An Indirect Competitive Enzyme-linked Immunosorbent Assay for Simultaneous Determination of Florfenicol and Thiamphenicol in Animal Meat and Urine

A high-affinity polyclonal antibody was prepared by immunizing animals with haptens FFD and FFM. Under the optimal combination of coating antigen and antibody, an indirect competitive enzyme-linked immunosorbent assay (icELISA) for simultaneous detection of florfenicol and thiamphenicol residues in animal meat and urine samples was developed. The icELISA showed an IC₅₀ value of 1.32 ng mL^?1 for florfenicol and 2.13 ng mL^?1 for thiamphenicol, respectively. The linear ranges were from 0.31 to 5.61 ng mL^?1 with a limit of detection of 0.12 ng mL^?1 for florfenicol, and 0.41 to 11.2 ng mL^?1 with a limit of detection of 0.15 ng mL^?1 for thiamphenicol, respectively. The average recoveries of florfenicol and thiamphenicol in spiked samples ranged from 77.2% to 116.0% with a relative standard deviation of less than 15%. Therefore, this proposed icELISA provided a valid detection method for florfenicol and thiamphenicol residues in animal tissue and urine samples.     

Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic assay for several (fluoro)quinolones in milk

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a visual colloidal gold-based immunochromatographic assay (CGIA) for simultaneous determination of ofloxacin, marbofloxacin, and fleroxacin residues in milk using polyclonal antibodies. The half-maximum inhibition concentrations (IC₅₀) of ofloxacin, marbofloxacin, fleroxacin, and limits of detection (LODs; calculated as IC₁₅ values) are between 0.20 and 0.53?ng mL^?1, and between 0.02 and 0.05?ng mL^?1, respectively. The average recoveries range from of 78% to 113%, and the coefficients of variation of intra- and inter-assays are between 2 and 11%, and 3 to 19%, respectively. The LODs for ofloxacin, marbofloxacin, fleroxacin in milk are between 3.5 and 8.9?ng mL^?1. The visual minimum detection limit of the optimized CGIA is 2?ng mL^?1 for milk samples. The detection process can be completed within 10?min. The strips can be stored at 4?°C for 8?weeks without significant loss of activity. The results of the analysis of spiked samples showed that the CGIA can be applied to preliminary, fast, and on-site screening of milk samples. The ELISA and CGIA allow for a rapid, sensitive, and low-cost determination of (fluoro)quinolones residues in milk samples.

Label-free immunosensor for monitoring vitellogenin as a biomarker for exogenous oestrogen compounds in amphibian species

A label-free, optical waveguide lightmode spectroscopy based immunosensor was developed for frog (Bombina orientalis) vitellogenin (Vtg) determination in biological samples as a biomarker for exogenous oestrogen compounds. Antibody against Vtg was produced in rabbits immunised with purified lipovitellin (Lpv), a precursor of Vtg, from the homogenised ovary of oriental fire-bellied toads (B. orientalis). The purified protein and Lpv/Vtg-specific serum were applied in both competitive and direct immunoassay formats using optical waveguide lightmode spectroscopy immunosensor. When measuring Vtg in direct manner, the Lpv antibody (1.76 µg mL^?1) was immobilised on the sensor surface, and the linear measuring range for Vtg was 0.1–10 µg mL^?1. During the competitive measurement, 100 ng mL^?1 Lpv was applied for the immobilisation. The linear measuring range for Vtg was 0.5–50 ng mL^?1. We studied the relative substrate specificity of the antibody, and it was concluded that the method is suitable for the sensitive and selective determination of Vtg levels in toads. Heart, liver and gonad samples from male animals were spiked with Vtg and were analysed using the newly developed method, and female toads and spawn samples were tested and compared to the calibration curve obtained by the spiked samples.     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

Monitoring chlorophenols in industrial effluents by solid-phase microextraction–gas chromatography–mass spectrometry

