutane, and reduced spectral dependence on reagent gas pressure. Electron capture negative chemical ionization using 1% i-C4H10 in argon tripled analyte signal compared with methane and was reproducible, unlike pure isobutane. The operative lifetime of the ion source using isobutane/argon mixtures was extended exponentially compared with pure isobutane, producing stable and reproducible CI signal throughout. By diluting the reagent gas in an inert buffer gas, isobutane CI-MS experiments were made as practical to use as methane CI-MS experiments but with superior analytical performance. Graphical Abstract ᅟ.

Analyte quantification with comprehensive two-dimensional gas chromatography: Assessment of methods for baseline correction, peak delineation, and matrix effect elimination for real samples

Comprehensive two-dimensional gas chromatography (GC×GC) is used widely to separate and measure organic chemicals in complex mixtures. However, approaches to quantify analytes in real, complex samples have not been critically assessed. We quantified 7 PAHs in a certified diesel fuel using GC×GC coupled to flame ionization detector (FID), and we quantified 11 target chlorinated hydrocarbons in a lake water extract using GC×GC with electron capture detector (μECD), further confirmed qualitatively by GC×GC with electron capture negative chemical ionization time-of-flight mass spectrometer (ENCI-TOFMS). Target analyte peak volumes were determined using several existing baseline correction algorithms and peak delineation algorithms. Analyte quantifications were conducted using external standards and also using standard additions, enabling us to diagnose matrix effects. We then applied several chemometric tests to these data. We find that the choice of baseline correction algorithm and peak delineation algorithm strongly influence the reproducibility of analyte signal, error of the calibration offset, proportionality of integrated signal response, and accuracy of quantifications. Additionally, the choice of baseline correction and the peak delineation algorithm are essential for correctly discriminating analyte signal from unresolved complex mixture signal, and this is the chief consideration for controlling matrix effects during quantification. The diagnostic approaches presented here provide guidance for analyte quantification using GC×GC.

Retracted: β-Carotene in Golden Rice is as good as β-carotene in oil at providing vitamin A to children

Golden Rice (GR) has been genetically engineered to be rich in β-carotene for use as a source of vitamin A. The objective was to compare the vitamin A value of β-carotene in GR and in spinach with that of pure β-carotene in oil when consumed by children. Children (n = 68; age 6-8 y) were randomly assigned to consume GR or spinach (both grown in a nutrient solution containing 23 atom% ²H₂O) or [²H₈]β-carotene in an oil capsule. The GR and spinach β-carotene were enriched with deuterium (²H) with the highest abundance molecular mass (M) at M(β-C)+²H₁₀. [¹³C₁₀]Retinyl acetate in an oil capsule was administered as a reference dose. Serum samples collected from subjects were analyzed by using gas chromatography electron-capture negative chemical ionization mass spectrometry for the enrichments of labeled retinol: M(retinol)+4 (from [²H₈]β-carotene in oil), M(retinol)+5 (from GR or spinach [²H₁₀]β-carotene), and M(retinol)+10 (from [¹³C₁₀]retinyl acetate). Using the response to the dose of [¹³C₁₀]retinyl acetate (0.5 mg) as a reference, our results (with the use of AUC of molar enrichment at days 1, 3, 7, 14, and 21 after the labeled doses) showed that the conversions of pure β-carotene (0.5 mg), GR β-carotene (0.6 mg), and spinach β-carotene (1.4 mg) to retinol were 2.0, 2.3, and 7.5 to 1 by weight, respectively. The β-carotene in GR is as effective as pure β-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of ~100 to 150 g cooked GR (50 g dry weight) can provide ~60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children.

Stable isotope gas chromatography–tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples

Aminoethylcysteine ketimine decarboxylated dimer (AECK–DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography–tandem mass spectrometry (GC–MS/MS) method for specific and sensitive determination of AECK–DD in biological samples. 13C2-labeled AECK–DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK–DD and m/z 330 to 270 for [13C2]AECK–DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK–DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK–DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK–DD were detected in mouse brain (∼1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK–DD (∼6.8pmol/g fresh tissue).

