Gas Chromatography-thermal Energy Analysis Research Articles

Evaluation of the influence of proline, hydroxyproline or pyrrolidine in the presence of sodium nitrite on N-nitrosamine formation when heating cured meat

N-nitrosamines are meant to be probable or possible carcinogenic components, possibly formed out of a reaction between nitrite and N-containing substances such as amino acids and secondary amines. Nitrite is often used for processing meat products because of its colouring and antimicrobial properties. During this experimental setup, the influence of proline, hydroxyproline or pyrrolidine on N-nitrosamine formation in meat samples was evaluated. The N-nitrosamines concentrations were measured with gas chromatography-thermal energy analyzer. Only the concentrations of N-nitrosodimethylamine and N-nitrosopyrrolidine were found above the limit of detection in a number of tested experimental conditions. The concentration of these two N-nitrosamines was modelled as a function of temperature and nitrite concentration for different situations (presence or absence of added natural N-containing meat components). It could be concluded that proline and pyrrolidine promoted the formation of N-nitrosopyrrolidine. It could also be confirmed that the higher the temperature of the meat processing procedure and the higher the sodium nitrite amounts added, the higher were the yields of the respective N-nitrosamines.

Presence of N-nitroso-L-thioproline and N-nitroso-L-methylthioprolines in human urine as major N-nitroso compounds.

Unknown N-nitroso compounds were found in human urine of healthy volunteers by gas chromatography-thermal energy analysis. These compounds were identified as N-nitroso-L-thioproline and cis- and trans-N-nitroso-L-methylthioprolines by gas chromatography-mass spectrometry. The precursors of these new N-nitroso compounds may be formed by the reactions of L-cysteine with formaldehyde and acetaldehyde in the human body.

Formation of volatile nitrosamines by drug-nitrite interactions under physiological conditions.

Twenty-eight drugs, most of which are tertiary amines, were tested for the formation of volatile nitrosamines by reaction with nitrite under physiological conditions; the drugs (10mM) were incubated with nitrite ( 40mM ) at pH 3.0, 37 degrees for 1 and 4 hr. The volatile nitrosamines formed were determined by gas chromatography-thermal energy analysis. Of the 28 drugs, 24 formed measurable amounts of volatile nitrosamines that are known carcinogens. The yields of nitrosodimethylamine (NDMA) from aminopyrine (55-65%) and minocycline (11%) were higher than that from dimethylamine under the same conditions. This result suggests that there may be a pathway not involving the secondary amine (dimethylamine) as an intermediate in the formation of NDMA from minocycline as well as from aminopyrine, Tolazamide gave rise to nitrosopiperidine ( NPIP ) in addition to nitrosohexamethyleneimine ( NHXI ), formation of which was expected from the chemical structure of tolazamide, and the yield of NPIP (2-7%) was higher than that of NHXI (0.2-1.2%). Ascorbic acid ( 40mM ) was effective in decreasing the formation of nitrosamines from drugs by reaction with nitrite, although the blocking effects varied between 88 and 100% depending on the drugs tested or on the nitrosamines formed.

Spectrophotometric determination of cyclotrimethylenetrinitramine (RDX) in explosive mixtures and residues with the Berthelot reaction

On-site colorimetric methods are a valuable, cost-effective tool to assess the nature and extent of contamination in remediated sites and to enable on-site screening for police criminology laboratories. The existing colorimetric method for cyclotrimethylenetrinitramine (RDX) based on a Griess reaction suffers from the non-quantitative reduction to nitrite and from the unstable character of HNO 2 in acidic medium. Thus we propose a novel spectrophotometric RDX assay in explosive mixtures and residues, based on (Zn + HCl) reduction of RDX in a microwave oven, followed by neutralization of the reduction products to ammonia and low molecular-weight amines, and Berthelot reaction of these amine-compounds with phenol and hypochlorite in alkaline medium to give an intensely blue indophenol dye absorbing at 631 nm. The molar absorptivity and limit of detection (LOD) for RDX were (1.08 ± 0.04) × 10 4 L mol −1 cm −1 and 0.18 mg L −1, respectively. Application of the method to synthetic mixture solutions of RDX and trinitrotoluene (TNT) at varying proportions showed that there was minimal interference from TNT (which could be compensated for by dicyclohexylamine colorimetry), since the Berthelot reaction was essentially non-responsive to m-substituted anilines derived from TNT upon (Zn + HCl) reduction. The proposed method was successfully applied to military-purpose explosive mixtures of (RDX + inert matter) such as Comp A5, Comp C4, and Hexal P30, and to (RDX + TNT) mixtures such as Comp B. The molar absorptivity of RDX was much higher than that of either ammonium or nitrate; RDX could be effectively separated from ammonium and nitrate in soil mixtures, based on solubility differences. The Berthelot method for RDX was statistically validated using Comp B mixtures against standard HPLC equipped with a Hypersil C-18 column with (40% MeOH–60% H 2O) mobile phase, and against gas chromatography–thermal energy analysis (GC–TEA) system.

