Chlorine Isotope Pattern Research Articles

Semi-Targeted Analysis of Complex Matrices by ESI FT-ICR MS or How an Experimental Bias may be Used as an Analytical Tool.

Ammonia is well suited to favor deprotonation process in electrospray ionization mass spectrometry (ESI-MS) to increase the formation of [M - H]-. Nevertheless, NH3 may react with carbonyl compounds (aldehyde, ketone) and bias the composition description of the investigated sample. This is of significant importance in the study of complex mixture such as oil or bio-oil. To assess the ability of primary amines to form imines with carbonyl compounds during the ESI-MS process, two aldehydes (vanillin and cinnamaldehyde) and two ketones (butyrophenone and trihydroxyacetophenone) have been infused in an ESI source with ammonia and two different amines (aniline and 3-chloronaniline). The (+) ESI-MS analyses have demonstrated the formation of imine whatever the considered carbonyl compound and the used primary amine, the structure of which was extensively studied by tandem mass spectrometry. Thus, it has been established that the addition of ammonia, in the solution infused in an ESI source, may alter the composition description of a complex mixture and leads to misinterpretations due to the formation of imines. Nevertheless, this experimental bias can be used to identify the carbonyl compounds in a pyrolysis bio-oil. As we demonstrated, infusion of the bio-oil with 3-chloroaniline in ESI source leads to specifically derivatized carbonyl compounds. Thanks to their chlorine isotopic pattern and the high mass measurement accuracy, (+) ESI Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) unambiguously highlighted them from the numerous CxHyOz bio-oil components. These results offer a new perspective into the detailed molecular structure of complex mixtures such as bio-oils. Graphical Abstract ᅟ.

Analysis of xyloglucans by ambient chloride attachment ionization tandem mass spectrometry

Xyloglucan oligomers obtained upon enzyme digestion from Hymenaea courbaril, Arabidopsis Columbia-0 and mur3 were ionized and analyzed by using chloride anion attachment electrospray ionization (ESI) and tandem mass spectrometry. MW determination and structural elucidation of several xyloglucan oligomers was performed directly from the mixture solutions without sample pretreatment or derivatization. Sodium cation attachment was used to determine the number of xyloglucans present in the mixtures and their MWs. However, tandem mass spectrometry results showed that structure elucidation based on the sodium adducts is ambiguous. Chloride anion also forms stable adducts with these xyloglucans upon ESI. These adducts can be readily identified due to the chlorine isotope pattern. The mass spectral profile of xyloglucans obtained for the mixtures matches the HPAEC results, thus validating this methodology for the determination of the xyloglucan composition and the MW of each xyloglucan. Upon collisional activation in MS2 experiments, the chloride anion adducts readily lose HCl, which helps verify the molecular weight of each xyloglucan. Isolating the resulting anion (deprotonated oligomer) and subjecting it to further collision-activated dissociation experiments (MSn; n=3–4) yields useful structural information that allows the differentiation between isomeric anions and hence determination of the sequence of the xyloglucan oligomers. The deprotonated oligomers fragment by a stepwise loss of sugar units from the reducing end.

Analysis of carbohydrates by atmospheric pressure chloride anion attachment tandem mass spectrometry

The ability to characterize oligosaccharides directly in complex mixtures would greatly benefit many research efforts focused on the development of biofuels from lignocellulosic biomass. We report here on the utility of chloride anion attachment in atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) tandem mass spectrometry for MW determination and structural elucidation of several mono-, di- and oligosaccharides directly without sample pretreatment or derivatization. Chloride anion forms stable adducts with these sugars upon ESI or APCI, which can be readily identified due to the chlorine isotope pattern. Fragment anions or other product anions are usually not observed, which is beneficial for mixture analysis. Upon collisional activation in MS2 experiments, these adducts readily lose HCl, which helps verify the molecular weight of each analyte. Isolating the resulting anion and subjecting it to further collision-activated dissociation experiments (MSn; n=3–4) until no ion signal remains yields useful structural information. Examination of equimolar mixtures of mono-, di- and oligosaccharides and mixtures of enzymatically digested biomass demonstrates the ability of this methodology to ionize all the components of the mixtures without bias and with nearly equal efficiency.

