Electroanalytical overview: the sensing of hydroxylamine.

One of the principal raw ingredients used in the manufacturing of pharmaceuticals, nuclear fuel, and semiconductors is hydroxylamine, a mutagenic and carcinogenic substance, ranking high on the list of environmental contaminants. Electrochemical methods for monitoring hydroxylamine have the advantage of being portable, quick, affordable, simple, sensitive, and selective enough to maintain adequate constraints in contrast with conventional yet laboratory based quantification methods. This review outlines the most recent advancements in electroanalysis directed toward the sensing of hydroxylamine. Potential future advancements in this field are also offered, along with a discussion of method validation and the use of such devices in real samples for the determination of hydroxylamine.

Degradation of chloramphenicol by α-FeOOH-activated two different double-oxidant systems with hydroxylamine assistance.

The pipe deposits from water distribution network are iron-wastes, which could be used as a catalyst of advanced oxidation processes (AOPs). This paper prepared one main composition (α-FeOOH) of pipe deposits and compared the difference of chloramphenicol (CAP) degradation by α-FeOOH-activated hydrogen peroxide/persulfate and α-FeOOH-activated hydrogen peroxide/peroxymonosulfate with hydroxylamine assistance. Several key affecting factors were investigated. The results revealed that the double-oxidant system has a synergy effect in CAP degradation process. The hydroxyl radicals were identified as the predominant radicals in two different degradation processes via electron paramagnetic resonance (EPR) technique. The possible degradation pathways and products were confirmed by liquid chromatography-mass spectrometry (LC-MS). This study provided a theoretic research for pollutant removal by taking full advantage of pipe deposits and advance the development of water quality security in water distribution network in future.

Nitrite accumulation in comammox-dominated nitrification-denitrification reactors: Effects of DO concentration and hydroxylamine addition.

In this study, nitrite accumulation was investigated under different DO conditions and different hydroxylamine addition methods during the domestic wastewater treatment. Two sequencing batch reactors in parallel were operated under cyclic aerobic and anoxic conditions with the DO concentration of 2.0 and 4.0 mg/L in aerobic phase. The nitrite accumulation rate during high DO conditions increased to 44.8 and 66.7% in 20 days. During hydroxylamine addition, the NAR increased over 90% under the continuous and intermittent hydroxylamine addition. Continuous hydroxylamine addition could result in a more efficient and rapid nitrite accumulation. The findings suggested that comammox could be the main reason for the failure of partial nitrification in a low DO environment (< 0.5 mg/L). The nitrogen variation during typical cycles showed that the continuous hydroxylamine addition suppressed the activity of NOB and the ammonia oxidation rate. Further, the qPCR results indicated that the abundance of comammox amoA (ranged from 6.25 × 107 to 4.16 × 108 copies/g VSS) was higher than those of AOB amoA and Nitrobacter in sludge samples. The findings from the current study may enrich our understanding of partial nitrification and its control strategy.

Förster Resonance Energy Transfer-Based Fluorescent Probe for the Selective Imaging of Hydroxylamine in Living Cells.

Hydroxylamine (HA) is an important product of cell metabolism and plays a significant role in many biological processes, and therefore, real-time imaging of HA is of great importance for the in-depth study of its physiological and pathological functions. However, a HA-specific fluorescent probe is currently lacking primarily because the highly selective HA-responsive site is undeveloped. To address this critical issue, we present a HA-specific FRET-based fluorescent probe (RhChr) for the selective detection of HA in living systems. Inspired by aza-Michael addition, the unsaturated system appended with an iminium ion was employed as the new HA-specific response site. In response to HA, RhChr provided a ratiometric signal output with excellent selectivity toward HA over biothiols and ammonia. We have demonstrated that RhChr could be applied for the ratiometric imaging of endogenous HA in living cells and the evaluation of xanthine oxidase (XOD) activity in living organs.

A High-Performance Liquid Chromatography Method for Determination of Genotoxic Impurity Hydroxylamine in Drug Substances.

Hydroxylamine (NH2OH) is widely used in pharmaceutical intermediates and final drug substances synthesis. Since hydroxylamine is a well-known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical compounds. It is very difficult to detect using conventional analytical techniques due to its physical-chemical properties like the lack of chromophore, low molecular weight, the absence of carbon atom and high polarity. In addition to that, the analysis of the pharmaceutical samples encounters considerable obstruction from matrix components that greatly overshadow the response of hydroxylamine. This report describes a simple, selective and sensitive high-performance liquid chromatography (HPLC)-UV derivatization method for the determination of hydroxylamine in drug substances. The HPLC method was detected up to 0.01 ppm of hydroxylamine with S/N > 3.0 and quantified up to 0.03 ppm of hydroxylamine with S/N ratio > 10.0. This validated method can be applied as a generic method to detect the hydroxylamine for pharmaceutical process control and drug substance release.