Optimization and development of analytical methods for the determination of new brominated flame retardants and polybrominated diphenyl ethers in sediments and suspended particulate matter

With more stringent legislation on brominated flame retardants, it is expected that increasing amounts of substitutes would replace polybrominated diphenylethers (PBDEs). Therefore, the development and optimization of analytical methodologies that allow their identification and quantification are of paramount relevance. This work describes the optimization of an analytical procedure to determine pentabromochlorocyclohexane, tetrabromo-o-chlorotoluene, 2,3,5,6-tetrabromo-p-xylene, tetrabromophthalic anhydride, 2,3,4,5,6-pentabromotoluene, tris(2,3-dibromopropyl)phosphate, decabromodiphenylethane and 1,2-bis(2,4,6-tribromophenoxy)ethane together with PBDEs in sediments and in suspended particulate matter. This method comprises a pressurized liquid extraction followed by three cleanup steps (gel permeation chromatography and solid phase extraction on Oasis™ HLB and on silica cartridges). Gas chromatography–mass spectrometry, using electron capture negative chemical ionization, is used for the final analysis. The proposed method provides recoveries >85%. The method was applied to sediment and suspended particulate matter samples from different locations in the Western Scheldt estuary (the Netherlands). To the best of our knowledge, this is the first time that the occurrence of the additive flame retardants 2,3,5,6-tetrabromo-p-xylene, 3,4,5,6-tetrabromo-o-chlorotoluene and 2,3,4,5,6-pentabromochlorocyclohexane is reported in the literature. The concentrations of these new flame retardants ranged from 0.05 to 0.30 μg/kg dry weight.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-4807-8) contains supplementary material, which is available to authorized users.

Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25 mL oral fluid and 0.75 mL buffer) was applied to conditioned CEREX ® Polycrom™ THC solid-phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N, O-bis-(trimethylsilyl)trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS–electron capture negative chemical ionization (NCI-MS). Linearity was 0.5–50 ng/mL for THC, 11-OH-THC, CBD and 1–50 ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5–500 pg/mL. Intra- and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3–6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex ® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.

Compatibility of electron ionization and soft ionization methods in gas chromatography/orthogonal time‐of‐flight mass spectrometry

Orthogonal acceleration time-of-flight (oa-TOF) mass spectrometry (MS) was coupled to gas chromatography (GC) to measure ion yields (ratio of ion counts to number of neutrals entering the ion source) and signal-to-noise (S/N) in the electron ionization (EI) mode (hard ionization) as well as in the soft ionization modes of chemical ionization (CI), electron capture negative ion chemical ionization (NICI) and field ionization (FI). Mass accuracies of the EI and FI modes were also investigated. Sixteen structurally diverse volatile organic compounds were chosen for this study. The oa-TOF mass analyzer is highly suited for FI MS and provided an opportunity to compare the sensitivity of this ionization method to the more conventional ionization methods. Compared to the widely used quadrupole mass filter, the oa-TOF platform offers significantly greater mass accuracy and therefore the possibility of determining the empirical formula of analytes. The findings of this study showed that, for the instrument used, EI generated the most ions with the exception of compounds able to form negative ions readily. Lower ion yields in the FI mode were generally observed but the chromatograms displayed greater S/N and in many cases gave spectra dominated by a molecular ion. Ion counts in CI are limited by the very small apertures required to maintain sufficiently high pressures in the ionization chamber. Mass accuracy for molecular and fragment ions was attainable at close to manufacturer's specifications, thus providing useful information on molecular ions and neutral losses. The data presented also suggests a potentially useful instrumental combination would result if EI and FI spectra could be collected simultaneously or in alternate scans during GC/MS.

