Insights into N2O turnovers under polyethylene terephthalate microplastics stress in mainstream biological nitrogen removal process.

The ubiquitous microplastics in wastewater have raised growing concerns due to their unintended effects on microbial activities. However, whether and how microplastics affect nitrous oxide (N2O) (a potent greenhouse gas) turnovers in mainstream biological nitrogen removal (BNR) process remain unclear. This work therefore aimed to fill such knowledge gap by conducting both long-term and batch tests. After over 100 days of feeding with wastewater containing polyethylene terephthalate (PET) microplastics (0-500 µg/L), the long-term results showed that both production and reduction of N2O during denitrification were reduced, as well as the N2O production during nitrification. Accordingly, 60% reduction in N2O accumulation and 70% reduction in N2O production were observed in the denitrification and nitrification batch tests, respectively. Nevertheless, the long-term N2O emission factors under PET microplastics stress were comparable to that in the control reactor, mainly because PET microplastics led to more nitrite accumulation in anoxic period. With the aid of online N2O sensors and site-preference analysis, it was demonstrated that the heterotrophic bacteria pathway and ammonia oxidizing bacteria denitrification pathway for N2O production were negatively affected by PET microplastics, whereas a clear increase in the contribution of hydroxylamine pathway (+ 22.9%) was observed. Further investigation revealed that PET microplastics even at environmental level (i.e. 10 µg/L) significantly reshaped the BNR sludge characteristics (e.g. much larger particle size) and microbial communities (e.g. Thauera, Rhodobacte and Nitrospira) as well as the nitrogen metabolism pathways, which were chiefly responsible for the changes of N2O turnovers and N2O production pathways under the PET microplastics stress.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 µg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-ß-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Highly sensitive analysis of low-molecular-mass aldehydes in beverages using a hydroxylamine reagent by high-performance liquid chromatography with fluorescence detection.

In this study, a fluorescent reagent, 4-((aminooxy)methyl)-7-hydroxycoumarin (AOHC), was for the first time applied to label the low-molecular-mass aldehydes (LMMAs) through reductive oxyamination reaction to afford single N,O-substituted oxyamine derivatives at room temperatures with derivatization efficiencies as high as 96.8%. In the following high-performance liquid chromatography with fluorescence detection analysis, 12 LMMAs, including furfurals, aromatic aldehydes, and aliphatic aldehydes, were baseline-separated on an ODS column and detected with low limits of detection (LODs) (0.2-50 nM), and good precisions (intraday relative standard deviations [RSDs] were 2.40-4.68%, and interday RSDs were 4.65-8.91%). This approach was then adopted to analyze six alcoholic beverages and five dairy products, and nine LMMAs with concentrations in the 0.28-798.16 µM range were successfully detected with excellent accuracies (recoveries were 92.2-106.2%). Finally, the results were statistically analyzed and discussed. The proposed method has several advantages, including high sensitivity, room-temperature labeling, and the avoidance of further extraction and/or enrichment procedures, demonstrating its great utility for monitoring LMMAs in various complex matrices.

Assessing the mutagens ethylnitrolic acid and 2-methyl-1,4-dinitro-pyrrole in meat products: Sample preparation and simultaneous analysis by LC-MS/MS.

The simultaneous use of nitrite and sorbate as preservatives in meat products may produce mutagenic compounds such as the ethylnitrolic acid and 2-methyl-1,4-dinitro-pyrrole. We developed a sensitive analytical method with high metrological reliability. After assessing several extraction approaches and chromatographic separation modes, a modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) approach was chosen for sample preparation, which were analyzed by reversed-phase liquid chromatography (with C18 as stationary phase) coupled to tandem mass spectrometry. After validation, we confirmed that this method is fit-for-purpose, since it was applied to the analysis of several meat products. Limits of detection were set from 5 to 20 µg kg-1. Satisfactory results were obtained for both compounds, such as precision (CV > 20%) and recoveries (77-92%). This method determine these carcinogenic compounds in processed meats, contributing to the preservation of public health and the improvement of food regulation and control.

Simultaneous analysis by LC-MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 µl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11ß-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11ß-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.

Analysis of Metabolites from the Tricarboxylic Acid Cycle for Yeast and Bacteria Samples Using Gas Chromatography Mass Spectrometry.