A method for the analysis of sucralose with electrospray LC/MS in recipient waters and in sewage effluent subjected to tertiary treatment technologies

A method for the analysis of the artificial sweetener sucralose in sewage water and recipient water was developed. Extraction and clean up was performed with solid-phase extraction utilising Oasis HLB columns. Detection was made by liquid chromatography electrospray mass spectrometry (LC/MS). The triple-quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. However, ‘pseudo MRM’ was used, a technique where the two quadrupoles monitor the same m/z. The sodium adduct of sucralose was used for quantification, since lower detection limits were obtained as compared to the sucralose quasi-molecular ion in negative ion mode. The two ions with highest intensity in the chlorine isotope pattern were monitored. The reduction of matrix effects with this approach is discussed. The method limit of quantification (MLOQ) for sewage water was 0.2 µg L−1, whereas for recipient water MLOQ was 0.02 µg L−1. The method was used to analyse effluent samples from an experimental sewage treatment plant (STP) to assess the efficiency of tertiary treatment techniques for removal of sucralose. Filtration through activated carbon was shown to be efficient, while ozonation, advanced oxidation techniques and membrane bioreactors were less efficient. Analyses of receiving waters showed low dilution of sucralose emitted from the STPs.

Urinary excretion profiles of toremifene metabolites by liquid chromatography-mass spectrometry. Towards targeted analysis to relevant metabolites in doping control

In the present study, toremifene urinary excretion studies were evaluated in order to examine main metabolic reactions and to select target metabolites in doping control analysis. Urine samples from three female subjects were collected every 3 h for at least 15 days after the oral administration of a single dose of Fareston® (60 mg). The elemental compositions of the compounds detected were determined by liquid chromatography-mass spectrometry using a time-of-flight system with accurate mass measurement. More detailed structure elucidation was obtained by monitoring the presence or absence of structure-specific ions, using product ion scan and neutral loss acquisition modes, whereas the metabolites urinary profiles were evaluated in selected reaction monitoring acquisition mode. The results showed that the main routes of phase-I modifications involved carboxylation of the chlorinated side chain, N-demethylation and hydroxylation in different positions. Fifteen metabolites were found in all subjects studied, most of them were detected for more than 10 days in the free, glucuronide and sulphate fractions, with a maximum of excretion generally after 9-22 and 34-47 h from drug administration. These metabolites can be divided in two groups: metabolites with the characteristic chlorine isotope pattern and metabolites without the characteristic chlorine isotope pattern. The most abundant and long-term compounds were the carboxylated metabolites followed by the hydroxylated metabolites. Their product ions originating after collision-induced dissociation were observed to occur prevalently in the dimethylaminoethoxy and in the chlorinated side chains. These structure-specific ions were used to design screening and confirmation procedures to positively identify toremifene administration in doping control analysis.

Simultaneous determination of ochratoxin A, mycophenolic acid and fumonisin B2 in meat products

Here we present a method for simultaneous determination of the fungal metabolites mycophenolic acid, ochratoxin A (OTA) and fumonisin B(2) (FB(2)) in meat products. Extraction was performed with water-acetonitrile, followed by acetone-induced precipitation of salts and proteins. Purification and identification of analytes was performed by mixed-mode reversed-phase anion-exchange chromatography in direct ion-exchange mode, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. Quantification was based on isotope dilution with fully (13)C-labelled FB(2) and OTA, and matrix-spiked calibration curves. Fermented sausages inoculated with an OTA- and FB(2)-producing strain of Aspergillus niger were analysed, but no analytes were detected. Analysis of 22 retail products showed one Parma meat with a very high level of OTA contamination (56-158 microg/kg) that clearly exceeded the Italian regulatory limit of 1 microg/kg. This sample and uninfected control samples were subsequently reanalysed, and the high OTA content was verified by two other techniques: (i) LC-time-of-flight MS confirmed the accurate mass as well as chlorine isotope pattern; and (ii) sample methylation in methanol-BF(3) and subsequent LC-MS/MS provided indirect confirmation by detection of the OTA methyl ester. In the contaminated Parma ham, the high OTA level most likely originated from growth of Penicillium nordicum on the meat.

A Mass Spectrometric Approach for the Study of the Metabolism of Clomiphene, Tamoxifen and Toremifene by Liquid Chromatography Time-of-Flight Spectroscopy