A Novel Method for the Analysis of Discharged Smokeless Powder Residues

A novel method for the estimation of intermediate-long firing distance range is proposed. The method is based on the characterization and chemical analysis of the smokeless powder particles on the target. An adhesive lifter is applied to collect the suspected gunshot residues (GSRs) from the surface of an object, and a Modified Griess Test (MGT) is carried out after alkaline hydrolysis on the adhesive lifter. Visualized particles are removed from the adhesive lifter under a microscope. Two systems are used for the analysis of organic discharge residues from the smokeless powder: (1) gas chromatography/thermal energy analysis (GC/TEA) for the analysis of nitroglycerine (NG) and 2,4-dinitrotoluene (2,4-DNT), (2) gas chromatography/mass spectrometry (GC/MS) for the identification of organic components such as DNT, NG, and some stabilizers. By using this procedure and confirming that the suspected particles are indeed GSR, one can estimate the intermediate-long firing distance of c. 0.75-3 m in the presence of very few particles and provide information for the classification of ammunition type in casework.

Migration of nitrosamines from rubber products - are balloons and condoms harmful to the human health?

Studies performed in 2001 and 2003 surveyed the release of carcinogenic nitrosamines and nitrosatable substances from rubber toy balloons by extraction with artificial saliva and gas chromatography-thermal energy analysis (GC-TEA). 81% of the 16 in 2001 sampled balloons and 93% of the 14 in 2003 sampled balloons released nitrosamines above the recommended level in Germany of 10 mug per kg material. Furthermore, 32 rubber condom samples collected in 2004 from the German market were surveyed for nitrosamines by determining the amount migrating into an artificial sweat test solution. The levels released from condoms varied from < 10 to 660 mug per kg material (i. e., up to 1.4 mug nitrosamines per condom). In a model calculation, not considering the differences that may exist in the resorption rate, we have calculated that the exposure from condoms may exceed the exposure from food 1.5-3 fold. To our knowledge so far no legal binding legislation exists worldwide concerning nitrosamine migration from toy balloons or condoms.

A Novel Method for Extraction and Analysis of Gunpowder Residues on Double-Side Adhesive Coated Stubs

A study was conducted to develop an efficient method for extraction and analysis of gunpowder (propellant) residues from double-side adhesive coated stubs, which are used for sampling suspects or their clothing for gunshot (primer) residues (GSR). Conductive and non-conductive double-side adhesives were examined, and the analysis was carried out by gas chromatography/thermal energy analyzer (GC/TEA) and ion mobility spectrometry (IMS). The optimal procedure for the extraction, as was developed in the present study, employs two stages: (1) extraction of the stubs with a mixture of 80% v/v aqueous solution of 0.1% w/v of sodium azide and 20% v/v of ethanol employing sonication at 80 degrees C for 15 min. and (2) residues from the obtained extract were further extracted with methylene chloride. The methylene chloride phase was concentrated by evaporation prior to analysis. Extraction efficiencies of 30-90% for nitroglycerine (NG) and for 2,4-dinitro toluene (2,4-DNT) were found. No significant interferences in the analysis were observed from the adhesives or skin. Interferences were observed in the analysis by the GC/TEA of the samples collected from hair. The method enables analysis of propellant residues on a double-side adhesive coated stub after it was examined for primer residues by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX). Thus, the probative value of the evidence may be increased.

