Fast screening immunoassay of sulfonamides in commercial fish samples

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed in plate to detect three sulfonamide residues (sulfamerazine (SMR), sulfadimetoxine (SDM), and sulfadiazine (SDZ)) in gilthead sea bream (Sparus aurata) samples. Different extraction methodologies—using methanol/water 1:1 (v/v) + ethylene diamine tetraacetic acid (EDTA) 0.5% (m/v), acetonitrile, phosphate-buffered saline (PBS) 10 mmol L⁻¹ pH 7 and acetate buffer 100 mmol L⁻¹ pH 5—and cleanup steps, based on solid-phase extraction (C₁₈, SCX, Si) or liquid extraction with hexane, were assayed. As optimum, a fast and simple method using acetonitrile was selected to extract the sulfonamide residues from the edible muscle of fish. Due to matrix effects, a standard addition calibration curve in fish extract is necessary for quantification purposes. Sulfonamide-free samples were spiked at different concentration levels (between 30 and 90 ng g⁻¹, 5–15 ng mL⁻¹ in plate) and average recoveries (n = 8), ranging from 71% to 95%, 65% to 79%, and 72% to 95%, were obtained for SMR, SDM, and SDZ, respectively. The assay detection limits for these antibiotics were lower than 100 μg kg⁻¹ (maximum residue level established by the European Union). The accuracy was evaluated by spiking blank fish extracts at different concentrations (10–40 ng mL⁻¹, 5–20 ng mL⁻¹ in plate), and the relative errors ranged between ±20%. Finally, in order to confirm the utility of the developed ELISA as a screening methodology, fish samples from different supermarkets were analyzed, and results were compared with those obtained by a validated high-performance liquid chromatography (HPLC) method. The correlation between the results obtained by both ELISA and HPLC methods is satisfactory.      

Development of a multichannel flow-through chemiluminescence microarray chip for parallel calibration and detection of pathogenic bacteria

Pathogen detection is important for health and safety reasons. Several outbreaks all over the world have shown the need for rapid, qualitative, quantitative, and, particularly, multianalyte detection systems. Hence, a multichannel flow-through chemiluminescence microarray chip for parallel detection of pathogenic bacteria was developed. The disposable chip made of acrylonitrile–butadiene–styrene (ABS) copolymer was devised as a support for a multiplexed sandwich immunoassay. Calibration and measurement was possible in one experiment, because the developed chip contains six parallel flow-through microchannels. Polyclonal antibodies against the pathogenic bacteria Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila were immobilized on the chip by microcontact printing in order to use them as specific receptors. Detection of the captured bacteria was carried out by use of specific detection antibodies labelled with biotin and horseradish peroxidase (HRP)–streptavidine conjugates. The enzyme HRP generates chemiluminescence after adding luminol and hydrogen peroxide. This signal was observed by use of a sensitive CCD camera. The limits of detection are 1.8 × 10⁴ cells mL⁻¹ for E. coli O157:H7, 7.9 × 10⁴ cells mL⁻¹ for L. pneumophila, and 2.0 × 10⁷ cells mL⁻¹ for S. typhimurium. The overall assay time for measurement and calibration is 18 min, enabling very fast analysis.      

Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection

To identify the explosive used in a terrorist attack, or to obtain an early sign of environmental pollution it is important to use simple and rapid assays able to detect analytes at low levels, possibly on-site. This is particularly true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because of its toxicity, a well known pollutant. In this work we describe the development of an indirect competitive ELISA with chemiluminescent detection (CL-ELISA) and of a lateral-flow immunoassay (LFIA) based on colloidal gold nanoparticle labels. A commercially available monoclonal antibody was used and 13 specially synthesized conjugates were tested. We optimized the assay by determining the optimal concentration of monoclonal antibody and conjugates and the influence of various non-specific factors such as: tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The sensitivity and reproducibility of the CL-ELISA were good (LOD and IC₅₀ values in the ng mL⁻¹ range, and CV value about 7%). It has been applied to real samples of various materials involved in a controlled explosion of an “improvised explosive device”. Three extraction procedures were tested on these samples, all employing methanol as the solvent. The lateral flow immunoassay (LFIA), developed by using the same immunoreagents, reached a detection limit of 1 μg mL⁻¹ when tested on the same samples analysed by CL-ELISA.      