Characteristics of N2O production and hydroxylamine variation in short-cut nitrification SBR process.

In order to study the characteristics of nitrous oxide (N2O) production and hydroxylamine (NH2OH) variation under oxic conditions, concentrations of NH2OH and N2O were simultaneously monitored in a short-cut nitrification sequencing batch reactor (SBR) operated with different influent ammonia concentrations. In the short-cut nitrification process, N2O production was increased with the increasing of ammonia concentration in influent. The maximum concentrations of dissolved N2O-N in the reactor were 0.11 mg/L and 0.52 mg/L when ammonia concentrations in the influent were 50 mg/L and 70 mg/L respectively. Under the low and medium ammonia load phases, the concentrations of NH2OH-N in the reactor were remained at a low level which fluctuated around 0.06 mg/L in a small range, and did not change with the variation of influent NH4+-N concentration. Based on the determination results, the half-saturation of NH2OH in the biochemical conversion process of NH2OH to NO2--N was very small, and the value of 0.05 mg NH2OH-N/L proposed in the published literature was accurate. NH2OH is an important intermediate in the nitrification process, and the direct determination of NH2OH in the nitrification process was beneficial for revealing the kinetic process of NH2OH production and consumption as well as the effects of NH2OH on N2O production in the nitrification process.

A Simple and Direct LC-MS Method for Determination of Genotoxic Impurity Hydroxylamine in Pharmaceutical compounds.

Hydroxylamine is a known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical processes. It is difficult to detect using conventional analytical techniques due to its physio-chemical properties like lack of chromophore, low molecular weight, absence of carbon atom and high polarity. In addition to that, analysis of the pharmaceutical samples encounters considerable obstruction from matrix components that greatly overshadow the response of hydroxylamine. This study describes a simple, sensitive and direct Liquid Chromatographic-Mass Spectrometric method (LC-MS) for detection of hydroxylamine in pharmaceutical compounds. The LC-MS method was detected up to 0.008 ppm of hydroxylamine with S/N > 3.0 and quantified up to 0.025 ppm of hydroxylamine with S/N ratio >10.0. This validated method can be applied as a generic method to detect the hydroxylamine for pharmaceutical process control and drug substance release.

N2O micro-profiles in biofilm from a one-stage autotrophic nitrogen removal system by microelectrode.

Emission of nitrous oxide (N2O), a greenhouse gas, is of growing concern in biological wastewater treatment. N2O emission from biofilm in a one-stage completely autotrophic nitrogen removal system was investigated using microelectrodes in this study. It is indicated that the pathways of nitrogen transformation in biofilm mainly included partial nitrification and anaerobic ammonium oxidation (anammox), also included nitrification and heterotrophic denitrification (HD). Ammonium-oxidizing bacteria (AOB) denitrification and HD were the main pathways resulting in N2O production in the biofilm, and hydroxylamine (NH2OH) oxidation was a subordinate pathway. In addition, the amount of N2O emission in test in which both NH4+ and NO2- were added (NH4+-N: NO2--N = 1:1) was about 2 times greater than that in test with NH4+ addition only. This result expressed that NO2- is an important factor affecting N2O production in the biofilm. In conclusion, the present study provides a theoretical support for reducing N2O production in one-stage completely autotrophic nitrogen removal system.

Iron and Carbon Dynamics during Aging and Reductive Transformation of Biogenic Ferrihydrite.

