Negative Ion Chemical Ionization Conditions Research Articles

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography–tandem mass spectrometry and evaluation of the excretion after oral intake

3-Nitrotyrosine (NO(2)Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)-tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[(2)H(3)]tyrosine, and GC-tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5+/-7.2) 3-nitrotyrosine levels were determined to be 8.4+/-10.4 nM (range, 1.6-33.2 nM) or 0.46+/-0.49 nmol/mmol creatinine (range, 0.05-1.30 nmol/mmol creatinine). The present GC-tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 microg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.

Unusual collision-induced dissociation of fluorated and non-fluorated α-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole mass spectrometer under electron-capturing negative-ion chemical ionization conditions

Unusual collision-induced dissociation (CID) of perfluorated and non-perfluorated α -nitrotoluene analogs in a gas chromatograph triple-stage quadrupole (TSQ) mass spectrometer (GC–QqQ-MS) under electron-capturing negative-ion chemical ionization conditions is reported. CID of [M − 1] − of α-nitro-2,3,4,5,6-pentafluorotoluene (C 6F 5CH 2 NO 2) and α-nitro-2,5-difluorotoluene (C 6H 3F 2CH 2 NO 2) produced an intense ion with m/ z 66. By using 15N- or 18O-labelled C 6F 5CH 2 NO 2 analogs, we found that this anion has the formula C 3NO. By contrast, CID of [M − 1] − of α-nitrotoluene (C 6H 5CH 2 NO 2) and α-nitro-3,5-difluorotoluene (C 6H 3F 2CH 2 NO 2) produced an anion with m/ z 86 with the formula C 3H 4NO 2. The expected CID of the C N-bond of all α-nitrotoluene analogs to form the nitrite anion (NO 2 −, m/ z 46) did not occur. We propose mechanisms for the formation of the anions C 3NO and C 3H 4NO 2 in the collision chamber of the TSQ mass spectrometer. The most likely structures for the anion C 3NO are :C C C N O − and N C C C O −. The unique CID behavior of C 6F 5CH 2 NO 2 can be utilized to unequivocally identify and accurately quantify nitrite in biological fluids by GC–tandem MS.

Quantitative determination of fluvastatin in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry using [18O2]‐fluvastatin as an internal standard

A sensitive and specific method for the quantitative determination of fluvastatin in human plasma is presented. The drug was isolated from plasma by extractive alkylation with pentafluorobenzyl bromide and further derivatized to the bis-trimethylsilyl derivative. [18O2]-Fluvastatin was prepared from unlabelled fluvastatin and used as an internal standard. Gas chromatography/mass spectrometry under negative ion chemical ionization conditions was used for quantitative measurement of the drug, using m/z 554.26 and 558.26 for target and internal standard, respectively. Calibration graphs were linear within a range of 2 and 512 ng mL(-1) plasma. Intra-day precision was 0.94% (2 ng mL(-1)), 2.53% (8.2 ng mL(-1)), 2.16% (81.9 ng mL(-1)) and 3.26% (409.6 ng mL(-1)); inter-day variability was found to be 1.64% (2 ng mL(-1)), 0.97% (8.2 ng mL(-1)), 1.97% (81.9 ng mL(-1)) and 2.01% (409.6 ng mL(-1)). Intra-day accuracy showed deviations of 0.6% (2 ng mL(-1)), 0.37% (8.2 ng mL(-1)), -1.52% (81.9 ng mL(-1)) and -1.67% (409.6 ng mL(-1)); inter-day accuracy was of -1.64% (2 ng mL(-1)), -1.13% (8.2 ng mL(-1)), -2.28% (81.9 ng mL(-1)) and -0.46% (409.6 ng mL(-1)). The stable isotope labelled standard was found to be stable under the analytical conditions.