Biological transformation, kinetics and dose-response assessments of bound musk ketone hemoglobin adducts in rainbow trout as biomarkers of environmental exposure

Low levels (ng/g) of musk ketone (MK), used as a fragrance additive in the formulation of personal care products, are frequently detected in the water and other environment. Thus, aquatic organisms can be continuously exposed to MK. In this study, kinetics and dose-response assessments of 2-amino-MK (AMK) metabolite, bound to cysteine-hemoglobin (Hb) in rainbow trout, formed by enzymatic nitro-reduction of MK have been demonstrated. Trout were exposed to a single exposure of 0.010, 0.030, 0.10, and 0.30 mg MK/g fish. Twenty-seven Hb samples were collected from exposed- and control fish subsequent to exposure intervals of 1 d (24 h), 3 d (72 h), and 7 d (168 h). Basic hydrolysis released bound AMK metabolite was extracted into n-hexane and then concentrated and analyzed by gas chromatography (GC) electron capture negative ion chemical ionization (NICI) mass spectrometry (MS) using selected ion monitoring (SIM). The presence of the AMK metabolite in Hb extracts was confirmed by agreement of similar mass spectral features and retention time with a standard. In the dose-response study, maximum adduct formation was obtained at the 0.10 mg/g dose with an average AMK metabolite concentration of 2.2 ng/g. For kinetics, the highest concentration of the AMK metabolite was found to be 32.0 ng/g at 0.030 mg/g dose in 3-d sample. Further elimination of the metabolite showed kinetics with a half-life estimated to be 2 d, assuming first-order kinetics. The metabolite was not detected in the control samples, non-hydrolyzed Hb, and reagent blank extracts. The detection limit for AMK in the Hb was approximately 0.30 ng/g, based on a signal to noise ratio of 3 (S/N = 3).

Biotransformation of musk xylene in trout haemoglobin: dose–response and toxicokinetics of musk xylene metabolites haemoglobin adducts by gas chromatography-mass spectrometry

Musk xylene (MX) is frequently used as a fragrance in commercial toiletries. Biotransformation of MX into 4-amino-MX (4-AMX) and 2-amino-MX (2-AMX) metabolites in rainbow trout haemoglobin (Hb) has been described. The dose–response relationship and toxicokinetics of the metabolites as adducts in the Hb were determined by gas chromatography (GC)–electron capture negative chemical ionization (NCI)–mass spectrometry (MS), and GC–electron ionization (EI)–MS/MS, using selected ion monitoring (SIM). The trout were subjected to a single exposure of 0.010, 0.030, 0.10, and/or 0.30 mg MX/g of fish. Hb samples were collected from exposed and control fish, and analysed subsequent to exposure at intervals of 24, 72, and 168 h. Alkaline hydrolysis released 4-AMX and 2-AMX metabolites from the Hb, and the solutes were extracted into n-hexane. The extracts were preconcentrated and analysed. The presence of the metabolites in the Hb extracts was confirmed based on agreement of similar mass spectral features from NCI/MS and EI-MS/MS spectra, and retention times of the metabolites with standards. The NCI/MS results were used for dose–response and toxicokinetics measurements. For dose–response, the concentrations of adducts of the metabolites increased with dosage, and a maximum adduct formation was observed at 0.10 mg g−1, beyond which it decreased. The average concentrations of 4-AMX and 2-AMX at a dosage of 0.10 mg g−1 were 700 and 7.4 ng g−1, respectively. For toxicokinetics, the concentration of the metabolites in the Hb reached a maximum in the 3 day sample after administration of MX. Further elimination of the metabolites exhibited kinetics with a half-life estimated to be 1–2 days, assuming first-order kinetics. Quantitations were made based on an internal standard and a calibration plot. In control samples, non-hydrolysed Hb, and reagent blank extracts, the metabolites were not detected. The limits of detection for 4-AMX and 2-AMX in the Hb were approximately 1.7 and 1.4 µg L−1, respectively, based on a signal-to-noise ratio of 3 with NCI/MS.

Analysis of 7‐methylguanine using isotope dilution and gas chromatography/electron‐capture negative chemical ionization mass spectrometry

This paper presents results obtained for in vivo endogenous and exogenous 7-methylguanine (7-MG) analyzed using a method incorporating gas chromatography with electron-capture negative chemical ionization mass spectrometry and isotope dilution (GC/EC-ID-MS). 13C4-Labeled 7-MG was synthesized to serve as an internal standard to improve accuracy of quantitation, and was used to analyze 7-MG in livers of control mice and dacarbazine-treated mice. The results confirm that 7-MG in tissue DNA can be measured using this GC/EC-ID-MS method with excellent sensitivity and specificity. Administration of 0, 30, and 60 mg/kg dacarbazine to mice led to dose-dependent increases in the formation of 7-MG. The results indicate that this method could be applied to the analysis of endogenous and exogenous 7-MG in human tissues for future molecular epidemiology studies on potential health effects caused by methylating agents.

