Chromatography Electron Capture Negative Ionization Research Articles

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.

Detection of Nitro-Substituted Polycyclic Aromatic Hydrocarbons in the Antarctic Airborne Particulate

Nitro-PAH have been determined in the Antarctic air particulate for the first time. This achievement was made possible by the use of an extremely sensitive and specific analytical procedure, which allowed us to investigate the composition of size-fractionated airborne particulates, collected in the Terra Nova Bay area (Antarctica) during two-days high-volume sampling periods. Eleven air-particulate samples and three blanks were extracted and analyzed by gas chromatography—electron capture negative ion—tandem mass spectrometry. The high specificity of the procedure relies on both the ionization method and the fixed setting of the second mass-analyzer, selectively filtering the NO2-ions (m/z 46). Only the most volatile nitro-PAHs (nitro-and dinitronaphtalenes) were detected in the Antarctic air particulate, unlike in temperate areas. Single nitro-PAH concentrations varied in the 1–200 fg/m3 range. Principal component analysis allowed to cluster the blanks, clearly distinguishing them from the samples, and to classify the samples, as a function of their composition. Nitro-PAH distribution proved to depend on the sampling day but not on the particle size-fraction within the same sampling period. For two air particulate samples the analytical data proved to be biased by sea-triggering the sampling device. The origin of Antarctic nitro-PAHs remains uncertain: while the abundance ratio of the two nitronaphtalene isomers supports the hypothesis of local direct emission, the high abundance of dinitrowith respect to mononitro-derivatives suggests that an efficient gas-phase nitration may take place on a substrate possibly transported from other continents.

Determination of Q1, an unknown organochlorine contaminant, in human milk, Antarctic air, and further environmental samples

Q1, an organochlorine component with the molecular formula C9H3Cl7N2 and of unknown origin was recently identified in seal blubber samples from the Namibian coast (southwest of Africa) and the Antarctic. In these samples, Q1 was more abundant than PCBs and on the level of DDT residues. Furthermore, Q1 was more abundant in seals from the Antarctic than the Arctic. To prove this assumption, gas chromatography–electron-capture negative ion mass spectrometry (GC/ECNI–MS), which is sensitive and selective for Q1, allowed for screening of traces of Q1 even in samples with particularly high levels of other organochlorine contaminants. Q1 was isolated by high-performance liquid chromatography (HPLC) from a skua liver sample. A 1:1 mixture with trans-nonachlor in electron-capture detectors (ECDs) was used to determine the relative response factor with ECNI–MS. The ECNI–MS response of Q1 turned out to be 4.5 times higher than that of trans-nonachlor in an ECD. With GC/ECNI–MS in the selected ion-monitoring mode, four Antarctic and four Arctic air samples were investigated for the presence of Q1. In the Antarctic air samples, Q1 levels ranged from 0.7 to 0.9 fg/m3. In Arctic air samples, however, Q1 was below the detection limit (<0.06 fg/m3 or 60 ag/m3). We also report on high Q1 levels in selected human milk samples (12–230 μg/kg lipid) and, therefore, suggested that the unknown Q1 is an environmental compound whose origin and distribution should be investigated in detail. Our data confirm that Q1 is a bioaccumulative natural organochlorine product. Detection of a highly chlorinated natural organochlorine compound in air and human milk is novel.

Occurrence of C10−C13 Polychlorinated n-Alkanes in Canadian Midlatitude and Arctic Lake Sediments

Sediment cores from six lakes in Canada ranging from 49°N to 81°N were analyzed for C10−C13 polychlorinated n-alkanes (PCAs) with the intent of (i) examining the depositional trends with increasing latitude, (ii) studying the historical profiles and fluxes of PCAs in dated sediment slices, and (iii) investigating possible in situ degradation. Sediment slices were dated using 210Pb and 137Cs, and extracts were analyzed for PCA concentrations by high resolution gas chromatography electron capture negative ion high resolution mass spectrometry (HRGC-ECNI/HRMS) in the selected ion monitoring (SIM) mode. Concentrations of total PCAs in surface sediments declined significantly from 135 ng/g (dry weight (dw)) in sediments from the southern basin of Lake Winnipeg (50°N/96°W, Manitoba) to 4.52 ng/g in Hazen Lake (81°N/71°W, high Arctic); corresponding surficial fluxes were 147 and 0.9 μg/m2 yr, respectively. The high flux of PCAs to the south Lake Winnipeg basin suggests local contamination. This was confirmed by ...

Amino Acid Analysis of Peptides and Proteins on the Femtomole Scale by Gas Chromatography/Mass Spectrometry

Amino acid analysis (AAA) is a useful aid in protein chemistry, but its routine application is limited by a modest limit of detection. Typically, 10 pmol of material is required, but even at this level the reproducibility can be poor. We have employed isotope dilution gas chromatography electron capture negative ionization mass spectrometry (GC/ECNI/MS) to provide accurate and reliable data on less than 100 fmol of material. Precision and accuracy are good, and all 20 non-hydrolyzed amino acids can be determined in this manner. The protein is hydrolyzed (HCl), and then a cocktail, composed of all 20 amino acids as stable isotope-labeled forms (i.e., (13)C and (2)H), is added. The mixture of protein-derived and stable isotope-labeled amino acids is then converted to volatile electron-capturing derivatives with a multistep approach employing heptafluorobutyric anhydride (HFBA), pentafluorobenzyl bromide (PFBBr), and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). These derivatives are then injected directly into the GC/MS system. Groups of selected ions, characteristic of each derivatized amino acid, are thereafter monitored at appropriate time intervals. The ratios of the ion current for the selected ions for the native amino acid and its labeled form are determined and converted to absolute amounts of the native amino acids in the protein hydrolyzate by reference to standard samples prepared at the time of the analysis.

