Determination of the steroid hormone levels in water samples by dispersive liquid-liquid microextraction with solidification of a floating organic drop followed by high-performance liquid chromatography

In this study, the steroid hormone levels in river and tap water samples were determined by using a novel dispersive liquid-liquid microextraction method based on the solidification of a floating organic drop (DLLME-SFO). Several parameters were optimized, including the type and volume of the extraction and dispersive solvents, extraction time, and salt effect. DLLME-SFO is a fast, cheap, and easy-to-use method for detecting trace levels of samples. Most importantly, this method uses less-toxic solvent. The correlation coefficient of the calibration curve was higher than 0.9991. The linear range was from 5 to 1000 μg L⁻¹. The spiked environmental water samples were analyzed using DLLME-SFO. The relative recoveries ranged from 87% to 116% for river water (which was spiked with 4 μg L⁻¹ for E1, 3 μg L⁻¹ for E2, 4 μg L⁻¹ for EE2 and 9 μg L⁻¹ for E3) and 89% to 102% for tap water (which was spiked with 6 μg L⁻¹ for E1, 5 μg L⁻¹ for E2, 6 μg L⁻¹ for EE2 and 10 μg L⁻¹ for E3). The detection limits of the method ranged from 0.8 to 2.7 μg L⁻¹ for spiked river water and 1.4 to 3.1 μg L⁻¹ for spiked tap water. The methods precision ranged from 8% to 14% for spiked river water and 7% to 14% for spiked tap water.     

Quantitative detection of tetracycline residues in honey by a simple sensitive immunoassay

Tetracyclines (TCs) are widely used for prevention and control of infectious diseases and have a great activity against variety of Gram-positive and Gram-negative bacteria. Due to the widespread use of TCs in animal husbandry, it can lead to an increase the risk of TCs remaining in human food. To protect consumers, many countries have set acceptable tolerance levels for these drugs. Therefore, it is necessary to establish a suitable analytical technique with specificity, sensitivity and simplicity.The biotin-avidin mediated ELISA method was performed to determine TC residues in honey quantitatively. By using PBS-EDTA assay buffer at pH 7.2, a honey solution of TC standard was prepared and diluted. And no additional pre-treatment of sample was required in this method. The limit of detection and limit of quantitation of the optimized method were 3.98 × 10⁻¹⁰ M (0.19 μg L⁻¹) and 7.94 × 10⁻¹⁰ M (0.38 μg L⁻¹), respectively, and the dynamic range was from 1.52 μg L⁻¹ to 152 μg L⁻¹ of TC in honey. No cross-reactivity was observed with the structurally similar compounds, and mean percent recoveries of TC spiked in honey ranged from 95% to 101%. Compared to other methods, this method was superior in terms of detection limit, dynamic range, and % recovery with simple sample-preparation.     

Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens

In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC₅₀ of the ELISA for enrofloxacin from 1.3 μg L⁻¹ to as low as 0.07 μg L⁻¹. The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step.     

Feasibility of ultra-high performance liquid and gas chromatography coupled to mass spectrometry for accurate determination of primary and secondary phthalate metabolites in urine samples

Phthalates (PAEs) are ubiquitous toxic chemical compounds. During the last few years, some phthalate metabolites (MPAEs) have been proposed as appropriate biomarkers in human urine samples to determine PAE human intake and exposure. So, it is necessary to have fast, easy, robust and validated analytical methods to determine selected MPAEs in urine human samples. Two different instrumental methods based on gas (GC) and ultra-high performance liquid (UHPLC) chromatography coupled to mass spectrometry (MS) have been optimized, characterized and validated for the simultaneous determination of nine primary and secondary phthalate metabolites in urine samples. Both instrumental methods have similar sensitivity (detection limits ranged from 0.03 to 8.89 pg μL⁻¹ and from 0.06 to 0.49 pg μL⁻¹ in GC–MS and UHPLC–MS², respectively), precision (repeatability, expressed as relative standard deviation, which was lower than 8.4% in both systems, except for 5OH-MEHP in the case of GC–MS) and accuracy. But some advantages of the UHPLC–MS² method, such as more selectivity and lower time in the chromatographic runs (6.8 min vs. 28.5 min), have caused the UHPLC–MS² method to be chosen to analyze the twenty one human urine samples from the general Spanish population. Regarding these samples, MEP showed the highest median concentration (68.6 μg L⁻¹), followed by MiBP (23.3 μg L⁻¹), 5cx-MEPP (22.5 μg L⁻¹) and MBP (19.3 μg L⁻¹). MMP (6.99 μg L⁻¹), 5oxo-MEHP (6.15 μg L⁻¹), 5OH-MEHP (5.30 μg L⁻¹) and MEHP (4.40 μg L⁻¹) showed intermediate levels. Finally, the lowest levels were found for MBzP (2.55 μg L⁻¹). These data are within the same order of magnitude as those found in other similar populations.     

Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I₅₀ and detection limits were 0.05-2.5 and 0.1-7.5 μg L⁻¹, 0.35 (±0.04) μg L⁻¹ and 0.9 (±0.1) μg L⁻¹, 60 and 100 μg L⁻¹ in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I₅₀ in real samples was 0.2 μg L⁻¹ corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.     

Application of a modified enzyme-linked immunosorbent assay for 3-amino-2-oxazolidinone residue in aquatic animals

Furazolidone has been banned from use in food animals because of its carcinogenicity and mutagenicity, but its continued misuse is widespread in aquacultures. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in aquatic products. In this regard, we modified a simplified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to address this need. A good linearity was achieved over a concentration range of 0.05-12.15 μg L⁻¹, and the IC₅₀ value was 0.96 μg L⁻¹. The sample preparation was simple and effective included water bath treatments, acid hydrolysis combined with overnight derivatization of AOZ by benzaldehyde. The limit of detection and the limit of quantification were 0.15 and 0.3 μg kg⁻¹. The recoveries of AOZ in all tissues were between 78.0-95.3% at the levels of 0.3, 1.0, and 2.0 μg kg⁻¹. The inter-assay variability was less than 19.1%. The modified ic-ELISA was applied in quantification of AOZ elimination in carp. The results showed that AOZ was quite difficult to eliminate. Good correlations of the results obtained by ELISA and LC-MS/MS were observed in incurred carp muscle (r = 0.9923) and carp plasma (r = 0.9915) at the levels of 2.5-571.8 μg kg⁻¹ (μg L⁻¹). Better results were obtained by modified ic-ELISA when compared with commercial ELISA kit. Therefore, the present assay is considered a rapid, accurate, reliable, and inexpensive method for the detection of furazolidone-residues in the edible tissues of aquatic animals.     

Sensitive determination of 17β-estradiol in river water using a graphene based electrochemical sensor

In this study, a novel material for the electrochemical determination of 17β-estradiol using an electrode based on reduced graphene oxide and a metal complex porphyrin has been applied to environmental monitoring. The electrochemical profile of the proposed electrode was analyzed by differential pulse voltammetry, which showed a shift of the oxidation peak potential of 17β-estradiol to 150 mV in a less positive direction compared to the bare reduced graphene oxide electrode. DPV experiments were performed in PBS at pH 7.0 to determine 17β-estradiol without any previous step of extraction, cleanup, or derivatization, in the range of 0.1–1.0 μmol L⁻¹ with a detection limit archived at 5.3 nmol L⁻¹ (1.4 μg L⁻¹). The proposed sensor was successfully applied in the determination of 17β-estradiol in a river water sample without any purification step and was successfully analyzed under the standard addition method. All the obtained results were in agreement with those from the HPLC procedure.     

Determination of 4-ethylcatechol in wine by high-performance liquid chromatography-coulometric electrochemical array detection

A HPLC method using a coulometric electrode array detector (CEAD) to analyse 4-ethylcatechol in wine was established. The procedure does not require any sample preparation or analyte derivatisation and performs chromatographic separation in a short time. The assay method is linear up to 1520 μg L⁻¹ and precise (R.S.D. < 3%), with limits of detection and quantitation of 1.34 μg L⁻¹ and 2.2 μg L⁻¹, respectively. Recoveries in spiked wine samples ranged from 95% to 104% with a median value of 102% and matrix effects were not observed. The method was applied to the evaluation of the concentration of 4-EC in 250 commercial Italian wines. The red wines analysed had median, 75° percentile and maximum values of 37 μg L⁻¹, 89 μg L⁻¹ and 1610 μg L⁻¹, respectively. For Sangiovese-based wines the mean ratios of 4-EP and 4-EG to 4-EC were 3.7:1 and 0.7:1, respectively. The feasibility of a cheaper fluorimetric approach to 4-EC quantification was investigated.     

Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity

A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides, including malathion, dimethoate, phenthoate, phosmet, methidathion, fenitrothion, methyl parathion and fenthion. Three haptens with different spacer-arms were synthesized. The haptens were conjugated to bovine serum albumin (BSA) for immunogens and to ovalbumin (OVA) for coating antigens. Rabbits were immunized with the immunogens and six polyclonal antisera were produced and screened against each of the coating antigens using competitive indirect enzyme-linked immunosorbent assay for selecting the proper antiserum. The effect of hapten heterology on immunoassay sensitivity was also studied. The antibody-antigen combination with the most selectivity for malathion was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC₅₀ values, under optimum conditions, were estimated to be 30.1 μg L⁻¹for malathion, 28.9 μg L⁻¹ for dimethoate, 88.3 μg L⁻¹ for phenthoate, 159.7 μg L⁻¹ for phosmet, 191.7 μg L⁻¹ for methidathion, 324.0 μg L⁻¹ for fenitrothion, 483.9 μg L⁻¹ for methyl parathion, and 788.9 μg L⁻¹ for fenthion. Recoveries of malathion, dimethoate, phenthoate, phosmet and methidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%. This assay can be used in monitoring studies for the multi-residue determination of O,O-dimethyl organophosphorus pesticides.     

Validation of an enzyme-linked immunosorbent assay (ELISA) for the determination of 4-nonylphenol and octylphenol in surface water samples by LC-ESI-MS

4-Nonylphenol (NP) and octylphenol (OP) were measured by direct ELISA in both laboratory-fortified and surface water samples collected monthly from 10 rivers. In this procedure, samples were concentrated by solid phase extraction (SPE) using Lichrolut RP-18 sorbent with good recoveries obtained for both LC-MS and ELISA, giving a low level of detection (LOD) at the range of low μg L⁻¹ and good reproducibility. Analysis of 40 surface water samples demonstrated that the ELISA may be a useful screening tool for the determination of the alkylphenols in surface water matrices. The concentration of NP and OP in surface waters ranged from 0.11 to 6.58 μg L⁻¹. A good correlation of results obtained by ELISA and LC-MS within the concentration range of 0.08-6.86 μg L⁻¹ was found in the river samples [R² = 0.924, n = 39]. The influence of various factors on assay determination was also discussed.     

The use of a polymer inclusion membrane for separation and preconcentration of orthophosphate in flow analysis

A highly sensitive flow analysis system has been developed for the trace determination of reactive phosphate in natural waters, which uses a polymer inclusion membrane (PIM) with Aliquat 336 as the carrier for on-line analyte separation and preconcentration. The system operates under flow injection (FI) and continuous flow (CF) conditions. Under optimal FI conditions the system is characterised by a linear concentration range between 0.5 and 1000 μg L⁻¹ P, a sampling rate of 10 h⁻¹, a limit of detection of 0.5 μg L⁻¹ P and RSDs of 3.2% (n = 10, 100 μg L⁻¹) and 7.7% (n = 10, 10 μg L⁻¹). Under CF conditions with 10 min stop-flow time and sample solution flow rate of 1.32 mL min⁻¹ the flow system offers a limit of detection of 0.04 μg L⁻¹ P, a sampling rate of 5 h⁻¹ and an RSD of 3.4% (n = 5, 2.0 μg L⁻¹). Interference studies revealed that anions commonly found in natural waters did not interfere when in excess of at least one order of magnitude. The flow system, operating under CF conditions, was successfully applied to the analysis of natural water samples containing concentrations of phosphate in the low μg L⁻¹ P range, using the multipoint standard addition method.     

ELISA for semicarbazide and its application for screening in food contamination

The development of a direct competitive enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies specific for semicarbazide (SEM) is described. Molecular modelling of the hapten mimics and other key components of the assay system was conducted to explain antibody properties in relation to hapten design. The small aliphatic molecule SEM was coupled to 3-carboxybenzaldehyde to produce carboxyphenyl-SEM (CPSEM), for the generation of specific antibodies. Five rabbits produced antibodies against NPSEM (used in direct and indirect ELISA formats) exhibiting a 50% binding inhibition level (IC₅₀ values) of 0.06-2.28 μg L⁻¹ in assay buffer for SEM. The most sensitive indirect assay based on the antibody MVK39 showed a high dynamic range providing a linear readout in the range of 0.01-0.2 μg L⁻¹. Antibody MVK31 (IgG) allowed specific SEM detection at an IC₅₀ = 0.14 μg L⁻¹ in direct ELISA and was evaluated using solvent extracted SEM-spiked porcine and baby food samples. Recovery levels determined from fortified samples (0.5, 1.0, 1.5, 5, 10 and 20 μg kg⁻¹) of porcine and baby food ranged from 82.9 to 105.3%, respectively, with a coefficient of variation less than 15.5%. Respective detection capability and threshold of the assay for porcine muscle, set on the basis of acceptance of no false negative results, was 0.3 and 0.11 μg kg⁻¹.     