A method for the determination of 19 chlorophenols in industrial effluents samples using solid-phase microextraction (SPME) coupled to gas chromatography–mass spectrometry has been developed. Four kinds of different SPME fibres have been studied. Among them, the polyacrylate and carbowax®-divinylbenzene fibres were the most adequate. The extraction process was optimized by means of the experimental design, which allows the study of a large number of factors with a reasonable number of experiments. The optimized method allows the determination of the studied chlorophenols in complex matrices with a high organic content with detection limits down to 0.07?ng?mL^?1 and RSD ranging from 4.4% to 13.8%. The recovery studies with spiked real effluent samples at low levels of chlorophenols ranged from 59.8% to 142.1% for the lowest level (0.5?ng?mL^?1) and from 79.6% to 115.8% for the highest spiked level (2?ng?mL^?1). These results show the suitability of the proposed method to monitor chlorophenols in complex samples. 2,4,5-TCP was detected at concentrations close to its limits of detection in effluents coming from an oil refinery.     

Voltametric and Flow Injection Determination of Oxytetracycline Residues in Food Samples Using Carbon Fiber Microelectrodes

A voltammetric method for the determination of the antibiotic oxytetracycline (OTC) in food samples is reported. Carbon fiber microelectrodes (CFMEs), which allow voltammetric measurements to be performed in a small volume (1 mL) of the analyte extract from the samples, are employed. Repeatable electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. Under the optimized square‐wave conditions, a linear calibration plot for OTC was obtained in the 1.0×10^?6–1.0×10^?4 mol L^?1 range, with a detection limit of 2.9×10^?7 mol L^?1 (150 ng mL^?1) OTC. The determination of OTC by a flow‐injection method with amperometric detection using a homemade flow cell specially designed to work with CFMEs, was also evaluated using pure acetonitrile as the carrier. The SW voltammetric method was applied to the determination of OTC in spiked milk and eggs samples, at 100 ng mL^?1 and 200 ng g^?1 levels, respectively. The procedure involved the extraction of the analyte in ethyl acetate, evaporation of the solvent and reconstitution of the residue in acetonitrile ?5.0×10^?4 mol L^?1 tetrabutylammonium perchlorate medium. Recoveries of 96±8 and 91±8% were obtained for milk and eggs, respectively, by applying the standard additions method.     

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Rabbit Monoclonal Antibody-Based Lateral Flow Immunoassay Platform for Sensitive Quantitation of Four Sulfonamide Residues in Milk and Swine Urine

Based on the available rabbit monoclonal antibody (RabMAb), a rapid and sensitive lateral flow immunoassay (LFA) platform has been developed for quantitative detection of four sulfonamide residues(SRs) of sulfadiazine (SD), sulfathiazole (STZ), sulfapyridine (SP), and sulfamethoxazole (SMX).Within the designed LFA competitive format assay, which was based on antigen-antibody properties, the hapten conjugate N¹-[4-(carboxymethyl)-2-thiazolyl] sulfanilamide linked to protein ovalbumin (TS-OVA) and goat anti-rabbit antibody were sprayed as capture and control reagents, respectively, and then the antibody was conjugated to colloidal gold particles as the detection reagent. With quantitative assessment aided by a colorimetric strip reader, the sensitivities of the established LFA method for SD, STZ, SP, and SMX were 0.91 ng mL^?1, 0.10ng mL^?1,0.12ng mL^?1, and 2.13ng mL^?1, and the half-maximum inhibition concentrations (IC₅₀) were 5.19 ng mL^?1, 1.25 ng mL^?1, 0.66 ng mL^?1, and 24.14 ng mL^?1, respectively. The recoveries at three spiked levels (5, 20, 50 ng mL^?1for SD, STZ, and SP; 20, 50, 100 ng mL^?1 for SMX) were in the range of 78.02–135.10% and 76.40–137.16% for milk and swine urine, respectively. More importantly, the detection performance of the established platform was consistent with that of in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed the potential for fast, sensitive and relatively accurate quantification of four sulfonamide residues in practical uses.     