A highly sensitive and specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of antibiotic olaquindox in animal feed samples

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N′N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC₅₀ values of 16 ng mL⁻¹ and 19 ng mL⁻¹, respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC₅₀ value for eight standard curves was in the range 12–18 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 ± 0.11 ng mL⁻¹. The ELISA tolerated 5% methanol without significant influence on IC₅₀ value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3–119.0% and the intra-assay relative standard deviation (RSD) was within 4.7–33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense. Figure Polyclonal antibody based ELISA for detection of olaquindox     

Development of a highly precise ID-ICP-SFMS method for analysis of low concentrations of lead in rice flour reference materials

Microwave digestion and isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-SFMS) has been applied to the determination of Pb in rice flour. In order to achieve highly precise determination of low concentrations of Pb, the digestion blank for Pb was reduced to 0.21 ng g⁻¹ after optimization of the digestion conditions, in which 20 mL analysis solution was obtained after digestion of 0.5 g rice flour. The observed value of Pb in a non-fat milk powder certified reference material (CRM), NIST SRM 1549, was 16.8 ± 0.8 ng g⁻¹ (mean ± expanded uncertainty, k = 2; n = 5), which agreed with the certified value of 19 ± 3 ng g⁻¹ and indicated the effectiveness of the method. Analytical results for Pb in three brown rice flour CRMs, NIST SRM 1568a, NIES CRM 10-a, and NIES CRM 10-b, were 7.32 ± 0.24 ng g⁻¹ (n = 5), 1010 ± 10 ng g⁻¹ (n = 5), and 1250 ± 20 ng g⁻¹ (n = 5), respectively. The concentration of Pb in a candidate white rice flour reference material (RM) sample prepared by the National Metrology Institute of Japan (NMIJ) was observed to be 4.36 ± 0.28 ng g⁻¹ (n = 10 bottles). Figure Digestion blank of Pb was carefully reduced to approximately 0.2 ng g^-1 which permitted the highly precise determination of Pb at low ng g^-1 level in foodstuff samples by ID-SFMS     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study

Radix Scrophulariae (Xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of Xuanshen. The purpose of this study was to develop an HPLC–UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of Xuanshen extract (760 mg kg⁻¹). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid–liquid extraction. Separation was achieved on a Kromasil C₁₈ column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7–103.5% and 96.9–102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of Xuanshen extract were as follows: C _max 1488.7 ± 205.9 and 556.8 ± 94.2 ng mL⁻¹, T _max 2.09 ± 0.31 and (1.48 ± 0.14 h, AUC_0–24 10336.4 ± 1426.8 and 3653.1 ± 456.4 ng h mL⁻¹, 11276.8 ± 1321.4 and 3704.5 ± 398.8 ng h mL⁻¹, and t _1/2 4.9 ± 1.3 and 2.5 ± 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of Radix Scrophulariae extract in rats. Figure Radix Scrophulariae     

Determination of melamine in animal feed based on liquid chromatography tandem mass spectrometry analysis and dynamic microwave-assisted extraction coupled on-line with strong cation-exchange resin clean-up

In this work, a new method was developed for the determination of melamine (MEL) in animal feed. The method was based on the on-line coupling of dynamic microwave-assisted extraction (DMAE) to strong cation-exchange (SCX) resin clean-up. The MEL was first extracted by 90% acidified methanol aqueous solution (v/v, pH = 3) under the action of microwave energy, and then the extract was cooled and passed through the SCX resin. Thus, the protonated MEL was retained on the resin through ion exchange interaction and the sample matrixes were washed out. Some obvious benefits were achieved, such as acceleration of analytical process, together with reduction in manual handling, risk of contamination, loss of analyte, and sample consumption. Finally, the analyte was separated by a liquid chromatograph with a SCX analytical column, and then identified and quantitatived by a tandem mass spectrometry with positive ionization mode and multiple-reaction monitoring. The DMAE parameters were optimized by the Box–Behnken design. The linearity of quantification obtained by analyzing matrix-matched standards is in the range of 50–5,000 ng g⁻¹. The limit of detection and limit of quantification obtained are 12.3 and 41.0 ng g⁻¹, respectively. The mean intra- and inter-day precisions expressed as relative standard deviations with three fortified levels (50, 250, and 500 ng g⁻¹) are 5.1% and 7.3%, respectively, and the recoveries of MEL are in the range of 76.1–93.5%. The proposed method was successfully applied to determine MEL in different animal feeds obtained from the local market. MEL was detectable with the contents of 279, 136, and 742 ng g⁻¹ in three samples.      