In this paper, we discuss the capabilities of liquid chromatography coupled to mass spectrometry with a time-of flight system with accurate mass measurement for the detection and characterisation of drug metabolites in biological samples for anti-doping purpose. Urinary excretion samples of three selective oestrogen receptor modulators (SERMs) with a common triphenylethylene structure: clomiphene, toremifene, and tamoxifen, obtained after oral administration of a single dose of each drug, were analysed using a time-of-flight system, after automatic tuning and calibration of the equipment, in positive full scan mode using an electrospray ionisation source. Following this approach we detected most of all significant metabolites reported by others and postulated new metabolites, especially for toremifene, have been characterised: N-demethyl-3-hydroxy-4-methoxy-toremifene and 3- hydroxy-4-methoxy-toremifene; in addtiona to this, in the urinary excretion samples of toremifene some metabolites, without the characteristic chlorine isotope pattern, discarded in previous studies, that are also metabolites of tamoxifen, were identified. The lack of certified reference materials does not allow an accurate determination of the limit of detection (LODs) of all metabolites; however an estimation taking into account the response factor of similar compounds allows to estimate that all metabolites are clearly detectable in a range of concentration comprised between 10 ng mL(-1) and 30 ng mL(-1).

In Vitro and in Vivo Studies on Acyl-Coenzyme A-Dependent Bioactivation of Zomepirac in Rats

Zomepirac [ZP, 5-(chlorobenzoyl)-1,4-dimethylpyrrole-2-acetic acid] was withdrawn from the market because of unpredictable allergic reactions that may have been caused by ZP-protein adducts formed by reaction of the reactive acyl glucuronide of ZP (ZP-O-G) with endogenous proteins. To test the hypothesis that the reactive ZP acyl coenzyme A thioester (ZP-CoA) was formed and potentially could contribute to formation of ZP-protein adducts, we investigated the acyl CoA-dependent metabolism of ZP in freshly isolated rat hepatocytes (1 mM) and in vivo (100 mg ZP/kg, ip) in rat livers (2 h after dose administration), rat bile (0-4 h), and rat urine (0-24 h). ZP-CoA was detected in freshly isolated hepatocytes and in vivo in rat livers by LC/MS/MS. In addition, the ZP glycine conjugate (ZP-Gly) and ZP taurine conjugates (ZP-Tau) were identified by LC/MS/MS in rat hepatocytes and in vivo in rat livers, rat urine, and rat bile. The identities of ZP-CoA, ZP-Gly, and ZP-Tau were confirmed by comparison of retention times and MS/MS spectra with those of authentic standards. Moreover, the ZP acyl carnitine ester was detected in rat urine and rat bile based upon (i) the chlorine isotope pattern, (ii) MS/MS spectra showing significant ions characteristic for carnitine (m/z 60, 144 and loss of m/z 59) and ZP (m/z 139), and (iii) accurate mass measurements with a mass accuracy of 0.2 ppm. ZP-CoA serves as an obligatory intermediate in the formation of ZP-Gly, ZP-Tau, and ZP carnitine ester, and it is therefore of mechanistic significance that these conjugates were identified. Finally, time-dependent concentration profiles obtained in experiments with rat hepatocytes and in vivo from quantitative analysis of rat livers indicate that ZP-CoA, in addition to ZP-O-G, may contribute to formation of the potentially toxic covalent ZP-protein adducts.

In-vitro characterization of the metabolism of prochlorperazine

Background Prochlorperazine (PCZ) is an antiemetic widely used in the management of nausea and vomiting. PCZ is reported to be extensively metabolized via the CYP450 system. However, the specific CYP450 isoforms responsible have not been identified and only two metabolites have been characterized (sulphoxide n-oxide, N-desmethyl). Purpose To identify the metabolites of PCZ and quantify the relative contribution of the CYP450 enzymes involved in their metabolism. Methods 100 μM PCZ was incubated in cDNA (control, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5, 2E1) with NADPH, at 37°C for 60 minutes. The reaction was halted with 100 μL of ice cold ACN. The samples were centrifuged at 2000g for 5 minutes and then the supernatant was analyzed by mass spectrometry. PCZ standard curve ranged from 6.25–100 μM. The detection of metabolites by LCMS was aided by the presence of a chlorine isotopic pattern (1:0.33). LC/MS/MS was used to postulate the identities of the metabolites formed by the CYP isoforms. Results The percent loss of PCZ compared to control for each of the CYP450 isoforms is illustrated below. The seven PCZ metabolites identified are: sulphoxide n-oxide, hydroxy-nor, desulfonated, hydroxide isomers (2), N-desmethyl, and sulphoxide. The latter three metabolites appear to be the most prevalent. Conclusions Of the various CYP450s that contribute to PCZ metabolism, CYP2D6 and CYP2C19 are the most efficient. N-desmethyl PCZ was the major metabolite formed by CYP2D6 and CYP2C19. Clinical Pharmacology & Therapeutics (2004) 75, P85–P85; doi: 10.1016/j.clpt.2003.11.324 Figure 1. Contribution of CYP450 Isoforms to the Metabbolism of ProchlorperazineOpen in figure viewerPowerPoint