Utilisation of polypyrrole modified electrode for the determination of pesticides

Cyclic voltammetric studies of isoproturon and carbendazim using polypyrrole modified glassy carbon electrode were carried out. The electrode and reaction conditions, which yielded maximum current signal, were selected for the development of stripping voltammetric procedure for the determination of the pesticides. The oxidation peak around 1.3?V obtained for isoproturon and carbendazim while employing polypyrrole modified electrode showed maximum current response. This peak was chosen for stripping analysis using square wave mode. The experimental parameters were optimized and the calibration plot was obtained. The LOD was 0.5?ng?mL^?1 for isoproturon and 5?ng?mL^?1 for carbendazim. The relative standard deviation for 5 identical measurements was 2.81% and 3.33% for isoproturon and carbendazim respectively. The applicability of the method was verified by determining the pesticides in spiked soil and water samples.     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Catalytic Spectrophotometric Determination of Titanium(IV) Using Methylene Blue‐Ascorbic Acid Redox Reaction

A highly selective and sensitive catalytic method for the determination of trace amounts of titanium(IV) was developed. The method is based on the catalytic effect of titanium(IV) on the methylene blue‐ascorbic acid redox reaction. The reaction was followed spectrophotometrically by measuring the change in absorbance of methylene blue at 665 nm, 5 minutes after the initiation of the reaction. In this study experimental parameters were optimized and the effect of the presence of various cations and some anions on the determination of titanium(IV) was examined. The calibration graph was linear in the range of 3‐25 ng mL^?1 of titanium(IV). The relative standard deviation for the determination of 10 and 20 ng mL^?1 of titanium(IV) were 2.64% and 1.51%, respectively (n = 8). The detection limit calculated from three times of standard deviation of blank 3S_b was 0.6 ng mL^?1. The method was successfully applied to the determination of titanium(IV) in tap water and ore samples.     

Synthesis of derivatives and production of antiserum for class specific detection of pyrethroids by indirect ELISA

Two series of haptens including 3-phenoxybenzoic acid (PBA) and 3-(2-chloro-3, 3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclo-propanecarboxylic acid (CF₃MPA) were used to prepare immunogens through attachment of 4-C or 6-C handles. Class selective antibodies were produced by immunising rabbits. Ab502 showed the highest reactivity towards tau-fluvalinate (IC₅₀ 1.3 ng mL^?1), λ-cyhalothrin (IC₅₀ 2.3 ng mL^?1), cyfluthrin (IC₅₀ 2.2 ng mL^?1) and fenpropathrin (IC₅₀ 18.5 ng mL^?1) among the antibodies in a competitive ELISA. The effects of methanol, pH and salt concentration were optimised for maximum efficiency of the ELISA (Enzyme-Linked ImmunoSorbent Assay). Ab502 (1:80000)/2-OVA-1(0.2 µg mL^?1) was chosen for ELISA optimisation. Finally, 0.05 M phosphate buffered saline (PBS) at pH 6.5 containing 30% methanol (v/v) was used to dilute the standards. Target analytes in honey samples were extracted with ethyl acetate by sonication. The samples were spiked with three different concentrations of each compound (tau-fluvalinate, 0.5 ng g^?1, 3 ng g^?1, 12 ng g^?1; λ-cyhalothrin and cyfluthrin 1 ng g^?1, 5 ng g^?1, 65 ng g^?1). The recoveries were 36–59% at the lowest spiking concentration and 61–81% at the higher concentration. This assay might be useful to screen pyrethroid residues in honey or other matrix.     

Simultaneous Determination of Ten Antibiotic Residues in Milk by UPLC

An analytical method for rapid screening and quantitative determination of ten antibiotics (chloramphenicol, thiamphenicol, tetracycline, oxytetracycline, chlortetracycline, metacycline, doxycycline, cefoperazone, ceftriaxone and cefaclor) residues in milk was developed using ultra performance liquid chromatography with photodiode array detector. After extraction with McIIvaine buffer + methanol (8 + 2), the extract was cleaned up with solid-phase extraction cartridge. The conditions of sample extraction, cleaning and separation were optimized. The average spiked recoveries of milk samples were 52.1–68.0, 70.1–81.0 and 76.2–101.0% at spiked levels of 0.1, 0.5, 2.5 μg g^?1, respectively with precisions of 3.3–15.9%. The limits of detection and quantification were 0.003–0.022 and 0.01–0.08 μg g^?1, respectively. The proposed method has been applied to the determination of antibiotics in actual milk samples with satisfactory results.     