We here explain step by step the implementation of gas chromatography coupled with tandem mass spectrometry for the quantitative analysis of intracellular metabolites from the tricarboxylic acid (TCA) cycle such as citrate, isocitrate, alpha-ketoglutarate, succinate, malate, and fumarate. Isotope dilution is used to correct for potential metabolite losses during sample processing, matrix effects, incomplete derivatization, and liner contamination. All measurements are performed in selected reaction monitoring (SRM) mode. Standards and samples are first diluted with a fixed volume of a mixture of fully 13 C-labeled internal standards and then derivatized to give trimethylsilyl-methoxylamine derivatives prior GC-MS/MS analysis.

Sequential derivatization of polar organic compounds in cloud water using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, N,O-bis(trimethylsilyl)trifluoroacetamide, and gas-chromatography/mass spectrometry analysis.

Cloud water samples from Whiteface Mountain, NY were used to develop a combined sampling and gas chromatography-mass spectrometric (GCMS) protocol for evaluating the complex mixture of highly polar organic compounds (HPOC) present in this atmospheric medium. Specific HPOC of interest were mono- and di keto-acids which are thought to originate from photochemical reactions of volatile unsaturated hydrocarbons from biogenic and manmade emissions and be a major fraction of atmospheric carbon. To measure HPOC mixtures and the individual keto-acids in cloud water, samples first must be derivatized for clean elution and measurement, and second, have low overall background of the target species as validated by GCMS analysis of field and laboratory blanks. Here, we discuss a dual derivatization method with PFBHA and BSTFA which targets only organic compounds that contain functional groups reacting with both reagents. The method also reduced potential contamination by minimizing the amount of sample processing from the field through the GCMS analysis steps. Once derivatized only gas chromatographic separation and selected ion monitoring (SIM) are needed to identify and quantify the polar organic compounds of interest. Concentrations of the detected total keto-acids in individual cloud water samples ranged from 27.8 to 329.3ngmL(-1) (ppb). Method detection limits for the individual HPOC ranged from 0.17 to 4.99ngmL(-1) and the quantification limits for the compounds ranged from 0.57 to 16.64ngmL(-1). The keto-acids were compared to the total organic carbon (TOC) results for the cloud water samples with concentrations of 0.607-3.350mgL(-1) (ppm). GCMS analysis of all samples and blanks indicated good control of the entire collection and analysis steps. Selected ion monitoring by GCMS of target keto-acids was essential for screening the complex organic carbon mixtures present at low ppb levels in cloud water. It was critical for ensuring high levels of quality assurance and quality control and for the correct identification and quantification of key marker compounds.

Multilayer chitosan-based open tubular capillary anion exchange column with integrated monolithic capillary suppressor.

We describe a multilayered open tubular anion exchange column fabricated by alternately pumping solutions of chitosan and glutaraldehyde. The column is terminated in an integrally bonded monolithic suppressor cast around a mandrel of a tungsten wire, composed of an acrylic acid (AA)-ethylene dimethacrylate (EDMA) monolith that is made with sufficient porogen for the monolith to function as a membrane. For a 4.5m long 75 µm bore column coated with 24 successive layers of the condensation polymer (estimated to contain ~72 molecular layers) and coupled to 1cm length of a suppressor fabricated with 55-60% AA, effective separation of several common anions (F(-), Cl(-), NO(2)(-), Br(-), NO(3)(-), average number of theoretical plates ~12,000) and adequate suppression of 1 mM KOH used as eluent was observed at a flow rate of 800 nL min(-1) to obtain sub-picomol detection limits at an operating pressure of ~1 bar. The separation is not time efficient but the system can be meritorious in unique niche applications where a small form factor is desired and liquid volume and power consumption are more important than separation speed.

Chemical discrimination between dC and 5MedC via their hydroxylamine adducts.

The presence of the methylated nucleobase (5Me)dC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of (5Me)dC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and (5Me)dC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the (5Me)dC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and (5Me)dC with high accuracy, leading towards a novel and mild chemistry for methylation analysis.

Role of human cyclooxygenase-2 in the bioactivation of dapsone and sulfamethoxazole.

Sulfamethoxazole (SMX) and dapsone (4,4'-diaminodiphenylsulfone, DDS) are believed to mediate their adverse effects subsequent to bioactivation to their respective arylhydroxylamine and arylnitroso metabolites, resulting in covalent adduct formation with intracellular proteins. Various bioactivating enzymes, such as cytochromes P450 and myeloperoxidase, have been shown to be capable of catalyzing the N-oxidation of these compounds. We assessed the role of human cyclooxygenase-2 (COX-2) in the metabolism and subsequent adduct formation of DDS and SMX using recombinant human COX-2. Using an adduct-specific enzyme-linked immunosorbent assay, we found that the complete enzyme system gave rise to covalent adducts. However, the nonspecific COX inhibitor indomethacin did not reduce the amount of covalent adduct formed. Formation of the arylhydroxylamine metabolites was demonstrated via high performance liquid chromatography coupled with UV absorption. Metabolite formation was found to be secondary to the H2O2 in the incubation mixture and was not enzyme-mediated. Hence, COX-2 does not play a direct role in the bioactivation of these parent drugs to their arylhydroxylamine metabolites.

