Determination of iron in blood serum using flow injection with luminol chemiluminescence detection.

A simple and rapid fl ow injection method is reported for the determination of iron in blood serum after acid digestion with HNO3 and HClO4, based on luminol CL detection in the absence of added oxidant. The detection limit (3 s) was 1.0 nmol/L with a sample throughput of 120/h. The calibration graph was linear over the range 0.001-1.0 micromol/L (r2 = 0.9974), with relative standard deviations (RSD) (n = 4) in the range 3.2-5%. The effect of interfering cations (Ca(II), Mg(II), Cu(II), Cd(II), Pb(II), Mn(II), Zn(II), Ni(II), Co(II) and Fe(III)) and anions (Cl-, SO4(2-), HCO3-, NO3-, NO2-) were studied using a luminol CL system for Fe(II) determination. The method was applied to normal blood serum and the results (1.32 +/- 0.08-1.74 +/- 0.05 mg/L) were compared with those from a spectrophotometric reference method (1.34 +/- 0.06-1.80 +/- 0.10 mg/L), which agree fairly well with the overall reference range in blood.     

Flow injection chemiluminescence determination of dihydralazine sulphate based on permanganate oxidation sensitized by rhodamine B.

A novel flow injection chemiluminescence (CL) method for the determination of dihydralazine sulphate (DHZS) is described. The method is based on the CL produced during the oxidation of DHZS by acidic permanganate solution in the presence of rhodamine B. Rhodamine B is suggested as a fluorescing compound for the energy-transferred excitation. The CL emission allows quantitation of DHZS concentration in the range 5-800 ng/mL, with a detection limit of 1.9 ng/mL (3sigma). The experimental conditions for the CL reaction are optimized and the possible reaction mechanism is discussed. The method has been applied to the determination of DHZS in pharmaceutical preparations and compares well with the high performance liquid chromatography (HPLC) method.     

Potential of the luminol reaction in the sensitive detection of pesticide residues by flow injection analysis.

This study presents the first analytical application of the luminol chemiluminescence (CL) reaction for the sensitive detection of carbamate residues. Some experiments have been carried out to check the influence of the presence of traces of a N-methylcarbamate (carbaryl) on the CL emission produced from the oxidation of luminol using different oxidants, showing a significant enhancing effect on the CL emission when the oxidation of luminol is produced by potassium permanganate in alkaline medium, this enhancement being proportional to the carbaryl concentration. This fact has permitted the establishment of a sensitive chemiluminescence flow-injection (CL-FIA) method for the direct determination of carbaryl. The optimization of instrumental and chemical variables influencing the CL response has been carried out by applying experimental designs. Under the optimal conditions, the CL intensity was linear for a carbaryl concentration over the range 5-100 ng/mL with a detection limit of 4.9 ng/mL. This luminol-KMnO4-based FIA-CL system in basic medium shows an easy, fast and cheap alternative detection mode for the analysis of carbaryl residues in environmental water samples.     

Hydrazine-induced post-chemiluminescence phenomenon of permanganate-luminol reaction and its applications.

The post-chemiluminescence phenomenon arising from the permanganate-luminol reaction induced by hydrazine and isoniazid was investigated. When hydrazine or isoniazid was injected into the mixture after the end of the reaction of permanganate with alkaline luminol, a new chemiluminescence (CL) reaction was initiated and strong CL signal was detected. A possible CL mechanism is suggested, based upon the studies of the kinetic characteristics of the CL reaction, the UV-visible spectra, the CL spectra and some other experiments. The present reactions allow the determination of 0.1-10.0 mg/L hydrazine and 0.02-1.0 mg/L isoniazid, with detection limits of 0.03 mg/L and 0.006 mg/L, respectively. The method was applied to the determination of isoniazid in pharmaceutical preparations.     

Chemiluminescence of the peroxomonosulphate-cobalt(II)-aliphatic monocarboxylic acids system.