Immersed solvent microextraction of aryloxyphenoxypropionate herbicides from aquatic media

An immersed solvent microextraction (SME) method was successfully developed for the trace enrichment of aryloxyphenoxypropionate herbicides from aquatic media. A microdrop of toluene was used as the extraction solvent. Some important extraction parameters such as type of solvent, solvent dropsize, stirring rate, ionic strength and extraction time were investigated and optimized. The microdrop volume of 1.5?µL, a sampling time of 25?min, and use of toluene were major parameters for achieving high enrichment factors. The linearity was studied by preconcentration of 4?mL of the water samples spiked with a standard solution of aryloxyphenoxypropionates at the concentration range of 0.15 to 30?ng?mL^?1. The coefficient of determination was satisfactory (r ^2?>?0.99) for all the studied analyte and the relative standard deviations (RSD%) values under the optimized condition were found to be 1.7 to 14.2% at the concentrations of 1 and 10?ng?mL^?1. The enrichment factors were from 217 to 403 for the samples spiked at 1?ng?mL^?1. Detection limits were obtained to be in the range of 0.05 to 0.15?ng?mL^?1 using time-scheduled selected ion monitoring (SIM). The EI mass spectra of these herbicides revealed that fenoxaprop-P-ethyl and quizalofop-P-ethyl exhibited [M-COOC₂H₅]⁺ as the base peak while, clodinafop-propargyl, haloxyfop-etotyl and haloxyfop-P-methyl showed [M-C₂H₄COOC₃H₃]⁺, [M-CH₂COOC₄H₈O]⁺ and [M-COOCH₃]⁺ as the base peaks, respectively. The developed method was successfully applied to the extraction and determination of aryloxyphenoxypropionates in river water samples.     

A Highly Sensitive Electrochemical Immunosensor for Fumonisin B1 Detection in Corn Using Single‐Walled Carbon Nanotubes/Chitosan

A sensitive electrochemical immunosensor was developed for detecting fumonisin B₁ (FB₁) in corn using the single‐walled carbon nanotubes/chitosan. The detection mechanism of immunosensor was based on an indirect competitive binding to a fixed amount of anti‐FB₁ between free FB₁ and FB₁‐bovine serum albumin, which was conjugated on covalently functionalized nanotubes/chitosan laid on the glass carbon electrode. The anti‐rabbit immunoglobulin G secondary antibody labeled with alkaline phosphatase was then bound to the electrode surface through reactisubstrate α‐naphthyl phosphate, which produced electrochemical signal. Under optimized conditions, this method could detect FB₁ from 0.01 to 1000 ng mL^?1 with a detection limit of 2 pg mL^?1. This is well below the detection limit required from European Union legislation, 2–4 mg L^?1. Moreover, good recoveries were obtained for the detection of spiked corn samples and actual corn samples. As the method has good sensitivity and recovery for detecting FB₁, it is a practical detection method.     

Molecularly imprinted polymers for the solid‐phase extraction of four fluoroquilones from milk and lake water samples

A method based on molecular crowding and ionic liquids as reaction solvents has been used for the synthesis of molecularly imprinted polymers. Levofloxacin was selected as the template, polymethyl methacrylate was the molecular crowding agent, and 1‐butyl‐3‐methylimidazolium tetrafluoroborate (ionic liquid) was selected as the reaction solvent and porogen. The optimized proportion for the mixed porogen was dimethyl sulfoxide/ionic liquid/polymethyl methacrylate 1:1.6:5 in chloroform (150 mg mL^?1). The morphology and chemical composition of levofloxacin imprinted polymers were assessed by scanning electron microscopy and Fourier transform infrared spectroscopy. The absorption experiments demonstrated that the levofloxacin imprinted polymers possess high selective recognition property to levofloxacin and analogs, including enrofloxacin, ciprofloxacin and gatifloxacin, which all belong to fluoroquinolones. An extraction method using levofloxacin imprinted polymers as sorbent followed by high‐performance liquid chromatography analysis was optimized for the determination of four fluoroquinolones in milk and lake water samples. Under the optimized conditions, good linearity was observed in a range of 5–1000 ng g^?1 with the limit of detection between 0.3 and 0.5 ng g^?1 for the four fluoroquinolones. The recoveries at three spiked levels ranged 82.4–98.3% with the relative standard deviation ≤4.9.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of glycyrrhizic acid

