Development of a non-competitive immunoassay for monitoring DDT, its metabolites and analogues in water samples

This report highlights the characteristics of a general method of performing non-competitive immunoassays for low-molecular-mass analytes, which was developed and applied to 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) determination in aqueous samples. The method is based on the separation of the analyte-bound antibody from the excess of the free antibody by a chromatographic step, followed by the dissociation of the complex and the capture of the previously bound antibody on a solid phase. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 8 ng l⁻¹) obtained by the above method enabled us to decidedly improve the sensitivity of the corresponding enzyme-linked immunosorbant assay (ELISA) and of all reported immunoassays for DDT.In addition, by applying this new format, even if a very selective antibody was used, a broad selectivity was observed, which allowed DDT + DDD + DDE to be determined instead of only p,p′-DDT as in the ELISA performed with the same antibody. In addition, real water samples were validated in a percentage recovery test. Very good recovery rates were obtained, highlighting the validity of the proposed method to accurately determine the total DDT content in water.     

Development of a voltammetric sensor for diospyrin determination in nanomolar concentrations

A sensor based on glassy carbon (GC) electrode modified with cobalt tetrasulfonated phthalocyanine (CoTSPc) and a poly-l-lysine (PLL) film is proposed for diospyrin determination in nanomolar concentrations with differential pulse voltammetry (DPV) technique. The modified electrode showed excellent catalytic activity presenting much higher peak currents than those measured on a bare GC electrode. Linear response range, sensitivity and limit of detection (LOD) were of 1-120 nmol l⁻¹, 220.46 nA l nmol⁻¹ cm⁻² and 0.3 nmol l⁻¹, respectively. The repeatability of the proposed sensor, evaluated in term of relative standard deviation (R.S.D.), was measured as 4.4% for 10 experiments in 50 μmol l⁻¹ diospyrin samples. The developed sensor was applied for the determination of diospyrin in the crude extracts of the stem-bark of Diospyros montana Roxb. and the average recovery for these samples was 101.9 (±3.1)%.     

Stacking and separation of urinary porphyrins in capillary electrophoresis: optimization of concentration efficiency and resolution

We demonstrated that anionic porphyrins could be stacked and separated in micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) by applying acetonitrile and high salt content in human urine sample matrix. The introduction of sample containing acetonitrile and sodium chloride into the CE capillary at more than 10% of the total capillary volume resulted in the improvement of peak resolution and the enhancement of detection sensitivity. The achieved acetonitrile stacking enrichment factors of six porphyrins ranged from 12 to 32 in MEKC and from 28 to 33 in MEEKC, respectively. The stacking technique was successfully applied for analyzing porphyrins present in urine samples that were deproteinized with acetonitrile. For the analysis of coproporphyrin isomers, addition of the sodium cholate (SC) into micelle and microemulsion solutions provided adequate resolution. Calibration curves obtained for the determination of coproporphyrin isomers were found linear between 30 and 400 nmol L⁻¹, and the limit of detection (LOD) was 20 nmol L⁻¹ in MEEKC. Intra- and interday precisions (n = 11) in the microemulsion separation system for the isomers at spiked concentrations of 40-400 nmol L⁻¹ in urine were in the range of 0.1-0.4% and 0.7-7.6% for migration time and peak area, respectively. Coproporphyrin III, coproporphyrin I and uroporphyrin were detected at levels of 80.7 nmol L⁻¹, 32.3 nmol L⁻¹ and 19.8 nmol L⁻¹, respectively, in the urine samples collected from healthy individuals. Different porphyrin profiles, however, were observed in urine samples from porphyria cutanea tarda (PCT) patients.     