Gas phase enantiomeric distinction of ( R)- and ( S)-aromatic hydroxy esters by negative ion chemical ionization mass spectrometry using a chiral reagent gas

Negative ion chemical ionization (NICI) using a chiral reagent gas such as (2 S,3 S) butanediol (G SS ) allows the differentiation of chiral α hydroxy esters ( M R or M S ). The distinction is significantly enhanced by using CID on the deprotonated hetero dimer [ M + (G SS −H)] −. The contribution of a non covalent [ M + (G SS −H)] − competitive form is very minor. In fact, a covalent form appears favored. To produce a covalent adduct ion, an enantioselectivity of the alkoxide attack on the electrophilic ester site to form a tetravalent adduct is suggested by the product ion abundances. This observed steric control is consistent with the one observed in solution (Cram–Felkin addition/reduction orientation). The dissociations under collision conditions of the product deprotonated diastereomeric compounds show a stereospecific effect in the elimination of alcohol from the tetravalent adduct ions rather than a regeneration of the deprotonated diol reagent as expected from non-covalent heterodimer. This study shows an orientation with a chiral compound that allows, from an analytical point of view, the distinction of enantiomers and the attribution of chirality in the gas phase under NICI conditions.

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography–mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography–mass spectrometry (GC–MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17β]- 2H 3-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C 18 with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l ( r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 μg/l ( r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 μg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.

Reactions of O −· with methyl benzoate: a negative ion chemical ionization and Fourier transform ion cyclotron resonance study

The reactions of O −· with methyl benzoate have been examined by the measurement of negative ion chemical ionization (NICI) mass spectra using a CI source, with confirmatory studies carried out on a Fourier transform ion cyclotron resonance mass spectrometer. Reaction mechanisms have been elucidated using isotopically labeled esters. Nucleophilic attack at the carbonyl carbon and the aromatic ring were important reaction pathways. Nucleophilic attack at the carbonyl carbon was followed by the production of products (C 6H 5CO 2 − and CH 3OCO 2 −) characteristic of radical, β-fragmentation. Using 18O-labeled methyl benzoate, the S N2 reaction was found to account for a smaller percentage, 21(±1)%, of the benzoate product. Aromatic ring attack resulted in formation of [M + O − H] − and [M − 2H] −· ions. Although aryl hydrogens accounted for most H 2 +· abstracted by O −·, evidence for abstraction of H arylH alkyl +· and H alkylH alkyl +· was also found. Although present at much lower abundance, dehydrobenzoate, dehydrophenoxy, and C 7H 6 −· ([M − 2H − CO 2] −·) radical anions were also observed. An H aryl/H alkyl exchange associated with formation of the benzoate anion was attributed to an H alkyl abstraction that occurred within the methanol/dehydrobenzoate ion–dipole complex. The [M − 2H] −·, dehydrobenzoate, dehydrophenoxy, and [M − 2H − CO 2] −· ion signals were quenched by reaction with O 2. Conditions required for production of O −· spectra under NICI conditions were also examined.

Determination of oestrone, 17α- and 17β-oestradiol in bovine aqueous humor using gas chromatography-negative ion chemical ionization mass spectrometry

Perfluorotolyl (PFT)-ether and perfluorotoly-trimethylsilyl (PFT-TMS) ether derivatives of oestrone, 17alpha- and 17beta-oestradiol were prepared under phase transfer conditions. The former derivatives under negative ion chemical ionization conditions gave significant ions in the mass spectrometer but 17alpha- and 17alpha-oestradiol gave poor resolution. However, the PFT-TMS derivatives of 17alpha- and 17beta-oestradiol showed good resolution. These derivatives were used for the analysis of oestrogens in bovine aqueous humour, vitreous humour and retina. The mean (+/-SEM) concentrations of oestrone in bovine aqueous humour (n=18), vitreous humour (n=18) and bovine retina (n=4) were 0.47+/-0.11, 0.46+/-0.14 and 1.10+/-0.24 ng.ml(-1), respectively. 17alpha-Oestradiol was detected in 16 out of 18 samples of bovine aqueous humour and vitreous humour and the mean (+/-SEM) concentrations were 0.30+/-0.10 and 0.08+/-0.02 ng.ml(-1), respectively. The mean (+/-SEM) concentration of 17beta-oestradiol in aqueous humour (n=7) and vitreous homour (n=11) 0.83+/-0.26 ng ml(-1) and 0.39+/-0.09 ng ml(-1), respectively. In retina the concentrations of both steroids were below the detection limit.