Mass spectrometric elucidation of chlorine location in dichloro fatty acids following 4,4-dimethyloxazoline derivatization, and its application to chlorinated fatty acids in fish

Fatty acids containing two vicinal chlorine atoms located between C-5 and C-15 were transformed to 4,4-dimethyloxazoline (DMOX) derivatives, which were subjected to electron-impact mass spectrometry (EIMS). The results showed that DMOX derivatization is a useful technique for MS structural analysis of dichloro fatty acids. In contrast to those commonly used derivatives such as alkyl esters which result in mass spectral features indistinguishable among positional isomers, DMOX derivatives of dichloro fatty acids display some distinctive EIMS features which are characteristic of positional isomers. Fragmentation mechanisms responsible for important fragment ions and patterns are inherently related to the position of the vicinal dichloro group on the acyl chain. Among those diagnostic ions and patterns, a homologous series of characteristic chlorodienyl ions, notable by their sizeable intensity and chlorine isotope pattern, are the most valuable structural indicators. The location of the vicinal chlorines in a chlorinated fatty acid can be readily deduced from these ions by equating the mass of the first ion in the series to “144+14 x”, where x represents the location of the first of the vicinal chlorine atoms. In contrast to the “12-mu” rule which, as in the case of monoenoic acids, is only applicable to molecules containing the dichloro group in the central region of the acyl chain, the “144+14 x” rule is applicable to almost all the positional isomers of vicinal dichloro fatty acids. Using this technique, the 5,6-location of chlorine atoms was confirmed for the previously identified threo-5,6-dichlorotetradecanoic acid in a fish lipid sample.

Determination of atomic isotope patterns from mass spectra of molecular ions containing multiple polyisotopic elements

A chemometrical method that allows one to obtain the isotopic abundances of the atoms composing a molecular ion, given its elemental composition and its mass spectrum, was developed. The method uses non-linear regression to fit the isotopic abundances of the elements, so that the experimental spectrum of the ion is reproduced. A least squares method with minimization by downhill simplex algorithm was implemented in C language as a library, but a graphical user interface application that allows an end-user to apply the method to his/her own spectra is also available. Examples of its application to electrospray mass spectra of NaAuCl 4 and CdCl 2 are given. The chlorine isotope pattern was obtained from the AuCl 4 − cluster in the former case and, simultaneously, chlorine and cadmium isotope patterns from the CdCl 3 − cluster. The performance of the method was extensively characterized by applying it to a great number of simulated spectra. Non-idealities, against which the method was evaluated, include noise, mass discrimination, detection non-linearity, base line offset (background counting), and low resolution. It has been demonstrated that, in spite of what intuition might tell one, the polyatomic approach to isotopic analyses has no a priori disadvantage to the common monatomic approach. Indeed, the results demonstrate that, in some cases, the figures of merit can be improved by using polyatomic species and that other polyisotopic elements can be included in the formula without significant loss of precision, as long as they have a dominant isotope, which is the case of carbon, hydrogen, oxygen, and nitrogen, for example. This means that polyatomic species derived from organic species could be useful for isotopic measurement purposes.

Analysis of chlorophenoxy acid herbicides in water by large-volume on-line derivatization and gas chromatography–mass spectrometry

This work presents a modified method to analyze chlorophenoxy acid herbicides in water samples. The herbicides 2,4-D (2,4-dichlorophenoxyacetic acid), Silvex (2,4,5-trichlorophenoxypropionic acid) and 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) were used to evaluate the method. The method involves extraction of samples by a graphitized carbon black cartridge, and on-line derivatization in the GC injection port using a large-volume (10–20 μl) direct sample introduction (DSI) device with tetraalkylammonium salts. The analytes were then identified and quantitated by ion-trap gas chromatography–mass spectrometry. The large-volume DSI injection-port derivatization technique provides sensitivity, fast and reproducible results for chlorophenoxy acid herbicides residues, to quantitation at 0.1 to 0.2 μg/l in 500-ml water samples. An enhanced characteristic mass chromatogram of molecular ions of butylated chlorophenoxy acid herbicides with a significant chlorine isotope pattern by electron impact ionization MS allows us to determine herbicides residues at trace levels in aqueous samples. Recovery of the herbicide residues in spiked various water samples ranged from 70 to 99% while RSDs ranged from 1 to 13%.

