Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

Abstract

A Chemiluminescence Enzyme‐Linked Immuno‐Sorbent Assay (CL‐ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti‐thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC₅₀ and the limit of detection (LOD) values were 60 ng mL^?1 and 9 ng mL^?1, respectively, similar to those of colorimetric ELISA with a calibration range of 9–15,000 ng mL^?1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid‐liquid extraction or the graphitized carbon‐based solid phase extraction.

The calibration curves in honeybee extracts from liquid‐liquid procedure gave an IC₅₀ of 141 ng mL^?1 and a LOD of 17 ng mL^?1. In case of extracts obtained by SPE these values were 139 ng mL^?1 and 15 ng mL^?1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL^?1 of thiram was 72% for SPE, higher than for liquid‐liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid‐liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia.     

Determination of Protoberberine Alkaloids in Coptis chinensis by Microextraction and High Performance Liquid Chromatography

Sample preparation technique based on an organic filter membrane (pH-resolved filter membrane microextraction) (pH-RFMME) was developed, coupled with high-performance liquid chromatography, and used to determine protoberberine alkaloids (jatrorrhizine, epiberberine, coptisine, palmatine, and berberine) in Coptis chinensis at different pH values through a one-step procedure. This green procedure provides a desirable sample pretreatment technology. The main variables affecting the extraction such as filter membrane area (or volumes of extraction solvents), sample pH, eluent pH, ionic strength, extraction stirring rate, extraction time, and sample volume were optimized. Under the optimized conditions, the enrichment factors of the analytes were 40.4–52.0, the linear ranges were 3.2–6250 ng · mL^?1 for jatrorrhizine and epiberberine, 6.0–12000 ng · mL^?1 for coptisine, 1.8–3600 ng · mL^?1 for palmatine, and 18.8–18800 ng · mL^?1 for berberine, with r ² ≥ 0.9945. The limits of detection were less than 0.3 ng · mL^?1. Satisfactory recoveries (84.8%–115.5%) and precision (1.8%–10.0%) were also achieved. These results confirmed that pH-RFMME is a simple, rapid, practical, and environmentally friendly method to isolate analytes that exhibit significant differences in acidity or alkalinity from complex samples.     

Determination of Alkylphenols in Water by Dispersive Liquid–Liquid Microextraction Based on Solid Formation without a Disperser

Dispersive liquid–liquid microextraction based on solid formation without a disperser combined with high-performance liquid chromatography has been developed for the determination of 4-tert-butylphenol, 4-n-nonylphenol, and 4-tert-octylphenol. This method is rapid, easy, and uses only 10 µL of a low toxicity organic solvent (1-hexadecanethiol) for the extraction solvent and no disperser solvent. The extraction time and centrifugation time require less than 10 min. The linear range was 1–500 ng mL^?1 for 4-tert-butylphenol, 2–1000 ng mL^?1 for 4-tert-octylphenol, and 5–500 ng mL^?1 for 4-n-nonylphenol with r² ≥ 0.9986. The detection limits were between 0.2 and 1.5 ng mL^?1. The recoveries of lake and river water samples were in the range of 79% to 108%, and the relative standard deviations were 5% to 10%.     

Determination of methylisothiocyanate in soil and water by HS-SPME followed by GC–MS–MS with a triple quadrupole

Methylisothiocyanate (MITC) is the main degradation product of metam sodium, a soil disinfectant widely used in agriculture, and is responsible for its disinfectant properties. Because MITC is highly toxic and volatile, metam sodium has to be applied in a manner that tries to reduce atmospheric emissions but still maintains adequate concentration of MITC in soil to ensure its disinfectant effect. Thus, monitoring of MITC concentrations in soil is required, and to this end sensitive, fast, and reliable analytical methods must be developed. In this work, a headspace solid-phase microextraction (HS-SPME) method was developed for MITC determination in water and soil samples using gas chromatography-tandem mass spectrometry (GC–MS–MS) with a triple-quadrupole analyzer. Two MS–MS transitions were acquired to ensure the reliable quantification and confirmation of the analyte. The method had linear behavior in the range tested (0.026–2.6 ng mL^?1 in water, 1–100 ng g^?1 in soil) with r ² over 0.999. Detection limits were 0.017 ng mL^?1 and 0.1 ng g^?1 in water and soil, respectively. Recoveries for five replicates were in the range 76–92 %, and RSD was below 7 % at the two spiking levels tested for each matrix (0.1 and 1 ng mL^?1 for water, 4 and 40 ng g^?1 for soil). The potential of using multiple HS-SPME for analyzing soil samples was also investigated, and its feasibility for quantification of MITC evaluated. The developed HS-SPME method was applied to soil samples from experimental plots treated with metam sodium following good agriculture practices. Figure