Simultaneous identification of different classes of hydrocarbons and determination of nitro-polycyclic aromatic hydrocarbons by means of particle beam liquid chromatography-mass spectrometry

LC-MS with a particle beam interface was used to separate and identify aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs) and their nitrated derivatives (nitro-PAHs). In particular, the behaviour of nitro-PAHs and of two nitrated biphenyls was checked in terms of instrumental response. A normal-phase chromatographic system was used, with a silica column and heptane-tetrahydrofuran as mobile phase. Electron impact and positive- and negative-ion chemical ionization (NICI) modes were used; for nitro-PAHs, the best results were obtained under NICI conditions. In the flow-injection mode, detection limits as low as pg levels were obtained for 9-nitroanthracene, 3-nitro-9-fluorenone, 1-nitropyrene, 1,8-dinitronaphthalene, 2,7-dinitrofluorene and 2,7-dinitro-9-fluorenone, whereas the low-molecular-mass 1- and 2-nitronaphthalene were not detected even at μg levels; using the analytical column, a detection limit five times higher was determined for 1-nitropyrene. For most of the nitro-PAHs considered, the dependences of the intensities on the amounts injected were found to be linear in a range of more than two orders of magnitude.

Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation.

We report that exposure to the chemical warfare agent sulfur mustard can be monitored by means of a modified Edman degradation involving selective release of the N-terminal valine adduct of hemoglobin with the agent. The degree of alkylation of the N-terminal valine in human hemoglobin is approximately 1-2% of the total alkylation induced in hemoglobin upon treatment of human blood with sulfur mustard. After modified Edman degradation, followed by derivatization with heptafluorobutyric anhydride, the obtained pentafluorophenyl thiohydantion derivative of the valine adduct could be analyzed at a > or = 0.5 fmol level by means of GC/MS under negative ion chemical ionization conditions. Applying this procedure, in vitro exposure of human blood to > or = 0.1 microM of sulfur mustard could be determined. In vivo exposure of guinea pigs could also be established at 48 h after intoxication intravenously with 0.5 mg/kg (0.06 LD50) of the agent.

Micro liquid chromatography—mass spectrometry with direct liquid introduction used for separation and quantitation of all- trans- and 13- cis-retinoic acids and their 4-oxo metabolites in human plasma

The separation and quantitation of the pentafluorobenzyl derivatives of all- trans- and 13- cis-retinoic acids and their 4-oxo metabolites in human plasma on micro high-performance liquid chromatographic columns (0.32 mm I.D.) is described. The column outlet was directly coupled to the source of a quadrupole mass spectrometer via a simple SFC-frit interface. Negative ion chemical ionization conditions were obtained by coaxial introduction of ammonia as a reagent gas. A signal-to-noise ratio well above 3 was obtained for 1 pg of each analyte injected. The limit of quantitation calculated from spiked biological plasma extracts was 0.3 ng/ml.

Identification of metabolites of halofantrine, a new candidate anti-malarial drug, by gas chromatography—mass spectrometry

Two previously unknown metabolites of halofantrine, a candidate anti-malarial drug, have been isolated by thin-layer chromatography from the plasma of dogs administered a single oral dose of 60 mg/kg. Their identities were investigated after trimethylsilylation by gas chromatography—mass spectrometry under electron-impact and negative-ion chemical ionization conditions. The structural assignment was further confirmed by using a combination of elemental composition analysis of all the isotope peaks at low mass resolution and isotope pattern matching. These two metabolites were formed by modification of the dibutylaminopropyl side-chain of the parent compound involving deamination and oxidation or reduction.

Structural dependence of anion/dipole complex upon the deprotonated site in [MH]− ions of 17β‐estradiol‐17‐stearate ester in ammonia negative‐ion chemical ionization

AbstractUnder ammonia negative‐ion chemical ionization (NICI), of 17β‐estradiol‐17‐stearate ester, a deprotonation reaction occurs competitively at two acidic positions(i.e., the phenol and enolizable sites), demonstrated by using gas phase labelling NICI conditions which yield both [MdD]− and [MdH]− ions. Investigation under low‐energy collisionally‐activated dissociation (CAD) of both molecular species indicates that prior to dissociation, each molecular species isomerizes into different ion/dipole intermediates. These complexes can lead to labile proton exchange. Dissociation of these complexes gives rise to the formation of complementary daughter ions whose abundance depends upon the relative gas phase acidity of the corresponding neutrals.