Speciation and preconcentration of inorganic antimony in waters by Duolite GT-73 microcolumn and determination by segmented flow injection-hydride generation atomic absorption spectrometry (SFI-HGAAS)

A selective matrix removal/separation/enrichment method, utilizing a microcolumn of a chelating resin with SH functional groups (Duolite GT-73), was proposed for the determination of Sb(III) in waters by segmented flow injection-hydride generation atomic absorption spectrometry (SFI-HGAAS). The resin was selective to Sb(III) at almost all pH and acidity values employed, whereas Sb(V) was not retained at all and could be determined after a pre-reduction step with l-cysteine. Spike recoveries were tested at various concentration levels in different water types and were found to vary between 85 and 118%. Accuracy of the proposed methodology was checked by analyzing a standard reference material and a good correlation was found between the determined (13.3 ± 1.1 μg l⁻¹) and the certified value (13.79 ± 0.42 μg l⁻¹). The method was applied to several bottled drinking water samples for antimony determination with and without preconcentration and none of the samples were found to contain antimony above the permissible level (5 μg l⁻¹). The characteristic concentration (the concentration of the analyte corresponding to an absorbance of 0.0044) was 0.55 μg l⁻¹ and the 3 s limit of detection (LOD) based on five times preconcentration was 0.06 μg l⁻¹. The applicability of the microcolumn separation/preconcentration/matrix removal method for flow injection systems was also demonstrated.     

Voltammetric determination of 4-nitrophenol at a lithium tetracyanoethylenide (LiTCNE) modified glassy carbon electrode

The reduction of 4-nitrophenol (4-NP) has been carried out on a modified glassy carbon electrode using cyclic and differential pulse voltammetry (DPV). The sensor was prepared by modifying the electrode with lithium tetracyanoethylenide (LiTCNE) and poly-l-lysine (PLL) film. With this modified electrode 4-NP was reduced at −0.7 V versus SCE. The sensor presented better performance in 0.1 mol l⁻¹ acetate buffer at pH 4.0. The other experimental parameters, such as concentration of LiTCNE and PLL, pulse amplitude and scan rate were optimized. Under optimized operational conditions, a linear response range from 27 up to 23200 nmol l⁻¹ was obtained with a sensitivity of 3.057 nA l nmol⁻¹ cm⁻². The detection limit for 4-NP determination was 7.5 nmol l⁻¹. The proposed sensor presented good repeatability, evaluated in term of relative standard deviation (R.S.D.=4.4%) for n=10 and was applied for 4-NP determination in water samples. The average recovery for these samples was 103.0 (± 0.7)%.     

Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l⁻¹ sodium borate buffer (pH 9.35) containing 20 mmol l⁻¹sodium deoxycholate (SDC) and 10 mmol l⁻¹ Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966-0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l⁻¹, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.     

Electrochemical detection of phenolic estrogenic compounds at carbon nanotube-modified electrodes

The use of a carbon nanotube-modified glassy carbon electrode (CNT-GCE) for the LC-EC detection of phenolic compounds with estrogenic activity is reported. Cyclic voltammograms for phenolic endocrine disruptors and estrogenic hormones showed, in general, an enhancement of their electrochemical oxidation responses at CNT-GCE attributable to the electrocatalytic effect caused by CNTs. Hydrodynamic voltammograms obtained under flow injection conditions lead to the selection of +700 mV as the potential value to be applied for the amperometric detection of the phenolic estrogenic compounds, this value being remarkably less positive than those reported in the literature using other electrode materials. Successive injections of these compounds demonstrated that no electrode surface fouling occurred. A mobile phase consisting of a 50:50 (v/v) acetonitrile:0.05 mol l⁻¹ phosphate buffer of pH 7.0 was selected for the chromatographic separation of mixtures of these compounds, with detection limits ranging between 98 and 340 nmol l⁻¹. Good recoveries were obtained in the analysis of underground well water and tap water samples spiked with some phenolic estrogenic compounds at a 14 nmol l⁻¹ concentration level.     