A weak chemiluminescence (CL) emission was observed due to the production of singlet oxygen ((1)O(2)) during the decomposition of peroxomonosulphate (HSO(5)(-)) catalysed by cobalt(II). Low molecular mass aliphatic monocarboxylic acids, such as formic, acetic, propionic, butyric and valeric acids, influenced the CL emission, and the reaction of aliphatic monocarboxylic acids with HSO(5)(-)/Co(2+) solution was further investigated using a flow injection analysis (FIA) CL method. The results indicated that the CL intensities of aliphatic monocarboxylic acids were improved with increase in the carbon chain length in the potassium peroxomonosulphate-cobalt(II) sulphate system. Generation of singlet oxygen was confirmed by the fact that the CL emission of aliphatic monocarboxylic acids with the HSO(5)(-)/Co(2+) solution was quenched by NaN(3), and from the CL spectrum of the reaction system. Additionally, a possible mechanism of aliphatic monocarboxylic acids CL emission enhancement was proposed.     

Enhancement of chemiluminescence of the KIO4–luminol system by gallic acid,acetaldehyde and Mn2+: application for the determination of catecholamines

Chemiluminescence (CL) from the oxidation of luminol with potassium periodate in strong alkaline solutions was greatly enhanced by the combined effect of gallic acid, acetaldehyde and Mn²⁺. The CL spectra exhibited only one emission band at 425 nm, indicating 3‐aminophthalate as the emitting species. Various scavengers for superoxide anion, hydroxyl radical and singlet oxygen quenched the CL emission very efficiently (74–100%), suggesting the possible involvement of these reactive oxygen species (ROS) in the CL reactions. It is postulated that oxidation of gallic acid and acetaldehyde by periodate catalyzed by Mn²⁺ generates these ROS, which then react with luminol to enhance the CL emission. We also found that the enhanced CL emission was strongly inhibited by catecholamines, probably because of their effective scavenging of ROS. Based on this observation, a simple, rapid and sensitive new CL method was developed for the determination of catecholamines. The detection limits (3σ) for dopamine, l‐ dopa, norepinephrine and epinephrine were 0.63, 1.37, 0.56 and 14.3 nmol/L, respectively. The linear range was 1–10 nmol/L; relative standard deviations were 0.71–1.34% for 0.1 µmol/mL catecholamines. This CL method was applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Scavenger effect of flavonols on HOCl-induced luminol chemiluminescence.

Hypochlorous acid (HOCl), the main product of the myeloperoxidase system, is a strong oxidant and a potent chlorinating agent, which can damage host tissues. In the present work, the scavenger effect of three aglycone flavonols (myricetin, quercetin and kaempferol) and of the natural glycoside flavonol, rutin, was studied towards HOCl using luminol-dependent chemiluminescence (CL). At 1 micro mol/L fi nal concentration, rutin was the most powerful scavenger of HOCl with an inhibitory luminol oxidation of 91.4% +/- 3.2%. Quercetin, kaempferol and myricetin inhibited the luminol-dependent CL at the same concentration only by 75.9% +/- 3.4%, 57.7% +/- 5.3% and 43.3% +/- 3.5%, respectively. With increasing concentration of these flavonols, a dose-dependent inhibition of luminol CL was observed. In order to prove to what extent flavonols scavenge HOCl, their concentrations that gave 50% inhibition of luminescence (IC50) were compared to IC50 values of the sulphur-containing compounds N-acetyl cysteine (NAC) and taurine. The scavenging activities of compounds tested decrease in the order: rutin > NAC > quercetin > kaempferol > taurine. The present study revealed that rutin was the most effective scavenger agent.     

Evidence for a super-reduced cobamide as the major corrinoid fraction in vivo and a histidine residue as a cobalt ligand of the p-cresolyl cobamide in the acetogenic bacterium Sporomusa ovata

The redox state of cobalt in p-cresolyl cobamide and one of its axial ligands were determined by EPR spectroscopy of Sporomusa ovata as harvested. The analyses revealed that less than 2% (less than 30 nmol/g dry cells) of the total corrinoids (greater than 2400 nmol/g dry cells) were in a low-spin Co(II) complex. The amount increased to about 15% (190-450 nmol/g dry cells) upon partial oxidation by air, indicating that the original valence state of cobalt was a Co(I) prior to this treatment. The cob(I)amide was quantified as Co(III)-CH3 after methylation by iodomethane. More than 45% (1100 nmol/g dry cells) of the extractable corrinoids were in the methylated form, whereas non-treated cells revealed less than 1% (less than 15 nmol g dry cells) of light-sensitive corrinoids. EPR spectra of the Co(II) complex exhibited a threefold N-hyperfine splitting in the gz region, which was similar to vitamin B12. Cells grown with [1.3-15N2]histidine showed a twofold N-hyperfine splitting, demonstrating that the axial N ligand of the corrinoid was derived from the imidazole group of histidine. It is concluded that the super-nucleophilic p-cresolyl cob(I)amide is the major corrinoid complex in vivo and that it is stabilized by its protein(s). The Co(II) ion of the prosthetic group was coordinated by one histidine residue of the apoprotein(s).     