Development of a new electrochemical hydride generator with tungsten wire cathode for the determination of As and Sb by atomic fluorescence spectrometry

A novel electrochemical hydride generator has been developed for the determination of As and Sb. This newly devised hydride generator is constructed from a flowing electrolytic cell, in which the tungsten wire is selected as cathode. Compared with some cathode material usually used in electrochemical hydride generator, the tungsten cathode is of better interference tolerance, corrosion-resistant and longer working time. The characteristics of the cathode material, hydride generating efficiency and interferences of concomitant have been studied in detail. The detection limits (3σ) of As and Sb in sample solution were 0.10 μg L⁻¹ and 0.15 μg L⁻¹, the precisions for 11 replicate measurements of 20 μg L⁻¹ As and Sb were 1.3% and 1.7%. The electrochemical hydride generator coupled with atomic fluorescence spectrometry has been applied to the determination of total As and Sb in tobacco samples.     

High-sensitivity determination of lead and cadmium based on the Nafion-graphene composite film

Graphene nanosheets, dispersed in Nafion (Nafion-G) solution, were used in combination with in situ plated bismuth film electrode for fabricating the enhanced electrochemical sensing platform to determine the lead (Pb²⁺) and cadmium (Cd²⁺) by differential pulse anodic stripping voltammetry (DPASV). The electrochemical properties of the composite film modified glassy carbon electrode were investigated. It is found that the prepared Nafion-G composite film not only exhibited improved sensitivity for the metal ion detections, but also alleviated the interferences due to the synergistic effect of graphene nanosheets and Nafion. The linear calibration curves ranged from 0.5 μg L⁻¹ to 50 μg L⁻¹ for Pb²⁺ and 1.5 μg L⁻¹ to 30 μg L⁻¹ for Cd²⁺, respectively. The detection limits (S/N = 3) were estimated to be around 0.02 μg L⁻¹ for Pb²⁺ and Cd²⁺. The practical application of the proposed method was verified in the water sample determination.     

Synthesis and application of a carbamazepine-imprinted polymer for solid-phase extraction from urine and wastewater

A molecularly imprinted polymer (MIP) designed to enable the selective extraction of carbamazepine (CBZ) from effluent wastewater and urine samples has been synthesised using a non-covalent molecular imprinting approach. The MIP was evaluated chromatographically in the first instance and its affinity for CBZ also confirmed by solid-phase extraction (SPE). The optimal conditions for SPE consisted of conditioning of the cartridge using acidified water purified from a Milli-Q system, loading of the sample under basic aqueous conditions, clean-up using acetonitrile and elution with methanol. The attractive molecular recognition properties of the MIP gave rise to good CBZ recoveries (80%) when 100 mL of effluent water spiked with 1 μg L⁻¹ was percolated through the polymer. For urine samples, 2 mL samples spiked with 2.5 μg L⁻¹ CBZ were extracted with a recovery of 65%. For urine, the linear range was 0.05-24 mg L⁻¹, the limit of detection was 25 μg L⁻¹ and precision, expressed as relative standard deviation at 0.5 mg L⁻¹ (n = 3), was 3.1% and 12.6% for repeatability and reproducibility between days, respectively.     

Purge efficiency in the determination of trihalomethanes in water by purge-and-trap gas chromatography

Purge-and-trap gas chromatography-mass spectrometry (PT-GC-MS) has become an accepted method for the analysis of trihalomethanes (THMs) in water. The purge-and-trap technique is based on an efficient transfer of volatile organic compounds from the liquid (contained in the purge chamber) to the gaseous phase by bubbling with an inert gas. The aim of this work was to study the purge system's efficiency by means of several consecutive purge cycles lasting 11 min each of the same liquid sample. The concentration range chosen of THMs was very wide [5-200 μg L⁻¹]. The inert gas flow rate was 40 mL min⁻¹, and experiments were performed at temperatures of 25, 35 and 50 °C. Bromoform (CHBr₃), the least volatile compound, needed 19 cycles to be purged quantitatively at a concentration of 200 μg L⁻¹ and only 7 cycles at 5 μg L⁻¹ for a 25 mL sample at 25 °C. Chloroform (CHCl₃), the most volatile compound, required 4 cycles to be fully extracted at 200 μg L⁻¹ and 2 at 5 μg L⁻¹. Finally, Novak's theoretical model, based on the distribution constant between gas and liquid phases, was used to correlate the THMs purging extraction data.     