Identification of the Dopaminergic Neurotoxin 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline in Human Blood after Intake of the Hypnotic Chloral Hydrate

1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), a potent toxin toward dopaminergic neurons, readily originatesin vitrofrom the biogenic amine tryptamine and the unnatural aldehyde chloral. For this reason, this heterocycle has been postulated to be formed endogenously in humans after administration of the hypnotic chloral hydrate or after exposure to the industrial solvent trichloroethylene by a spontaneous chemical ring closure reaction. In this paper, we report on the first identification of TaClo in blood samples of patients treated orally with chloral hydrate. Using a specific and sensitive gas chromatographic screening procedure based upon electron-capture and mass-selective detection, TaClo was determined after conversion to its volatile trifluoroacetyl derivative. The identity of TaClo in humans was clearly demonstrated by GC–MS analysis in selected-ion-monitoring mode, by the characteristic chlorine isotopic pattern of the molecular ion.

Structure, bonding and force fields of mercurated methanes

Complete force field studies of C(HgX) 4 (X = F, Cl, Br, I, CH 3 and CN), CH 2(HgCl) 2 and CD 2(HgCl) 2 have been performed. The C-Hg stretching force constants of C(HgX) 4 are 0.6 to 0.7 N cm −1 lower than in related compounds CH 3HgX, suggesting some interaction between mercury atoms. The Hg⋯Hg bond order is around 0.4/mercury atom. The splittings of the degenerate stretching modes ν a(CHg 4) in the vibrational spectra of the solid compounds C(HgX) 4 were reproduced in calculations with distortion of the tetrahedron and corresponding force constant corrections. The complex structure of the mercury-chlorine stretching bands was reproduced by a calculated chlorine isotope pattern. The CHg force constants 2.55, 2.15 and 1.87 N cm −1 for CH 3HgCl, CH 2(HgCl) 2 and C(HgCl) 4, respectively, reflect a considerable weakening of the CHg bonds with increasing mercuration.

A vibrational analysis of the gas phase i.r. spectrum of CHFCl 2

The gas-phase i.r. spectrum of natural CHFCl 2 was recorded under moderate resolution and a number of regions between 3100 and 350 cm −1 were analysed. All the fundamentals but ν 6 (lying at 277 cm −1) were observed and characterized; the band centre of the latter was evaluated by difference of suitable vibrational levels. Numerous combinations, ‘hot’ and ‘difference’ bands were assigned. The chlorine isotopic splitting pattern, often originating from a complex spectral structure, was also interpreted by the support of a normal coordinate analysis. Examples of possible Fermi resonance interactions were considered.

Combination of the matrix isolation and the isotopic substitution techniques: Vibrational spectra and molecular constants of tintetrachloride

The infrared spectra of gaseous 116SnCl 4, 124SnCl 4, 116Sn 35Cl 4, and 116Sn 37Cl 4 have been measured. The ν 3( F 2) modes of gaseous 116Sn 35Cl 4 and 116Sn 37Cl 4 have a well-resolved R branch and a broad P branch, overlapped by “hot bands”, while the ν 4( F 2) modes have a well-resolved P- Q- R structure. Using the Q-R-branch spacing of ν 3( F 2) and the P-R-branch spacing of ν 4( F 2), Coriolis coupling constants have been calculated. The chlorine isotopic splitting pattern of the ν 3( F 2) mode of 116SnCl 4 and 124SnCl 4, isolated in an Ar matrix at 20 K, were recorded, and the isotopic frequencies were assigned. The isotopic shifts, obtained from the gas phase spectra, and from Ar and N 2 matrix spectra were almost identical. Thus the combination of the matrix isolation and heavy atom isotopic substitution techniques is a good method in determining accurate isotopic shifts for simple volatile molecules with one isotopically pure central atom and chlorine in natural abundance. The force constants of tintetrachloride have been determined using nine additional and independent data.

Infrared Study of Matrix-Isolated Chlorinated Tetrahedral Molecules

The infrared spectra of CH3Cl, CH3CH2CH2Cl, CH2Cl2, HCCl3, (CH3)–CCl3, FCCl3, BrCCl3, CCl4, and SiCl4 in an argon matrix at 20°K were studied. The chlorine isotopic splitting pattern of the bands in the 500–1300-cm−1 region were recorded, and most of the isotopic frequencies were assigned. Normal-coordinate calculations have been performed on these molecules to establish complete chlorine isotopic splitting patterns of all the fundamental vibration bands. Different isotopic species have different Fermi resonance interactions in the same band system; examples of this were observed in the spectra of FCCl3 and CCl4.