Determination of protein surface excess on a liquid/solid interface by single-molecule counting

Determination of protein surface excess is an important way of evaluating the properties of biomaterials and the characteristics of biosensors. A single-molecule counting method is presented that uses a standard fluorescence microscope to measure coverage of a liquid/solid interface by adsorbed proteins. The extremely low surface excess of lysozyme and bovine serum albumin (BSA), in a bulk concentration range from 0.3 nmol L⁻¹ (0.02 μg mL⁻¹) to 3 nmol L⁻¹ (0.2 μg mL⁻¹), were measured by recording the counts of spatially isolated single molecules on either hydrophilic (glass) or hydrophobic (polydimethylsiloxane, PDMS) surfaces at different pH. The differences observed in amounts of adsorbed proteins under different experimental conditions can be qualitatively explained by the combined interactions of electrostatic and hydrophobic forces. This, in turn, implies that single-molecule counting is an effective way of measuring surface coverage at a liquid/solid interface. Figure Adsorption fraction of proteins on different surfaces changed with pH.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.     

GC–MS analysis of low-molecular-weight dicarboxylic acids in atmospheric aerosol: comparison between silylation and esterification derivatization procedures

This paper describes methods for the determination of low-molecular-weight (LMW) dicarboxylic acids in atmospheric aerosols as important chemical tracers for source apportionment of aerosol organics and for studying atmospheric processes leading to secondary organic aerosol formation. The two derivatization procedures most widely used in GC analysis of dicarboxylic acids were compared: esterification using BF₃/alcohol reagent and silylation using N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). The advantages and drawbacks of the two methods are investigated and compared in terms of (1) precision and accuracy of the results and (2) sensitivity and detection limit of the procedure. The comparative investigation was performed on standard solutions containing target C₃–C₉ dicarboxylic acids and on experimental particulate matter (PM) samples. Attention was focused on low-volume sampling devices that collect small amounts of sample for organic speciation. The results show that, overall, both the techniques appear suitable for the analysis of LMW dicarboxylic acids in atmospheric aerosols since they provide low detection limits (≤4 ng m⁻³) and satisfactory reproducibility (RSD% ≤ 15%). Between them, BSTFA should be the reagent of choice under the most limiting conditions of PM filters collected by low-volume air samplers: It provides determination of all the target C₃–C₉ dicarboxylic acids with lower detection limits (≤2 ng m⁻³) and higher reproducibility (RSD% ≤ 10%)      

Electrochemical deposition of silicate–cetyltrimethylammonium bromide nanocomposite film on glassy carbon electrode for sensing of methyl parathion

A novel electrochemical sensor for methyl parathion based on silicate– cetyltrimethylammonium bromide nanocomposite film has been fabricated by electro-assisted deposition onto glassy carbon electrode in one-step via an electrochemical modulation of pH at the electrode/solution interface to promote controlled gelification of tetraethylorthosilicate sol, and was characterized with scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy. The electrochemical sensing of methyl parathion on the film-modified electrode was investigated applying cyclic voltammetry and square wave voltammetry. Compared to the unmodified electrode, the shapes of the redox peaks were improved and the peak currents significantly increased. Experimental parameters such as deposition time, pH value, and accumulation conditions have been optimized. A linear relationship between the peak current and methyl parathion concentration was obtained in the range from 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.04 × 10 ⁻⁸ mol L⁻¹ (S/N = 3) after accumulation at 0 V for 120 s. The film electrode shows great promise for determination of methyl parathion in real samples.      

Determination of diquat by flow injection–chemiluminescence

A simple, economic, sensitive and rapid method for the determination of the pesticide diquat was described. This new method was based on the coupling of flow injection analysis methodology and direct chemiluminescent detection; to the authors’ knowledge, this approach had not been used up to now with this pesticide. It was based on its oxidation with ferricyanide in alkaline medium; significant improvements in the analytical signal were achieved by using high temperatures and quinine as sensitiser. Its high throughput (144 h⁻¹), together with its low limit of detection (2 ng mL⁻¹), achieved without need of preconcentration steps, permitted the reliable quantification of diquat over the linear range of (0.01–0.6) μg mL⁻¹ in samples from different origins (river, tap, mineral and ground waters), even in the presence of a 40-fold concentration of paraquat, a pesticide commonly present in the commercial formulations of diquat. Figure Quartz luminometer cell     

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 × 10⁻⁴ mol L⁻¹ (ΔA = 5.2 × 10⁻⁶ Abs.) for (+)-CSA and 8.4 × 10⁻⁴ mol L⁻¹ (ΔA = 5.0 × 10⁻⁶ Abs.) for (−)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified. Figure Sensitive chiral analysis by thermal lens microscope (TLM)     

Application of chitosan functionalized with 3,4-dihydroxy benzoic acid moiety for on-line preconcentration and determination of trace elements in water samples