Trace Quantification of the Oxidative Damage Products,meta- andortho-Tyrosine, in Biological Samples by Gas Chromatography–Electron Capture Negative Ionization Mass Spectrometry

Oxygen radicals damage biomolecules and may contribute to cellular aging and degenerative disease. We describe a sensitive method for the quantification of two endogenous biomarkers of oxidative damage:meta-tyrosine (m-Tyr) andortho-tyrosine (o-Tyr). The assay can be applied to direct analysis of free amino acids or protein-bound amino acids following hydrolysis. The assay involves derivatization with pentafluorobenzyl bromide and extraction inton-decane, followed by gas chromatography–mass spectrometry. Stable isotope labeledm- ando-Tyr (2H4) and phenylalanine [i.e., Phe (2H5)] were added as internal standards to improve analytical accuracy. Quantification of as little as 50 pg ofm- ando-Tyr in 100 μg protein is possible and the data are expressed as a molar ratio ofm- ando-Tyr to native Phe. The assay was used to determine the levels ofm- ando-Tyr in freshly isolated human plasma protein (4.05 ± 0.67m-Tyr per 104Phe, 0.35 ± 0.07o-Tyr per 104Phe). Exposure of human plasma to reactive oxygen species significantly increased the levels ofm-Tyr (56.4 ± 1.1m-Tyr per 104Phe,P< 0.0001) ando-Tyr (48.9 ± 1.3o-Tyr per 104Phe,P< 0.0001). The mild hydrolysis and derivatization conditions caused no artifactual formation of eitherm- oro-Tyr.

Determination of ethylenethiourea in water by single-step extractive derivization and gas chromatography—negative ion chemical ionization mass spectrometry

A method for the determination of ethylenethiourea (ETU) in water samples is described that involves a single-step derivatization and extraction by means of phase transfer-catalysed reaction with 3,5-bis(trifluoromethyl)benzyl bromide followed by gas chromatography—electron-capture negative ion mass spectrometric separation and detection. The reaction resulted in three ETU derivatives, identified by GC—Fourier transform spectrometry as the mono-N-substituted form and two forms substituted at the N,N′- and the N,S-positions. Quantification in the multiple-ion detection mode was performed on the abundant [M — 227] − ions of the N,N′-isomer in low-level (<5 μg/l) and at the N,S-isomer in high-level samples (>5 μg/l). The limit of determination in surface water samples was 0.05 μg/l with recoveries ranging between 60 and 110%. The method was applied for confirmation purposes for the presence of ETU as analysed by HPLC with UV detection. In general, a good correlation was found the results from both methods.

KO2 chemical transformation/mass spectrometry detection of covalent damage to the DNA of cultured human lymphocytes exposed to benzo[a]pyrene.

Cultured human lymphocytes were exposed to benzo[a]pyrene (B[a]P), and diol epoxide-type DNA adducts arising from this chemical were detected by a method consisting of the following sequence of steps: (1) isolate the DNA; (2) subject the DNA to mild acid hydrolysis to release the polyaromatic moiety as a tetrahydrotetrol; (3) add an internal standard; (4) oxidize the tetrahydrotetrol with potassium superoxide to pyrene-2,3-dicarboxylic acid; (5) derivatize the latter with pentafluorobenzyl bromide; (6) purify the diester product on a silica cartridge; and (7) detect this product by gas chromatography electron capture negative ion mass spectrometry. From the dose (1 microgram/mL) of B[a]P applied, five adducts in 10(7) normal nucleotides were found. Largely because steps 2-5 of the method take place sequentially in a single vial, the procedure is convenient and affords precise results. To demonstrate the potential of the method to detect KO2-susceptible polyaromatic hydrocarbon DNA adducts in general, including unknowns, it was also applied to picomole and femtomole amounts of a standard of chrysene-1,4-quinone using scanning and selected ion monitoring conditions, respectively, in the MS. Since standard products can be detected with selected ion monitoring at levels 10(4) below those encountered here (prior work), it should be possible in the future to extend the method to samples containing smaller amounts of such adducts.

Measurement of MeIQx and DiMeIQx in fried beef by capillary column gas chromatography electron capture negative ion chemical ionisation mass spectrometry.

A gas chromatographic-mass spectrometric assay has been developed for the simultaneous measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) in fried beef. The method employs capillary column gas chromatography, electron capture negative ion chemical ionisation mass spectrometry and a stable isotope labelled analogue of MeIQx (the synthesis of which is described) as common internal standard. Two patties of lean minced beef which had been cooked separately were analysed and found to contain both compounds (patty 1-2.4 ng MeIQx/g meat, 1.2 ng DiMeIQx/g meat; patty 2-1.3 ng MeIQx/g meat, 0.5 ng DiMeIQx/g meat). Neither compound was present in the meat prior to cooking.