Natural organic matter is often associated with Fe(III) oxyhydroxides, and may be stabilized as a result of coprecipitation or sorption to their surfaces. However, the significance of this association in relation to Fe and C dynamics and biogeochemical cycling, and the mechanisms responsible for organic matter stabilization as a result of interaction with minerals under various environmental conditions (e.g., pH, Eh, etc.) are not entirely understood. The preservation of mineral-bound OM may be affected by OM structure and mineral identity, and bond types between OM and minerals may be central to influencing the stability, transformation and composition of both organic and mineral components under changing environmental conditions. Here we use bulk and submicron-scale spectroscopic synchrotron methods to examine the in situ transformation of OM-bearing, biogenic ferrihydrite stalks (Gallionella ferruginea-like), which formed following injection of oxygenated groundwater into a saturated alluvial aquifer at the Rifle, CO field site. A progression from oxidizing to reducing conditions during an eight-month period triggered the aging and reductive transformation of Gallionella-like ferrihydrite stalks to Fe (hydroxy)carbonates and Fe sulfides, as well as alteration of the composition and amount of OM. Spectromicroscopic measurements showed a gradual decrease in reduced carbon forms (aromatic/alkene, aliphatic C), a relative increase in amide/carboxyl functional groups and a significant increase in carbonate in the stalk structures, and the appearance of organic globules not associated with stalk structures. Biogenic stalks lost ∼30% of their initial organic carbon content. Conversely, a significant increase in bulk organic matter accompanied these transformations. The character of bulk OM changed in parallel with mineralogical transformations, showing an increase in aliphatic, aromatic and amide functional groups. These changes likely occurred as a result of an increase in microbial activity, or biomass production under anoxic conditions. By the end of this experiment, a substantial fraction of organic matter remained in identifiable Fe containing stalks, but carbon was also present in additional pools, for example, organic matter globules and iron carbonate minerals.

Simultaneous determination of hydroxylamine and phenol using a nanostructure-based electrochemical sensor.

The electrochemical oxidation of hydroxylamine on the surface of a carbon paste electrode modified with carbon nanotubes and 2,7-bis(ferrocenyl ethyl)fluoren-9-one is studied. The electrochemical response characteristics of the modified electrode toward hydroxylamine and phenol were investigated. The results showed an efficient catalytic activity of the electrode for the electro-oxidation of hydroxylamine, which leads to lowering its overpotential. The modified electrode exhibits an efficient electron-mediating behavior together with well-separated oxidation peaks for hydroxylamine and phenol. Also, the modified electrode was used for determination of hydroxylamine and phenol in some real samples.

A genetically encoded aldehyde for rapid protein labelling.

Using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling.

Discriminative EPR detection of NO and HNO by encapsulated nitronyl nitroxides.

Nitric oxide, •NO, is one of the most important molecules in the biochemistry of living organisms. By contrast, nitroxyl, NO-, one-electron reduced analog of •NO which exists at physiological conditions in its protonated form, HNO, has been relatively overlooked. Recent data show that HNO might be produced endogenously and display unique biological effects. However, there is a lack of specific and quantitative methods of detection of endogenous HNO production. Here we present a new method for discriminative •NO and HNO detection by nitronyl nitroxides (NNs) using electron paramagnetic resonance (EPR). It was found that NNs react with •NO and HNO with similar rate constants of about 10(4) M(-1) s(-1) but yield different products: imino nitroxides and the hydroxylamine of imino nitroxides, correspondingly. An EPR approach for discriminative •NO and HNO detection using liposome-encapsulated NNs was developed. The membrane barrier of liposomes protects NNs against reduction in biological systems while is permeable to both analytes, •NO and HNO. The sensitivity of this approach for the detection of the rates of •NO/HNO generation is about 1 nM/s. The application of encapsulated NNs for real-time discriminative •NO/HNO detection might become a valuable tool in nitric oxide-related studies.

Development and characterization of a novel semiautomated arrangement for electrochemically assisted injection in combination with capillary electrophoresis time-of-flight mass spectrometry.

Electrochemically assisted injection (EAI) is an attractive injection concept for CE that enables the separation of neutral analytes via electrochemical generation of charged species during the injection process. A new semiautomated EAI configuration was developed and applied in conjunction with CE-MS (EAI-CE-MS). The EAI cell arrangement consists of an integrated buffer reservoir for CE separations and a compartment holding screen-printed electrodes. A drop of sample solution (50 µL) was sufficient to cover the three-electrode structures. A piezo motor provided a fast and precise capillary positioning over the screen-printed electrode assembly. Using ferrocene methanol as a model system, the EAI arrangement was characterized regarding coulometric efficiency, precision, and sensitivity of electrospray ionization-time-of-flight-MS. The formation of the cationic oxidation product of ferrocene methanol enhanced the sensitivity of CE-MS determination by two orders of magnitude and the electrochemically formed product showed a migration time corresponding to its individual electrophoretic mobility. Preliminary studies of EAI-CE-MS in the field of the analysis of nitroaromatic compounds were carried out. The formation of corresponding hydroxylamines and amines paved the way for selective and sensitive CE-MS determinations without the need of adding surfactants to the electrophoresis buffer.