Eco-friendly nonconjugated polymer dots for chemiluminometric determination of 4-nitrophenol

Polymer dots (PDs) are a new family of quantum dots for which their behavior and potential applications have not yet been completely explored. In this study, nonconjugated PDs were synthesized using a simple pyrolysis method and used for the chemiluminescence (CL) assay of 4-nitrophenol (4-NP). PDs increased the CL signal of the Ce(IV)–Na₂SO₃ reaction 39-fold. Using the CL spectrum, it was concluded that the emission at 434 nm was generated by excited PDs (PDs*), which are produced by energy transfer from SO₂* to PDs. Our experiments showed that 4-NP enhanced the CL signal of the Ce(IV)–Na₂SO₃–PDs reaction. The mechanism of this effect was explored by obtaining CL, ultraviolet–visible, and Fourier transform infrared spectra. Due to the high sensitivity and selectivity of the CL system for 4-NP, a probe was designed to determine 4-NP in the linear range 1.0–500 nmol/L with a detection limit of 0.33 nmol/L. Different spiked real samples were successfully analyzed using this probe.     

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.     

Synthesis and in vitro and in vivo evaluation of [11C]methyl-BIII277CL for imaging the PCP-binding site of the NMDA receptor by pet

A new benzomorphane derivative, [11C]methyl-BIII277CL, was evaluated as a potential radiotracer for visualizing the PCP-binding site of the N-methyl-D-aspartate (NMDA) receptor by positron emission tomography (PET). Methyl-BIII277CL was prepared by reacting the desmethyl compound (BIII277CL) with dimethylsulfate. The pharmacological profile of methyl-BIII277CL was determined by in vitro receptor-screening assays. At a concentration of 100 nM, methyl-BIII277CL showed a significant interaction with the PCP-binding site of the NMDA receptor (79% inhibition of specific binding) and the sigma-binding site (46% inhibition). In displacement assays using mice cortical membranes, methyl-BIII277CL displayed a high affinity at the PCP-binding site of the NMDA receptor (Ki = 49 +/- 14 nmol/L) and a 130-fold lower interaction with the sigma1-binding site (Ki = 6.35 +/- 0.26 micromol/L). For saturation experiments and in vivo studies, methyl-BIII277CL was radiolabeled with 11C at the O-position of the desmethyl precursor (BIII277CL) using [11C]methyliodide with a specific activity of 35-70 GBq/micromol at the end of synthesis (EOS). In saturation assays using rat whole brain membranes [11C]methyl-BIII277CL showed a Kd of 6 +/- 1 nmol/L and a Bmax of 670 +/- 154 fmol/mg protein. Biodistribution and PET studies in rats and pigs, however, indicated a lack of specific binding and unfavorable pharmacokinetics. Kinetic modeling using the 1-tissue compartment model demonstrated for [11C]methyl-BIII277CL a low distribution volume (Dv = 0.98 mL/mL(tissue)) and very high values for the kinetic parameters K1 and k2 (K1 = 0.36 mL/mL(tissue)/min and k2 = 0.37min(-1)) in pig cortex. [11C]methyl-BIII277CL, due to the lack of specificity in vivo, may not be a candidate for imaging the PCP-binding site of the NMDA receptor.     

Flow injection chemiluminescent determination of tetracycline using a tris(2,2'-bipyridine)ruthenium(II)-cerium(IV) sulphate system.

A flow-injection chemiluminescence method for the determination of tetracycline was developed. The method is based on an enhancement by tetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine)ruthenium(II). In sulphuric acid medium, the chemiluminescence is generated by the continuous oxidation of tris(2,2'-bipyridine)ruthenium(II) by cerium (IV) sulphate. The light-emission intensity is greatly enhanced in the presence of tetracycline. Under the optimum conditions, the calibration curve is linear over the range 3.75 x 10(-8) g/mL-1.5 x 10(-5) g/mL for tetracycline with the linear equation: deltaINT = 205.898 x C - 20.442 (R2 = 0.9974). The detection limit is 3.27 x 10(-8) g/mL. The proposed method was also successfully used to determine tetracycline in pharmaceutical formulation (mean recovery of tetracycline, 100.7%).     