Vacuum Collection of Gunpowder Residues from Clothing Worn by Shooting Suspects, and Their Analysis by GC/TEA, IMS, and GC/MS

Experiments were conducted to collect gunpowder (propellant) residues from shooters' clothing by vacuum and to analyze them by gas chromatography/thermal energy analyzer (GC/TEA), ion mobility spectrometry (IMS), and gas chromatography/mass spectrometry (GC/MS). The residues were collected on fiberglass and Teflon filters using the portable vacuum sampler, all supplied with the IMS instrument. Several solvents were examined for the extraction of the propellant components from the filters. The extracts were centrifuged and/or filtered, concentrated by evaporation, and analyzed without any additional clean-up procedure. Based on the results of the study, an operational method for analysis of gunpowder residues was introduced into casework without changing the present operational technique for gunshot (primer) residue (GSR) analysis on clothing implemented by the Israel Police. In the modified method, the clothing is first sampled by double-side adhesive-coated aluminum stubs (the tape-lift method) for GSR analysis (the existing method), followed by vacuum collection for propellant residue examination. The issue of interpretation of the analytical results is discussed.

N-Nitroso Compounds and Mutagens in Chinese Fermented (Sour) Corn Pancakes

Stomach cancer rates in rural Linqu County, Shandong Province, China, are exceptionally high. A previous case-control study revealed that the risk of stomach cancer was 30% higher among those who consumed sour (fermented) corn pancakes at least daily. A previous study of the sour pancakes reported volatile nitrosamines in most specimens, and almost half reportedly showed mutagenic activity. Few households currently consume sour pancakes, and the duration of fermentation has been shortened. We tested specimens of pancake batter and sour pancakes from Linqu County for mutagenic activity using the Ames test; for N-nitroso compounds (NOC) we used the Nitrolite-thermal energy analysis (TEA) method. Results of the Ames test were inconclusive: only 1 out of 15 cooked pancakes showed a positive mutagenic response, and all 15 batter specimens were negative; however, several batter specimens showed a weakly positive trend of mutagenicity with extract concentration. Our assay for total nitroso compounds was weakly positive in only 1 out of 15 specimens of sour pancake batter. That specimen was also tested by gas chromatography-TEA for nitrosaminoacids and volatile nitrosamines, but none were detected. It seems unlikely that the Chinese sour pancakes are significantly contaminated by NOC or other mutagens.

Inhibition of Methyl-n-Amylnitrosamine Hydroxylation by Diallyl Sulfide and Phenethylisothiocyanate in the Rat

Formation of the stable 2-, 3-, and 4-hydroxy derivatives of methyl-n-amylnitrosamine (MNAN) probably reflects cytochrome P-450-catalyzed activation of MNAN by 1-hydroxylation. Here we studied inhibition of the oxidation of MNAN to hydroxy-MNANs (HO-MNANs) by freshly excised tissues from MRC-Wistar rats treated with the vegetable-derived chemicals diallyl sulfide (DAS) and phenethylisothiocyanate (PEITC). Rats were gavaged with DAS (200 mg/kg), PEITC (163 mg/kg), or vehicle (corn oil) alone. After various times, the rats were killed, the esophagus, nasal mucosa, and liver were removed, and the tissues/tissue slices were incubated for two hours with 23 μM MNAN. HO-MNAN formation was measured by gas chromatography-thermal energy analysis. Significant (p < 0.01) 72-75%, 40%, and 44% inhibitions of total HO-MNAN formation were observed for nasal mucosa removed at 3-18 hours, for esophagus at 18 hours, and for liver at 3 hours, respectively, after gavage of DAS. Significant (p < 0.03) 46-75% inhibition of HO-MNAN formations was observed for the esophagus at 2-24 hours after gavage of PEITC. In disposition studies, rats were treated with DAS (200 mg/kg) in corn oil and sacrificed after various intervals. DAS was determined by gas chromatography of tissue homogenate extracts. After gavage of DAS, its total recovery from all tissues studied was 27% of the dose after 45 minutes and 15-19% after 90 and 180 minutes, with >80% of the recovered DAS in the stomach contents. Up to 2% per tissue of the recovered DAS was found in the stomach wall, liver, and blood. After intraperitoneal injection of DAS, ≤2% of the dose was recovered in the blood and ≤0.7% in the liver. Hence, gavage of DAS and PEITC significantly inhibited HO-MNAN formation for up to 18 and 24 hours, respectively, whereas DAS was >80% metabolized 90 minutes after its gavage. These findings suggest that long-lasting inhibitors or their metabolites, or inactivation of P-450 enzymes, were responsible for the persistence of inhibition of MNAN metabolism.