Chitosan resin functionalized with 3,4-dihydroxy benzoic acid (CCTS-DHBA resin) was used as a packing material for flow injection (FI) on-line mini-column preconcentration in combination with inductively coupled plasma-atomic emission spectrometry (ICP-AES) for the determination of trace elements such as silver, bismuth, copper, gallium, indium, molybdenum, nickel, uranium, and vanadium in environmental waters. A 5-mL aliquot of sample (pH 5.5) was introduced to the minicolumn for the adsorption/preconcentration of the metal ions, and the collected analytes on the mini-column were eluted with 2 M HNO₃, and the eluates was subsequently transported via direct injection to the nebulizer of ICP-AES for quantification. The parameters affecting on the sensitivity, such as sample pH, sample flow rate, eluent concentration, and eluent flow rate, were carefully examined. Alkali and alkaline earth metal ions commonly existing in river water and seawater did not affect the analysis of metals. Under the optimum conditions, the method allowed the determination of metal ions with detection limits of 0.08 ng mL⁻¹ (Ag), 0.9 ng mL⁻¹ (Bi), 0.07 ng mL⁻¹ (Cu), 0.9 ng mL⁻¹ (Ga), 0.9 ng mL⁻¹ (In), 0.08 ng mL⁻¹ (Mo), 0.09 ng mL⁻¹ (Ni), 0.9 ng mL⁻¹ (U), and 0.08 ng mL⁻¹ (V). By using 5 mL of sample solution, the enrichment factor and collection efficiency were 8–12 fold and 96–102%, respectively, whereas the sample throughput was 7 samples/hour. The method was validated by determining metal ions in certified reference material of river water (SLRS-4) and nearshore seawater (CASS-4), and its applicability was further demonstrated to river water and seawater samples.     

DNA Quantification in Nanoliter Volumes

A novel method for the determination of proteins at nanogram levels was proposed based on the decrease of resonance light scattering (RLS) signal resulting from the interaction of dibromo-o-nitrophenylfluorone (DBONPF)-sodium lauroyl glutamate (SLG) with proteins. At pH 2.97, the decrease RLS intensity was proportional to the concentration of proteins in the range of nanogram levels with 3σ detection limits being 3.4 ng mL⁻¹ for bovine serum albumin (BSA), 1.7 ng mL⁻¹ for human serum albumin (HSA), 4.1 ng mL⁻¹ for γ-globulin (γ-IgG), 4.4 ng mL⁻¹ for egg albumin, 6.2 ng mL⁻¹ for pepsin (Pep) and 3.7 ng mL⁻¹ for α-chymotrypsin (Chy). The method is no protein-to-protein variability, simple, rapid, practical and relatively free from interference from coexisting substance, as well as much more sensitive than most of the reported methods. The proposed method was successfully applied to determine total protein in human serum samples.     

Molecularly imprinted solid-phase extraction and flow-injection chemiluminescence for trace analysis of 2,4-dichlorophenol in water samples

Highly sensitive flow-injection chemiluminescence (CL) combined with molecularly imprinted solid-phase extraction (MISPE) has been used for determination of 2,4-dichlorophenol (2,4-DCP) in water samples. The molecularly imprinted polymer (MIP) for 2,4-DCP was prepared by non-covalent molecular imprinting methods, using 4-vinylpyridine (4-VP) and ethylene glycol dimethacrylate (EGDMA) as the monomer and cross-linker, respectively. 2,4-DCP could be selectively adsorbed by the MIP and the adsorbed 2,4-DCP was determined by its enhancing effect on the weak chemiluminescence reaction between potassium permanganate and luminol. The enhanced CL intensity was linear in the range from 1 × 10⁻⁷ to 2 × 10⁻⁵g mL⁻¹. The LOD (S/N = 3) was 1.8 × 10⁻⁸g mL⁻¹, and the relative standard deviation (RSD) was 3.0% (n = 11) for 1.4 × 10⁻⁶g mL⁻¹. The proposed method had been successfully applied to the determination of 2,4-DCP in river water. Figure Effect of 4-VP content on the ultraviolet spectrum of 2,4-DCP in chloroform     

Dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry for the rapid and sensitive determination of UV filters in environmental water samples