Preparation, characterization and evaluation of water-soluble l-cysteine-capped-CdS nanoparticles as fluorescence probe for detection of Hg(II) in aqueous solution

Water-soluble l-cysteine-capped-CdS nanoparticles were prepared in aqueous solution at room temperature through a straightforward one-pot process by using safe and low-cost inorganic salts as precursors, and characterized by transmission electron microscopy, X-ray diffraction spectrometry, Fourier transform infrared spectrometry, spectrofluorometry and ultraviolet-visible spectrometry. The prepared l-cysteine-capped-CdS nanoparticles were evaluated as fluorescence probe for Hg(II) detection. The fluorescence quenching of the l-cysteine-capped-CdS nanoparticles depended on the concentration and pH of Hg(II) solution. Maximum fluorescence quenching was observed at pH 7.4 with the excitation and emission wavelengths of 360 nm and 495 nm, respectively. Quenching of its fluorescence due to Hg(II) at the 20 nmol l⁻¹level was unaffected by the presence of 5 × 10⁶-fold excesses of Na(I) and K(I), 5 × 10⁵-fold excesses of Mg(II), 5 × 10⁴-fold excesses of Ca(II), 500-fold excesses of Al(III), 91-fold excesses of Mn(II), 23.5-fold excesses of Pb(II), 25-fold excesses of Fe(III), 25-fold excesses of Ag(I), 8.5-fold excesses of Ni(II) and 5-fold excesses of Cu(II). Under optimal conditions, the quenched fluorescence intensity increased linearly with the concentration of Hg(II) ranging from 16 nmol l⁻¹ to 112 nmol l⁻¹. The limit of detection for Hg(II) was 2.4 nmol l⁻¹. The developed method was applied to the detection of trace Hg(II) in aqueous solutions.     

Method for monitoring urea and ammonia in wine and must by flow injection-pervaporation

An easy to automate flow injection-pervaporation method for monitoring urea and ammonia in must and wine was developed. The method is based on separation of the ammonia from the sample matrix by pervaporation followed by its reaction with salicylate, hypochlorite and nitroprusside to form a diazonium salt with maximum absorption at 647 nm. Conversion of urea into ammonia catalysed by urease was mandatory before pervaporation. After optimisation by either the univariate or multivariate approaches as required, the linear range was established (between 0 and 25 mg l⁻¹) for both analytes. Then, the assessment of the proposed method versus a reference one for urea and ammonia was studied in terms of repeatability (0.52 and 0.43 mg l⁻¹, respectively), reproducibility (1.34 and 1.21 mg l⁻¹, respectively), detection and quantification limits (LOD=0.9 and 0.6 mg l⁻¹, LQ=1.02 and 0.67 mg l⁻¹, respectively) and traceability. The sample throughput was 16 samples h⁻¹. The method can be applied to the monitoring of the target analytes in must and young wine in order to control their contents, preventing formation of ethyl carbamate.     

Ultrasensitive and selective determination of trace amounts of nitrite ion with a novel fluorescence probe mono[6-N(2-carboxy-phenyl)]-β-cyclodextrin

A novel fluorescence probe, mono[6-N(2-carboxy-phenyl)]-β-cyclodextrin (OACCD), has been developed for the determination of trace nitrite, In dilute HCl medium, the fluorescence intensity of the newly synthesized fluorescence probe OACCD was quenched in presence of trace nitrite at room temperature. Based on this, a simple, sensitive, and selective method for rapid determination of nitrite was described. Furthermore, common ions do not interfere the determination of trace amounts of nitrite. The fluorescence quenching intensity was linear over a nitrite concentration of 0.02-1.7 μmol l⁻¹ with a detection limit of 0.2 nmol l⁻¹ (S/N = 3). The method was applied to the determination of nitrite in different water samples, soil samples, and food samples with satisfactory results.     

Development and validation of a high-throughput high-performance liquid chromatographic assay for the determination of caffeine in food samples using a monolithic column

The present study reports the development and validation of a high-throughput high-performance liquid chromatographic (HPLC) assay for the determination of caffeine in food samples. The analyte was separated rapidly from sample matrix using a short monolithic column (50 mm × 4.6 mm i.d.). The flow rate was 3.0 mL min⁻¹, while the mobile phase consisted of ACN/water (10:90, v/v). Caffeine was detected directly at 274 nm. Under the optimal HPLC conditions, the sampling rate was 60 h⁻¹. The assay was validated for linearity, LOD and LOQ, precision, selectivity and ruggedness. The case of external calibration versus standard addition for the analysis of real samples was also examined. The proposed assay was applied to the analysis of beverages and coffee samples.     

Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis

The consumption of ethanol is known to increase the likelihood of oral cancer. In addition, there has been a growing concern about possible association between long term use of ethanol-containing mouthwashes and oral cancer. Acetaldehyde, known to be a carcinogen, is the first metabolite of ethanol and it can be produced in the oral cavity after consumption or exposure to ethanol. This paper reports on the development of a gas-diffusion flow injection method for the online determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Acetaldehyde samples and standards (80 μL) were injected into the donor stream containing NaCl from which acetaldehyde diffused through the hydrophobic Teflon membrane of the gas-diffusion cell into the acceptor stream containing the two reagents mentioned above. The resultant intense green coloured dye was monitored spectrophotometrically at 600 nm. Under the optimum working conditions the method is characterized by a sampling rate of 9 h⁻¹, a linear calibration range of 0.5–15 mg L⁻¹ (absorbance = 5.40 × 10⁻² [acetaldehyde, mg L⁻¹], R² = 0.998), a relative standard deviation (RSD) of 1.90% (n = 10, acetaldehyde concentration of 2.5 mg L⁻¹), and a limit of detection (LOD) of 12.3 μg L⁻¹. The LOD and sampling rate of the proposed method are superior to those of the conventional gas chromatographic (GC) method (LOD = 93.0 μg L⁻¹ and sampling rate = 4 h⁻¹). The reliability of the proposed method was illustrated by the fact that spiked with acetaldehyde saliva samples yielded excellent recoveries (96.6–101.9%), comparable to those obtained by GC (96.4–102.3%) and there was no statistically significant difference at the 95% confidence level between the two methods when non-spiked saliva samples were analysed.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

Spectrofluorimetric determination of trace nitrite in food products with a new fluorescent probe 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene

A new fluorescent probe 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene (TMDCDABODIPY) has been developed to detect nitrite in meat products and vegetables. The fluorescence of TMDCDABODIPY is very weak, but when it reacts with nitrite, a strong fluorescent triazole forms in aqueous medium at room temperature, which offers the advantage of specificity and sensitivity for the determination of nitrite. The fluorescence intensity was linear over a nitrite concentration of 9-300 nmol l⁻¹ with a detection limit of 0.21 nmol l⁻¹ (S/N = 3). The proposed method has been used for the determination of trace nitrite in food products with the recoveries of 94.62-105.48%.     

Indirect competitive immunoassay for trichlorophenol determination: Rational evaluation of the competitor heterology effect

An improved competitive indirect immunoassay for the detection of 2,4,6-trichlorophenol (2,4,6-TCP) has been developed and optimized by preparing heterologous haptens that have been evaluated as coating antigens. The relation between the degree of heterology and immunoassay detectability has been investigated according to the geometric and electronic distribution similarities between the haptens and the analyte using molecular modeling tools. The assay has been characterized according to different physicochemical parameters such as the incubation time, the ionic strength, the effect of detergents and the pH. The resulting assay has an IC₅₀ of 1.44 μg l⁻¹ and a limit of detection (LOD) of 0.2 μg l⁻¹ and it shows a good accuracy and suitability to analyze trichlorophenol in drinking water.     

Determination of hydrogen peroxide by using a flow injection system with immobilized peroxidase and long pathlength capillary spectrophotometry

The development of a highly sensitive method for the determination of nanomolar concentrations of hydrogen peroxide in the liquid phase is described. This paper demonstrates for the first time a flow injection analysis (FIA) system with immobilized enzyme reactor combined with a total internal reflective cell (a liquid waveguide capillary cell (LWCC)) and spectrophotometric detection, for the development of an improved procedure for the determination of hydrogen peroxide. Moreover, the newly synthesized 4-aminopyrazolone derivative, 4-amino-5-(p-aminophenyl)-1-methyl-2-phenyl-pyrazol-3-one (DAP), is used as a color coupler in its oxidative condensation with the sodium salt of N-ethyl-N-sulphopropylaniline sodium salt (ALPS) which acts as a hydrogen donor. Immobilization of peroxidase is achieved by coupling the periodate-treated enzyme to aminopropyl controlled-pore glass (CPG) beads. The determination of hydrogen peroxide is carried out in a 0.1 M phosphate buffer and the product is monitored at 590 nm with a charge-coupled device (CCD) detector equipped with fiber optics in a fully computerized system. The interference of different species, mainly ionic, was investigated.The method permits detection down to 4 nmol l⁻¹ hydrogen peroxide (signal-to-noise ratio=3). A linear calibration graph was obtained over the range 20-700 nmol l⁻¹. The relative standard deviation (R.S.D.) at 300 nmol l⁻¹ H₂O₂ is 1.7% (n=7). The method was successfully applied for the determination of hydrogen peroxide in samples from a vat-cleaning process.     