Application of capillary supercritical fluid chromatography–double focusing mass spectrometry under negative ion chemical ionization conditions

AbstractThe application of mass spectrometry in the negative ion mode with capillary column supercritical fluid chromatography is described. The supercritical fluid chromatography mobile phase (CO2) was used as the primary reagent gas to generate thermalized electrons and reagent anions (such as O− and CO2−). When trace amounts of CH4 or NH3 gas were also introduced into the chemical ionization source, the mass spectrometer sensitivity was increased. Detection limits (S/N = 5) for heptachlor were determined to be 20 pg and 200 fg for full scan and selected ion monitoring modes, respectively. Mass spectra of a cyclic peptide, valinomycin, obtained with these mixed reagents (CH4 + CO2 and NH3 + CO2) gave the molecular anion at m/z 1110 as the most abundant ion and additional fragment ions that yielded peptide sequence information.

Sensitivity study of perfluorocarbons and heteroatom-containing fluorocarbons by negative ion chemical ionization mass spectrometry

The relative response of various perfluorocarbons (PFCs) such as perfluorinated alkanes, alkenes, aromatics and heteroatom-containing fluorocarbons such as perfluorinated 2-butyltetrahydrofuran, tert-butanol, bis(isopropyl) ketone and 1H-heptane, with respect to the internal standard, perfluoro-cis-decalin, was studied using different negative ion chemical ionization (NICI) reagent gases. Under the NICI conditions used in our laboratory, methane was found to be the best NICI reagent gas for the optimization of the electron attachment (in terms of total ion current) for perfluorinated alkanes, alkenes, aromatics and 1-methyladamantane. Carbon dioxide and ammonia were found to be good NICI reagent gases for optimizing the response of hydrogen- and oxygen-containing fluorocarbons and highly symmetric PFCs such as perfluoroadamantane and perfluoro-trans-decalin. On the other hand, CF4 was found to yield the largest linear range in the calibration curve for perfluoro-C11-cycloalkanes (PCA) when perfluoro-cis-decalin was used as the internal standard. The employment of NH3 as the NICI reagent gas gave the most sensitive response to the increase of trace PCA concentration.

Gas chromatographic and mass spectrometric determination of some resin and fatty acids in pulpmill effluents as their pentafluorobenzyl ester derivatives

A sensitive gas chromatographic method for the determination of resin and fatty acids commonly found in pulpmill effluents is presented. The acids are extracted from effluent samples at pH 8 by methyl tert.-butyl ether and converted into their respective pentafluorobenzyl ester derivatives. After silica gel column cleanup, sample extracts are analyzed by gas chromatography with an electron-capture detector using a 30-m DB-17 column. Mass spectral data of these esters obtained under electron impact and electron capture negative ion chemical ionization conditions are also described. The abundant and characteristic (M — 181) − ions are used for the identification and quantitation of resin and fatty acids using a selected ion monitoring technique. Using an effluent with a low blank, spiked recovery of a mixture of 15 acids at 1000-, 100- and 10-μg/1 levels is quantitative. Based on a 25-ml sample and a concentration factor of 10, the method detection limit is 1 μg/1 for all acids. Application of this procedure to some Canadian pulpmill samples is also presented.

Gas-phase reactions of nucleophiles with α,β-unsaturated nitriles

Gas-phase reactions of nucleophiles with a number of α,β-unsaturated nitriles were studied in a high-pressure negative ion chemical ionization (NICI) source. Both weak and strong nucleophiles react with these molecules giving rise to adduct ions. Under NICI conditions using Cl − and Br − ions the spectra show predominantly M −· and [M + Cl] − or [M + Br] − ions formed by hydrogen bonding. In the hydroxy compounds, [M − H] − is predominant instead of M −·. With stronger nucleophiles, such as MeO − and CH 2CN −, deprotonation competes with adduct formation and electron attachment. The adduct ions observed are [M + MeO] −, [M + O − H] −, [M + CH 2CN] − and [M + CN − H] −. These ions are absent in the saturated nitriles suggesting that unlike the weak nucleophiles, Cl − and Br −, stronger nucleophiles, such as MeO − and CH 2CN −, form adduct ions by covalent bonding with the α,β-unsaturated system. A ring substitution by CN − is suggested for the formation of [M + CN − H] − ions. Deuterium- and 13C-labelled nitriles were also used in this study.