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Determination of DNA Methylation and Hydroxymethylation Levels in Biological Samples by Field-Amplified Sample Injection-Capillary Zone Electrophoresis with UV Detection

In this study, a method of field-amplified sample injection coupled with capillary zone electrophoresis with ultraviolet detection was established for evaluation of DNA methylation and hydroxymethylation levels in biological materials. By modifying an existing method, the separation of cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) was performed on an uncoated capillary column (40 cm × 75 μm I.D.) using 300 mmol L⁻¹ tris solution (pH 2.90) as running buffer and detected at 280 nm. The detection limits (S/N = 3) of the method were 0.004 ng mL⁻¹ for cytosine (C), 0.01 ng mL⁻¹ for 5-methylcytosine (5-mC), and 0.02 ng mL⁻¹ for 5-hydroxymethylcytosine (5-hmC). The proposed method has been successfully applied to the evaluation of DNA methylation and hydroxymethylation levels of blood samples from 15 hepatocellular carcinoma patients and 5 liver cirrhosis patients and liver tissues from 50 pairs of tumor and matched tumor-adjacent samples.

     

Determination of Estrone and 17β-Estradiol in Water Samples Using Dispersive Liquid–Liquid Microextraction Followed by LC

A new method, termed dispersive liquid–liquid microextraction (DLLME), was developed for the extraction and pre-concentration of estrone (E1) and 17β-estradiol (E2) in water samples. The samples were extracted by 0.50 mL methanol (disperser solvent) containing 25.0 μL tetrachloroethane (extraction solvent). Important factors such as the volume and type of extraction and disperser solvent, extraction time and salt effect were studied. Under optimum conditions, the enrichment factors and the limits of detection were 347 and 0.2 ng mL^?1 for E1, and 203 and 0.1 ng mL^?1 for E2, respectively. The linear range was 0.5–5,000 ng mL^?1. Compared to other methods, DLLME–LC–VWD has advantages for E1 and E2 analysis in water: high enrichment factor, low cost, simplicity, quick and easy operation.     

Simultaneous Determination of Abacavir,Efavirenz and Valganciclovir in Human Serum Samples by Isocratic HPLC-DAD Detection

A rapid, sensitive, and specific reverse phase high performance liquid chromatography with diode array detection procedure for the simultaneous determination of abacavir, efavirenz and valganciclovir in spiked human serum is described. Separation was performed on a 5 μm Waters Spherisorb column (250 × 4.6 mm ID) with acetonitrile: methanol:KH₂PO₄ (at pH 5.00) (40:20:40 v/v/v) isocratic elution at a flow rate of 1.0 mL min⁻¹. Calibration curves were constructed in the range of 50–30,000 ng mL⁻¹ for abacavir and efavirenz, and 10–30,000 ngmL⁻¹ for valganciclovir in serum samples. The limit of detection and limit of quantification concentrations of the HPLC method were 3.80 and 12.68 ng mL⁻¹ for abacavir, 2.61 and 8.69 ng mL⁻¹ for efavirenz, 1.30 and 4.32 ng mL⁻¹ for valganciclovir. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in human serum.

     

Determination of silver(I) ion in water samples by graphite furnace atomic absorption spectrometry after preconcentration with dispersive liquid-liquid microextraction

A method was developed for the determination of silver ion (Ag) by combining dispersive liquid-liquid microextraction preconcentration with graphite furnace atomic absorption spectrometry. Diethyldithiocarbamate was used as a chelating agent, and carbon tetrachloride and methanol as extraction and dispersive solvent. Factors influencing the extraction efficiency of Ag and its subsequent determination were studied and optimized. The detection limit is 12 ng L^?1 (3 s) with an enrichment factor of 132, and the relative standard deviation is 3.5% (n?=?7, at 1.0 ng mL^?1). The method was successfully applied to the determination of trace amounts of Ag in water samples.     