Determination of N-nitrosodiethanolamine in urine by gas chromatography thermal energy analysis: application in workers exposed to aqueous metalworking fluids.

The aim of this study was to describe a detailed and validated methodology designed for the analysis of carcinogenic N-nitrosodiethanolamine (NDELA) down to sub-microgram/l levels in urine and its application to a number of workers exposed to NDELA-contaminated aqueous metalworking fluids (MWF). Following a work-up procedure based on solid-phase extraction of NDELA, the urinary extracts were analysed without derivatization by gas chromatography on a polar wide-bore column with chemiluminescent detection using a thermal energy analyser (TEA). N-Nitroso-(2-hydroxypropyl)amine was used as an internal standard. The method was applied to 12 workers using "nitrite-free" or "nitrite-formulated" MWF and to 15 unexposed subjects. The NDELA content of the MWF was also determined using a similar, but simpler method able to easily quantify NDELA down to at least 0.1 mg/l. Contamination by NDELA traces of some chemicals used for the sample preparation, particularly ethyl formate, must be carefully checked since it can give rise to false-positive results of up to 1 or 2 micrograms/l. The response was linear in the range of 0-500 micrograms/l. Between 0.5 and 10 micrograms/l, the recovery rate was close to 95%, while repeatability ranged from 12.5 to 6.4% (n = 5). The detection limit was 0.3 microgram/l (Signal/noise = 3). No detectable NDELA could be observed in the control workers. There was no significant increase in NDELA levels at the end of shift spot samples from an exposed worker over 1 week. Higher NDELA concentrations were found in two workers (4.3 and 10.7 micrograms/l) exposed to "nitrite-formulated" fluids (contaminated with 65 and 18 mg NDELA per 1, respectively) than in nine workers (range, 0.4-1.3 micrograms/l exposed to "nitrite-free" fluids with lower levels of NDELA (range, 0.5-6.6 mg/l). The detailed methodology described in this work and applied to a limited industrial situation was found to be suitable for monitoring NDELA in the urine of workers exposed to aqueous MWF. A much larger screening has been undertaken with the aim of obtaining better information on the real exposure of workers sometimes exposed to "nitrite-formulated" fluids that are still used.

Diffusion of dialkylnitrosamines into the rat esophagus as a factor in esophageal carcinogenesis.

To indicate how readily nitrosamines (NAms) diffuse into the esophagus, we measured diffusion rate (flux) through rat esophagus of dialkyl-NAms using side-by-side diffusion apparatuses. Mucosal and serosal flux at 37 degrees C of two NAms, each at 50 microM, was followed for 90 min by gas chromatography-thermal energy analysis of NAms in the receiver chamber. Mucosal flux of one or two NAms at a time gave identical results. Mucosal flux was highest for the strong esophageal carcinogens methyl-n-amyl-NAm (MNAN) and methylbenzyl-NAm. Mucosal esophageal flux of 11 NAms was 18-280 times faster and flux of two NAms through skin was 13-28 times faster than that predicted for skin from the molecular weights and octanol:water partition coefficients, which were also measured. Mucosal: serosal flux ratio was correlated (P < 0.05) with esophageal carcinogenicity and molecular weight. For seven NAms tested for carcinogenicity by Druckrey et al. [(1967) Z. Krebsforsch., 69, 103-201], mucosal flux was correlated with esophageal carcinogenicity with borderline significance (P = 0.07). The MNAN:dipropyl-NAm ratio for mucosal esophageal flux was unaffected when rats were treated with phenethylisothiocyanate and was similar to that for forestomach, indicating no involvement by cytochromes P450. Mucosal esophageal flux of MNAN and dimethyl-NAm was reduced by >90% on enzymic removal of the stratum corneum, was unaffected by 0.1 mM verapamil and was inhibited 67-94% by 1.0 mM KCN and 82-93% by 0.23% ethanol. NAm flux through rat skin and jejunum was 5-17% of that through esophagus. Flux through skin increased 5-13 times after enzymic or mechanical removal of the epidermis; the histology probably explained this difference from esophagus. Hence, NAms could be quite rapidly absorbed by human esophagus when NAm-containing foods or beverages are swallowed, the esophageal carcinogenicity of NAms may be partly determined by their esophageal flux and NAm flux probably occurs by passive diffusion.