The performance of the dispersive liquid–liquid microextraction (DLLME) technique for the determination of eight UV filters and a structurally related personal care species, benzyl salicylate (BzS), in environmental water samples is evaluated. After extraction, analytes were determined by gas chromatography combined with mass spectrometry detection (GC-MS). Parameters potentially affecting the performance of the sample preparation method (sample pH, ionic strength, type and volume of dispersant and extractant solvents) were systematically investigated using both multi- and univariant optimization strategies. Under final working conditions, analytes were extracted from 10 mL water samples by addition of 1 mL of acetone (dispersant) containing 60 μL of chlorobenzene (extractant), without modifying either the pH or the ionic strength of the sample. Limits of quantification (LOQs) between 2 and 14 ng L⁻¹, inter-day variability (evaluated with relative standard deviations, RSDs) from 9% to 14% and good linearity up to concentrations of 10,000 ng L⁻¹ were obtained. Moreover, the efficiency of the extraction was scarcely affected by the type of water sample. With the only exception of 2-ethylhexyl-p-dimethylaminobenzoate (EHPABA), compounds were found in environmental water samples at concentrations between 6 ± 1 ng L⁻¹ and 26 ± 2 ng mL⁻¹.     

Flow-injection solid surface lanthanide-sensitized luminescence sensor for determination of <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

A single optosensing device based on lanthanide-sensitized luminescence was developed for determination of p-aminobenzoic acid (PABA). The method is based on the formation of a complex between PABA and Tb(III) immobilized on the solid phase (QAE A-25 resin) placed inside the flow cell. NaCl (1 M) was used as carrier solution and HCl (0.05 M) as eluent. The sample solutions of PABA (100 μL) containing Tb(III) and buffered at pH = 6.0 were injected into the carrier stream and the luminescence was measured at λ _ex = 290 nm and λ _em = 546 nm. The method shows a linear range from 0.2 to 6.0 μg mL⁻¹ with an RSD of 1.2% (n = 10) and a sampling frequency of 22 h⁻¹. A remarkable characteristic of the method is its high selectivity which allows it to be satisfactorily applied to the analysis of PABA in pharmaceutical samples without prior treatment. Figure Typical emission bands of Tb(III) in a solid-phase PABA–Tb(III) luminescence spectrum     

Comparative study of screening methodologies for ochratoxin A detection in winery by-products

The worldwide contamination of winery by-products by mycotoxins may present a serious hazard to human and animal health. Mycotoxins are secondary metabolites of fungi with possible adverse effects on humans, animals, and crops that result in illnesses and economic losses. Mycotoxins are under continuous survey in Europe, but the regulatory aspects still need to be set up for winery by-products, which may be used in animal feed. The aim of this study was to implement a simple but reliable analytical methodology for ochratoxin A (OTA) quantification in grape pomaces in order to perform a survey of samples from the Douro Demarcated Region, Portugal. The method involved a unique preparation step, solvent extraction, followed by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection. A comparative study was performed with two extraction solvents (ethyl acetate and methanol) as well as using extraction on an immunoaffinity column. The linearity range for OTA analysis was 0.05–23.5 μg L⁻¹ with a detection limit of 0.05 μg L⁻¹ and a precision (expressed by the coefficient of variation under repeatability conditions) of 0.4–14.7%. The percentage of recovery was on average 23.5 ± 3.6% (extraction with ethyl acetate) or 70.1 ± 2.5% (extraction with 70% methanol). Accounting for the recovery factor and the chromatographic detection limit, as well as the preconcentration factor, the limit of detection in grape pomaces is 0.04 μg kg⁻¹ (ethyl acetate extraction) and 0.33 μg kg⁻¹ (methanol extraction). Samples from 12 out of 13 sites in the Douro Demarcated Region showed OTA presence with concentrations not exceeding 0.4 μg kg⁻¹. Both developed methods for evaluation of OTA in grape pomace are simple but efficient. Figure Extraction of ochratoxin A (OTA) from grape pomaces allows simple but efficient quantification of OTA in winery by-products by HPLC-FL     

Spectrophotometric determination of total protein in serum using a novel near-infrared cyanine dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine

The application of near-infrared (NIR) dyes (λ _em > 750 nm) to the analysis of biological samples shows much promise, because the long emission wavelengths of such dyes allow interferences from biomolecule matrices to be minimized. In this paper, a novel NIR dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric determination of total protein in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportional to the concentration of protein. The linear range of the method was found to be 0.04–0.5 μg mL⁻¹ for bovine serum albumin (BSA) and human serum albumin (HSA), and detection limits of 5 ng mL⁻¹ were obtained for these substances. The maximum binding number of BSA with DCDSTCY was measured to be 133. The method proposed here has been applied to the quantitation of total protein in serum, and recoveries of 96.6–104% were achieved. Figure Near-infrared probe for protein determination