Determination of Biogenic Amines in Cheese Using Simultaneous Derivatization and Microextraction Method Followed by Gas Chromatography–Mass Spectrometry

Microwave-assisted extraction and dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry as a sensitive and efficient method was applied to extract and determine four biogenic amines (BAs) in Iranian Lighvan cheese samples. Carrez solutions were used for the sedimentation of proteins. Effective factors on the performance of microextraction were studied and optimized. The proposed method showed good linear ranges from 5 to 500 ng mL^?1, with the coefficients of determination higher than 0.9929. Average recoveries were between 97 and 103%. Limits of detection for all analyzed BAs ranged from 5.9 to 14.0 ng g^?1, and limits of quantitation ranged between 19.7 and 46.2 ng g^?1. Compared with previous methods, the proposed method is simple, fast, accurate, and precise and gives low detection limits for investigating trace amounts of BAs in Iranian Lighvan cheese samples. The levels of four BAs were determined in five Lighvan cheese samples. Cadaverine was found as prevailing amine in the cheese samples. Putrescine, tyramine, and histamine were present at the second, third, and fourth highest levels, respectively.     

Accuracy Profile Theory for the Validation of an LC–MS-MS Method for the Determination of Risperidone and 9-Hydroxyrisperidone in Human Plasma

A sensitive and specific LC–MS-MS method is described for the simultaneous quantification of risperidone and 9-hydroxyrisperidone in human plasma. After extraction with tert-butyl methyl ether, plasma samples were separated on an Atlantis HILIC Silica C18 column (4.6 × 150 mm, 5 μm)with a mobile phase of ammonium formate buffer (10 mM, pH 4.0)/acetonitrile (40/60, v/v). Detection was by MS-MS. The method was fully validated according to the accuracy profile theory. It is based on β-expectation tolerance interval for the total measurement error which includes trueness and intermediate precision. The measurement uncertainty derived from β-expectation tolerance interval was estimated at each of the validation standards. The linearity fitted well over the range of 0.11–26.75 ng mL⁻¹ for risperidone with an LLOQ of 0.11 ng mL⁻¹, and for 9-hydroxyrisperidone, at a range of 0.15–37.8 ng mL⁻¹ with an LLOQ of 0.15 ng mL⁻¹. The intra- and inter-batch precision of risperidone were <5.71 and 8.22%, respectively. For 9-hydroxyrisperidone, the data were 5.78 and 6.48%. The recoveries were 88.78% (risperidone) and 70.35% (9-hydroxyrisperidone). The developed method was applied to a pharmacokinetic study of risperidone.

     

Utilization of water-contained surfactant-based ultrasound-assisted microextraction followed by liquid chromatography for determination of polycyclic aromatic hydrocarbons and benzene in commercial oil sample

In this work, a microextraction method, water-contained surfactant-based ultrasound-assisted, followed by high-performance liquid chromatography (HPLC) was developed for determination of five polycyclic aromatic hydrocarbons (PAHs) and benzene in commercial oil samples. During the microextraction method, a micellar solution as the only extraction solvent was injected into the oil sample in a conical bottom glass tube and formed a cloudy solution. The dispersion process was accelerated by applying ultrasound irradiation. Phase separation was done by centrifugation and then the lower sediment phase was directly analyzed by HPLC. A chemometrics approach was applied for the optimization of the extraction condition. Under the optimum conditions, the proposed method showed good linearity within the different ranges for different analytes (e.g., 0.10–200 ng mL^?1 for phenanthrene), the square of the correlation coefficient was higher than 0.999 and the appropriate limit of detection was in the range of 0.04–0.41 ng mL^?1. The recoveries in all cases were above 95 %.     

Simultaneous derivatization and extraction of iodine from milk samples by hollow fiber liquid-phase microextraction followed by gas chromatography-electron capture detection

A simple, low-cost and sensitive method is demonstrated for derivatization and extraction of iodine from milk samples using hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography-electron capture detection. Iodide ions are converted to iodine under acidic medium and in the presence of an oxidant. The generated iodine reacted with 3-pentanone in extraction vial to give 2-iodo-3-pentanone and was extracted into 4 μL of 1-octanol located in the lumen of a hollow fiber. Organic solvent was selected using one variable at a time optimization method and the other main factors affecting derivatization and HF-LPME procedures were evaluated using a Taguchi’s L₁₆ (4⁵) orthogonal array. Under optimal conditions, the method showed low limit of detection (0.5 ng mL^?1), wide linear range (1–2,000 ng mL^?1) with good correlation coefficient (0.9997) and acceptable relative standard deviation (4.6 %, n = 5). Finally, the developed method was successfully applied for determination of iodide in real samples including infant milk formulas and cow milk with reasonable relative recoveries (99.8–110.5 %).     