A preliminary study of the analysis of explosives using packed-column supercritical fluid chromatography with atmospheric pressure chemical ionisation mass spectrometric detection

A packed-column supercritical fluid chromatographic (SFC) separation of explosive compounds hyphenated to atmospheric pressure chemical ionisation (APCI) mass spectrometric (MS) detection has been developed. Nitroaromatics, nitramines and nitrate esters can be resolved and identified, with theoretical limits of detection of approximately 100 ng on column. This represents a development over previously described gas chromatography–thermal energy analysis (GC–TEA), gas chromatography– electron capture detection (GC–ECD) and SFC methods for the analysis of explosives due to the molecular identification afforded by the mass spectrometry. Explosives in the combinations expected in commercially available mixtures can be separated and identified. A successful application to a laboratory trial simulating casework is described.

Rapid semi-quantitative estimation of N-nitrosodibutylamine and N-nitrosodibenzylamine in smoked hams by solid-phase microextraction followed by gas chromatography-thermal energy analysis

A solid-phase microextraction (SPME) analytical method has been developed for the determination of N-nitrosodi- n-butylamine (NDBA) and N-nitrosodibenzylamine (NDBzA) in hams that is based on: (a) isolation of the compounds by steam distillation, (b) SPME from the distillate headspace using a polycrylate coated silica fibre and (c) determination by gas chromatography-thermal energy analyzer technique or confirmation by gas chromatography-mass spectrometry. Recoveries of both NDBA and NDBzA from hams spiked at 5 to 160 μg/kg levels ranged between 41 to 112%. The overall method is fast, sensitive (detection limits, 1 to 3 μg/kg), precise (within 10%) and fairly accurate (average recoveries 86% and 70%, respectively). The results obtained by this technique for seven ham samples agreed fairly well with those obtained by an existing method ( r 2 = 0.97). The new method is solventless, environmetally friendly and useful for rapid monitoring purposes.

Detection of methylating activity due to nitrosamide in some nitrosated nigerian foodstuffs

Popular vegetables, condiments and some Nigerian staple foods were evaluated for their relative methylating potential due to nitrosamide formation following nitrosation under standardized conditions. Methylating activity of nitrosated foodstuffs, expressed as N-nitroso- N-methylurea equivalents, was determined by gas chromatography-thermal energy analysis. In positive samples (detection limit 10 μg/kg) methylating activity detected was in the range of 50–1200 μg/kg, the highest activity being found in Telfairia occidentalis (ugwu). This value constitutes the highest amount ever detected for a fresh vegetable. The data suggest that some commonly consumed local foodstuffs might contribute to overall human burden of environmental carcinogens in Nigeria.

Tobacco-specific nitrosamines in some Nigerian cigarettes

Ten popular brands of cigarettes on the Nigerian market were analyzed for tobacco-specific nitrosamines (TSNA) in tobacco and in mainstream smoke, as well as nitrate in tobacco. TSNA was analyzed using a gas chromatography/thermal energy analyzer (GC-TEA), while nitrate was determined spectrophotometrically as nitrite following on-line reduction with copper, diozatization with sulfanilamide and coupling with N-(1-naphthyl) ethylene diamine to form an azo dye. In mainstream smoke, the concentration of NNN, NAB/NAT and NNK were respectively, between 8 and 90 ng, 10 and 65 ng, and between 15 and 72 ng/cigarette. Preformed NNN ranged between 64 and 565 ng/cigarette, while preformed NAB/NAT and NNK ranged respectively from 109 to 476 ng/cigarette, and from 55 to 317 ng/cigarette. Nitrate levels ranged between 1.5 and 6.1 mg/g tobacco. In general, the results indicate that the TSNA content of Nigerian cigarettes are within the range found for European and American cigarettes.