Glycyrrhizic acid (GL) is a major active compound of licorice. The specific monoclonal antibody (MAb) (designated as 8F₈A₈H₄2H₇) against GL was produced with the immunogen GL–BSA conjugate. The dissociation constant (K _d) value of the MAb was approximately 9.96×10⁻¹⁰ M. The cross reactivity of the MAb with glycyrrhetic acid was approximately 2.6%. The conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA adapted with a modified procedure were established using the MAb. The IC₅₀ value and the detect range by the conventional icELISA were 1.1 ng mL⁻¹ and 0.2–5.1 ng mL⁻¹, respectively. The IC₅₀ value and the detect range by the simplified icELISA were 5.3 ng mL⁻¹ and 1.2–23.8 ng mL⁻¹, respectively. The two icELISA formats were used to analyze GL contents in the roots of wild licorice and different parts of cultivated licorice (Glycyrrhiza uralensis Fisch). The results obtained with the two icELISAs agreed well with those of the HPLC analysis. The correlation coefficient was more than 0.98 between HPLC and the two icELISAs. The two icELISAs were shown to be appropriate, simple, and effective for the quality control of raw licorice root materials.     

Simultaneous Determination of Aluminum,Cadmium and Lead in Whole Blood by Simultaneous Atomic Absorption Spectrometry with Oxygen Charring

A method for the simultaneous determination of aluminum (Al), cadmium (Cd) and lead (Pb) in whole blood has been developed by using simultaneous atomic absorption spectrometry (SIMAAS) with oxygen charring. The optimized conditions for the simultaneous determination of Al, Cd and Pb were obtained in the presence of palladium (Pd) as the chemical modifier, using 600 °C and 2400 °C as the pyrolysis and the atomization temperature, respectively. The whole blood samples were diluted 1+5 (v/v) directly with 0.1% (v/v) Triton X‐100. Oxygen was employed to eliminate the interference of carbonaceous residues in the charring step before pyrolysis. The calibration curves were carried out with aqueous standard solutions and the linear ranges were 0–40 ng mL⁻¹, 0–4 ng mL⁻¹ and 0–40 ng mL⁻¹ for Al, Cd and Pb, respectively. The detection limits were 0.96 ng mL⁻¹ (19.2 pg) for Al, 0.03 ng mL⁻¹ (0.6 pg) for Cd and 0.60 ng mL⁻¹ (12.0 pg) for Pb. The spiked recoveries of Al, Cd and Pb in whole blood were 98.0%, 100.0% and 101.7%, respectively. The accuracy of the proposed method was evaluated with the analysis of a whole blood certified reference material (Seronorm, level 2). The found concentrations were in agreement with the recommended values. The proposed method has been successfully applied to the simultaneous determination of Al, Cd and Pb in whole blood of healthy volunteers before and after eating barbecued foods.     

A Monoclonal Antibody-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Olaquindox in Animal Feed

A reliable indirect competitive enzyme-linked immunosorbent assay (ELISA) based on a new specific monoclonal antibody was developed to determine olaquindox in animal feed. The influence of several physicochemical factors (nonfat dried milk solution, organic solvent, incubation time) on the immunoassay was investigated. In the optimized system, the 50% inhibition concentration was 9.66 ± 1.81 µ g L^?1. The limits of detection for porcine, chicken, and fish feed were 0.28, 0.46, and 0.48 µg kg^?1. The limits of quantification were 1.00 µg kg^?1 for the feed samples. The recoveries from porcine, chicken, and fish feed spiked with olaquindox were 90–104%, 77–103%, and 78–107%, respectively, with coefficients of variation (CVs) between 3.8 and 14.1%. The cross-reactivity was less than 2.08% with four structurally related compounds and no recognition of five other restricted or forbidden drugs was observed. Parallel analysis of the three spiked feed samples showed comparable results between the indirect competitive ELISA and the standard high-performance liquid chromatography method in China (R² = 0.9985 for porcine feed, R² = 0.9896 for chicken feed, and R² = 0.9987 for fish feed). These data suggest that the developed indirect competitive ELISA is a specific and convenient method and is suitable for olaquindox determination in animal feed.     

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.