Analysis of dextromethorphan and pseudoephedrine in human plasma and urine samples using hollow fiber-based liquid–liquid–liquid microextraction and corona discharge ion mobility spectrometry

We report on the use of hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) followed by corona discharge ion mobility spectrometry for the determination of dextromethorphan and pseudoephedrine in urine and plasma samples. The effects of pH of the donor phase, stirring rate, ionic strength and extraction time on HF-LLLME were optimized. Under the optimized conditions, the linear range of the calibration curves for dextromethorphan in plasma and urine, respectively, are from 1.5 to 150 and from 1 to 100 ng mL^?1. The ranges for pseudoephedrine, in turn, are from 30 to 300 and from 20 to 200 ng mL^?1. Correlation coefficients are better than 0.9903. The limits of detection are 0.6 and 0.3 ng mL^?1 for dextromethorphan, and 8.6 and 4.2 ng mL^?1 for pseudoephedrine in plasma and urine samples, respectively. The relative standard deviations range from 6 to 8%.

Simultaneous preconcentration and determination of malachite green and fuchsine dyes in seafood and environmental water samples using nano-alumina-based molecular imprinted polymer solid-phase extraction

Molecular imprinted polymer for determination of malachite green (MG) and fuchsine basic (FU) dyes by spectrophotometry has been used, to develop a novel simultaneous extraction and preconcentration method. Molecularly imprinted layer-coated nano-alumina (MIP@Nano-Al₂O₃) as adsorbent was prepared by surface molecular imprinting technique, and characterised by FTIR spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis (EDAX) and thermogravimetric analysis (TGA). The method is based on simultaneous extraction of MG and FU dyes from aqueous solution by using molecularly imprinted polymer and measuring the absorbance at 617 and 546 nm for MG and FU, respectively. Parameters which affect the extraction efficiency such as pH, volume of eluent and amount of adsorbent were investigated and optimised. Linear calibration curves were obtained in the range of 2–750 ng mL^?1 for MG and 1–240 ng mL^?1 for FU under optimum conditions. Detection limit based on three times the standard deviation of the blank (3S_b) was 0.655 and 0.245 ng mL^?1 (n = 10) for MG and FU, respectively. The relative standard deviation (RSD) for 100 ng mL^?1 of MG and FU was 2.35 and 3.06% (n = 7), respectively. The method was applied to the simultaneous determination of the dyes in different seafood and environmental water samples.     

Determination of migrated phthalic acid residues into edible oils using a green mode of air-assisted liquid–liquid microextraction followed by high-performance liquid chromatography–diode array detector

In this study, for the first time, an organic solvent-free air-assisted liquid–liquid microextraction method has been reported for the extraction and preconcentration of phthalic acids (o-phthalic acid, m-phthalic acid, and p-phthalic acid) from edible oil samples. The method is based on the repeated aspirating/injection of an alkaline aqueous solution and the oil sample mixture in a conical bottom centrifuge tube to form a cloudy solution. After phase separation by centrifuging, the sedimented phase is directly analyzed by high-performance liquid chromatography–diode array detection. Under the optimum extraction conditions, the method showed low limits of detection and quantification between 0.11–0.29 and 0.28–0.91 ng mL^?1, respectively. Extraction recoveries and enrichment factors were from 81 to 97% and 406 to 489, respectively. The relative standard deviations for the analysis of 5 ng mL^?1 of each analyte were less than 5.9% for intraday (n = 6) and interday (n = 5) precisions. Finally, different oil samples were successfully analyzed using the proposed method and m-phthalic acid, and p-phthalic acid were determined in some of them at ng mL^?1 level.     

Solvent Bar Microextraction with HPLC for Determination and Protein-Binding Characteristics of Oleanolic Acid and Ursolic Acid

An improved solvent bar microextraction (SBME) combined with HPLC was applied to rapidly determine oleanolic acid (OA) and ursolic acid (UA) in Chinese medicinal herbs (CMHs), and to investigate drug–protein binding. Variables influencing SBME were investigated and optimized. Under optimal conditions, the linear ranges of 3.6–1,820 ng mL^?1 for OA and 4.2–2,080 ng mL^?1 for UA were obtained with square correlation coefficients of 0.9996 and 0.9997, respectively. The detection limits were 1.3 ng mL^?1 for OA and 1.5 ng mL^?1 for UA. The relative standard deviations were lower than 9.4 %. The protein-binding rates, the numbers of binding sites, and the binding constants of OA and UA were also obtained via the method. It has been demonstrated that SBME can not only be a simple, rapid and efficient sample preparation method for determination of active components from CMHs but also be a potential research protocol for protein-binding interactions.     