Chemiluminometric determination of the pesticide 3-indolyl acetic acid by a flow injection analysis assembly

A new method is proposed for the chemiluminescent determination of the pesticide 3-indolyl acetic acid by means of an flow injection analysis system. The chemiluminescence emission is obtained by oxidation of the analyte with Ce (IV) in nitric acid and presence of β-cyclodextrine.The continuous-flow method allows the determination of 159 samples h⁻¹ of 3-indolyl acetic acid in an interval of concentrations over the range 0.5-15.0 mg l⁻¹. The limit of detection was 0.1 μg l⁻¹ and the R.S.D. (n, 17) at 2.0 mg l⁻¹ of the pesticide level was 2.7%. The method was applied to water samples.     

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L⁻¹ and 1.3 ng L⁻¹ using two different up-converting phosphors and 0.16 ng L⁻¹ using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.     

Factorial design for optimization of experimental variables in preconcentration of copper by a chromotropic acid loaded Q-Sepharose adsorbent

An agarose-based anion exchanger (Q-Sepharose) was loaded with chromotropic acid (CTA) and used for column preconcentration and determination of copper by flame AAS. Preliminary experiments indicated that a sample pH of 5.7-6.5 is best suited for accumulation of copper and a 2.5 ml portion of a 0.02 mol l⁻¹ HCl solution can efficiently desorb the analyte from the column. An incomplete factorial design was used for optimization of five different variables that affect recovery of copper. The results indicated that ionic strength, pH and sample volume variables are the most important effects, respectively. Hence, these variables and their possible interactions were studied more carefully. In optimized conditions, the column could tolerate up to 0.18 mol l⁻¹ sodium nitrate in the matrix. A 5 ml portion of a 0.02 mol l⁻¹ CTA was sufficient for loading of a 0.5 ml column prior to preconcentration of copper from a 150 ml sample solution. Matrix ions of Ca²⁺, Mg²⁺, Na⁺ and K⁺ and potentially interfering ions of Pb²⁺, Ni²⁺, Cd²⁺, Co²⁺, Zn²⁺ and Mn²⁺ with relatively high concentrations did not have any significant effect on the recovery of the analyte. A preconcentration factor of 60 and a detection limit of 1.0 μg l⁻¹ was obtained for the determination of copper by the flame AAS method. A precision better than 2.5%, expressed as R.S.D., was also achieved. Application of the method to tap water and two different river water samples resulted in values well confirmed by direct determinations with ET-AAS.     

Determination of some phthalate acid esters in artificial saliva by gas chromatography-mass spectrometry after activated carbon enrichment

The determination of six phthalate acid esters was achieved in artificial saliva using gas chromatography-mass spectrometry following activated carbon enrichment of samples. Central composite experimental design was applied to optimize method parameters, such as pH, adsorption time and amount of activated carbon. The best compromise of analytical conditions for the simultaneous determination of analytes from spiked artificial saliva were found to be: pH (3), adsorption time (30 min), activated carbon amount (1.8 g L⁻¹) and elution solvent (chloroform). These conditions were applied to study the migration of phthalate acid esters from different children's toys into saliva. A horizontal agitation method was applied to extract the analytes from plastic toys into saliva for 2 h at 37 °C. The detection limits of the method were in the range of 1.3-5.1 μg L⁻¹, while the relative standard deviation (%) values for the analysis of 100 μg L⁻¹ of the analytes were below 3.0% (n = 5). Di-2-ethylhexyl phthalate was the main analyte found in these samples.