BIS-trifluoromethylbenzoyl derivatives for steroid analysis by gas chromatography electron capture negative ion chemical ionisation mass spectrometry

3,5- Bis-trifluoromethylbenzoyl derivatives of eight monohydroxy steroids and eight dihydroxy steroids were synthesised. Under the reaction conditions described, the monohydroxy steroids each gave a single derivative while the dihydroxy steroids, with the exception of corticosterone, showed multiple product formation. The reactivity of hydroxyl groups relative to their stereochemistry is discussed. The electron capture negative ion chemical ionisation mass spectra of these derivatives were recorded. When the derivatised hydroxyl group was alicyclic or aromatic, a molecular ion was normally the base peak in the mass spectrum. Selected ion monitoring of molecular ions indicated that, in certain cases, as little as 1 pg of the parent steroid could be detected. The potential use of this derivative in the quantitative analysis of steroids by gas chromatography-mass spectrometry is discussed.

Bistrifluoromethylaryl derivatives for drug analysis by gas chromatography electron capture negative ion chemical ionization mass spectrometry. Application to the measurement of (N-dicyclopropylmethyl)amino-2-oxazoline in plasma.

A gas chromatographic mass spectrometric assay for (N-dicyclopropylmethyl) amino-2-oxazoline in plasma with a detection limit of 0.1 ng ml-1 was required. Various fluoroaryl derivatives of this compound (code name S3341) were synthesized and their positive ion chemical ionization and electron capture negative ion chemical ionization mass spectra recorded. While fluorobenzyl derivatives of S3341 could be made by heating with the requisite benzyl bromide and diisopropylethylamine in acetonitrile, initial efforts to synthesize corresponding fluorobenzoyl derivatives using a benzoyl chloride in dry ethyl acetate at 60 degrees C were unsuccessful. Mass spectral data indicated that only a fragment of the oxazoline ring was retained in the reaction product and that an N-(2-chloroethyl)benzamide was formed. However, when diisopropylethylamine was included in the reaction mixture, a benzoyl derivative of the complete molecule was obtained. The mechanisms of these reactions are discussed. The negative ion mass spectrum of the 3,5-bistrifluoromethylbenzoyl derivative of S3341 has a base peak at m/z 420 (the molecular ion) and, when this ion is specifically monitored, an amount of derivative equivalent to 1 pg of S3341 can be detected. This allowed the development of an assay for S3341 in plasma with a precision of 9% (SD) at 0.2 ng ml-1 and a lower limit for quantitative determination of 0.1 ng ml-1.

Bis(trifluoromethyl)aryl derivatives for drug analysis by gas chromatography electron capture negative ion chemical ionization mass spectrometry. Application to the measurement of low levels of clonidine in plasma.

A gas chromatographic mass spectrometric assay for clonidine in plasma with a detection limit of a few picograms per ml was required. The p-trifluoromethylbenzyl, pentafluorobenzyl and pentafluorobenzoyl derivatives of clonidine were synthesized and the electron capture negative ion chemical ionization mass spectra of these compounds show extensive fragmentation with prominent ions at m/z 35 and 37 due to the two chlorine atoms in the clonidine molecule. Selected ion monitoring of specific high mass ions in these mass spectra indicated that the required sensitivity could not be obtained with these derivatives. Several bis(trifluoromethyl)pyrimidines were synthesized and these compounds were found to give an intense negative ion current under conditions of resonance electron capture. Consequently, a derivative of clonidine containing a bis(trifluoromethyl)aryl group was synthesized by reacting the drug with 3,5-bis(trifluoromethyl)benzoyl chloride. The negative ion mass spectrum of the reaction product has a base peak at m/z 673 and, when this ion is specifically monitored, an amount of derivative equivalent to 1 picogram of clonidine can be detected. This allowed the development of an assay for clonidine in plasma with a precision of 8% (SD) at 50 pg ml-1, 22% (SD) at 20 pg ml-1 and a lower limit for quantitative determination of 10 pg ml-1. Plasma concentrations of clonidine in 10 subjects given a single 50 micrograms oral dose are reported.

Hydroxypolychlorodibenzofurans in technical pentachlorophenol: Electron capture negative ion chemical ionization mass spectra of alkoxypolychlorodibenzofurans

A mixture of hydroxypolychlorodibenzofurans was isolated from technical pentachlorophenol. The alkyl ethers were prepared, and analyzed by fused silica capillary column gas chromatography electron capture negative ion chemical ionization mass spectrometry. The appearance of intense molecular ion peaks for the alkyl ethers apparently depends on the ring position of alkoxy substitution. Loss of chlorine atoms from the ring, followed by protonation of the resultant anion as the alkyl group is eliminated, is postulated on the basis of deuterium labeling experlments. Evidence is also presented for the presence of a hydroxyheptachlorodibenzodioxin in technical pentachlorophenol.