Cobalt (II) imprinted chitosan for selective removal of cobalt during nuclear reactor decontamination

The selectivity of chitosan has been modified through metal ion imprinting technique for its potential application in nuclear industry. Considerable reduction in radioactive waste volume, generated during the chemical decontamination of nuclear power plants, can be achieved through the selective removal of the radionuclides. In this context, a Co(II) imprinted chitosan was synthesized using epichlorohydrin as the crosslinker. The selective removal of Co(II) in presence of Fe(II), which is the major non-radioactive ion present in excess during decontamination, was studied. The imprinted chitosan showed selective sorption of Co(II) over Fe(II), while the raw chitosan was selective to Fe(II) over Co(II). The imprinted chitosan was found to retain the enhanced selectivity towards Co(II) under various solution conditions, including typical nuclear reactor decontamination formulations containing strong complexants. The highest uptake by the imprinted chitosan, with maximum selectivity for Co(II) over Fe(II), was obtained in citrate medium at pH 4.8.     

Chemiluminescence accompanied by the reaction of acridinium ester and manganese (II)

An acridinium ester (AE) alkaline solution can react with Mn(II) to generate a strong chemiluminescence (CL) centered at 435 nm. The effects of reaction conditions such as pH and Mn(II) concentration on CL intensity were examined. In order to explore the CL mechanism, the effect of oxygen on the CL reaction was examined and an X‐ray photoelectron spectroscopy study of the reaction precipitate was carried out. The results indicated that oxygen participated in the CL reaction and Mn(IV) was the primary product in the system. A possible mechanism was proposed that involved two pathways: (1) dissolved oxygen was reduced to reactive oxygen radicals by Mn(II), these reactive intermediates then reacted with AE to produce excited state acridone; (2) Mn(II) could reduce AE to partly reduced AE, which then reacted with oxygen to form excited state acridone. The reactions of other metal ions with AE were also tested, and only Mn(II) was shown to trigger strong CL emission of AE, which indicated that the system had good selectivity for Mn(II). Copyright © 2014 John Wiley & Sons, Ltd.     

Spectral and redox studies on mixed ligand complexes of cobalt(III) phenanthroline/bipyridyl and benzoylhydrazones, their DNA binding and antimicrobial activity

Cobalt(III) complexes of the type [Co(N-N)2L](ClO4)2.H2O [where L=anionic form of para-substituted benzaldehyde-benzoylhydrazone (BHBX-); X=H, Me, OMe, OH, Cl or NO2; N-N=2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen)] have been synthesized and characterized through UV-Vis, IR, NMR and electrochemical studies. The IR spectral frequencies support the mode of coordination of BHBX to the metal through the imino nitrogen and enolic oxygen atoms. The electronic absorption spectra exhibit metal to ligand charge transfer (MLCT) transition around 450 nm together with intraligand (IL) bands that are comparable to that of [Co(phen/bpy)3]3+. In acetonitrile solution these complexes show two well defined redox couples corresponding to Co(III/II) and Co(II/I) processes. Binding of these complexes with herring sperm DNA have been investigated by spectroscopic and voltammetric methods. The lower binding constant values of these complexes with respect to the [Co(phen/bpy)3]3+ are ascribed to the polar interaction of the substituted benzoylhydrazone moiety with the sugar-phosphate backbone of the DNA. The UV spectrum shows reasonable hypochromism with slight (2-4 nm) red shift, while the cyclic voltammogram shows decrease in current intensity along with a very small shift in the formal potential of the Co(III/II) redox couple. These experimental results indicate that phen mixed ligand complexes bind to DNA through an intercalative mode more effectively than their bpy counterparts. These complexes are also found to have good antimicrobial activity.     