Dispersive Liquid–Liquid Microextraction Coupled with High-Performance Liquid Chromatography for Determination of Coumarin Compounds in Radix Angelicae Dahuricae

In this paper, two methods, organic solvent dispersive liquid–liquid microextraction (OS-DLLME) and ionic liquid dispersive liquid–liquid microextraction (IL-DLLME), coupled with high-performance liquid chromatography have been critically compared and introduced for the analysis of the eight coumarin compounds (psoralen, isopsoralen, bergapten, isobergapten, oxypeucedanin, imperatorin, osthole, and isoimperatorin) in Radix Angelicae Dahuricae samples. Experimental conditions have been investigated for both OS-DLLME and IL-DLLME. Under optimal conditions, the detection limits of the eight coumarin compounds obtained by OS-DLLME and IL-DLLME ranged between 0.002–0.026 ng mL⁻¹ and 0.013–0.66 ng mL⁻¹, respectively. The relative standard deviations (RSDs, n = 9) were lower than 8.7 and 8.4% with enrichment factors in the range of 145–380 and 130–230 folds for OS-DLLME and IL-DLLME, respectively. The results showed that there were no significant deviations between the two DLLME methods for the determination of the eight coumarin compounds. Both methods were simple, fast, efficient, and inexpensive. However, compared with IL-DLLME, the OS-DLLME technique exhibited a higher extraction capacity for the eight target analytes.

     

Oxidized Multiwalled Carbon Nanotubes as an SPME Fiber Coating for Rapid LC–UV Analysis of Benzimidazole Fungicides in Water

The potential of oxidized multiwalled carbon nanotubes as a fiber coating for solid-phase microextraction of carbendazim, thiabendazole, and thiophanate-methyl from aqueous solution has been investigated, and the mechanism of adsorption has been explored. When an analytical method combining SPME with high-performance liquid chromatography was applied to real environmental water samples, recoveries of the spiked compounds was 75.7–104.5%, the linear range was 5–500 ng mL^?1, the squared correlation coefficients (r ²) were from 0.9978 to 0.9984, LOD were from 0.3 to 1.5 ng mL^?1, and RSD were from 2.3 to 10.5%.     

Multivariate optimization of the hollow fiber-based liquid phase microextraction of lead in human blood and urine samples using graphite furnace atomic absorption spectrometry

The present study developed a liquid-phase microextraction based on hollow fiber coupled with graphite furnace atomic absorption spectrometry for the effective extraction and quantitation of lead from urine and blood samples. A multivariate design was used for the optimization of the experimental conditions to ensure high extraction efficiency. Six factors (solvent type, chelating agent, time extraction, temperature, donor phase pH, and acceptor phase pH) were obtained by screening eleven factors of the Plackett–Burman design; these were optimized using the central composite design of response surface methodology. The optimum conditions of donor phase pH, acceptor phase pH, temperature, and extraction time were 5, 9.5, 40 °C, and 120 min, respectively. In addition, oleic acid containing dicyclohexyl-18-krone-6 was used for the membrane phase. Under optimal conditions, the enrichment factor, limit of detection, and limit of quantification were obtained in the ranges of 21.3–18.7, 0.001–0.002 ng mL^?1, and 0.008–0.01 ng mL^?1, respectively, in urine and blood samples. The linearity of the calibration curve was established for the concentration of Pb in the range of 1–50 ng mL^?1 (r²?=?0.9983). Finally, the performance of the developed method was evaluated for the determination of lead in urine and blood samples, and satisfactory results were obtained (RSDs <?10% with recovery >?95).     

Determination of Anilines and Toluidines in Water by Salt-Assisted Dispersive Liquid–Liquid Microextraction Combined with GC-FID

Dispersive liquid–liquid microextraction (DLLME) assisted with salting-out was applied for the determination of five aromatic amines in water samples by using gas chromatography with flame ionization detection. In this extraction method, several factors influencing the extraction efficiency of the target analytes, such as extraction and disperser solvent type and their volume, salt addition and amount, and pH, were studied and optimized. Under the optimal DLLME conditions, good linearity was observed in the range of 4–1,000 ng mL⁻¹ with the RSDs from 1.2 to 7.9 %. The LODs based on S/N of 3 ranged from 0.2 to 3.4 ng mL⁻¹ and the enrichment factors ranged from 207 to 4,315. The proposed method was successfully applied to the water samples collected from the tap and the lake, and the relative recoveries were in the range of 87.7–108.4 %.