Gas-phase chemistry of (alpha-terpineol with ozone and OH radical: rate constants and products.

A bimolecular rate constant, kOH+alpha-terpineol, of (1.9 +/- 0.5) x 10(-10) cm3 molecule(-1) s(-1) was measured using gas chromatography/mass spectrometry and the relative rate technique for the reaction of the hydroxyl radical (OH) with alpha-terpineol (1-methyl-4-isopropyl-1-cyclohexen-8-ol) at (297 +/- 3) K and 1 atm total pressure. Additionally, a bimolecular rate constant, kO3+alpha-terpineol, of (3.0 +/- 0.2) x 10(-16) cm3 molecule(-1) s(-1) was measured by monitoring the first order decrease in ozone concentration as a function of excess alpha-terpineol. To better understand alpha-terpineol's gas-phase transformation in the indoor environment, the products of the alpha-terpineol + OH and alpha-terpineol + 03 reactions were also investigated. The positively identified alpha-terpineol/OH reaction products were acetone, ethanedial (glyoxal, HC(=O)C(=O)H), and 2-oxopropanal (methyl glyoxal, CH3C(=O)C(=O)H). The positively identified alpha-terpineol/O3 reaction product was 2-oxopropanal (methyl glyoxal, CH3C(=O)C(=O)H). The use of derivatizing agents O-(2,3,4,5,6-pentalfluorobenzyl)hydroxylamine (PFBHA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) clearly indicated that several other reaction products were formed. The elucidation of these other reaction products was facilitated by mass spectrometry of the derivatized reaction products coupled with plausible alpha-terpineol/OH and alpha-terpineol/O3 reaction mechanisms based on previously published volatile organic compound/ OH and volatile organic compound/O3 gas-phase reaction mechanisms.

Impact of ferrihydrite and anthraquinone-2,6-disulfonate on the reductive transformation of 2,4,6-trinitrotoluene by a gram-positive fermenting bacterium.

Batch studies were conducted to explore differences in the transformation pathways of 2,4,6-trinitrotoluene (TNT) reduction by a Gram-positive fermenting bacterium (Cellulomonas sp. strain ES6) in the presence and absence of ferrihydrite and the electron shuttle anthraquinone-2,6-disulfonate (AQDS). Strain ES6 was capable of TNT and ferrihydrite reduction with increased reduction rates in the presence of AQDS. Hydroxylaminodinitrotoluenes, 2,4-dihydroxylamino-6-nitrotoluene (2,4-DHANT), and tetranitroazoxytoluenes were the major metabolites observed in ferrihydrite- and AQDS-free systems in the presence of pure cell cultures. Ferrihydrite enhanced the production of amino derivatives because of reactions with microbially produced surface-associated Fe(ll). The presence of AQDS in the absence of ferrihydrite promoted the fast initial formation of arylhydroxylamines such as 2,4-DHANT. However, unlike in pure cell systems, these arylhydroxylamines were transformed into several unidentified polar products. When both microbially reduced ferrihydrite and AQDS were present simultaneously, the reduction of TNT was more rapid and complete via pathways thatwould have been difficult to infer solely from single component studies. This study demonstrates the complexity of TNT degradation patterns in model systems where the interactions among bacteria, Fe minerals, and organic matter have a pronounced effect on the degradation pathway of TNT.

Development and validation of an HPLC-UV method for the analysis of methoxyamine using 4-(diethylamino)benzaldehyde as a derivatizing agent.