Indirect determination of hexavalent chromium ion in complex matrices by adsorptive stripping voltammetry at a mercury electrode

A sensitive method for the determination of chromium ion(VI) in complex matrices such as crude oil and sludge is presented based on the decreasing effect of Cr(VI) on cathodic adsorptive stripping peak height of Cu-adenine complex. Under the optimum experimental conditions (pH 7.5 Britton-Robinson buffer, 5 × 10⁻⁵ M copper, 8 × 10⁻⁶ M adenine and accumulation potential −250 mV versus Ag/AgCl), a linear decrease of the peak current of Cu-adenine was observed, when the chromium(VI) concentration was increased from 5 μg L⁻¹ to 120 μg L⁻¹. Detection limit of 2 μg L⁻¹ was achieved for 120 s accumulation time. The relative standard deviations (R.S.D., %) were 1.8% and 4% for chromium(VI) concentrations of 18 μg L⁻¹ and 100 μg L⁻¹, respectively. The method was applied to the determination of chromium(VI) in the presence of high levels of chromium(III), in various real samples such as crude oil, crude oil tank button sludge, waste water and tap water samples. Effects of foreign ions and surfactants on the voltammetric peak and the influences of instrumental and analytical parameters were investigated in detail. The accuracy of the results was checked by ICP and/or AA.     

Headspace liquid-phase microextraction using ionic liquid as extractant for the preconcentration of dichlorodiphenyltrichloroethane and its metabolites at trace levels in water samples

A novel technique, high temperature headspace liquid-phase microextraction (HS-LPME) with room temperature ionic liquid (RTIL), 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄MIM][PF₆]) as extractant, was developed for the analysis of dichlorodiphenyltrichloroethane (p,p′-DDT and o,p′-DDT) and its metabolites including 4,4′-dichlorodiphenyldichloroethylene (p,p′-DDE) and 4,4′-dichlorodiphenyldichloroethane (p,p′-DDD) in water samples by high performance liquid chromatography with ultraviolet detection. The parameters such as salt content, sample pH and temperature, stirring rate, extraction time, microdrop volume, and sample volume, were found to have significant influence on the HS-LPME. The conditions optimized for extraction of target compounds were as follows: 35% NaCl (w/v), neutral pH condition, 70 °C, 800 rpm, 30 min, 10 μL [C₄MIM][PF₆], and 25 mL sample solutions. Under the optimized conditions, the linear range, detection limit (S/N = 3), and precision (R.S.D., n = 6) were 0.3-30 μg L⁻¹, 0.07 μg L⁻¹, and 8.0% for p,p′-DDD, 0.3-30 μg L⁻¹, 0.08 μg L⁻¹, and 7.1% for p,p′-DDT, 0.3-30 μg L⁻¹, 0.08 μg L⁻¹, and 7.2% for o,p′-DDT, and 0.2-30 μg L⁻¹, 0.05 μg L⁻¹, and 6.8% for p,p′-DDE, respectively. Water samples including tap water, well water, snow water, reservoir water, and wastewater were analyzed by the proposed procedure and the recoveries at 5 μg L⁻¹ spiked level were in the range of 86.8-102.6%.     

Determination of organophosphorus pesticides in ecological textiles by solid-phase microextraction with a siloxane-modified polyurethane acrylic resin fiber

A novel solid-phase microextraction (SPME) fiber coating was prepared with siloxane-modified polyurethane acrylic resin by photo-cured technology. The ratio of two monomers was investigated to obtain good microphase separation structure and better extraction performance. The self-made fiber was then applied to organophosphorus pesticides (OPPs) analysis and several factors, such as extraction/desorption time, extraction temperature, salinity, and pH, were studied. The optimized conditions were: 15 min extraction at 25 °C, 5% Na₂SO₄ content, pH 7.0 and 4 min desorption in GC inlet. The self-made fiber coating exhibited better extraction efficiency for OPPs, compared with three commercial fiber coatings. Under the optimized conditions, the detection limits of 11 OPPs were from 0.03 μg L⁻¹ to 0.5 μg L⁻¹. Good recoveries and repeatabilities were obtained when the method was used to determine OPPs in ecological textile.