Design and preparation of a nanoprobe for imaging inflammation sites.

To image inflammation sites, we developed a novel nanoparticle, hydroxylamine-containing nanoparticle (HANP), which emits an intense electron spin resonance (ESR)-signal triggered by enzymatic oxidation reaction and pH-sensitive self-disintegration. The nanoparticle was prepared from an amphiphilic block copolymer, poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-hydroxyl)aminomethylstyrene] (PEG-b-PMNT-H), which spontaneously forms a core-shell type polymeric micelle (particle diameter = ca. 50 nm) in aqueous media. Because the PMNT-H segment in the block copolymer possesses amino groups in each repeating unit, the particle can be disintegrated by protonation of the amino groups in an acidic pH environment such as inflammation sites, which is confined to the hydrophobic core of HANP. Mixing HANP with horseradish peroxidase (HRP)/H(2)O(2) mixture resulted in enzymatic oxidization of the hydroxylamines in the PEG-b-PMNT-H and converted the hydroxylamine to the stable nitroxide radical form in PEG-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT), which shows an intense ESR signal. It is interesting to note that the ESR signal increased at a greater rate under acidic conditions (pH 5.6) than that under neutral conditions (pH 7.4), although the enzymatic activity of HRP under neutral conditions is known to be much higher than that under acidic conditions. This indicates that enzymatic oxidation reaction was accelerated by synchronizing the disintegration of HANP under acidic conditions. On the basis of these results, HANP can be used as a high-performance ESR probe for imaging of inflammation sites.

Sensitive detection of hydroxylamine at a simple baicalin carbon nanotubes modified electrode.

A baicalin multi-wall carbon nanotubes (BaMWCNT) modified glassy carbon electrode (GCE) for the sensitive determination of hydroxylamine was described. The BaMWCNT/GCE with dramatic stability was firstly fabricated with a simple adsorption method. And it showed excellent catalytic activity toward the electrooxidation of hydroxylamine. The amperometric response at the BaMWCNT/GCE modified electrode increased linearly to hydroxylamine concentrations in the range of 0.5 µM to 0.4mM with a detection limit of 0.1 µM. The modified electrode was applied to detection hydroxylamine in the tap water, and the average recovery for the standards added was 96.0%.

Linking community profiles, gene expression and N-removal in anammox bioreactors treating municipal anaerobic digestion reject water.

Anaerobic ammonium oxidation (anammox) requires 60% less oxygen and no external organic carbon compared to conventional biological nitrogen removal (BNR). Nevertheless, full-scale installations of anammox are uncommon, primarily owing to the lack of well-established process monitoring and control strategies that result in stable anammox reactor performance. The overarching goal of this study was to develop and apply molecular biomarkers that link microbial community structure and activity to anammox process performance in a bioreactor fed with actual anaerobic digestion centrate from a full-scale operational wastewater treatment facility. Over long-term operation, Candidatus "Brocadia sp. 40" emerged as the dominant anammox population present in the reactor. There was good correspondence between reactor nitrogen removal performance and anammox bacterial concentrations. During the period of reactor operation, there was also a marked shift in biomass morphology from discrete cells to granular aggregates, which was paralleled by a shift also to more stable nitrogen removal and the succession and establishment of bacteria related to the Chlorobi/Bacteroidetes superfamily. Based on batch assays, hydrazine oxidoreductase (hzo) expression and concentrations of the 16S-23S rRNA intergenic spacer region (ISR) were good quantitative biomarkers of oxygen- and nitrite-mediated inhibition. When applied to a continuous anammox reactor, both molecular biomarkers show promise as monitoring tools for "predicting" reactor performance.

Highly sensitive determination of hydroxylamine using fused gold nanoparticles immobilized on sol-gel film modified gold electrode.