Biosorption of cobalt by fungi from serpentine soil of Andaman

Fungi belonging to Aspergillus, Mortierella, Paecilomyces, Penicillium, Pythium, Rhizopus and Trichoderma, isolated from serpentine soil of Andaman (India) were screened for cobalt-resistance. Eleven out of total 38 isolated fungi which tolerated > 6.0 mM Co(II) were evaluated for cobalt biosorption using dried mycelial biomass. Maximum Co(II)-loading (1036.5 microM/g, 60 min) was achieved with Mortierella SPS 403 biomass, which removed almost 50% of 4.0 mM cobalt from the aqueous solution. Co(II)-sorption kinetics of Mortierella SPS 403 biomass was fast and appreciable quantities of metal [562.5 microM/g] was adsorbed during first 10 min of incubation. The metal biosorption capacity of the isolate was accelerated with increasing cobalt concentration, while it was reverse with increase of initial biomass. The optimum pH and temperature for Co(II) removal were 7.0 and 30 degrees C, respectively. However, Co(II)-uptake was inhibited in presence of other metals (Pb, Cd, Cu, Ni, Cr and Zn). Freundlich adsorption isotherm appropriately describes Mortierella SPS 403 biomass as an efficient Co(II)-biosorbent.     

Oxidation and chemiluminescence of catechol by hydrogen peroxide in the presence of Co(II) ions and CTAB micelles.

The oxidation of catechol in neutral and slightly alkaline aqueous solutions (pH 7-9.6) by excess hydrogen peroxide (0.002-0.09 mol/L) in the presence of Co(II) (2.10(-7)-2.10(-5) mol/L) is accompanied by abrupt formation of red purple colouration, which is subsequently decolourized within 1 h. The electron spectra of the reaction mixture are characterized by a broad band covering the whole visible range (400-700 nm), with maximum at 485 nm. The reaction is initiated by catechol oxidation to its semiquinone radical and further to 1,2-benzoquinone. By nucleophilic addition of hydrogen peroxide into the p-position of benzoquinone C=O groups, hydroperoxide intermediates are formed, which decompose to hydroxylated 1,4-benzoquinones. It was confirmed by MS spectroscopy that monohydroxy-, dihydroxy- and tetrahydroxy-1,4-benzoquinone are formed as intermediate products. As final products of catechol decomposition, muconic acid, its hydroxy- and dihydroxy-derivatives and crotonic acid were identified. In the micellar environment of hexadecyltrimethylammonium bromide the decomposition rate of catechol is three times faster, due to micellar catalysis, and is accompanied by chemiluminescence (CL) emission, with maxima at 500 and 640 nm and a quantum yield of 1 x 10(-4). The CL of catechol can be further sensitized by a factor of 8 (maximum) with the aid of intramicellar energy transfer to fluorescein.     

Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.     

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.     

Determination of nitrate and nitrite in freshwaters using flow‐injection with luminol chemiluminescence detection

A simple and sensitive flow‐injection (FI) method for the determination of nitrate and nitrite in natural waters, based on luminol chemiluminescence (CL) detection, is reported. Nitrate was reduced online to nitrite via a copperized cadmium (Cu–Cd) column and then reacted with acidic hydrogen peroxide to form peroxynitrous acid. CL emission was observed from the oxidation of luminol in an alkaline medium in the presence of the peroxynitrite anion. The limits of detection (S:N = 3) were 0.02 and 0.01 µg N/L, with sample throughputs of 40 and 90 /h for nitrate and nitrite, respectively. Calibration graphs were linear over the range 0.02–50 and 0.01–50 µg N/L [R² = 0.9984 (n = 8) and R² = 0.9965 (n = 7)] for nitrate and nitrite, respectively, with relative standard deviations (RSDs; n = 3) in the range 1.8–4.6%. The key chemical and physical variables (reagent concentrations, buffer pH, flow rates, sample volume, Cu–Cd reductor column length) were optimized and potential interferences investigated. The effect of cations [Ca(II), Mg(II), Co(II), Fe(II) and Cu(II)] was masked online with EDTA. Common anions (PO₄^3?, SO₄^2? and HCO₃^?) did not interfere at their maximum admissible concentrations in freshwaters. The effect of salinity on the luminol CL reaction with and without nitrate and nitrite (2 and 0.5 µg N/L, respectively) was also investigated. The method was successfully applied to freshwaters and the results obtained were in good agreement with those obtained by an automated segmented flow analyser reference method. Copyright © 2011 John Wiley & Sons, Ltd.