Nitro musk metabolites bound to carp hemoglobin: determination by GC with two MS detection modes: EIMS versus electron capture negative ion MS

Nitroaromatic compounds including synthetic nitro musks are important raw materials and intermediates in the synthesis of explosives, dyes, pesticides, and pharmaceutical and personal-care products (PPCPs). The nitro musks such as musk xylene (MX) and musk ketone (MK) are extensively used as fragrance ingredients in PPCPs and other commercial toiletries. Identification and quantification of a bound 4-amino-MX (4-AMX) metabolite as well as a 2-amino-MK (2-AMK) metabolite were carried out by gas chromatography–mass spectrometry (GCMS), with selected ion monitoring (SIM) in both the electron ionization (EIMS) and electron capture (EC) negative ion chemical ionization (NICIMS) modes. Detection of 4-AMX and 2-AMK occurred after the cysteine adducts in carp hemoglobin, derived from the nitroso metabolites, were released by alkaline hydrolysis. The released metabolites were extracted into n-hexane. The extract was preconcentrated by evaporation, and analyzed by GC-SIM-MS. A comparison between the EI and EC approaches was made. EC NICIMS detected both metabolites whereas only 4-AMX was detected by EIMS. The EC NICIMS approach exhibited fewer matrix responses and provided a lower detection limit. Quantitation in both approaches was based on an internal standard and a calibration plot.

Determination of quinoxaline-2-carboxylic acid, the major metabolite of carbadox, in porcine liver by isotope dilution gas chromatography–electron capture negative ionization mass spectrometry

A sensitive and robust method using isotope dilution gas chromatography–electron capture negative chemical ionization mass spectrometry (GC–ECNI-MS) was developed and validated for the analysis of quinoxaline-2-carboxylic acid (QCA) in porcine liver. [ 2H 4]QCA was added to liver samples which were then deproteinated with 2% metaphosphoric acid in 20% methanol. Followed by sequential extraction with water-saturated ethyl acetate and phosphate buffer, the buffer extracts were subject to solid-phase extraction clean-up by mixed mode anion-exchange columns. QCA was derivatized with N-methyl- N- tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) prior to GC–ECNI-MS determination. For unambiguous identification, a second GC–ECNI-MS experiment was performed on suspected positive samples which were derivatized independently with another derivatization agent, trimethylsilyldiazomethane. Excellent recovery and precision were obtained and the limit of quantitation was 0.7 μg/kg (S/N>60). Method ruggedness by Taguchi orthogonal array technique is also presented.

Tobacco smoke and formation of N-(2-hydroxyethyl)valine in human hemoglobin.

Human exposure to ethylene oxide (EtO) occurs mainly through inhalation of polluted air in occupational workplaces and/or via tobacco smoke. A significant biochemical reaction of EtO converts the terminal valine of hemoglobin into N-(2-hydroxyethyl)valine (HOEtVal). In the present study, the extent of HOEtVal formation in 360 healthy adults who were not occupationally exposed to EtO was measured with a gas chromatograph/mass spectrometer in the electron-capture negative chemical ionization mode. This parameter was correlated with smoking habits and urinary cotinine concentration, exhibiting a positive relationship between HOEtVal and the number of cigarettes smoked (r2 = .4416). Urinary cotinine measurements also correlated with HOEtVal and the number of cigarettes smoked. This positive correlation between urinary cotinine and HOEtVal (r2 = .3893) provides a new perspective on the early stages of carcinogenic processes.

Artifact-Free Quantification of Free 3-Chlorotyrosine, 3-Bromotyrosine, and 3-Nitrotyrosine in Human Plasma by Electron Capture–Negative Chemical Ionization Gas Chromatography Mass Spectrometry and Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Our results indicate that electron capture–negative chemical ionization–gas chromatography/mass spectrometry (EC–NCI GC/MS) is 100-fold more sensitive than liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.