We are reporting the highly sensitive determination of hydroxylamine (HA) using 2-mercapto-4-methyl-5-thiazoleacetic acid (TAA) capped fused spherical gold nanoparticles (AuNPs) modified Au electrode. The fused TAA-AuNPs were immobilized on (3-mercaptopropyl)-trimethoxysilane (MPTS) sol-gel film, which was pre-assembled on Au electrode. The immobilization of fused TAA-AuNPs on MPTS sol-gel film was confirmed by UV-vis absorption spectroscopy and atomic force microscopy (AFM). The AFM image showed that the AuNPs retained the fused spherical morphology after immobilized on sol-gel film. The fused TAA-AuNPs on MPTS modified Au electrode were used for the determination of HA in phosphate buffer (PB) solution (pH=7.2). When compared to bare Au electrode, the fused AuNPs modified electrode not only shifted the oxidation potential of HA towards less positive potential but also enhanced its oxidation peak current. Further, the oxidation of HA was highly stable at fused AuNPs modified electrode. Using amperometric method, determination of 17.5 nM HA was achieved for the first time. Further, the current response of HA increases linearly while increasing its concentration from 17.5 nM to 22 mM and a detection limit was found to be 0.39 nM (S/N=3). The present modified electrode was also successfully used for the determination of 17.5 nM HA in the presence of 200-fold excess of common interferents such as urea, NO(2)(-), NH(4)(+), oxalate, Mn(2+), Na(+), K(+), Mg(2+), Ca(2+), Ba(2+) and Cu(2+). The practical application of the present modified electrode was demonstrated by measuring the concentration of HA in ground water samples.

Spectrophotometric method for the determination of chromium (VI) in water samples.

A simple and sensitive spectrophotometric method for the determination of chromium has been developed. The method is based on the diazotization of Dapsone in hydroxylamine hydrochloride medium and coupling with N-(1-Napthyl) Ethylene Diamine Dihydrochloride by electrophilic substitution to produce an intense pink azo-dye, which has absorption maximum at 540 nm. The Beer's law is obeyed from 0.02-1.0 microg mL(-1) and the molar absorptivity is 3.4854 L mol(-1) cm(-1). The Limits of quantification and Limit of detection of the proposed method are 0.0012 microg mL(-1) and 0.0039 microg mL(-1) respectively. The method has been successfully applied for the determination of chromium in water samples and the results were statistically evaluated with that of the reference method.

Detection and characterization of cyclic hydroxylamine adducts by mass spectrometry.

Two cyclic hydroxylamines (cHA) bearing pyrrolidine (CPH) and piperidine moieties (TMTH) were evaluated to trap hydroxyl, peptide and phospholipid free radicals using mass spectrometry for their detection. The cHA ionized as [M+H](+) ions, showing higher relative abundances when compared to the DMPO, probably due to higher ionization efficiency. In the presence of hydroxyl radicals, both cHA generated new ions that could be attributed to loss of (*)H and (*)CH(3), most likely deriving from decomposition reactions of the nitroxide spin adduct. Addition of cHA to Leucine-enkephalin and palmitoyl-lineloyl-glycerophosphatidylcholine free radicals promoted the formation of cHA biomolecule adducts, which were confirmed by MS/MS data. Results suggest that the cHA are not suitable for hydroxyl radical trapping but can be used for trapping biomolecule radicals, having the advantage, over the most used cyclic nitrones, of being water soluble. The biomolecule adducts identified by MS are ESR silent, evidencing the importance of MS detection.

Thioredoxin and lipoic acid catalyze the denitrosation of low molecular weight and protein S-nitrosothiols.

The nitrosation of cellular thiols has attracted much interest as a regulatory mechanism that mediates some of the pathophysiological effects of nitric oxide (NO). In cells, virtually all enzymes contain cysteine residues that can be subjected to S-nitrosation, whereby this process often acts as an activity switch. Nitrosation of biological thiols is believed to be mediated by N2O3, metal-nitrosyl complexes, and peroxynitrite. To date, however, enzymatic pathways for S-denitrosation of proteins have not been identified. Herein, we present experimental evidence that two ubiquitous cellular dithiols, thioredoxin and dihydrolipoic acid, catalyze the denitrosation of S-nitrosoglutathione, S-nitrosocaspase 3, S-nitrosoalbumin, and S-nitrosometallothionenin to their reduced state with concomitant generation of nitroxyl (HNO), the one-electron reduction product of NO. In these reactions, formation of NO and HNO was assessed by ESR spectrometry, potentiometric measurements, and quantification of hydroxylamine and sodium nitrite as end reaction products. Nitrosation and denitrosation of caspase 3 was correlated with its proteolytic activity. We also report that thioredoxin-deficient HeLa cells with mutated thioredoxin reductase denitrosate S-nitrosothiols less efficiently. We conclude that both thioredoxin and dihydrolipoic acid may be involved in the regulation of cellular S-nitrosothiols.