Nitrosatable amines and nitrosamide formation in natural stimulants: Cola acuminata, C. nitida and Garcinia cola

Three varieties of kola nut, Cola acuminata, C. nitida and Garcinia cola, of Nigerian origin, were analysed for their content of primary and secondary amines, and assessed for their relative methylating potential due to nitrosamide formation. Primary and secondary amines were determined as benzene sulfonamides by gas chromatography/thermal energy analysis (GC/TEA). Dimethylamine, methylamine, ethylamine and isopentylamine were detected in all kola nut varieties, while pyrrolidine, piperidine and isobutylamine were detected in one or more varieties. Estimated average total daily intake of aliphatic amines by a typical kola nut chewer varied from 260 to 1040 μg/day for secondary amines and from 2430 to 9710μg/day for primary amines. Methylating activity of the nitrosated kola nuts, expressed as N-nitroso- N-methylurea equivalents, was also determined by GC/TEA. Methylating activity was significantly higher in kola nuts (170–490 μg/kg) than has ever been reported for a fresh plant product. These data suggest that the possible role of kola nut chewing in human cancer aetiology should be explored in countries where kola nuts are widely consumed as stimulants.

Determination of N-nitrosodimethylamine in beer by gas chromatography-stable isotope dilution chemical ionization mass spectrometry

A sensitive method for the determination of N-nitrosodimethylamine in beer samples is described. The analyte was isolated by distillation and subsequent extraction from the distillate using dichloromethane. Analysis was carried out by gas chromatography combined with positive-ion chemical ionization mass spectrometry. Quantification was performed by stable isotope dilution using [ 2H 6]-N-nitrosodimethylamine as the internal standard. The measurements exhibited excellent linearity in the range 0.10–4.00 μg kg −1 ( r = 0.99999) and a detection limit of 0.04 μg kg −1 was achieved. The between-sample relative standard deviation of the method was 0.73% ( n = 5) at a concentration level of 0.86 μg kg −1. The recovery of the compound was 78 ± 2.1%. The proposed procedure seems to be a good alternative to the frequently used gas chromatography-thermal energy analysis approach for the determination of N-nitrosodimethylamine.

A Sensitive and Specific Method for the Determination ofN-Nitrosodimethylamine in Drinking Water and Fruit Drinks

Abstract A method is described for the determination of N-nitrosodimethylamine (NDMA) in drinking water and fruit drinks/juices. It is highly sensitive and specific, and is practically free of artifactual formation. Such formation, in this method, is minimized by distilling or extracting the samples in the presence of sulfamic acid, an excellent inhibitor of N-nitrosation. Both gas chromatography‐thermal energy analyzer (GC-TEA) and gas chromatography‐high resolution mass spectrometry selected ion monitoring (GC-MS-SIM) were used for the determination of NDMA. The minimum detection limits of the two methods were 15 pg/g and 1 pg/g, respectively. The recoveries of NDMA and that of N-nitrosodi-n-propylamine (NDPA), the internal standard, at 6.6–133 pg/g spiking levels ranged between 74–105%. The efficiency of the method in preventing artifactual formation was verified by carrying out a few analyses with added marker amines such as ethylbutylamine, morpholine, and deuterium labelled dimethylamine. Several samples of contaminated water and fruit drinks contained 0.09–5.5 ng/g levels of NDMA, but those procured from retail outlets or natural sources were negative.

Absence of volatile N-nitrosamines in cooked nitrite-free cured muscle foods

Nitrite-free cured pork systems were prepared using the preformed cooked cured-meat pigment (CCMP) and sodium ascorbate. Absence of volatile N-nitrosamines in cooked nitrite-free systems was confirmed using a gas chromatography-thermal energy analyzer (GC-TEA) methodology. Similar results were obtained when cod, cod surimi or mixtures containing pork with 15 or 50% cod or cod surimi were used. However, counterpart samples cured with sodium nitrite (156 ppm) and sodium ascorbate (550 ppm) produced N-nitrosodimethylamine at 1·0 ppb levels or less. Results demonstrate that nitrite-free curing of meat and meat/fish systems containing the preformed CCMP is successful in yielding products devoid of volatile N-nitrosamines.