Methoxyamine (MX) is a potential new anti-cancer drug. In this paper, a quantitative HPLC-UV method for MX using 4-(diethylamino)benzaldehyde (DEAB) as a derivatizing agent has been developed and validated. The studies showed that MX reacts with DEAB under acidic conditions to form protonated 4-(diethylamino)benzaldehyde o-methyloxime (DBMOH+). The equilibrium between DBMOH+ and its conjugate base 4-(diethylamino)benzaldehyde o-methyloxime (DBMO) is affected by both buffer concentration and organic solvent content in the solution. The method developed uses a reversed phase C18 column for the separation of MX derivatives, an internal standard benzil for method calibration, and a UV detector at a wavelength of 310 nm for analyte detection. The MX derivatives can be resolved in ca. 20 min. The method has a linear calibration range from 0.100 to 10.0 microM with a correlation coefficient of 0.999 for MX and a detection limit of 5 pmol with a 50 microl sample size. The intra-assay and inter-assay precision expressed in terms of percent relative standard deviation were < or =5 and 8%; and the intra-assay and inter-assay accuracy defined as the measured value divided by the accepted value multiplied by 100% were 94.2-100 and 92.6-111%, respectively. This method may be used for the analysis of MX in pharmaceutical preparations.

Rapid determination of C6-aldehydes in tomato plant emission by gas chromatography-mass spectrometry and solid-phase microextraction with on-fiber derivatization.

A simple, rapid, sensitive, and solvent-free method was developed for determination of plant-signalling compounds, the three C6-aldehydes hexanal, (Z)-3-hexenal, and (E)-2-hexenal, in tomato plant emission by gas chromatography-mass spectrometry (GC-MS) and solid-phase microextraction (SPME) with on-fiber derivatization. In this method, O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine (PFBHA) in aqueous solution was first headspace adsorbed onto a 65 microm poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber at 25 degrees C for 5 min, and then the fiber with adsorbed PFBHA was used for headspace extraction of tomato plant emission at 25 degrees C for 6 min. Finally, the resulting oximes adsorbed on the fiber were desorbed and analyzed by GC-MS. Extraction conditions and method validation were studied. The proposed method had low detection limit values for the three aldehydes from 0.1 to 0.5 ng/L and good precision (RSD less than 10%). In this work, the method was applied to investigation of tomato plant defense response to Helicoverpa armigera.

Determination of carbonyl compounds in pool water with O-(2,3,4,5,6-pentafluorobenzyl)hydroxyamine hydrochloride and gas chromatographic-tandem mass spectrometric analysis.

To avoid microbiological decay pool water is disinfected, a procedure which results into a lot of disinfection by-products, like carbonyl compounds, as well as a large number of others. The carbonyl compounds dissolved in pool water were derivatisized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxyamine hydrochloride (PFBHA) and extracted using n-hexane. Measuring with the help of GC-electron-capture detection is hardly possible because of interferents like halogenated organics. Another method to detect the PFBHA derivates is the use of tandem mass spectrometry. Calibration ranges and precision are applicable and sufficient to determine carbonyl compounds in pool water.

A new sensitive HPLC assay for methoxyamine and its analogs.

Methoxyamine (MOA) and its analogs are polymerization regulators, building blocks and intermediates for agrichemicals and pharmaceuticals. MOA induces mutagenesis of nucleic acids and has been considered for anti-cancer and anti-virus therapy. It has been studied as a DNA repair modifier in anti-cancer therapy. HPLC procedures available in the literature for MOA are all based on electrochemical detection, which is not commonly available. This paper describes the development and validation of a HPLC assay with UV detection for MOA and its analogs. The analytes are first reacted with o-phthalaldehyde to form an oxime derivative before chromatography with an ODS column. Detection is achieved by UV at 254 nm. The chromatography resolves MOA from its decomposition products and analogs. The assay is reproducible (R.S.D. < 0.8%), linear (r(2) = 0.9997), and accurate (error < 1%). The method is sensitive and has a lower detection limit of 5 pmol (0.4 ng of MOA.HCl), which is comparable to that of electrochemical detection.

Reactivity of partially reduced arylhydroxylamine and nitrosoarene metabolites of 2,4,6-trinitrotoluene (TNT) toward biomass and humic acids.