Determination of captopril in human blood by gas chromatography—negative-ion chemical ionization mass spectrometry with [ 18O 4]captopril as internal standard

A method for the quantitative measurement of captopril in human blood is described. Blood was immediately treated with N-ethylmaleimide to prevent oxidative degradation. The carboxyl moiety was derivatized to the pentafluorobenzyl ester, which shows excellent properties for negative-ion chemical ionization mass spectrometry. A stable isotope-labelled standard was prepared from the intact target molecule in quantitative yield by exchanging the oxygen atoms of the free carboxylic acid and the imide moiety against 18O. The detection limit under negative-ion chemical ionization conditions is ca. 100 times lower than under electron-impact or positive-ion chemical ionization conditions, therefore only very small amounts of the original sample have to be analysed. The method was applied to be quantitative determination of unchanged captopril in human plasma after oral administration of a 25-mg dose.

Reactivity in the gas phase. Behaviour of isoxazoles under negative ion chemical ionization conditions

Abstract[M  H+]− ions of isoxazole (la), 3‐methylisoxazole (1b), 5‐methylisoxazole (1c), 5‐phenylisoxazole (1d) and benzoylacetonitrile (2a) are generated using NICI/OH− or NICI/NH2− techniques. Their fragmentation pathways are rationalized on the basis of collision‐induced dissociation and mass‐analysed ion kinetic energy spectra and by deuterium labelling studies. 5‐Substituted isoxazoles 1c and 1d, after selective deprotonation at position 3, mainly undergo N  O bond cleavage to the stable α‐cyanoenolate NC  CH  CR  O− (R = Me, Ph) that fragments by loss of RCN, or RH, or H2O. The same α‐cyanoenolate anion (R = Ph) is obtained from 2a with OH−, or NH2−, confirming the structure assigned to the [M  H+]− ion of 1d, On the contrary, 1b is deprotonated mainly at position 5 leading, via NO and C(3)C(4) bond cleavages, to HC ≡ C O − and CH3CN. Isoxazole (1a) undergoes deprotonation at either position and subsequent fragmentations. Deuterium labelling revealed an extensive exchange between the hydrogen atoms in the ortho position of the phenyl group and the deuterium atom in the α‐cyanenolate NC  CD = CPh  O−.

Gas‐phase reactions of electrons, halide ions and radicals with (meso‐tetraphenylporphinato)metal(II) complexes under negative ion chemical ionization conditions

AbstractThe negative ion chemical ionization mass spectra of a series of (meso‐tetraphenylporphinato)metal(II) (metal = Mg, Co, Ni, Cu, Zn) complexes obtained with the electron‐energy‐moderating/reagent gases argon, NF3, CF2Cl2 and CF3Br are presented. Formation of the negative ions identified in the mass spectra is accounted for in terms of the chemistry which occurs in the gaseous plasmas. In argon plasmas, the metal complexes undergo resonance electron capture with comparable facility to produce stable molecular ions. In the NF3, CF2Cl2 and CF3Br plasmas, ionization occurs as a result of ion–molecule reactions as well as resonance electron capture, and is quite selective. Only fluoride ions react with each of the metal complexes, producing metal‐containing ions by nucleophilic addition and proton abstraction as well as by inducing ring closure. The less reactive chloride and bromide ions react readily by nucleophilic addition only with the magnesium and zinc complexes. Negative ions are also produced in the plasmas by radical‐molecule reactions followed by ionization of the neutral products of these encounters.

Electron capture negative ion chemical ionization analysis of arachidonic acid

A highly sensitive (subpicomole level) and structural specific method for the analysis of arachidonic acid esterified to complex glycerophospholipids has been developed using combined capillary gas chromatography and mass spectrometry. The methodology is based upon the formation of the pentafluorobenzyl ester of arachidonic acid, which is efficiently ionized using electron capture negative ion chemical ionization conditions to yield an abundant carboxylate anion at m/z 301. Quantification is carried out following hydrolysis of the complex glycerophospholipid in the presence of a known amount of (2H8) arachidonic acid. The use of this method is illustrated by the quantification of arachidonic acid within the glycerophospholipid classes isolated from resident peritoneal macrophage cells isolated from HS mice.