Sequential anaerobic/aerobic treatment of 2,4,6-trinitrotoluene (TNT) generally results in the incorporation of residues into biomass and natural organic matter fractions of a system. To better understand the potential contribution of hydroxylamine and nitroso moieties in these reactions, studies were conducted using model systems taking advantage of the biocatalytic-activity of Clostridium acetobutylicum that does not produce aminated TNT derivatives. To evaluate binding to biomass only, systems containing cell-free extracts of C. acetobutylicum and molecular hydrogen as a reductant were employed. At the end of treatment, mass balance studies showed that 10% of the total 14C was associated with an insoluble protein-containing precipitate that could not be extracted with organic solvents. Model reactions were conducted between a mixture of 2,4-dihydroxylamino-6-nitrotoluene (DHA6NT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) and 1-thioglycerol to test the involvement of the nitroso-thiol reaction in binding to biomass. It was demonstrated that DHA6NT formed a new and relatively polar product with 1-thioglycerol only in the presence of oxygen. The oxygen requirement confirmed that the nitroso functionality was responsible for the binding reaction. The reactivity of arylhydroxylamino and nitrosoarene functionalities toward International Humic Substance Society (IHSS) peat humic acid was evaluated under anaerobic and aerobic conditions, respectively. 4HADNT showed no appreciable reactivity toward peat humic acid. Conversely, the nitrosoarene compound, nitrosobenzene, showed rapid reactivity with peat humic acid (50% removal in 48 h). When tested with two other humic acids (selected on the basis of their protein content), it became apparent that the proteinaceous fraction was responsible at least in part for the nitrosoarene's removal from solution. Furthermore, the pretreatment of the humic acids with a selective thiol derivatizing agent had a considerable effect on their ability to react with nitrosobenzene. Finally, molecular modeling tools were used to compare the electrophilic characteristics of potential nitroso intermediates forming from the oxidation of arylhydroxylamino metabolites of TNT. Molecular modeling analysis demonstrated that the more reduced TNT derivative containing nitroso groups were more likely to react with nucleophiles in humic substances than the less reduced nitroso intermediates.

Spectrophotometric method for the determination of nifedipine with 4-(methylamino)phenol and potassium dichromate.

A new simple, sensitive and reproducible spectrophotometric method for the determination of nifedipine in pure and dosage forms has been proposed. It is based on the reduction of nifedipine with Zn/NNH4Cl, followed by coupling with N-methyl-1,4-benzoquinoneimine--the oxidation product of 4-(methylamino)phenol, to give a chromophore which absorbed maximally at 525 nm. The experimental conditions were optimised and Beer's law was obeyed over the concentration range of 5-175 microg ml(-1). The molar absorptivity, detection limit, recovery and RSD were found to be 1.9 x 10(3) l mol(-1) cm(-1), 1.1 microg ml(-1), 99.7-100.5% and 0.3-0.8%, respectively. The proposed method was compared favourably with the official B.P. method.

Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions.

A previous report that the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) allows DEPMPO radical cation formation to be detected via the production of a carbon-centred radical adduct (assigned as the cis-hydroxyethyl species, formed by an intramolecular process) is shown to be incorrect. Rather, this and other paramagnetic species arise from the facile oxidation of trace hydroxylamine impurities present in commercial DEPMPO samples. As a result, techniques for the detection and elimination of such hydroxylamine impurities from DEPMPO solutions were developed and are described; these should prove to be of general use in EPR spin trapping experiments.

[Biochemical diagnosis of pheochromocytoma and neuroblastomas]. / Diagnostic biochinique du phéochromocytome et du neuroblastome.

Pheochromocytoma and neuroblastoma are distinct tumours, but their biological diagnosis is based on secretion increase of one or several catecholamines. Assays have to be very sensible and specific for an early diagnosis. 24 hours urinary catecholamines and metabolites are currently measured, but technical improvements permit plasma metanephrine assay, an excellent indicator of pheochromocytoma. HPLC coupled to electrochemical detection represents the most efficient methodology. After a review of urinary and plasma assay methods, the authors show usual values of catecholamines, metanephrines, HVA and VMA, according to ages, and give examples of results encountered in classical or not tumours and in falsely positive cases. Urinary metanephrine assay is the most sensible and specific in biological diagnosis of pheochromocytoma, while catecholamines and VMA assays lack of sensibility. Results have to be given by 24 hours and by creatinine ratio. Metanephrine assay can be performed also in plasma and exhibits the same interest. However, in urine as in plasma, in case of renal failure, results cannot be interpreted. Neuroblastoma biological diagnosis is based classically on HVA, VMA, and dopamine assays, nowadays only in 24 hours urine (or in urinary micturition for screening), and results are also expressed as creatinine ratio. But even if several assays are advisable, 5% of the neuroblastoma cases do not produce increased catecholamine values. In some cases, metanephrine assay could be of interest. After the age of 12 months, clinical expression of neuroblastoma is dramatic in 70% of cases. So, a biological screening has been experimented in several countries including France. A French translation of the consensus conference report (1998) is appended, which shows the complexity of neuroblastoma screening. Now, there is no evidence that early tumour detection by screening lessens the mortality rate, but a weak benefit is not excluded.